Patent application title: Compositions and Methods for Treating Disorders Associated with Abnormal Phosphate Metabolism
Inventors:
Eli Sprecher (Kiryat Tivon, IL)
Reuven Bergman (Haifa, IL)
IPC8 Class: AA61K3500FI
USPC Class:
424 931
Class name: Drug, bio-affecting and body treating compositions whole live micro-organism, cell, or virus containing
Publication date: 2008-08-28
Patent application number: 20080206195
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Patent application title: Compositions and Methods for Treating Disorders Associated with Abnormal Phosphate Metabolism
Inventors:
Eli Sprecher
Reuven Bergman
Agents:
Martin D. Moynihan;PRTSI
Assignees:
Origin: ARLINGTON, VA US
IPC8 Class: AA61K3500FI
USPC Class:
424 931
Abstract:
The present invention uncovers that mutations in GALNT3 gene encoding
UDP-N-acetyl-alpha-D-galactosamine:polypeptide
N-acetylgalactosaminyltransferase (GalNAc-T3) cause familial tumoral
calcinosis (FTC). Methods and pharmaceutical compositions useful for
treating disorders associated with abnormal phosphate metabolism are
provided. Specifically, inducers of GalNAc-T3 can be used to treat
hyperphosphatemia related disorders such as FTC, and on the other hand,
inhibitors of GalNAc-T3 can be used to treat disorders associated with
hypophosphatemia, such as hypophosphatemic rickets. The present invention
further provides methods and kits for diagnosing familial tumoral
calcinosis as well as for identifying agents suitable for treating
disorders associated with abnormal phosphate metabolism.Claims:
1-34. (canceled)
35. A method of treating a disorder associated with abnormal phosphate metabolism comprising providing to an individual in need thereof an agent capable of regulating an expression level and/or activity of GalNAc-T3, thereby treating the disorder associated with abnormal phosphate metabolism in said individual.
36. The method of claim 35, wherein said disorder is associated with hyperphosphatemia.
37. The method of claim 36, wherein said disorder associated with hyperphosphatemia is selected from the group consisting of familial tumoral calcinosis (FTC), hyperphosphatemic calcinosis, hemodialysis and chronic renal failure.
38. The method of claim 35, wherein said regulating is upregulating said expression level and/or activity of said GalNAc-T3, said upregulating is effected by an agent selected from the group consisting of:(a) an exogenous polynucleotide encoding at least a functional portion of GALNT3;(b) an agent capable of increasing expression of endogenous GalNAc-T3 in an individual;(c) an agent capable of increasing endogenous GalNAc-T3 activity in an individual;(d) an exogenous polypeptide including at least a functional portion of GalNAc-T3;(e) a GalNAc-T3 substrate; and(f) a GalNAc-T3-expressing cell.
39. The method of claim 38, wherein said exogenous polynucleotide encoding at least a functional portion of GALNT3 is set forth in SEQ ID NO:29.
40. The method of claim 35, wherein said disorder is associated with hypophosphatemia.
41. The method of claim 40, wherein said disorder associated with said hypophosphatemia is selected from the group consisting of X-linked vitamin D resistant hypophosphatemic rickets (HYP), hereditary hypercalciuria with hypophosphatemic rickets (HHRH), oncogenic hypophosphatemic osteomalacia (OHO) and X-linked hypophosphatemic rickets (PHEX).
42. The method of claim 35, wherein said regulating is downregulating said expression level and/or said activity of said GalNAc-T3, said downregulating is effected by an agent selected from the group consisting of:(a) a molecule which binds said GalNAc-T3;(b) an enzyme which cleaves said GalNAc-T3;(c) an antisense polynucleotide capable of specifically hybridizing with at least part of an mRNA transcript encoding GALNT3;(d) a ribozyme which specifically cleaves at least part of an mRNA transcript encoding GALNT3;(e) a small interfering RNA (siRNA) molecule which specifically cleaves at least part of a transcript encoding GALNT3;(f) a non-functional analogue of at least a catalytic or binding portion of said GalNAc-T3;(g) a molecule which prevents GalNAc-T3 activation or substrate binding.
43. The method of claim 42, wherein said mRNA transcript encoding GALNT3 is set forth in SEQ ID NO:29.
44. A method of diagnosing familial tumoral calcinosis (FTC) in an individual, the method comprising identifying in a polynucleotide sequence of the individual at least one nucleic acid substitution resulting in downregulation of an expression level and/or activity of GalNAc-T3, thereby diagnosing familial tumoral calcinosis in the individual.
45. The method of claim 44, wherein said polynucleotide sequence is an mRNA sequence encoding GALNT3 or a genomic sequence region including the GALNT3 gene.
46. A method of diagnosing familial tumoral calcinosis in an individual, the method comprising identifying in a polypeptide sequence of the individual at least one amino acid substitution capable of downregulating expression level and/or activity of GalNAc-T3, thereby diagnosing familial tumoral calcinosis in the individual.
47. The method of claim 46, wherein said polypeptide sequence is set forth in SEQ ID NO:28.
48. The method of claim 46, wherein said identifying said at least one amino acid substitution is effected using an antibody capable of differentially binding to at least one polymorph of said GalNAc-T3, said at least one polymorph includes said amino acid substitution capable of downregulating said expression level and/or said activity of GalNAc-T3.
49. A kit for diagnosing familial tumoral calcinosis in an individual, the kit comprising a packaging material packaging at least one reagent and instructions for use in diagnosing familial tumoral calcinosis, said at least one reagent for identifying in a polynucleotide sequence of the individual at least one nucleic acid substitution resulting in downregulation of an expression level and/or activity of GalNAc-T3, thereby diagnosing familial tumoral calcinosis in the individual.
50. The kit of claim 49, wherein said polynucleotide sequence is an mRNA sequence encoding GALNT3 or a genomic sequence region including the GALNT3 gene.
51. A kit for diagnosing familial tumoral calcinosis in an individual, the kit comprising a packaging material packaging at least one reagent and instructions for use in diagnosing familial tumoral calcinosis, said at least one reagent for identifying in a polypeptide sequence of the individual at least one amino acid substitution capable of downregulating expression level and/or activity of GalNAc-T3, thereby diagnosing familial tumoral calcinosis in the individual.
52. The kit of claim 51, wherein said polypeptide sequence is set forth in SEQ ID NO:28.
53. The kit of claim 51, wherein said reagent for identifying said at least one amino acid substitution comprises an antibody capable of differentially binding to at least one polymorph of said GalNAc-T3, said at least one polymorph includes said amino acid substitution capable of downregulating said expression level and/or said activity of GalNAc-T3.
Description:
FIELD AND BACKGROUND OF THE INVENTION
[0001]The present invention relates to methods of treating disorders associated with abnormal phosphate metabolism, and more particularly to the use of regulators of GalNAc-T3 in the treatment of disorders associated with hyperphosphatemia such as Familial tumoral calcinosis (FTC), hyperphosphatemic calcinosis, hemodialysis, and chronic renal failure, as well as disorders associated with hypophosphatemia, e.g., X-linked vitamin D resistant hypophosphatemic rickets (HYP), hereditary hypercalciuria with hypophosphatemic rickets (HHRH), oncogenic hypophosphatemic osteomalacia (OHO), and X-linked hypophosphatemic rickets (PHEX).
[0002]Phosphate-related abnormalities (i.e., hypophosphatemia or hyperphosphatemia) characterize a class of metabolic disorders manifesting with hyper- and dys-lipidemia, rickets and/or serious metastatic calcification, which can eventually lead to death. These disorders result from disrupted phosphate metabolism. Inorganic phosphate is absorbed in the intestinal tract in a process regulated by 1α,25-dihydroxyvitamin D3 (vitamin D3). On the other hand, phosphate excretion, which is regulated by the parathyroid hormone, takes place in both kidney and intestinal tract (i.e., fecal excretion). Moreover, the liver, skin and kidney are involved in the conversion of vitamin D3 to its active metabolite, calcitriol, which plays an active role in maintaining phosphate balance and bone mineralization.
[0003]Under normal conditions, a decrease in plasma phosphate level stimulates the production of vitamin D3 in the renal proximal tubule, resulting in increased absorption of calcium and phosphate. The increase in calcium level leads to a secondary suppression of the parathyroid hormone (PTH), which results in upregulation of the sodium-dependent phosphate transport in the renal proximal tubule.
[0004]Hyperparathyroidism, a condition characterized by overproduction of PTH in the parathyroid glands, results in hypophosphatemia and increased phosphate excretion due to inhibition of sodium-dependent phosphate transport in the kidney.
[0005]Other conditions which involve hypophosphatemia include vitamin D deficiency, which causes rickets in children and osteomalacia in adults, X-linked vitamin D resistant hypophosphatemic rickets (HYP), hereditary hypercalciuria with hypophosphatemic rickets (HHRH), Dent's disease including certain types of renal Fanconi syndrome, renal I alpha-hydroxylase deficiency (VDDR 1), defects in 1,25-dihydroxy vitamin D3 receptor (end organ resistance, VDDR II), oncogenic hypophosphatemic osteomalacia (OHO), and X-linked hypophosphatemic rickets (PHEX) [Francis, Nat. Genet. (1995), 11: 130-136; Rowe, Hum. Genet. (1996), 97: 345-352; Rowe, Hum. Mol. Genet. (1997), 6: 539-549).
[0006]On the other hand, hyperphosphatemia, i.e., increased plasma level of PO4, is often a result of renal insufficiency. End-stage renal insufficiency, a condition affecting approximately 250,000 individuals in the USA, can lead to metastatic calcification, i.e., the deposition of calcium phosphate in previously healthy connective tissues and solid organs. Thus, advanced renal failure, i.e., a glomerular filtration rate of less than 20 mL/min, causes a decrease in PO4 excretion and an increase of plasma PO4. However, other conditions may also decrease PO4 excretion. These include pseudohypoparathyroidism or hypoparathyroidism. Hyperphosphatemia can also result from excess administration of oral PO4, or from overuse of enema containing phosphate salts. Furthermore, hyperphosphatemia may result from migration of intracellular PO4 to the cell exterior. Such migration frequently occurs in diabetic ketoacidosis (regardless of systemic PO4 loss), bruise, non-traumatic rhabdomyolysis, systemic infection and tumor lysis syndrome. Moreover, hyperphosphatemia plays a critical role in the onset of secondary hyperparathyroidism, and the onset of renal osteodystrophy in patients under dialysis treatment for a long period.
[0007]Familial tumoral calcinosis (FTC; MIM211900) is a severe autosomal recessive metabolic disorder manifesting with hyperphosphatemia and massive calcium deposits in the skin and subcutaneous tissues, especially of the hips and knees. Hyperphosphatemia, secondary to increased renal phosphate retention, is the major metabolic abnormality associated with familial tumoral calcinosis (FTC) and is accompanied by inappropriately normal or elevated levels of PTH and 1,25-dihydroxyvitamin D3, two essential regulators of phosphate metabolism [Steinherz, 1985 (Supra)]. While hyperphosphatemia appears in FTC patients as early as 21 months of age, the calcium deposits are noted later in childhood. Thus, all FTC patients have elevated serum phosphorus levels, and in some patients, elevated levels of 1,25-vitamin D are also detected. FTC represents the metabolic mirror image of hypophosphatemic rickets caused by mutations in PHEX (MIM307800) and in FGF23 (MIM193100) genes [Schiavi, S. C. and Kumar, R. Kidney Int. 65, 1-14 (2004); Quarles, L. D. Am. J. Physiol. Endocrinol. Metab. 285, 1-9 (2003)] and which is characterized by decreased phosphate levels, decreased renal tubular phosphate reabsorption and inappropriately normal or decreased levels of 1,25-dihydroxyvitamin D3 [Prince M. J. et al. Ann Intern Med. 96, 586-591 (1982)].
[0008]Current treatment regimens of hypophosphatemia related disorders [e.g., hypophosphatemic rickets (PHEX)] include active vitamin D analogues (e.g., calcitriol) and oral phosphate supplementation. However, both of these nutrition supplements often fail to normalize serum phosphate level and in many cases, the patients fail to reach normal adult height. The recent use of recombinant human growth hormone (rhGH) was reported to benefit children with PHEX, however, is often associated with disproportional growth of the trunk (Reviewed in Reusz G, 2001, Orv Hetil. 142: 2659-65; Wilson D M 2000, J. Pediatr. Endocrinol. Metab. Suppl 2: 993-8).
[0009]Treatment of hyperphosphatemia involves the use of aluminum- or calcium-based phosphate-binding agents, which effectively lower serum phosphorus levels. However, while the use of aluminum-based agents can be associated with bone toxicity, renal osteodystrophy and encephalopathy, the use of calcium-based agents is often associated with hypercalaemia and cardiovascular calcification. To overcome these limitations, non-calcium-, non-aluminium-based alternative agents were developed. These include the sevelamer hydrochloride and lanthanum carbonate (Hutchison A J, 2004, Nephrol Dial Transplant. 19 Suppl 1:i19-24; Chertow G M., 2003, J Am Soc Nephrol. 14: S310-4). However, while lanthanum was found to be effective and well-tolerated (Joy M S et al., 2003, Am. J. Kidney Dis. 42: 96-107), sevelamer was found to be less effective than aluminum (Cizman B. 2003, Nephrol. Dial. Transplant. 18 Suppl 5:v47-9) and the use of both of these agents is limited by their high cost. Moreover, both of these agents treat only the symptoms by suppressing phosphate re-absorption in the intestinal track and not the causes which lead to hyperphosphatemia.
[0010]Thus, there is a need to develop pharmaceutical compositions and methods of treating disorders associated with phosphate metabolism devoid of the above limitations.
[0011]Phosphatonin is a novel circulating phosphaturic factor, postulated to be primarily responsible for modulating urinary phosphate excretion in a variety of hypophosphatemic disorders.
[0012]Quarles, 2003 (J Clin Invest. 112: 642-646), suggested that phosphatonin is a circulating protein that inhibits sodium-dependent phosphate reabsorption in the renal proximal tubule via mechanisms which are distinct from PTH, and vitamin D3. The fibroblast growth factor 23 (FGF23), secreted frizzled-related protein 4 (SFRP4) and matrix extracellular phosphoglycoprotein (MEPE) [Schiavi, S. C. and Kumar, R. Kidney Int. 65, 1-14 (2004); Quarles, L. D. Am. J. Physiol. Endocrinol. Metab. 285, 1-9 (2003)] were suggested as the putative phosphatonin proteins since they modulate circulating phosphate levels [Shimada, T. et al. Proc Natl Acad. Sci. 98, 6500-6505 (2001); Bowe A. E. et al. Bioch Biophys Res Comm. 284, 977-981 (2001); Rowe P. S. N. et al. Bone. 34, 303-319 (2003); Berndt, T. et al. J. Clin. Invest. 112, 785-794 (2003)].
[0013]Thus, FGF23 and other phosphatonin genes have been considered as prime candidates for the FTC gene [Jan De Beur, S. M., and Levine, M. A. (2002), J. Clin. Endocrinol. Metab. 87: 2467-2473].
[0014]While reducing the present invention to practice, the present inventors have uncovered that mutations in GALNT3 gene encoding UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-T3) cause FTC. Thus, inducers of GalNAc-T3 can be used to treat hyperphosphatemia related disorders such as FTC, and on the other hand, inhibitors of GalNAc-T3 can be used to treat disorders associated with hypophosphatemia, such as hypophosphatemic rickets.
SUMMARY OF THE INVENTION
[0015]According to one aspect of the present invention there is provided a method of treating a disorder associated with abnormal phosphate metabolism comprising providing to an individual in need thereof an agent capable of regulating an expression level and/or activity of GalNAc-T3 thereby treating the disorder associated with abnormal phosphate metabolism in the individual.
[0016]According to another aspect of the present invention there is provided a use of an agent capable of regulating an expression level and/or activity of GalNAc-T3 for the treatment of a disorder associated with abnormal phosphate metabolism.
[0017]According to yet another aspect of the present invention there is provided a use of an agent capable of regulating an expression level and/or activity of GalNAc-T3 for the manufacture of a medicament identified for the treatment of a disorder associated with abnormal phosphate metabolism.
[0018]According to still another aspect of the present invention there is provided method of diagnosing familial tumoral calcinosis in an individual, the method comprising identifying in a polynucleotide sequence of the individual at least one nucleic acid substitution resulting in downregulation of an expression level and/or activity of GalNAc-T3, thereby diagnosing familial tumoral calcinosis in the individual.
[0019]According to an additional aspect of the present invention there is provided a kit for diagnosing familial tumoral calcinosis in an individual, the kit comprising a packaging material packaging at least one reagent, the at least one reagent for identifying in a polynucleotide sequence of the individual at least one nucleic acid substitution resulting in downregulation of an expression level and/or activity of GalNAc-T3, thereby diagnosing familial tumoral calcinosis in the individual.
[0020]According to yet an additional aspect of the present invention there is provided a method of diagnosing familial tumoral calcinosis in an individual, the method comprising identifying in a polypeptide sequence of the individual at least one amino acid substitution capable of downregulating expression level and/or activity of GalNAc-T3, thereby diagnosing familial tumoral calcinosis in the individual.
[0021]According to yet another aspect of the present invention there is provided a method of identifying an agent suitable for treating a disorder associated with abnormal phosphate metabolism, comprising exposing GalNAc-T3 or cells expressing GalNAc-T3 to a plurality of molecules and selecting from the plurality of molecules at least one molecule capable of regulating the expression level and/or the activity of the GalNAc-T3, the at least one molecule being the agent suitable for treating the disorder associated with abnormal phosphate metabolism.
[0022]According to still an additional aspect of the present invention there is provided a kit for diagnosing familial tumoral calcinosis in an individual, the kit comprising a packaging material packaging at least one reagent, the at least one reagent for identifying in a polypeptide sequence of the individual at least one amino acid substitution capable of downregulating expression level and/or activity of GalNAc-T3, thereby diagnosing familial tumoral calcinosis in the individual.
[0023]According to a further aspect of the present invention there is provided a method of identifying an agent suitable for treating a disorder associated with abnormal phosphate metabolism, comprising exposing GalNAc-T3 or cells expressing GalNAc-T3 to a plurality of molecules and selecting from the plurality of molecules at least one molecule capable of regulating the expression level and/or the activity of the GalNAc-T3, the at least one molecule being the agent suitable for treating the disorder associated with abnormal phosphate metabolism.
[0024]According to yet a further aspect of the present invention there is provided a kit for identifying an agent suitable for treating a disorder associated with abnormal phosphate metabolism, the kit comprising a packaging material packaging at least one reagent, the at least one reagent include GalNAc-T3 and/or cells expressing GalNAc-T3 and a plurality of molecules, wherein at least one molecule of the plurality of molecules is capable of regulating the expression level and/or activity of the GalNAC-T3, thereby identifying the agent suitable for treating a disorder associated with abnormal phosphate metabolism.
[0025]According to further features in preferred embodiments of the invention described below, the disorder is associated with hyperphosphatemia.
[0026]According to still further features in the described preferred embodiments the disorder associated with the hyperphosphatemia is selected from the group consisting of Familial tumoral calcinosis (FTC), hyperphosphatemic calcinosis, hemodialysis, and chronic renal failure.
[0027]According to still further features in the described preferred embodiments regulating is upregulating the expression level and/or activity of the GalNAc-T3.
[0028]According to still further features in the described preferred embodiments, upregulating the expression level and/or activity of the GalNAc-T3 is effected by an agent selected from the group consisting of:
[0029](a) an exogenous polynucleotide encoding at least a functional portion of GALNT3;
[0030](b) an agent capable of increasing expression of endogenous GalNAc-T3 in an individual;
[0031](c) an agent capable of increasing endogenous GalNAc-T3 activity in an individual;
[0032](d) an exogenous polypeptide including at least a functional portion of GalNAc-T3;
[0033](e) a GalNAc-T3 substrate; and
[0034](f) a GalNAc-T3-expressing cell.
[0035]According to still further features in the described preferred embodiments the exogenous polynucleotide encoding at least a functional portion of GALNT3 is set forth in SEQ ID NO:29.
[0036]According to still further features in the described preferred embodiments the GalNAc-T3 is set forth in SEQ ID NO:28.
[0037]According to still further features in the described preferred embodiments the disorder is associated with hypophosphatemia.
[0038]According to still further features in the described preferred embodiments the disorder associated with the hypophosphatemia is selected from the group consisting of X-linked vitamin D resistant hypophosphatemic rickets (HYP), hereditary hypercalciuria with hypophosphatemic rickets (HHRH), oncogenic hypophosphatemic osteomalacia (OHO), and X-linked hypophosphatemic rickets (PHEX).
[0039]According to still further features in the described preferred embodiments regulating is downregulating the expression level and/or the activity of the GalNAc-T3.
[0040]According to still further features in the described preferred embodiments downregulating the expression level and/or the activity of the GalNAc-T3 is effected by an agent selected from the group consisting of:
[0041](a) a molecule which binds the GalNAc-T3;
[0042](b) an enzyme which cleaves the GalNAc-T3;
[0043](c) an antisense polynucleotide capable of specifically hybridizing with at least part of an mRNA transcript encoding GALNT3;
[0044](d) a ribozyme which specifically cleaves at least part of an mRNA transcript encoding GALNT3;
[0045](e) a small interfering RNA (siRNA) molecule which specifically cleaves at least part of a transcript encoding GALNT3;
[0046](f) a non-functional analogue of at least a catalytic or binding portion of the GalNAc-T3;
[0047](g) a molecule which prevents GalNAc-T3 activation or substrate binding.
[0048]According to still further features in the described preferred embodiments the mRNA transcript encoding GALNT3 is set forth in SEQ ID NO:29.
[0049]According to still further features in the described preferred embodiments the agent is formulated for systemic administration.
[0050]According to still further features in the described preferred embodiments the polynucleotide sequence is an mRNA sequence encoding GALNT3 or a genomic sequence region including the GALNT3 gene.
[0051]According to still further features in the described preferred embodiments the polynucleotide sequence is set forth by SEQ ID NO:29 or 33.
[0052]According to still further features in the described preferred embodiments identifying at least one nucleic acid substitution in the GALNT3 gene is effected using a method selected from the group consisting of DNA sequencing, restriction fragment length polymorphism (RFLP analysis), allele specific oligonucleotide (ASO) analysis, Denaturing/Temperature Gradient Gel Electrophoresis (DGGE/TGGE), Single-Strand Conformation Polymorphism (SSCP) analysis, Dideoxy fingerprinting (ddF), pyrosequencing analysis, acycloprime analysis, Reverse dot blot, GeneChip microarrays, Dynamic allele-specific hybridization (DASH), Peptide nucleic acid (PNA) and locked nucleic acids (LNA) probes, TaqMan, Molecular Beacons, Intercalating dye, FRET primers, AlphaScreen, SNPstream, genetic bit analysis (GBA), Multiplex minisequencing, SNaPshot, MassEXTEND, MassArray, GOOD assay, Microarray miniseq, arrayed primer extension (APEX), Microarray primer extension, Tag arrays, Coded microspheres, Template-directed incorporation (TDI), fluorescence polarization, Colorimetric oligonucleotide ligation assay (OLA), Sequence-coded OLA, Microarray ligation, Ligase chain reaction, Padlock probes, Rolling circle amplification, and Invader assay.
[0053]According to still further features in the described preferred embodiments identifying the at least one nucleic acid substitution in the mRNA sequence encoding the GALNT3 is effected using DNA sequencing of a GLANT3 RT-PCR product.
[0054]According to still further features in the described preferred embodiments the polypeptide sequence is set forth in SEQ ID NO:28.
[0055]According to still further features in the described preferred embodiments identifying the at least one amino acid substitution is effected using an antibody capable of differentially binding to at least one polymorph of the GalNAc-T3, the at least one polymorph includes the amino acid substitution capable of down-regulating the expression level and/or the activity of GalNAc-T3.
[0056]According to still further features in the described preferred embodiments the GalNAc-T3 includes at least a catalytic or binding portion of the GalNAc-T3.
[0057]According to still further features in the described preferred embodiments the GalNAc-T3 is set forth in SEQ ID NO:28.
[0058]According to still further features in the described preferred embodiments the cells expressing GalNAc-T3 are selected from the group consisting of kidney cells, fibroblasts, epithelial cells, lymphocytes, bone marrow cells, lung cells, liver cells and brain cells.
[0059]According to still further features in the described preferred embodiments the expression level is detected using an immunological detection method and/or an RNA detection method.
[0060]According to still further features in the described preferred embodiments the immunological detection method is selected from the group consisting of a radio-immunoassay (RIA), an enzyme linked immunosorbent assay (ELISA), a western blot, an immunohistochemical analysis, and a fluorescence activated cell sorting (FACS).
[0061]According to still further features in the described preferred embodiments the RNA detection method is selected from the group consisting of Northern Blot, RT-PCR, RNA in situ hybridization, and in situ RT-PCR.
[0062]According to still further features in the described preferred embodiments the activity is determined using an activity assay selected from the group consisting of in situ activity assay and in vitro activity assays.
[0063]According to still further features in the described preferred embodiments the activity assay is effected using a substrate selected from the group consisting of HIVH1Bgp120, Fibronectin, Prion-a, CD59, Muc1a, Muc2, EA2, Muc7.
[0064]The present invention successfully addresses the shortcomings of the presently known configurations by providing methods of treating disorders associate with abnormal phosphate metabolism.
[0065]Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0066]The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.
[0067]In the drawings:
[0068]FIGS. 1a-b are diagrams illustrating mapping of the familial tumoral calcinosis (FTC) critical region. Shown are haplotype analyses of a consanguinity FTC family (Family 1, FIG. 1a) and a non-consanguinity FTC family (Family 2, FIG. 1b) using polymorphic microsatellite markers on chromosome 2q24-q31.1. The shared disease-associated haplotypes of all participating individuals are indicated by boxes. The noted DNA markers correspond to the following GenBank Accession numbers: G08198 (D2S1399), G07884 (D2S1353); Z16431 (D2S111), Z54008 (D2S2330), G08153 (D2S1379), Z24635 (D2S399), Z50933 (D2S2345), G08180 (D2S1776), Z53666 (D2S2299), Z16821 (D2S142), Z53511 (D2S2284), Z52323 (D2S2177), and G08168 (D2S1391).
[0069]FIGS. 2a-b are photographs illustrating the clinical features of FTC. FIG. 2a--a large subcutaneous tumor over the left outer thigh of patient No. 01001 of family 1. FIG. 2b--a periarticular calcified mass over the left acetabulum of patient No. 01006 of family 1.
[0070]FIGS. 3a-b are multipoint LOD score analyses of a consanguinity FTC family (Family 1, FIG. 3a) and a non-consanguinity FTC family (Family 2, FIG. 3b). FIG. 3a--homozygosity mapping for 12 informative markers spanning 18.1 cM. Note the maximum multipoint LOD score of 6.7 at D2S111 (HOMOZ). FIG. 3b--multipoint linkage map using 7 microsatellite markers. Note the peak LOD of 3.4 for markers D2S2363 and D2S1379 (GeneHunter).
[0071]FIG. 4 illustrates RT-PCR determination of GALNT3 gene expression. RNA samples were extracted from various human tissues and the RT-PCR reaction was performed using the GALex6F (SEQ ID NO:23 and GALex9R (SEQ ID NO:25) primers followed by nested PCR using the GALex6F and GALex8R (SEQ ID NO:24) primers which are specific for GALNT3 transcript. Note the high-intensity bands obtained in RNA samples obtained from the skin, bone marrow and kidney. The RT-PCR product of the β-actin transcript (obtained using the Actin 5' and Actin 3' primers, SEQ ID NOs:26 and 27) was used as a measure of the level of RNA in each sample.
[0072]FIGS. 5a-d illustrate GALNT3 mutation analysis in FTC families. Shown are DNA sequencing chromatograms depicting the genomic sequence of exon 1 (FIGS. 5a-b) and exon 7 (FIG. 5c-d) of the GALNT3 gene in two FTC families. FIG. 5a--the wildtype sequence of exon 1 in family 1; FIG. 5b--the presence of a heterozygous C→T transition at position 484 of the GALNT3 mRNA sequence (GenBank Accession No. NM--004482, SEQ ID NO:29) creating a TGA termination codon (R162X) in exon 1 of affected individuals of family 2; FIG. 5c--the presence of a homozygous G→A transition at position 1524+1 (GALNT3 genomic contig GenBank Accession No. NT--005403) in affected individuals of family 1, FIG. 5d--the presence of a heterozygous G→A transition at position 1524+5 (GenBank Accession No. NT--005403) in all affected individuals of family 2.
[0073]FIGS. 6a-b are RFLP analyses depicting the segregation of the pathogenic mutations in families 1 and 2. FIG. 6a--segregation of the 1524+1G→A mutation in family 1. The 1524+1G→A nucleic acid change abolishes a recognition site for BsaAI; consequently, digestion of a PCR amplicon encompassing exon 7 generates an homozygous (uncut) 423 bp product in affected individuals, while heterozygous carriers of the mutation display an additional fragment of 314 bp. FIG. 6b--segregation of the 1524+5G→A and R162X mutations in family 2. The 1524+5G→A and R162X nucleic acid changes create novel recognition sites for endonucleases SspI (upper gel) and DdeI (lower gel) respectively; hence, affected individuals in family 2 display an additional fragment upon digestion of the relevant PCR amplicons (exons 7 and 1, respectively) with the corresponding restriction enzymes.
[0074]FIG. 7 is an RT-PCR analysis depicting the expression of GALNT3. RNA samples extracted from skin (lanes 1, 3, 5, 7) or blood lymphocytes (lanes 2, 4, 6, 8) of a healthy individual (lanes 1-4) and an FTC affected individual (patient No. 01001 of family 1, lanes 5-8) were subjected to RT-PCR analysis using the GALNT3 (lanes 1-2, 5-6) or β-actin (ACTB, lanes 3-4, 7-8) PCR primers as described in FIG. 4 hereinabove. Note the aberrant low-intensity, low-molecular weight GALNT3 RT-PCR products obtained using RNA from skin (lane 5, arrow) and blood lymphocytes (lane 6) of the FTC affected individual as compared with the high-intensity GALNT3 RT-PCR products obtained using RNA from skin (lane 1) and blood lymphocytes (lane 2) of the healthy individual. Also note the similar intensity bands of β-actin RT-PCR products in both affected and healthy individuals (compare lanes 3-4 to lanes 7-8).
[0075]FIG. 8 is a DNA sequencing chromatogram depicting the absence of exon 7 in a blood RNA sample of an FTC affected individual. Sequence analysis was performed on the aberrant low-molecular weight RT-PCR product observed in FIG. 7 lane 6 using the GALex6F (SEQ ID NO:23) and GALex8R (SEQ ID NO:24) nested PCR primers. Note the absence of exon 7 sequence in the aberrant low-molecular weight splice product from the blood sample of the affected individual.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0076]The present invention is of upregulators and downregulators of GalNAc-T3 which can be used in treating disorders associated with abnormal phosphate levels. Specifically, the present invention can be used to treat familial tumoral calcinosis and other disorders associated with hyperphosphatemia, as well as disorders associated with hypophosphatemia.
[0077]The principles and operation of the methods of treating disorders associated with abnormal phosphate metabolism according to the present invention may be better understood with reference to the drawings and accompanying descriptions.
[0078]Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
[0079]Familial tumoral calcinosis (FTC; MIM211900) is a severe autosomal recessive metabolic disorder manifesting with hyperphosphatemia and massive calcium deposits in the skin and subcutaneous tissues. Current methods of treating patients affected with this disease and other hyperphosphatemia-related disorders (e.g., end-stage renal failure, pseudohypoparathyroidism, hypoparathyroidism) include the use of aluminum- or calcium-based phosphate-binding agents. However, these phosphate-binders can cause bone toxicity, renal osteodystrophy, encephalopathy, hypercalcaemia and cardiovascular calcification. To overcome these limitations, aluminum and calcium free agents such as sevelamer hydrochloride and lanthanum carbonate were suggested for treatment of hyperphosphatemia (FOSRENOL®, AnorMED Inc.; Hutchison A J, 2004, Nephrol Dial Transplant. 19 Suppl 1:i19-24; Chertow G M., 2003, J Am Soc Nephrol. 14: S310-4). These agents suppress phosphate re-absorption in the intestinal track. Although safer than aluminum, sevelamer was found to be less effective than the aluminum-based phosphate binder (Cizman B. 2003, Nephrol. Dial. Transplant. 18 Suppl 5:v47-9). On the other hand, lanthanum was found to be effective and well-tolerated (Joy M S et al., 2003, Am. J. Kidney Dis. 42: 96-107). However, both of these expensive agents are used for treating the symptoms and not the cause of hyperphosphatemia.
[0080]While reducing the present invention to practice, the present inventors have uncovered deleterious mutations in the GALNT3 gene encoding GalNAc-T3 in individuals affected with familial tumoral calcinosis (FTC). Moreover, the present inventors have uncovered that inducers of GalNAc-T3 can be used in treating FTC and other hyperphosphatemia-related disorders, and that inhibitors of GalNAc-T3 can be used to treat hypophosphatemia-related disorders.
[0081]Thus, according to one aspect of the present invention there is provided a method of treating a disorder associated with abnormal phosphate metabolism in an individual.
[0082]As used herein, the term "individual" refers to a human being suffering from a disease associated with abnormal phosphate level, i.e., hyperphosphatemia or hypophosphatemia due to a genetic disease (e.g., an individual having familial tumoral calcinosis), hormonal imbalance (e.g., hyperparathyroidism), renal disease (as a result of e.g., hemodialysis) and the like.
[0083]The phrase "treating" refers to inhibiting or arresting the development of a disease, disorder or condition and/or causing the reduction, remission, or regression of a disease, disorder or condition in an individual suffering from, or diagnosed with, the disease, disorder or condition. Those of skill in the art will be aware of various methodologies and assays which can be used to assess the development of a disease, disorder or condition, and similarly, various methodologies and assays which can be used to assess the reduction, remission or regression of a disease, disorder or condition.
[0084]As used herein "abnormal phosphate metabolism" refers to abnormal levels of serum phosphate, which may reflect abnormal absorption of phosphate in the intestinal tract or abnormal phosphate excretion in both the kidney and intestinal tract (i.e., via fecal excretion). Thus, any deviation from the normal range of phosphate in the plasma (i.e., 2.5-4.5 mg/dl), can be referred to as being abnormal.
[0085]The phrase "disorder associated with abnormal phosphate metabolism" refers to hyper- or hypophosphatemia-related disorders, i.e., the presence of excess of phosphate in the serum (i.e., above 4.5 mg/dl) or insufficient levels of phosphate (i.e., under 2.5 mg/dl), respectively.
[0086]The method according to this aspect of the present invention is effected by providing to an individual in need thereof an agent capable of regulating an expression level and/or activity of GalNAc-T3.
[0087]The term "GalNAc-T3" as used herein refers to the UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3, which a member of the GalNAc-transferases family. Protein members of this family (e.g., GalNAc-T2, -T4, -T6, -T8) transfer N-acetyl galactosamine to the hydroxyl group of a serine or threonine residue in the first step of O-linked oligosaccharide biosynthesis. Individual GalNAc-transferase proteins have distinct activities and initiation of O-glycosylation is regulated by a repertoire of GalNAc-transferases. It will be appreciated that although these proteins share high sequence homology, each of the GalNAc-transferase proteins exhibits different substrate specificities. For example, the GalNAc-T3 exhibits substrate specificity towards HIV.sub.HIBgp120, Fibronectin, Prion-a, CD59, Muc1a, Muc2, EA2, and Muc7.
[0088]The term "regulating" as used herein refers to upregulating (i.e., increasing) or downregulating (i.e., inhibiting or decreasing) of the expression and/or activity of GalNAc-T3.
[0089]According to preferred embodiments of the present invention the disorder associated with abnormal phosphate metabolism is hyperphosphatemia and thus the method of the present invention is effected by upregulating expression or activity of GalNAc-T3. Non-limiting examples of hyperphosphatemia-related disorders which can be treated according to this aspect of the present invention include familial tumoral calcinosis (FTC), hyperphosphatemic calcinosis, hemodialysis, chronic renal failure and end-stage renal failure.
[0090]Upregulation of GalNAc-T3 can be effected at the genomic level (i.e., activation of transcription via promoters, enhancers, regulatory elements), at the transcript level (i.e., correct splicing, polyadenylation, activation of translation) or at the protein level (i.e., post-translational modifications, interaction with substrates and the like).
[0091]Following is a list of agents capable of upregulating the expression level and/or activity of GalNAc-T3.
[0092]An agent capable of upregulating expression level of a GalNAc-T3 may be an exogenous polynucleotide sequence designed and constructed to express at least a functional portion of the GalNAc-T3 protein. Accordingly, the exogenous polynucleotide sequence may be a DNA or RNA sequence encoding a GalNAc-T3 molecule, capable of modulating phosphate metabolism.
[0093]The phrase "functional portion" as used herein refers to part of the GalNAc-T3 protein (i.e., a polypeptide) which exhibits functional properties of the enzyme such as binding to a substrate. According to preferred embodiments of the present invention the functional portion of GalNAc-T3 is a polypeptide sequence including amino acids 188-374 (region of glycosyl transferase) and/or 507-629 (region of ricin-type beta trefoil) as set forth in SEQ ID NO:28. Preferably, the functional portion of GalNAc-T3 is a polypeptide sequence including amino acids 13-633, more preferably, amino acids 1-633 as set forth in SEQ ID NO:28.
[0094]GalNAc-T3 has been cloned from human and mouse sources. Thus, coding sequences information for GalNAc-T3 is available from several databases including the GenBank database available through http://www.ncbi.nlm.nih.gov/.
[0095]To express exogenous GalNAc-T3 in mammalian cells, a polynucleotide sequence encoding a GalNAc-T3 (GenBank Accession number NM--004482, SEQ ID NO:29) is preferably ligated into a nucleic acid construct suitable for mammalian cell expression. Such a nucleic acid construct includes a promoter sequence for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner.
[0096]It will be appreciated that the nucleic acid construct of the present invention can also utilize GalNAc-T3 homologues which exhibit the desired activity (i.e., transferring N-acetyl galactosamine to the hydroxyl group of a serine or threonine residue in the first step of O-linked oligosaccharide biosynthesis). Such homologues can be, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:29, as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals 50, length weight equals 3, average match equals 10 and average mismatch equals -9.
[0097]Constitutive promoters suitable for use with the present invention are promoter sequences which are active under most environmental conditions and most types of cells such as the cytomegalovirus (CMV) and Rous sarcoma virus (RSV). Inducible promoters suitable for use with the present invention include for example the inducible promoter of the alkaline phosphates gene (Dollard M A and Billard P J. 2003. Whole-cell bacterial sensors for the monitoring of phosphate bioavailability. Microbiol. Methods, 55: 221-9) and the tetracycline-inducible promoter (Zabala M, et al., Cancer Res. 2004, 64(8): 2799-804).
[0098]The nucleic acid construct (also referred to herein as an "expression vector") of the present invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). In addition, a typical cloning vectors may also contain a transcription and translation initiation sequence, transcription and translation terminator and a polyadenylation signal.
[0099]Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements. The TATA box, located 25-30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis. The other upstream promoter elements determine the rate at which transcription is initiated.
[0100]Enhancer elements can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer/promoter combinations that are suitable for the present invention include those derived from polyoma virus, human or murine cytomegalovirus (CMV), the long term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1983, which is incorporated herein by reference.
[0101]In the construction of the expression vector, the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
[0102]Polyadenylation sequences can also be added to the expression vector in order to increase the efficiency of GalNAC-T3 mRNA translation. Two distinct sequence elements are required for accurate and efficient polyadenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream. Termination and polyadenylation signals that are suitable for the present invention include those derived from SV40.
[0103]In addition to the elements already described, the expression vector of the present invention may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant DNA. For example, a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.
[0104]The vector may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid.
[0105]The expression vector of the present invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.
[0106]Examples for mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
[0107]Expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses can be also used. SV40 vectors include pSVT7 and pMT2. Vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5. Other exemplary vectors include pMSG, pAV009/A.sup.+, pMTO10/A.sup.+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
[0108]As described above, viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types. The targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell. Thus, the type of vector used by the present invention will depend on the cell type transformed. The ability to select suitable vectors according to the cell type transformed is well within the capabilities of the ordinary skilled artisan and as such no general description of selection consideration is provided herein. For example, bone marrow cells can be targeted using the human T cell leukemia virus type I (HTLV-I) and kidney cells may be targeted using the heterologous promoter present in the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) as described in Liang C Y et al., 2004 (Arch Virol. 149: 51-60).
[0109]Recombinant viral vectors are useful for in vivo expression of GalNAC-T3 since they offer advantages such as lateral infection and targeting specificity. Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny. Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
[0110]Various methods can be used to introduce the expression vector of the present invention into stem cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.
[0111]Introduction of nucleic acids by viral infection offers several advantages over other methods such as lipofection and electroporation, since higher transfection efficiency can be obtained due to the infectious nature of viruses.
[0112]It will be appreciated that upregulation of GalNAc-T3 can be also effected by administration of GalNAc-T3-expressing cells into the individual.
[0113]GalNAc-T3-expressing cells can be any suitable cells, such as kidney, bone marrow, keratinocyte and lymphocyte cells which are derived from the individuals and are transfected ex vivo with an expression vector containing the polynucleotide designed to express GalNAc-T3 as described hereinabove.
[0114]Administration of the GalNAc-T3-expressing cells of the present invention can be effected using any suitable route such as intravenous, intra peritoneal, intra kidney, intra gastrointestinal track, subcutaneous, transcutaneous, intramuscular, intracutaneous, intrathecal, epidural and rectal. According to presently preferred embodiments, the GalNAc-T3-expressing cells of the present invention are introduced to the individual using intravenous, intra kidney, intra gastrointestinal track and/or intra peritoneal administrations.
[0115]GalNAc-T3-expressing cells of the present invention can be derived from either autologous sources such as self bone marrow cells or from allogeneic sources such as bone marrow or other cells derived from non-autologous sources. Since non-autologous cells are likely to induce an immune reaction when administered to the body several approaches have been developed to reduce the likelihood of rejection of non-autologous cells. These include either suppressing the recipient immune system or encapsulating the non-autologous cells or tissues in immunoisolating, semipermeable membranes before transplantation.
[0116]Encapsulation techniques are generally classified as microencapsulation, involving small spherical vehicles and macroencapsulation, involving larger flat-sheet and hollow-fiber membranes (Uludag, H. et al. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64).
[0117]Methods of preparing microcapsules are known in the arts and include for example those disclosed by Lu M Z, et al., Cell encapsulation with alginate and alpha-phenoxycinnamylidene-acetylated poly(allylamine). Biotechnol Bioeng. 2000, 70: 479-83, Chang T M and Prakash S. Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms. Mol Biotechnol. 2001, 17: 249-60, and Lu M Z, et al., A novel cell encapsulation method using photosensitive poly(allylamine alpha-cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245-51.
[0118]For example, microcapsules are prepared by complexing modified collagen with a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 μm. Such microcapsules can be further encapsulated with additional 2-5 μm ter-polymer shells in order to impart a negatively charged smooth surface and to minimize plasma protein absorption (Chia, S. M. et al. Multi-layered microcapsules for cell encapsulation Biomaterials. 2002 23: 849-56).
[0119]Other microcapsules are based on alginate, a marine polysaccharide (Sambanis, A. Encapsulated islets in diabetes treatment. Diabetes Thechnol. Ther. 2003, 5: 665-8) or its derivatives. For example, microcapsules can be prepared by the polyelectrolyte complexation between the polyanions sodium alginate and sodium cellulose sulphate with the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride.
[0120]It will be appreciated that cell encapsulation is improved when smaller capsules are used. Thus, the quality control, mechanical stability, diffusion properties, and in vitro activities of encapsulated cells improved when the capsule size was reduced from 1 mm to 400 μm (Canaple L. et al., Improving cell encapsulation through size control. J Biomater Sci Polym Ed. 2002; 13: 783-96). Moreover, nanoporous biocapsules with well-controlled pore size as small as 7 nm, tailored surface chemistries and precise microarchitectures were found to successfully immunoisolate microenvironments for cells (Williams D. Small is beautiful: microparticle and nanoparticle technology in medical devices. Med Device Technol. 1999, 10: 6-9; Desai, T. A. Microfabrication technology for pancreatic cell encapsulation. Expert Opin Biol Ther. 2002, 2: 633-46).
[0121]An agent capable of upregulating a GalNAc-T3 may also be any compound which is capable of increasing the transcription and/or translation of an endogenous DNA or mRNA encoding the GalNAc-T3 and thus increasing endogenous GalNAc-T3 activity.
[0122]An agent capable of upregulating a GalNAc-T3 may also be an exogenous polypeptide including at least a functional portion (as described hereinabove) of the GalNAc-T3.
[0123]Upregulation of GalNAc-T3 can be also achieved by introducing at least one GalNAc-T3 substrate. Non-limiting examples of such agents include HIV.sub.HIBgp120, Fibronectin, Prion-a, CD59, Muc1a, Muc2, EA2, Muc7.
[0124]It will be appreciated that since GalNAc-T3 participates in phosphate metabolism downregulation thereof can be utilized to treat disorders which involve in hypophosphatemia, i.e., reduced levels of plasma phosphate.
[0125]Thus, according to another preferred embodiments of the present invention the disorder associated with abnormal phosphate metabolism is hypophosphatemia and the method of the present invention is effected by downregulating GalNAc-T3 expression or activity. Non-limiting examples of hypophosphatemia-related disorders which can be treated according to the method of the present invention include X-linked vitamin D resistant hypophosphatemic rickets (HYP), hereditary hypercalciuria with hypophosphatemic rickets (HHRH), oncogenic hypophosphatemic osteomalacia (OHO), X-linked hypophosphatemic rickets (PHEX) and hyperparathyroidism.
[0126]Downregulation of GalNAc-T3 can be effected on the genomic and/or the transcript level using a variety of molecules which interfere with transcription and/or translation (e.g., antisense, siRNA, Ribozyme, DNAzyme), or on the protein level using e.g., antagonists, enzymes that cleave the polypeptide and the like.
[0127]Following is a list of agents capable of downregulating expression level and/or activity of GalNAc-T3.
[0128]One example, of an agent capable of downregulating a GalNAc-T3 is an antibody or antibody fragment capable of specifically binding GalNAc-T3. Preferably, the antibody specifically binds at least one epitope of a GalNAc-T3. As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
[0129]Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
[0130]The term "antibody" as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, and Fv that are capable of binding to macrophages. These functional antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
[0131]Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).
[0132]Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
[0133]Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.
[0134]Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
[0135]Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
[0136]Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
[0137]Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).
[0138]Another agent capable of downregulating a GalNAc-T3 is a small interfering RNA (siRNA) molecule. RNA interference is a two step process. The first step, which is termed as the initiation step, input dsRNA is digested into 21-23 nucleotide (nt) small interfering RNAs (siRNA), probably by the action of Dicer, a member of the RNase III family of dsRNA-specific ribonucleases, which processes (cleaves) dsRNA (introduced directly or via a transgene or a virus) in an ATP-dependent manner. Successive cleavage events degrade the RNA to 19-21 bp duplexes (siRNA), each with 2-nucleotide 3' overhangs [Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002); and Bernstein Nature 409:363-366 (2001)].
[0139]In the effector step, the siRNA duplexes bind to a nuclease complex to from the RNA-induced silencing complex (RISC). An ATP-dependent unwinding of the siRNA duplex is required for activation of the RISC. The active RISC then targets the homologous transcript by base pairing interactions and cleaves the mRNA into 12 nucleotide fragments from the 3' terminus of the siRNA [Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002); Hammond et al. (2001) Nat. Rev. Gen. 2:110-119 (2001); and Sharp Genes. Dev. 15:485-90 (2001)]. Although the mechanism of cleavage is still to be elucidated, research indicates that each RISC contains a single siRNA and an RNase [Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002)].
[0140]Because of the remarkable potency of RNAi, an amplification step within the RNAi pathway has been suggested. Amplification could occur by copying of the input dsRNAs which would generate more siRNAs, or by replication of the siRNAs formed. Alternatively or additionally, amplification could be effected by multiple turnover events of the RISC [Hammond et al. Nat. Rev. Gen. 2:110-119 (2001), Sharp Genes. Dev. 15:485-90 (2001); Hutvagner and Zamore Curr. Opin. Genetics and Development 12:225-232 (2002)]. For more information on RNAi see the following reviews Tuschl ChemBiochem. 2:239-245 (2001); Cullen Nat. Immunol. 3:597-599 (2002); and Brantl Biochem. Biophys. Act. 1575:15-25 (2002).
[0141]Synthesis of RNAi molecules suitable for use with the present invention can be effected as follows. First, the GalNAc-T3 mRNA sequence is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl, T. 2001, ChemBiochem. 2:239-245]. It will be appreciated though, that siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5' UTR mediated about 90% decrease in cellular GAPDH mRNA and completely abolished protein level (www.ambion.com/techlib/tn/91/912.html).
[0142]Second, potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server (www.ncbi.nlm.nih.gov/BLAST/). Putative target sites which exhibit significant homology to other coding sequences are filtered out.
[0143]Qualifying target sequences are selected as template for siRNA synthesis. Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55%. Several target sites are preferably selected along the length of the target gene for evaluation. For better evaluation of the selected siRNAs, a negative control is preferably used in conjunction. Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome. Thus, a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.
[0144]For example, a suitable GalNAc-T3 siRNA can be the siRNA ID 14834 (Ambion Inc., Austin, Tex.).
[0145]Another agent capable of downregulating a GalNAc-T3 is a DNAzyme molecule capable of specifically cleaving an mRNA transcript or DNA sequence of the GalNAc-T3. DNAzymes are single-stranded polynucleotides which are capable of cleaving both single and double stranded target sequences (Breaker, R. R. and Joyce, G. Chemistry and Biology 1995; 2:655; Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 1997; 943:4262). A general model (the "10-23" model) for the DNAzyme has been proposed. "10-23" DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each. This type of DNAzyme can effectively cleave its substrate RNA at purine:pyrimidine junctions (Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 199; for rev of DNAzymes see Khachigian, L M [Curr Opin Mol Ther 4:119-21 (2002)].
[0146]Examples of construction and amplification of synthetic, engineered DNAzymes recognizing single and double-stranded target cleavage sites have been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymes of similar design directed against the human Urokinase receptor were recently observed to inhibit Urokinase receptor expression, and successfully inhibit colon cancer cell metastasis in vivo (Itoh et al, 20002, Abstract 409, Ann Meeting Am Soc Gen Ther. www.asgt.org). In another application, DNAzymes complementary to bcr-abl oncogenes were successful in inhibiting the oncogenes expression in leukemia cells, and lessening relapse rates in autologous bone marrow transplant in cases of CML and ALL.
[0147]Downregulation of a GalNAc-T3 can also be effected by using an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding the GalNAc-T3.
[0148]Design of antisense molecules which can be used to efficiently down-regulate a GalNAc-T3 must be effected while considering two aspects important to the antisense approach. The first aspect is delivery of the oligonucleotide into the cytoplasm of the appropriate cells, while the second aspect is design of an oligonucleotide which specifically binds the designated mRNA within cells in a way which inhibits translation thereof.
[0149]The prior art teaches of a number of delivery strategies which can be used to efficiently deliver oligonucleotides into a wide variety of cell types [see, for example, Luft J Mol Med 76: 75-6 (1998); Kronenwett et al. Blood 91: 852-62 (1998); Rajur et al. Bioconjug Chem 8: 935-40 (1997); Lavigne et al. Biochem Biophys Res Commun 237: 566-71 (1997) and Aoki et al. (1997) Biochem Biophys Res Commun 231: 540-5 (1997)].
[0150]In addition, algorithms for identifying those sequences with the highest predicted binding affinity for their target mRNA based on a thermodynamic cycle that accounts for the energetics of structural alterations in both the target mRNA and the oligonucleotide are also available [see, for example, Walton et al. Biotechnol Bioeng 65: 1-9 (1999)].
[0151]Such algorithms have been successfully used to implement an antisense approach in cells. For example, the algorithm developed by Walton et al. enabled scientists to successfully design antisense oligonucleotides for rabbit beta-globin (RBG) and mouse tumor necrosis factor-alpha (TNF alpha) transcripts. The same research group has more recently reported that the antisense activity of rationally selected oligonucleotides against three model target mRNAs (human lactate dehydrogenase A and B and rat gp130) in cell culture as evaluated by a kinetic PCR technique proved effective in almost all cases, including tests against three different targets in two cell types with phosphodiester and phosphorothioate oligonucleotide chemistries.
[0152]In addition, several approaches for designing and predicting efficiency of specific oligonucleotides using an in vitro system were also published (Matveeva et al., Nature Biotechnology 16: 1374-1375 (1998)].
[0153]For example, a suitable antisense oligonucleotides targeted against the GALNT3 mRNA (which is coding for the GalNAc-T3 protein) would be of the following sequences: CTGGCACATACACCTCTGG (SEQ ID NO:30).
[0154]Several clinical trials have demonstrated safety, feasibility and activity of antisense oligonucleotides. For example, antisense oligonucleotides suitable for the treatment of cancer have been successfully used [Holmund et al., Curr Opin Mol Ther 1:372-85 (1999)], while treatment of hematological malignancies via antisense oligonucleotides targeting c-myb gene, p53 and Bcl-2 had entered clinical trials and had been shown to be tolerated by patients [Gerwitz Curr Opin Mol Ther 1:297-306 (1999)].
[0155]More recently, antisense-mediated suppression of human heparanase gene expression has been reported to inhibit pleural dissemination of human cancer cells in a mouse model [Uno et al., Cancer Res 61:7855-60 (2001)].
[0156]Thus, the current consensus is that recent developments in the field of antisense technology which, as described above, have led to the generation of highly accurate antisense design algorithms and a wide variety of oligonucleotide delivery systems, enable an ordinarily skilled artisan to design and implement antisense approaches suitable for downregulating expression of known sequences without having to resort to undue trial and error experimentation.
[0157]Another agent capable of downregulating a GalNAc-T3 is a ribozyme molecule capable of specifically cleaving an mRNA transcript encoding a GalNAc-T3. Ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest [Welch et al., Curr Opin Biotechnol. 9:486-96 (1998)]. The possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications. In the therapeutics area, ribozymes have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders [Welch et al., Clin Diagn Virol. 10:163-71 (1998)]. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials. More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation. Several ribozymes are in various stages of clinical trials. ANGIOZYME was the first chemically synthesized ribozyme to be studied in human clinical trials. ANGIOZYME specifically inhibits formation of the VEGF-r (Vascular Endothelial Growth Factor receptor), a key component in the angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well as other firms have demonstrated the importance of anti-angiogenesis therapeutics in animal models. HEPTAZYME, a ribozyme designed to selectively destroy Hepatitis C Virus (HCV) RNA, was found effective in decreasing Hepatitis C viral RNA in cell culture assays (Ribozyme Pharmaceuticals, Incorporated--WEB home page).
[0158]Another agent capable of downregulating GalNAc-T3 would be any molecule which binds to and/or cleaves GalNAc-T3. Such molecules can be GalNAc-T3 antagonists, or GalNAc-T3 inhibitory peptide.
[0159]It will be appreciated that a non-functional analogue of at least a catalytic or binding portion of GalNAc-T3 can be also used as an agent which down-regulates GalNAc-T3.
[0160]Another agent which can be used along with the present invention to downregulate GalNAc-T3 is a molecule which prevents GalNAc-T3 activation or substrate binding.
[0161]Each of the upregulating or downregulating agents described hereinabove or the expression vector encoding GalNAc-T3 can be administered to the individual per se or as part of a pharmaceutical composition which also includes a physiologically acceptable carrier. The purpose of a pharmaceutical composition is to facilitate administration of the active ingredient to an organism.
[0162]As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
[0163]Herein the term "active ingredient" refers to the upregulating or downregulating agent or the expression vector encoding GalNAc-T3 which are accountable for the biological effect.
[0164]Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.
[0165]Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
[0166]Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.
[0167]Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
[0168]Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.
[0169]Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
[0170]Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
[0171]For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
[0172]For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
[0173]Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
[0174]Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
[0175]For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
[0176]For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
[0177]The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
[0178]Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
[0179]Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
[0180]The pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
[0181]Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (the upregulating or downregulating agent or the expression vector encoding GalNAc-T3) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., hyperphosphatemia or hypophosphatemia) or prolong the survival of the subject being treated.
[0182]Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
[0183]For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
[0184]Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p. 1).
[0185]Dosage amount and interval may be adjusted individually to provide plasma levels of the active ingredient are sufficient to prevent hyperphosphatemia or hypophosphatemia (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
[0186]Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
[0187]The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
[0188]Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as if further detailed above.
[0189]Thus, the teachings of the present invention can be used to treat individuals suffering from familial tumoral calcinosis (i.e., an hyperphosphatemia disorder). For example, an expression vector (e.g., a viral vector) including a polynucleotide sequence encoding the GALNT3 mRNA (SEQ ID NO:29) and the suitable promoter sequences to enable expression in kidney and skin cells is introduced into the individual via intravenous administration. Expression of such a vector in kidney and/or skin is expected to upregulate the expression level and/or activity of GalNAc-T3 in those tissues and thus to correct the hyperphosphatemia. Dosage of such an expression vector should be calibrated using cell culture experiments and animal models. Success of treatment is preferably evaluated by determining plasma phosphate levels and the individual general health status.
[0190]It will be appreciated, that if such a treatment is employed early in childhood, i.e., prior to the appearance of calcium deposits, or even prior to the detection of hyperphosphatemia (generally at 21 months of age), it may prevent the complications associated with such a disease (i.e., massive calcium deposits in the skin and subcutaneous tissues). In addition, since the expression vector is targeted to somatic cells which exhibit limited half-life (depending upon the cell line transduced), such a treatment is expected to be repeated periodically in order to prevent hyperphosphatemia.
[0191]As is shown in FIG. 5c and Example 2 of the Examples section which follows, the present inventors have uncovered that a 1524+1G→A mutation in the GALNT3 gene, in a homozygous form, causes FTC in a consanguinity family (family 1). In addition, as is shown in FIGS. 5b and d and Example 2 of the Examples section which follows, FTC affected members of the non-consanguinity family (family 2) are double heterozygous for the R162X and 1524+5G→A mutations in the GALNT3 gene. Thus, the presence of deleterious mutations in the GALNT3 sequence encoding GalNAc-T3 can be used in diagnosing FTC in an individual.
[0192]Thus, according to another aspect of the present invention there is provided a method of diagnosing familial tumoral calcinosis in an individual.
[0193]The method is effected by identifying in a polynucleotide sequence of the individual at least one nucleic acid substitution resulting in downregulation of an expression level and/or activity of GalNAc-T3.
[0194]As used herein the "polynucleotide sequence" refers to any DNA or RNA sequence which is derived from cells of the individual. DNA and RNA samples can be obtained from any source of cells. Preferably, the DNA is derived from peripheral blood cells (obtained using a syringe), skin cells (obtained from a skin biopsy) or mouth epithelial cells (obtained from a mouth wash), and the RNA is derived from blood or skin cells. Methods of extracting such DNA and RNA samples are well known in the art.
[0195]As is mentioned before, deleterious mutations in the GALNT3 gene cause FTC. Thus, according to preferred embodiments of the present invention the polynucleotide sequence is an mRNA sequence encoding GALNT3 (e.g., GenBank Accession No. NM--004482, SEQ ID NO:29) or a genomic sequence region including the GALNT3 gene (e.g., SEQ ID NO:33).
[0196]As used herein, the phrase "nucleic acid substitution" refers to any mutation in the DNA sequence of an individual which can result in downregulation of the expression level and/or activity of GalNAc-T3. Non-limiting examples of such nucleic acid changes include a missense mutation (i.e., a mutation which changes an amino acid residue in the protein with another amino acid residue), a nonsense mutation (i.e., a mutation which introduces a stop codon in a protein), a frameshift mutation (i.e., a mutation, usually, deletion or insertion of nucleic acids which changes the reading frame of the protein, and may result in an early termination or in a longer amino acid sequence), a readthrough mutation (i.e., a mutation which results in an elongated protein due to a change in a coding frame or a modified stop codon), a promoter mutation (i.e., a mutation in a promoter sequence, usually 5' to the transcription start site of a gene, which result in up-regulation or down-regulation of a specific gene product), a regulatory mutation (i.e., a mutation in a region upstream or downstream, or within a gene, which affects the expression of the gene product), a deletion (i.e., a mutation which deletes coding or non-coding nucleic acids in a gene sequence), an insertion (i.e., a mutation which inserts coding or non-coding nucleic acids into a gene sequence), an inversion (i.e., a mutation which results in an inverted coding or non-coding sequence), a splice mutation (i.e., a mutation which results in abnormal splicing or poor splicing) and a duplication (i.e., a mutation which results in a duplicated coding or non-coding sequence).
[0197]It will be appreciated that certain nucleic acid substitutions can be present in non-affected individuals [e.g., single nucleotide polymorphism (SNP)]. Such an SNP can cause, for example, a missense mutation in the GalNAc-T3, which, when present in the heterozygous form can be harmless, however, when present together with another, deleterious mutation such as a nonsense mutation, can lead to FTC at various degrees. Thus, according to preferred embodiments of the present invention the nucleic acid substitution is an SNP.
[0198]The nucleic acid substitution of the present invention can be identified using a variety of approaches suitable for identifying sequence alterations. One option is to determine the entire gene sequence of a PCR reaction product. Alternatively, a given segment of nucleic acid may be characterized on several other levels. At the lowest resolution, the size of the molecule can be determined by electrophoresis by comparison to a known standard run on the same gel. A more detailed picture of the molecule may be achieved by cleavage with combinations of restriction enzymes prior to electrophoresis, to allow construction of an ordered map. The presence of specific sequences within the fragment can be detected by hybridization of a labeled probe, or the precise nucleotide sequence can be determined by partial chemical degradation or by primer extension in the presence of chain-terminating nucleotide analogs.
[0199]Following is a non-limiting list of methods which can be used to identify the nucleic acid substitution and/or SNPs in the GALNT3 gene.
[0200]Direct sequencing of a PCR product: This method is based on the amplification of a genomic sequence using specific PCR primers in a PCR reaction following by a sequencing reaction utilizing the sequence of one of the PCR primers as a sequencing primer. Sequencing reaction can be performed using, for example, the Applied Biosystems (Foster City, Calif.) ABI PRISM® BigDye® Primer or BigDye® Terminator Cycle Sequencing Kits.
[0201]Restriction fragment length polymorphism (RFLP): This method uses a change in a single nucleotide (the SNP nucleotide) which modifies a recognition site for a restriction enzyme resulting in the creation or destruction of an RFLP.
[0202]For example, RFLP can be used to detect the R162X mutation (C→T substitution at nucleotide 484 as set forth in SEQ ID NO:29) in a genomic DNA of an individual. Briefly, genomic DNA is amplified using the GALe1bR (SEQ ID NO:1) and GALe1bF (SEQ ID NO:2) PCR primers, and the resultant PCR product is subjected to digestion using a restriction enzyme such as BstDEI, DdeI (see e.g., FIG. 6b) or TspRI which are capable of differentially digesting a PCR product containing the T allele (and not the C allele) at position 484 of SEQ ID NO:29.
[0203]Single nucleotide mismatches in DNA heteroduplexes are also recognized and cleaved by some chemicals, providing an alternative strategy to detect single base substitutions, generically named the "Mismatch Chemical Cleavage" (MCC) (Gogos et al., Nucl. Acids Res., 18:6807-6817, 1990). However, this method requires the use of osmium tetroxide and piperidine, two highly noxious chemicals which are not suited for use in a clinical laboratory.
[0204]Allele specific oligonucleotide (ASO): In this method, an allele-specific oligonucleotide (ASO) is designed to hybridize in proximity to the polymorphic nucleotide, such that a primer extension or ligation event can be used as the indicator of a match or a mis-match. Hybridization with radioactively labeled allelic specific oligonucleotides (ASO) also has been applied to the detection of specific SNPs (Conner et al., Proc. Natl. Acad. Sci., 80:278-282, 1983). The method is based on the differences in the melting temperature of short DNA fragments differing by a single nucleotide. Stringent hybridization and washing conditions can differentiate between mutant and wild-type alleles.
[0205]It will be appreciated that ASO can be applied on a PCR product generated from genomic DNA. For example, to detect the R162X mutation, genomic DNA is amplified using the GALe1bR (SEQ ID NO:1) and the GALe1bF (SEQ ID NO:2) PCR primers, and the resultant PCR product is subjected to an ASO hybridization using the following oligonucleotide probe: 5'-CTTTGCACtGAGATCTTGG (SEQ ID NO:31) which is capable of hybridizing to the thymidine nucleotide at position 484 of SEQ ID NO:29. As a control for the hybridization, the CTTTGCACCGAGATCTTGG (SEQ ID NO:32) oligonucleotide probe is applied to detect the presence of the wildtype allele.
[0206]Denaturing/Temperature Gradient Gel Electrophoresis (DGGE/TGGE): Two other methods rely on detecting changes in electrophoretic mobility in response to minor sequence changes. One of these methods, termed "Denaturing Gradient Gel Electrophoresis" (DGGE) is based on the observation that slightly different sequences will display different patterns of local melting when electrophoretically resolved on a gradient gel. In this manner, variants can be distinguished, as differences in melting properties of homoduplexes versus heteroduplexes differing in a single nucleotide can detect the presence of SNPs in the target sequences because of the corresponding changes in their electrophoretic mobilities. The fragments to be analyzed, usually PCR products, are "clamped" at one end by a long stretch of G-C base pairs (30-80) to allow complete denaturation of the sequence of interest without complete dissociation of the strands. The attachment of a GC "clamp" to the DNA fragments increases the fraction of mutations that can be recognized by DGGE (Abrams et al., Genomics 7:463-475, 1990). Attaching a GC clamp to one primer is critical to ensure that the amplified sequence has a low dissociation temperature (Sheffield et al., Proc. Natl. Acad. Sci., 86:232-236, 1989; and Lerman and Silverstein, Meth. Enzymol., 155:482-501, 1987). Modifications of the technique have been developed, using temperature gradients (Wartell et al., Nucl. Acids Res., 18:2699-2701, 1990), and the method can be also applied to RNA:RNA duplexes (Smith et al., Genomics 3:217-223, 1988).
[0207]Limitations on the utility of DGGE include the requirement that the denaturing conditions must be optimized for each type of DNA to be tested. Furthermore, the method requires specialized equipment to prepare the gels and maintain the needed high temperatures during electrophoresis. The expense associated with the synthesis of the clamping tail on one oligonucleotide for each sequence to be tested is also a major consideration. In addition, long running times are required for DGGE. The long running time of DGGE was shortened in a modification of DGGE called constant denaturant gel electrophoresis (CDGE) (Borrensen et al., Proc. Natl. Acad. Sci. USA 88:8405, 1991). CDGE requires that gels be performed under different denaturant conditions in order to reach high efficiency for the detection of SNPs.
[0208]A technique analogous to DGGE, termed temperature gradient gel electrophoresis (TGGE), uses a thermal gradient rather than a chemical denaturant gradient (Scholz, et al., Hum. Mol. Genet. 2:2155, 1993). TGGE requires the use of specialized equipment which can generate a temperature gradient perpendicularly oriented relative to the electrical field. TGGE can detect mutations in relatively small fragments of DNA therefore scanning of large gene segments requires the use of multiple PCR products prior to running the gel.
[0209]Single-Strand Conformation Polymorphism (SSCP): Another common method, called "Single-Strand Conformation Polymorphism" (SSCP) was developed by Hayashi, Sekya and colleagues (reviewed by Hayashi, PCR Meth. Appl., 1:34-38, 1991) and is based on the observation that single strands of nucleic acid can take on characteristic conformations in non-denaturing conditions, and these conformations influence electrophoretic mobility. The complementary strands assume sufficiently different structures that one strand may be resolved from the other. Changes in sequences within the fragment will also change the conformation, consequently altering the mobility and allowing this to be used as an assay for sequence variations (Orita, et al., Genomics 5:874-879, 1989).
[0210]The SSCP process involves denaturing a DNA segment (e.g., a PCR product) that is labeled on both strands, followed by slow electrophoretic separation on a non-denaturing polyacrylamide gel, so that intra-molecular interactions can form and not be disturbed during the run. This technique is extremely sensitive to variations in gel composition and temperature. A serious limitation of this method is the relative difficulty encountered in comparing data generated in different laboratories, under apparently similar conditions.
[0211]Dideoxy fingerprinting (ddF): The dideoxy fingerprinting (ddF) is another technique developed to scan genes for the presence of mutations (Liu and Sommer, PCR Methods Appli., 4:97, 1994). The ddF technique combines components of Sanger dideoxy sequencing with SSCP. A dideoxy sequencing reaction is performed using one dideoxy terminator and then the reaction products are electrophoresed on nondenaturing polyacrylamide gels to detect alterations in mobility of the termination segments as in SSCP analysis. While ddF is an improvement over SSCP in terms of increased sensitivity, ddF requires the use of expensive dideoxynucleotides and this technique is still limited to the analysis of fragments of the size suitable for SSCP (i.e., fragments of 200-300 bases for optimal detection of mutations).
[0212]In addition to the above limitations, all of these methods are limited as to the size of the nucleic acid fragment that can be analyzed. For the direct sequencing approach, sequences of greater than 600 base pairs require cloning, with the consequent delays and expense of either deletion sub-cloning or primer walking, in order to cover the entire fragment. SSCP and DGGE have even more severe size limitations. Because of reduced sensitivity to sequence changes, these methods are not considered suitable for larger fragments. Although SSCP is reportedly able to detect 90% of single-base substitutions within a 200 base-pair fragment, the detection drops to less than 50% for 400 base pair fragments. Similarly, the sensitivity of DGGE decreases as the length of the fragment reaches 500 base-pairs. The ddF technique, as a combination of direct sequencing and SSCP, is also limited by the relatively small size of the DNA that can be screened.
[0213]Pyrosequencing® analysis (Pyrosequencing, Inc. Westborough, Mass., USA): This technique is based on the hybridization of a sequencing primer to a single stranded, PCR-amplified, DNA template in the presence of DNA polymerase, ATP sulfurylase, luciferase and apyrase enzymes and the adenosine 5' phosphosulfate (APS) and luciferin substrates. In the second step the first of four deoxynucleotide triphosphates (dNTP) is added to the reaction and the DNA polymerase catalyzes the incorporation of the deoxynucleotide triphosphate into the DNA strand, if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of pyrophosphate (PPi) in a quantity equimolar to the amount of incorporated nucleotide. In the last step the ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5' phosphosulfate. This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by a charge coupled device (CCD) camera and seen as a peak in a Pyrogram®. Each light signal is proportional to the number of nucleotides incorporated.
[0214]Acycloprime® analysis (Perkin Elmer, Boston, Mass., USA): This technique is based on fluorescent polarization (FP) detection. Following PCR amplification of the sequence containing the SNP of interest, excess primer and dNTPs are removed through incubation with shrimp alkaline phosphatase (SAP) and exonuclease I. Once the enzymes are heat inactivated, the Acycloprime-FP process uses a thermostable polymerase to add one of two fluorescent terminators to a primer that ends immediately upstream of the SNP site. The terminator(s) added are identified by their increased FP and represent the allele(s) present in the original DNA sample. The Acycloprime process uses AcycloPol®, a novel mutant thermostable polymerase from the Archeon family, and a pair of AcycloTerminators® labeled with R110 and TAMRA, representing the possible alleles for the SNP of interest. AcycloTerminator® non-nucleotide analogs are biologically active with a variety of DNA polymerases. Similarly to 2',3'-dideoxynucleotide-5'-triphosphates, the acyclic analogs function as chain terminators. The analog is incorporated by the DNA polymerase in a base-specific manner onto the 3'-end of the DNA chain, and since there is no 3'-hydroxyl, is unable to function in further chain elongation. It has been found that AcycloPol has a higher affinity and specificity for derivatized AcycloTerminators than various Taq mutant have for derivatized 2',3'-dideoxynucleotide terminators.
[0215]Reverse dot blot: This technique uses labeled sequence specific oligonucleotide probes and unlabeled nucleic acid samples. Activated primary amine-conjugated oligonucleotides are covalently attached to carboxylated nylon membranes. After hybridization and washing, the labeled probe, or a labeled fragment of the probe, can be released using oligomer restriction, i.e., the digestion of the duplex hybrid with a restriction enzyme. Circular spots or lines are visualized colorimetrically after hybridization through the use of streptavidin horseradish peroxidase incubation followed by development using tetramethylbenzidine and hydrogen peroxide, or via chemiluminescence after incubation with avidin alkaline phosphatase conjugate and a luminous substrate susceptible to enzyme activation, such as CSPD, followed by exposure to x-ray film.
[0216]It will be appreciated that advances in the field of SNP detection have provided additional accurate, easy, and inexpensive large-scale SNP genotyping techniques, such as dynamic allele-specific hybridization (DASH, Howell, W. M. et al., 1999. Dynamic allele-specific hybridization (DASH). Nat. Biotechnol. 17: 87-8), microplate array diagonal gel electrophoresis [MADGE, Day, I. N. et al., 1995. High-throughput genotyping using horizontal polyacrylamide gels with wells arranged for microplate array diagonal gel electrophoresis (MADGE). Biotechniques. 19: 830-5], the TaqMan system (Holland, P. M. et al., 1991. Detection of specific polymerase chain reaction product by utilizing the 5'→3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci USA. 88: 7276-80), as well as various DNA "chip" technologies such as the GeneChip microarrays (e.g., Affymetrix SNP chips) which are disclosed in U.S. Pat. No. 6,300,063 to Lipshutz, et al. 2001, which is fully incorporated herein by reference, Genetic Bit Analysis (GBA®) which is described by Goelet, P. et al. (PCT Appl. No. 92/15712), peptide nucleic acid (PNA, Ren B, et al., 2004. Nucleic Acids Res. 32: e42) and locked nucleic acids (LNA, Latorra D, et al., 2003. Hum. Mutat. 22: 79-85) probes, Molecular Beacons (Abravaya K, et al., 2003. Clin Chem Lab Med. 41: 468-74), intercalating dye [Germer, S, and Higuchi, R. Single-tube genotyping without oligonucleotide probes. Genome Res. 9:72-78 (1999)], FRET primers (Solinas A et al., 2001. Nucleic Acids Res. 29: E96), AlphaScreen (Beaudet L, et al., Genome Res. 2001, 11(4): 600-8), SNPstream (Bell P A, et al., 2002. Biotechniques. Suppl.: 70-2, 74, 76-7), Multiplex minisequencing (Curcio M, et al., 2002. Electrophoresis. 23: 1467-72), SnaPshot (Turner D, et al., 2002. Hum Immunol. 63: 508-13), MassEXTEND (Cashman J R, et al., 2001. Drug Metab Dispos. 29: 1629-37), GOOD assay (Sauer S, and Gut I G. 2003. Rapid Commun. Mass. Spectrom. 17: 1265-72), Microarray minisequencing (Liljedahl U, et al., 2003. Pharmacogenetics. 13: 7-17), arrayed primer extension (APEX) (Tonisson N, et al., 2000. Clin. Chem. Lab. Med. 38: 165-70), Microarray primer extension (O'Meara D, et al., 2002. Nucleic Acids Res. 30: e75), Tag arrays (Fan J B, et al., 2000. Genome Res. 10: 853-60), Template-directed incorporation (TDI) (Akula N, et al., 2002. Biotechniques. 32: 1072-8), fluorescence polarization (Hsu T M, et al., 2001. Biotechniques. 31: 560, 562, 564-8), Colorimetric oligonucleotide ligation assay (OLA, Nickerson D A, et al., 1990. Proc. Natl. Acad. Sci. USA. 87: 8923-7), Sequence-coded OLA (Gasparini P, et al., 1999. J. Med. Screen. 6: 67-9), Microarray ligation, Ligase chain reaction, Padlock probes, Rolling circle amplification, Invader assay (reviewed in Shi M M. 2001. Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies. Clin Chem. 47: 164-72), coded microspheres (Rao K V et al., 2003. Nucleic Acids Res. 31: e66) and MassArray (Leushner J, Chiu N H, 2000. Mol Diagn. 5: 341-80).
[0217]It will be appreciated that nucleic acid substitutions can be also identified in mRNA molecules of the individual. Such mRNA molecules are first subjected to an RT-PCR reaction following which they are either directly sequenced or be subjected to any of the SNP detection methods described hereinabove.
[0218]As is shown in FIG. 8 and Example 2 of the Examples section which follows, sequencing of the RT-PCR product generated using the GALex6F (SEQ ID NO:23) and the GALex8R (SEQ ID NO:24) primers revealed the absence of exon 7 from the mature GALNT3 mRNA.
[0219]Thus, according to preferred embodiments of the present invention, identifying at least one nucleic acid substitution in the mRNA sequence encoding GALNT3 is effected using DNA sequencing of a GLANT3 RT-PCR product.
[0220]Downregulation or upregulation of the expression level and/or activity of GalNAc-T3 can be determined using molecular and immunological methods known in the art.
[0221]Following is a list of methods useful for detecting GALNT3 RNA level in cells of the individual.
[0222]Northern Blot analysis: This method involves the detection of a particular RNA in a mixture of RNAs. An RNA sample is denatured by treatment with an agent (e.g., formaldehyde) that prevents hydrogen bonding between base pairs, ensuring that all the RNA molecules have an unfolded, linear conformation. The individual RNA molecules are then separated according to size by gel electrophoresis and transferred to a nitrocellulose or a nylon-based membrane to which the denatured RNAs adhere. The membrane is then exposed to labeled DNA probes. Probes may be labeled using radio-isotopes or enzyme linked nucleotides. Detection may be using autoradiography, colorimetric reaction or chemiluminescence. This method allows both quantitation of an amount of particular RNA molecules and determination of its identity by a relative position on the membrane which is indicative of a migration distance in the gel during electrophoresis.
[0223]RT-PCR analysis: This method uses PCR amplification of relatively rare RNAs molecules. First, RNA molecules are purified from the cells and converted into complementary DNA (cDNA) using a reverse transcriptase enzyme (such as an MMLV-RT) and primers such as, oligo dT, random hexamers or gene specific primers. Then by applying gene specific primers and Taq DNA polymerase, a PCR amplification reaction is carried out in a PCR machine. Those of skills in the art are capable of selecting the length and sequence of the gene specific primers and the PCR conditions (i.e., annealing temperatures, number of cycles and the like) which are suitable for detecting specific RNA molecules. It will be appreciated that a semi-quantitative RT-PCR reaction can be employed by adjusting the number of PCR cycles and comparing the amplification product to known controls.
[0224]RNA in situ hybridization stain: In this method DNA or RNA probes are attached to the RNA molecules present in the cells. Generally, the cells are first fixed to microscopic slides to preserve the cellular structure and to prevent the RNA molecules from being degraded and then are subjected to hybridization buffer containing the labeled probe. The hybridization buffer includes reagents such as formamide and salts (e.g., sodium chloride and sodium citrate) which enable specific hybridization of the DNA or RNA probes with their target mRNA molecules in situ while avoiding non-specific binding of probe. Those of skills in the art are capable of adjusting the hybridization conditions (i.e., temperature, concentration of salts and formamide and the like) to specific probes and types of cells. Following hybridization, any unbound probe is washed off and the slide is subjected to either a photographic emulsion which reveals signals generated using radio-labeled probes or to a colorimetric reaction which reveals signals generated using enzyme-linked labeled probes.
[0225]In situ RT-PCR stain: This method is described in Nuovo G J, et al. [Intracellular localization of polymerase chain reaction (PCR)-amplified hepatitis C cDNA. Am J Surg Pathol. 1993, 17: 683-90] and Komminoth P, et al. [Evaluation of methods for hepatitis C virus detection in archival liver biopsies. Comparison of histology, immunohistochemistry, in situ hybridization, reverse transcriptase polymerase chain reaction (RT-PCR) and in situ RT-PCR. Pathol Res Pract. 1994, 190: 1017-25]. Briefly, the RT-PCR reaction is performed on fixed cells by incorporating labeled nucleotides to the PCR reaction. The reaction is carried on using a specific in situ RT-PCR apparatus such as the laser-capture microdissection PixCell I LCM system available from Arcturus Engineering (Mountainview, Calif.).
[0226]Following is a list of immunological detection methods which can be used to detect the level of GalNAc-T3 protein in cells of the individual.
[0227]Enzyme linked immunosorbent assay (ELISA): This method involves fixation of a sample (e.g., fixed cells or a proteinaceous solution) containing a protein substrate to a surface such as a well of a microtiter plate. A substrate specific antibody coupled to an enzyme is applied and allowed to bind to the substrate. Presence of the antibody is then detected and quantitated by a calorimetric reaction employing the enzyme coupled to the antibody. Enzymes commonly employed in this method include horseradish peroxidase and alkaline phosphatase. If well calibrated and within the linear range of response, the amount of substrate present in the sample is proportional to the amount of color produced. A substrate standard is generally employed to improve quantitative accuracy.
[0228]Western blot: This method involves separation of a substrate from other protein by means of an acrylamide gel followed by transfer of the substrate to a membrane (e.g., nylon or PVDF). Presence of the substrate is then detected by antibodies specific to the substrate, which are in turn detected by antibody binding reagents. Antibody binding reagents may be, for example, protein A, or other antibodies. Antibody binding reagents may be radiolabeled or enzyme linked as described hereinabove. Detection may be by autoradiography, calorimetric reaction or chemiluminescence. This method allows both quantitation of an amount of substrate and determination of its identity by a relative position on the membrane which is indicative of a migration distance in the acrylamide gel during electrophoresis.
[0229]Radio-immunoassay (RIA): In one version, this method involves precipitation of the desired protein (i.e., the substrate) with a specific antibody and radiolabeled antibody binding protein (e.g., protein A labeled with I125) immobilized on a precipitable carrier such as agarose beads. The number of counts in the precipitated pellet is proportional to the amount of substrate.
[0230]In an alternate version of the RIA, a labeled substrate and an unlabelled antibody binding protein are employed. A sample containing an unknown amount of substrate is added in varying amounts. The decrease in precipitated counts from the labeled substrate is proportional to the amount of substrate in the added sample.
[0231]Fluorescence activated cell sorting (FACS): This method involves detection of a substrate in situ in cells by substrate specific antibodies. The substrate specific antibodies are linked to fluorophores. Detection is by means of a cell sorting machine which reads the wavelength of light emitted from each cell as it passes through a light beam. This method may employ two or more antibodies simultaneously.
[0232]Immunohistochemical analysis: This method involves detection of a substrate in situ in fixed cells by substrate specific antibodies. The substrate specific antibodies may be enzyme linked or linked to fluorophores. Detection is by microscopy and subjective or automatic evaluation. If enzyme linked antibodies are employed, a colorimetric reaction may be required. It will be appreciated that immunohistochemistry is often followed by counterstaining of the cell nuclei using for example Hematoxyline or Giemsa stain.
[0233]Following is a list of enzymatic activity assays which can be used to determine GalNAc-T3 activity in cells of the individual.
[0234]In situ activity assay: According to this method, a chromogenic substrate is applied on the cells containing an active enzyme and the enzyme catalyzes a reaction in which the substrate is decomposed to produce a chromogenic product visible by a light or a fluorescent microscope.
[0235]In vitro activity assays: In these methods the activity of a particular enzyme is measured in a protein mixture extracted from the cells. The activity can be measured in a spectrophotometer well using colorimetric methods or can be measured in a non-denaturing acrylamide gel (i.e., activity gel). Following electrophoresis the gel is soaked in a solution containing a substrate and calorimetric reagents. The resulting stained band corresponds to the enzymatic activity of the protein of interest. If well calibrated and within the linear range of response, the amount of enzyme present in the sample is proportional to the amount of color produced. An enzyme standard is generally employed to improve quantitative accuracy.
[0236]The activity assays described hereinabove can use any of the GalNAc-T3 specific substrates, including, but not limited to, HIV.sub.HIBgp120, Fibronectin, Prion-a, CD59, Muc1a, Muc2, EA2, Muc7.
[0237]It will be appreciated that mutations which downregulate the expression level and/or activity level of GalNAc-T3 can be also identified in a polypeptide sequence of the individual.
[0238]Thus, according to another aspect of the present invention there is provided a method of diagnosing familial tumoral calcinosis in an individual.
[0239]The method is effected by identifying in a polypeptide sequence of the individual at least one amino acid substitution capable of downregulating the expression level and/or activity of GalNAc-T3.
[0240]As used herein, the phrase "one amino acid substitution" refers to any change in a polypeptide sequence (e.g., substitution, deletion, duplication, methylation, acetylation, glycosylation, phosphorylation of an amino acid) which can cause downregulation of the expression level and/or activity of the GalNAc-T3.
[0241]According to preferred embodiments of the present invention the amino acid substitution is identified in the GalNAc-T3 protein as set forth in SEQ ID NO:28.
[0242]Amino acid substitutions can be identified using any method known in the art, preferably, using an antibody which is capable of identifying between two polymorphs of the same protein.
[0243]The term "polymorph" as used herein refers to one form of a protein which is different in at least one amino acid or a modification on at least one amino acid from another form of the same protein. For example, the substitution of a methionine residue with a valine residue of a certain protein. In this case one protein polymorph contains the methionine and the other protein polymorph contains the valine. Other protein polymorphs can be identified by the presence or absence of specific post-translational modifications such as the phosphorylation of a serine, threonine or tyrosine residue in a protein.
[0244]Determination of at least one amino acid substitution between two polymorphs of the GalNAc-T3 can be accomplished directly, by analyzing the GalNAc-T3 protein, or portions thereof. Such a direct analysis is often accomplished using an immunological detection method as described hereinabove.
[0245]As is mentioned before, agents which upregulate or downregulate GalNAc-T3 expression level and/or activity can be used to treat hyperphosphatemia or hypophosphatemia, respectively. Additional examples of such agents can be identified via in vitro and/or ex vivo assays.
[0246]Thus, according to another aspect of the present invention there is provided a method of identifying an agent suitable for treating a disorder associated with abnormal phosphate metabolism.
[0247]The method is effected by exposing GalNAc-T3 or cells expressing GalNAc-T3 to a plurality of molecules and selecting from them at least one molecule capable of regulating the expression level and/or the activity of the GalNAc-T3, such a molecule being the agent suitable for treating the disorder associated with abnormal phosphate metabolism.
[0248]As used herein, "exposing GalNAc-T3" refers to subjecting a GalNAc-T3 protein preparation to various test molecules. A GalNAc-T3 protein preparation can be obtained by extracting proteins from cells or tissues exhibiting high expression level of GalNAc-T3 (e.g., skin, kidney, blood) or by purifying a protein extract of eukaryotic cells which over-express the GalNAc-T3 protein. Preferably, the GalNAc-T3 protein includes at least a catalytic (i.e., the region in the protein which is responsible for the catalytic activity of the protein) or binding (i.e., the region in the protein which is responsible for binding a substrate, receptor and the like) portion of the GalNAc-T3. For example, the catalytic domain of the GalNAc-T3, which includes the amino acids at positions 566-1122 of the GalNAc-T3 protein (GenBank Accession No. NP--004473, SEQ ID NO:28) can be used along with the present invention.
[0249]According to preferred embodiments of the present invention the GalNAc-T3 protein is set forth in SEQ ID NO:28.
[0250]The phrase "cells expressing GalNAc-T3" refers to eukaryotic cells, preferably mammalian cells, more preferably, human cells, which were transfected with an expression vector containing the GALNT3 polynucleotide (e.g., GenBank Accession No. NM--004482, SEQ ID NO:29) and are cultured in a tissue culture flask. Non-limiting examples of such cells include bone marrow cell, kidney cells, fibroblasts, epithelial cells and lymphoblastoid cells.
[0251]Once the GalNAc-T3 or cells expressing GalNAc-T3 are obtained, the test molecules (e.g., drugs, minerals, vitamins, and the like) are applied on the GalNAc-T3 or cells expressing GalNAc-T3 and the expression level and/or activity of GalNAc-T3 is detected using the molecular, immunological and biochemical methods which are fully described hereinabove. Molecules which exert significant modulations of GalNAc-T3 activity and/or expression level (i.e., upregulation or downregulation) become candidates for additional evaluations as suitable for treating disorders associated with abnormal phosphate metabolism.
[0252]The agents of the present invention which are described hereinabove may be included in a diagnostic kit/article of manufacture preferably along with appropriate instructions for use and labels indicating FDA approval for use in diagnosing familial tumoral calcinosis and/or identifying agents suitable for treating a disorder associated with abnormal phosphate metabolism.
[0253]Such a kit can include, for example, at least one container including at least one of the above described diagnostic agents (e.g., for identifying nucleic acid substitution) and an imaging reagent packed in another container (e.g., enzymes, buffers, chromogenic substrates, fluorogenic material). The kit may also include appropriate buffers and preservatives for improving the shelf-life of the kit.
[0254]As used herein the term "about" refers to ±10%.
[0255]Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.
EXAMPLES
[0256]Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.
[0257]Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., Ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (Eds.) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., Ed. (1994); "Culture of Animal Cells--A Manual of Basic Technique" by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., Ed. (1994); Stites et al. (Eds.), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (Eds.), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., Ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., Eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R. I., Ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, Calif. (1990); Marshak et al., "Strategies for Protein Purification and Characterization--A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
Example 1
Characterization of a Familial Tumoral Calcinosis (FTC) Critical Region
[0258]Familial tumoral calcinosis (FTC; MIM211900) is a severe autosomal recessive metabolic disorder manifesting with hyperphosphatemia and massive calcium deposits in the skin and subcutaneous tissues. To identify the genetic basis of FTC, linkage analysis was performed using DNA from informative FTC families, as follows.
[0259]Materials and Experimental Methods
[0260]FTC affected individuals--A total of 12 FTC affected individuals belonging to two large kindred of Druze and African American origin (FIGS. 1a-b), which have been extensively described in the literature (Steinherz, R. et al. 1985, Am. J. Dis. Child. 139: 816-819; Slavin, R. E., et al., 1993, Am. J. Surg. Path. 17: 788-802) were included in the study.
[0261]Linkage analysis--A genome wide scan using 362 microsatellite markers (InVitrogen, Cat. No. 20508, Huntsville, Ala., USA) was employed in a consanguinity FTC family (family 1, FIG. 1a).
[0262]RT-PCR analysis--RNA was extracted using the QIAGEN RNeasy kit (QIAGEN Inc., CA USA) from the following human cells or tissues: liver, heart, kidney, pancreas, skeletal muscle, bone marrow, lung, brain, placenta, retina, and skin. RT-PCR reactions were performed using the TITAN One Tube RT-PCR kit (Roche Molecular Biochemicals, Mannheim, Germany) and the GALex6F (SEQ ID NO:23) and GALex9R primers, following by a nested PCR reaction using the GALex6F and GALex8R (SEQ ID NO:24) primers (see Table 1, hereinbelow).
TABLE-US-00001 TABLE 1 RT-PCR primers and conditions Anneal. Primer Temp. (SEQ ID NO:) Sequence 5'→3' (° C.) GALex6F GGCAGTTGGAGATTATGCCTTG 60° C. (SEQ ID NO:23) GALex8R CAACATCCAGACATAGAGGCTG 60° C. (SEQ ID NO:24) GALex9R CTGCTCTCCAGTGACAACTGTC 60° C. (SEQ ID NO:25) Beta Actin 5' CGACGAGGCCCAGAGCAAGAGA 60° C. (SEQ ID NO:26) Beta Actin 3' TCCAGGGCGACGTAGCACAGCTT 60° C. (SEQ ID NO:27)
[0263]Experimental Results
[0264]Clinical characteristics of FTC affected individuals--All twelve FTC affected individuals reported recurrent painful, calcified subcutaneous masses of up to 1 kg (FIG. 2a), often resulting in secondary infection and incapacitating mutilation. Three patients developed deep periarticular tumors (FIG. 2b) and one patient succumbed to the disease. All patients displayed hyperphosphatemia [range (mg/dl): family 1: 6.2-8.5; family 2: 5.2-6.6] but normal levels of calcium, parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 (Vitamin D3).
[0265]Mapping of the FTC gene to chromosome 2q24-q31--Consanguinity in family 1 enabled the application of homozygosity mapping to identify a 15 Mb segment identical by descent in all affected individuals. This region is flanked by D2S142 and D2S2284/D2S2177 markers on 2q24-q31 (FIG. 1a). As is shown in FIG. 3a, a maximum multipoint LOD score of 6.7 was observed for the 15 Mb region using the MAPMAKER/HOMOZ (Kruglyak L, et al., 1995. Rapid multipoint linkage analysis of recessive traits in nuclear families, including homozygosity mapping. Am. J. Hum. Genet. 56: 519-27). Multipoint linkage analysis in family 2 using 7 markers in this critical region further reduced the interval to 3 Mb, flanked by D2S111 and D2S1776 (FIG. 1b) and yielded a maximum multipoint LOD score of 3.4 (FIG. 3b) using the GeneHunter (Kruglyak, L., et al., 1996, Am. J. Hum. Genet. 58: 1347-1363).
[0266]Analysis of FTC putative genes in the FTC critical region--Using the Mapviewer tool available via the NCBI web site (www.ncbi.nlm.nih.gov/), 11 genes were identified within the FTC region, among which B3GALT1, SCN7A, SCN9A, SCN1A, STK39 are thought to play a role in neural or neuroendocrine tissues, while the function of TAIP-2, CMYA3, FLJ11457, LOC90643, LOC253782 is mostly unknown. The last positional candidate gene, GALNT3, encodes the UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GalNAc-T3) (Bennett, E. P., et al., 1996, Biol. Chem. 271: 17006-17012). The GalNAc-T3 protein belongs to a large family of Golgi-associated biosynthetic enzymes that transfer GalNac from the sugar donor UDP-GalNac to serine and threonine residues, and thereby are responsible for initiating O-glycan synthesis, a prevalent form of post-translational modification (Ten Hagen, K. G., et al., 2003. Glycobiology 13: IR-16R).
[0267]Tissue specific expression of the GALNT3 gene in skin and kidney--To determine if the GALNT3 gene is a putative FTC candidate gene, the expression pattern of the gene was determined using RT-PCR analysis. As is shown in FIG. 4, high level of expression was detected in RNA from both kidney and skin, two tissues of functional relevance to the pathogenesis of FTC [Steinherz, 1985 (Supra); Slavin, 1993 (Supra)].
[0268]These results demonstrate the identification of a 3 MB FTC critical region on chromosome 2q24-q31. Furthermore, the high expression level of GALNT3 in both kidney and skin suggest its involvement in the pathogenesis of FTC.
Example 2
Biallelic Deleterious Mutations in GALNT3, Encoding a Protein Involved in O-Linked Glycosylation, Cause Familial Tumoral Calcinosis
[0269]To test whether mutations in the GALNT3 gene underlie the molecular basis of FTC, genomic DNA from FTC cases was subjected to sequence analysis of the GALNT3 coding sequence.
[0270]Materials and Experimental Methods
[0271]Sequencing analysis--Genomic DNA was amplified using PCR primers designed specific for the amplification of the GALNT3 coding exons. PCR primers and conditions are listed in Table 2, hereinbelow.
TABLE-US-00002 TABLE 2 PCR primers and conditions Anneal. Primer Temp. (SEQ ID NO:) Sequence 5'→3' (° C.) GALe1bR GCTCACCCCTCTCTCCCCTG 60° C. (SEQ ID NO:1) GALe1bF CATTGATGCTGGTGAGAG (SEQ ID NO:2) GALe1aR CTGAGGTGGACGGTCAAGGACAG 60° C. (SEQ ID NO:3) GALe1Af GTAGGACTGAATAGCTACTAATAC (SEQ ID NO:4) GALe2R CTGAGATGGCATACAGAGAGTAC 60° C. (SEQ ID NO:5) GALe2F CTCTGGGTGAGTGATTTGCTTG (SEQ ID NO:6) GALe3R CACAGAGCTGTTACCTGCTTGG 60° C. (SEQ ID NO:7) GALe3F GCTCTGTGGTTTCATTAGCTTTC (SEQ ID NO:8) GALe4R GCTATAAAGCAAACAGTGTGTAC 60° C. (SEQ ID NO:9) GALe4F CAATAAATCTGAGGAAGAAAGAAATC (SEQ ID NO:10) GALe5R GTGCACACATCTGTAATCATATG 60° C. (SEQ ID NO:11) GALe5F CAATGGGAGAGGACACGAAGTAC (SEQ ID NO:12) GALe6R GAATCGACGCAAAAGGACGTG 60° C. (SEQ ID NO:13) GALe6F GAAATGGCAGGGGACAGAGAC (SEQ ID NO:14) GALe7R GTAAAATCTCAAAAGCAATAAAGAAAG 60° C. (SEQ ID NO:15) GALe7F CAGAAATGAACAGGCAGGCATG (SEQ ID NO:16) GALe8R GCAACATCTCACTTGTGCTTG 60° C. (SEQ ID NO:17) GALe8F GATACGTGAGTATTTTCCTGTTCC (SEQ ID NO:18) GALe9R GATATATTCTCTTATCACATGGG 60° C. (SEQ ID NO:19) GALe9F GGCTATTGTATCGTCTATCAC (SEQ ID NO:20) GALe10R CTACAGTGTATGCCTAGTCACAG 60° C. (SEQ ID NO:21) GALe10F CTGTCTGCCTCTCTTCATTATG (SEQ ID NO:22)
[0272]Experimental Results
[0273]FTC patients of family 2 are compound heterozygous of a nonsense mutation in exon 1 and a splice mutation in intron 7 of GALNT3--As is shown in FIGS. 5a-b, affected individuals of family 2 were found to be carriers of a nonsense mutation in exon 1, in which a C→T substitution resulted in a change of the CGA codon of Arginine at position 162 of the GALNT3 protein (GenBank Accession No. NP--004473 SEQ ID NO:28) with a termination codon (i.e., TGA). Moreover, as is shown in FIG. 5c-d, the same affected individuals of family 2 were carriers of a splice mutation in intron 7 at position 1524+5G→A from the ATG translation start site (GLANT3 mRNA sequence--GenBank Accession No. NM--004482, SEQ ID NO:29; GLANT3 genomic contig--GenBank Accession No. NT--005403).
[0274]Homozygous splice mutation in intron 7 of the consanguinity Druze FTC family 1--Sequence analysis of all 10 coding exons of the GALNT3 gene from the Druze family revealed the presence of a homozygous G→A transition at position 1524+1 (from the ATG translation start site GenBank Accession No. NM--004482, SEQ ID NO: 29), resulting in the disruption of the intron 7 donor splice site consensus sequence (FIGS. 5c-d).
[0275]Noteworthy, all three mutations (i.e., 1524+1G→A; 1524+5G→A, R162X) were excluded from a panel of at least 290 chromosomes derived from healthy unrelated individuals.
[0276]Furthermore, as can be seen in FIGS. 6a-b, a complete co-segregation of the mutations with the disease phenotype was confirmed by PCR-RFLP in both FTC families.
[0277]Analysis and Discussion--The nonsense R162X mutation is expected to result in a non-functional null allele due to premature termination of protein translation. Mutations 1524+1G→A and 1524+5G→A alter the same splice donor site in intron 7. In contrast to the normal splicing score of 0.93 obtained for the intron 7 splice donor site predicted by the Splice Site Prediction by Neural Network software (http://www.fruitfly.org/seq_tools/splice.html), the calculated score of this sequence carrying a G→A mutation at position 1524+1 or 1524+5 was 0.00.
[0278]The 1524+1G→A splice mutation results in an absence of exon 7 from the GALNT3 mRNA transcript--To further assess the consequences of the 1524+1G→A splice site mutation, the expression pattern of GALNT3 was assessed using RT-PCR. As shown in FIG. 7, while a normal band of approximately 600 bp was detected in skin and blood samples of a healthy individual, no wildtype GALNT3 transcript was detected in RNA from affected individuals. In addition, low amounts of an aberrant splice variant were detected in RNA derived from both skin and blood samples. Sequence analysis of the aberrant shorter transcript revealed the presence of a shorter GALNT3 transcript lacking exon 7 nucleotide sequence (FIG. 8). This shorter mRNA transcripts results in an in-frame deletion of 44 amino acid residues in the mature polypeptide, destroying most of the linker region located between the catalytic domain and the ricin-like domain of the glycosyltransferase.
[0279]Altogether, these results demonstrate that deleterious mutations in the GALNT3 sequence underlie the molecular basis of FTC.
[0280]Analysis and Discussion--Since the original description of FTC more than a century ago by Giard, J. M, 1898 (Sur la calcification hibernale. Compes. Rend. Seanes. Soc. Biol. So. 1013-1015), the pathogenesis of this disease has stirred up a large number of investigations but has remained mostly elusive.
[0281]The results presented here suggest a role for GalNAc-T3-mediated glycosylation in the control of phosphatonin activity. Although the NetOGlyc 3.0 software (http://www.cbs.dtu.dk/services/NetOGlyc) identified potential O-glycosylation sites in FGF23 (setting O-glycosylation score significance at >0.5), this molecule is unlikely to mediate the deleterious effects of GALNT3 mutations in FTC. Indeed, impaired FGF23 activity in a murine model was recently shown to lead to prominent bone tissue abnormalities [Shimada, T et al. J Clin Invest. 113, 561-568 (2004)], which are absent in FTC patients. Of note, FGF23 circulating levels measured by ELISA (Immutopics, CA) were significantly elevated in 6 FTC patients (1710+864 RU/ml) as compared with 6 healthy controls (56+38 RU/ml), possibly reflecting a compensatory response to hyperphosphatemia. Thus GalNAc-T3 may affect phosphate homeostasis by modulating the activity of another phosphatonin or PHEX [Jan De Beur, 2002 (Supra)]. Alternatively, it may directly regulate non-circulating elements within tissues where GALNT3 is expressed such as the skin, where calcium deposition occurs [Steinherz, 1985 (Supra); Slavin, 1993 (Supra)], the bone, where candidate phosphatonins are expressed [Schiavi, 2004 (Supra); Quarles, 2003. (Supra)], and/or the kidneys and gastrointestinal tract, where phosphate transport occurs [Jan De Beur, 2002, (Supra)]. Given the existence of more than 20 ppGaNTase isoforms [Ten Hagen, 2003 (Supra)] substrate specificity and/or functional redundancy may account for the restricted nature of the FTC phenotype despite GALNT3 widespread tissue expression.
[0282]GalNAc-T3 may not be the sole regulator of phosphate homeostasis in peripheral tissues. Prince et al [Prince M. J. et al. Ann Intern Med. 96, 586-591 (1982)] established 2 decades ago that FTC can also present with normal phosphate levels. Using haplotype analysis in four families with normophosphatemic FTC, this FTC variant was excluded from linkage to 2q24-q31 (not shown), suggesting that normophosphatemic and hyperphosphatemic FTC are non-allelic disorders.
[0283]In summary, these results establish autosomal recessive mutations in GALNT3 as the molecular cause of hyperphosphatemic FTC and demonstrate the pathological consequences of a genetic defect in a mucin-type O-glycosylation pathway. The identification of the FTC gene should not only benefit the affected families, to which molecular testing can now be offered, but may also shed new light on the mechanisms regulating phosphate metabolism in health and disease, with obvious implications for the treatment of acquired disorders manifesting with hyperphosphatemic calcinosis, such as chronic renal failure.
[0284]It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.
[0285]Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.
Sequence CWU
1
33120DNAArtificial sequenceSingle strand DNA oligonucleotide 1gctcacccct
ctctcccctg
20218DNAArtificial sequenceSingle strand DNA oligonucleotide 2cattgatgct
ggtgagag
18323DNAArtificial sequenceSingle strand DNA oligonucleotide 3ctgaggtgga
cggtcaagga cag
23424DNAArtificial sequenceSingle strand DNA oligonucleotide 4gtaggactga
atagctacta atac
24523DNAArtificial sequenceSingle strand DNA oligonucleotide 5ctgagatggc
atacagagag tac
23622DNAArtificial sequenceSingle strand DNA oligonucleotide 6ctctgggtga
gtgatttgct tg
22722DNAArtificial sequenceSingle strand DNA oligonucleotide 7cacagagctg
ttacctgctt gg
22823DNAArtificial sequenceSingle strand DNA oligonucleotide 8gctctgtggt
ttcattagct ttc
23923DNAArtificial sequenceSingle strand DNA oligonucleotide 9gctataaagc
aaacagtgtg tac
231026DNAArtificial sequenceSingle strand DNA oligonucleotide
10caataaatct gaggaagaaa gaaatc
261123DNAArtificial sequenceSingle strand DNA oligonucleotide
11gtgcacacat ctgtaatcat atg
231223DNAArtificial sequenceSingle strand DNA oligonucleotide
12caatgggaga ggacacgaag tac
231321DNAArtificial sequenceSingle strand DNA oligonucleotide
13gaatcgacgc aaaaggacgt g
211421DNAArtificial sequenceSingle strand DNA oligonucleotide
14gaaatggcag gggacagaga c
211527DNAArtificial sequenceSingle strand DNA oligonucleotide
15gtaaaatctc aaaagcaata aagaaag
271622DNAArtificial sequenceSingle strand DNA oligonucleotide
16cagaaatgaa caggcaggca tg
221721DNAArtificial sequenceSingle strand DNA oligonucleotide
17gcaacatctc acttgtgctt g
211824DNAArtificial sequenceSingle strand DNA oligonucleotide
18gatacgtgag tattttcctg ttcc
241923DNAArtificial sequenceSingle strand DNA oligonucleotide
19gatatattct cttatcacat ggg
232021DNAArtificial sequenceSingle strand DNA oligonucleotide
20ggctattgta tcgtctatca c
212123DNAArtificial sequenceSingle strand DNA oligonucleotide
21ctacagtgta tgcctagtca cag
232222DNAArtificial sequenceSingle strand DNA oligonucleotide
22ctgtctgcct ctcttcatta tg
222322DNAArtificial sequenceSingle strand DNA oligonucleotide
23ggcagttgga gattatgcct tg
222422DNAArtificial sequenceSingle strand DNA oligonucleotide
24caacatccag acatagaggc tg
222522DNAArtificial sequenceSingle strand DNA oligonucleotide
25ctgctctcca gtgacaactg tc
222622DNAArtificial sequenceSingle strand DNA oligonucleotide
26cgacgaggcc cagagcaaga ga
222723DNAArtificial sequenceSingle strand DNA oligonucleotide
27tccagggcga cgtagcacag ctt
2328633PRTArtificial sequenceSingle strand DNA oligonucleotide 28Met Ala
His Leu Lys Arg Leu Val Lys Leu His Ile Lys Arg His Tyr1 5
10 15His Lys Lys Phe Trp Lys Leu Gly
Ala Val Ile Phe Phe Phe Ile Ile 20 25
30Val Leu Val Leu Met Gln Arg Glu Val Ser Val Gln Tyr Ser Lys
Glu 35 40 45Glu Ser Arg Met Glu
Arg Asn Met Lys Asn Lys Asn Lys Met Leu Asp 50 55
60Leu Met Leu Glu Ala Val Asn Asn Ile Lys Asp Ala Met Pro
Lys Met65 70 75 80Gln
Ile Gly Ala Pro Val Arg Gln Asn Ile Asp Ala Gly Glu Arg Pro
85 90 95Cys Leu Gln Gly Tyr Tyr Thr
Ala Ala Glu Leu Lys Pro Val Leu Asp 100 105
110Arg Pro Pro Gln Asp Ser Asn Ala Pro Gly Ala Ser Gly Lys
Ala Phe 115 120 125Lys Thr Thr Asn
Leu Ser Val Glu Glu Gln Lys Glu Lys Glu Arg Gly 130
135 140Glu Ala Lys His Cys Phe Asn Ala Phe Ala Ser Asp
Arg Ile Ser Leu145 150 155
160His Arg Asp Leu Gly Pro Asp Thr Arg Pro Pro Glu Cys Ile Glu Gln
165 170 175Lys Phe Lys Arg Cys
Pro Pro Leu Pro Thr Thr Ser Val Ile Ile Val 180
185 190Phe His Asn Glu Ala Trp Ser Thr Leu Leu Arg Thr
Val His Ser Val 195 200 205Leu Tyr
Ser Ser Pro Ala Ile Leu Leu Lys Glu Ile Ile Leu Val Asp 210
215 220Asp Ala Ser Val Asp Glu Tyr Leu His Asp Lys
Leu Asp Glu Tyr Val225 230 235
240Lys Gln Phe Ser Ile Val Lys Ile Val Arg Gln Arg Glu Arg Lys Gly
245 250 255Leu Ile Thr Ala
Arg Leu Leu Gly Ala Thr Val Ala Thr Ala Glu Thr 260
265 270Leu Thr Phe Leu Asp Ala His Cys Glu Cys Phe
Tyr Gly Trp Leu Glu 275 280 285Pro
Leu Leu Ala Arg Ile Ala Glu Asn Tyr Thr Ala Val Val Ser Pro 290
295 300Asp Ile Ala Ser Ile Asp Leu Asn Thr Phe
Glu Phe Asn Lys Pro Ser305 310 315
320Pro Tyr Gly Ser Asn His Asn Arg Gly Asn Phe Asp Trp Ser Leu
Ser 325 330 335Phe Gly Trp
Glu Ser Leu Pro Asp His Glu Lys Gln Arg Arg Lys Asp 340
345 350Glu Thr Tyr Pro Ile Lys Thr Pro Thr Phe
Ala Gly Gly Leu Phe Ser 355 360
365Ile Ser Lys Glu Tyr Phe Glu Tyr Ile Gly Ser Tyr Asp Glu Glu Met 370
375 380Glu Ile Trp Gly Gly Glu Asn Ile
Glu Met Ser Phe Arg Val Trp Gln385 390
395 400Cys Gly Gly Gln Leu Glu Ile Met Pro Cys Ser Val
Val Gly His Val 405 410
415Phe Arg Ser Lys Ser Pro His Ser Phe Pro Lys Gly Thr Gln Val Ile
420 425 430Ala Arg Asn Gln Val Arg
Leu Ala Glu Val Trp Met Asp Glu Tyr Lys 435 440
445Glu Ile Phe Tyr Arg Arg Asn Thr Asp Ala Ala Lys Ile Val
Lys Gln 450 455 460Lys Ala Phe Gly Asp
Leu Ser Lys Arg Phe Glu Ile Lys His Arg Leu465 470
475 480Arg Cys Lys Asn Phe Thr Trp Tyr Leu Asn
Asn Ile Tyr Pro Glu Val 485 490
495Tyr Val Pro Asp Leu Asn Pro Val Ile Ser Gly Tyr Ile Lys Ser Val
500 505 510Gly Gln Pro Leu Cys
Leu Asp Val Gly Glu Asn Asn Gln Gly Gly Lys 515
520 525Pro Leu Ile Met Tyr Thr Cys His Gly Leu Gly Gly
Asn Gln Tyr Phe 530 535 540Glu Tyr Ser
Ala Gln His Glu Ile Arg His Asn Ile Gln Lys Glu Leu545
550 555 560Cys Leu His Ala Ala Gln Gly
Leu Val Gln Leu Lys Ala Cys Thr Tyr 565
570 575Lys Gly His Lys Thr Val Val Thr Gly Glu Gln Ile
Trp Glu Ile Gln 580 585 590Lys
Asp Gln Leu Leu Tyr Asn Pro Phe Leu Lys Met Cys Leu Ser Ala 595
600 605Asn Gly Glu His Pro Ser Leu Val Ser
Cys Asn Pro Ser Asp Pro Leu 610 615
620Gln Lys Trp Ile Leu Ser Gln Asn Asp625
630293874DNAArtificial sequenceSingle strand DNA oligonucleotide
29atggctcacc taaagcgact agtaaaatta cacattaaaa gacattacca taaaaagttc
60tggaagcttg gtgcagtaat ttttttcttt ataatagttt tggttttaat gcaaagagaa
120gtaagtgttc aatattccaa agaggaatca aggatggaaa ggaacatgaa aaacaaaaac
180aagatgttgg atttaatgct agaagctgta aacaatatta aggatgccat gccaaaaatg
240caaataggag cacctgtcag gcaaaacatt gatgctggtg agagaccttg tttgcaagga
300tattatacag cagcagaatt gaagcctgtc cttgaccgtc cacctcagga ttcaaatgca
360cctggtgctt ctggtaaagc attcaagaca accaatttaa gtgttgaaga gcaaaaggaa
420aaggaacgtg gggaagctaa acactgcttt aatgctttcg caagtgacag gatttctttg
480caccgagatc ttggaccaga cactcgacct cctgaatgta ttgaacaaaa atttaagcgc
540tgccctcccc tgcccaccac cagtgtcata atagtttttc ataatgaagc gtggtccacg
600ttgcttagaa ctgtccacag tgtgctctat tcttcacctg caatactgct gaaggaaatc
660attttggtgg atgatgctag tgtagatgag tacttacatg ataaactaga tgaatatgta
720aaacaatttt ctatagtaaa aatagtcaga caaagagaaa gaaaaggtct gatcactgct
780cggttgctag gagcaacagt cgcaacagct gaaacgctca catttttaga tgctcactgt
840gagtgtttct atggttggct agaacctctg ttggccagaa tagctgagaa ctacacggct
900gtcgtaagtc cagatattgc atccatagat ctgaacacgt ttgaattcaa caaaccttct
960ccttatggaa gtaaccataa ccgtggaaat tttgactgga gtctttcatt tggctgggag
1020tcgcttcctg atcatgagaa gcaaagaagg aaagatgaaa cctacccaat taaaacaccc
1080acttttgcag gaggactttt ttccatatca aaagaatatt ttgagtatat tggaagctat
1140gatgaagaaa tggaaatctg gggaggtgaa aatatagaaa tgtctttcag agtatggcaa
1200tgtggtgggc agttggagat tatgccttgc tctgttgttg gacatgtttt tcgcagcaaa
1260agccctcata gctttccaaa aggcactcag gtgattgcta gaaaccaagt tcgccttgca
1320gaagtctgga tggatgaata caaggaaata ttttatagga gaaatacaga tgcagcaaaa
1380attgttaaac aaaaagcatt tggtgatctt tcaaaaagat ttgaaataaa acaccgtctt
1440cggtgtaaaa attttacatg gtatctgaac aacatttatc cagaggtgta tgtgccagac
1500cttaatcctg ttatatctgg atacattaaa agcgttggtc agcctctatg tctggatgtt
1560ggagaaaaca atcaaggagg caaaccatta attatgtata catgtcatgg acttggggga
1620aaccagtact ttgaatactc tgctcaacat gaaattcggc acaacatcca gaaggaatta
1680tgtcttcatg ctgctcaagg tctcgttcag ctgaaggcat gtacctacaa aggtcacaag
1740acagttgtca ctggagagca gatatgggag atccagaagg atcaacttct atacaatcca
1800ttcttaaaaa tgtgcctttc agcaaatgga gagcatccaa gtttagtgtc atgcaaccca
1860tcagatccac tccaaaaatg gatacttagc caaaatgatt aagtgttcct taaaattaag
1920ttgaaaaagg aaatattctt tctcataaaa ctgtgactag gcatacactg tagtttttga
1980aaattatgca aaagcagcta aatgtaactt attccaagtg catttttctt atttatatct
2040ttatgtagca ctactacaga aattctgcaa gtttctgttt caaagcacaa taactagtaa
2100taccaaagac tatttcaaaa tgtccagatg taggggaaga gatgtttaca gtatgatgaa
2160aataattttc caagtaaagt gatgtttgtg tgttttgtac acttagggat atatatatat
2220agctacattc acacactcac aatttaaaat atttccccta gttttttggg gggataggaa
2280gaaagatttg ttactgtatt tttttaacta cataaaaata gatcaataaa tgtcagcatt
2340ggcctctgtg tacaaaccaa gagcttttac agatccagaa tttattagtt taaaatgcag
2400gtgaactttt ttttgcgttt ggtttacttg tctgtcaaat gtttccttaa acatgaaact
2460gaataaggag aagagtattt ttaacactta aatttcttgg caaattttaa aacatttttt
2520agtctgtaat acactccact tgaagcactt aagtcttcct taaatgactt ttcttaagta
2580atgatactgt gtgttttccc aaagcacttt taaaaaaatt tttataaatt actatctgtt
2640gaaaaggtgt ccttttcctt tcttctagta ttttttttct taccaaaatt cactaatctt
2700gaatgtttgt gatattaaat ttcaaatgca gaatacttga ctcatttaaa gctaaatttt
2760gttactgatt caattataat tgtaatggat ttttgacttt gtaatggatt cttttcatca
2820aaaagcctta ttatttttta tctatgtgga aaacacaata aaaaatcctc aacactattg
2880taatcatttg gttaagtgct tattcctctt ttgggtaaaa tctgtaattg ataataggtg
2940ggggaaaatg aattttgtat gctgaatttc taagcgccta ttgtttgtaa aaccatcaga
3000tatttcttat ggcacaaaaa atgaggaata gcaaaattcc tgtgttcaat atttagaaaa
3060ttttgtatta atttctgata aagttcctta agcatctgat agaatgatgt tttaaaaaaa
3120tttgacgctt gcttaggaga tttaccactt tttttttttg tttttcgtca ttttatattt
3180agatctcctg tattcttgtt cccgaagtaa aatacgatcg gtttcatatt ttaaatctgg
3240cagagcctca gctgtacgaa aaagagcata tactggttat tgaccctatc ttctcattgt
3300ttgtttgtaa gtttgaattt gtattaaaaa gcctgcattc tgagctggac atggtggctc
3360agcttctaat cccagcactt tggtaggcaa aggtgggagg atcatttgag ctcaggagtt
3420ccagaccagc ctgggcaaca tagcaaaatc tcatctctac aaaaagtaaa aattaaaaaa
3480tgaaattaaa aataaaatta cctaggtgtg gtggcacgca tctgtagttc cagctataca
3540ggaaggtgag gcagaagcat tgcttgagct tgggagatcg aggctacagt gagctatgat
3600tacaccactg cacttcagtc tgtgtgactg agcaagactc tttcaaaaaa aaaaaaaagc
3660ctacattctc cagttgatta tttccaacta atgtgtatta tgtgcctaat tttctatcag
3720aagttgtatt aagcccgttt tcacactgct gttaaagaca tacctgagac tgggtaattt
3780ataaagaaaa ataggttcaa tggacccaca ggtccgcgtg gctggggaag cttcacaatc
3840atggcggaag gtgaaagcat gtcttacgtg gaag
38743019DNAArtificial sequenceAntisense oligonucleotide targeted against
the GALNT3 mRNA 30ctggcacata cacctctgg
193119DNAArtificial sequenceAllele specific probe
31ctttgcactg agatcttgg
193219DNAArtificial sequenceAllele specific probe 32ctttgcaccg agatcttgg
1933157775DNAHomo sapiens
33ttaaaaagat ctttaactat tataaggttc atataattaa tatagaggaa tcatccaatg
60atacttatga agggaaaatg acctattttc cttcaccact ctgaggacct aactcagtaa
120atgcagagga ggctgaggaa acatggaaga gacacgacct cagcaaccag ccttttctcc
180agcgtgatca atccatctct acaaaatcag tgtataatga gatttccact tagtgactag
240ctgattgaag gggggcctct acccaaatac tcaactaggc ctttcttttc tgaagcattt
300tgatttgtct tgcctgggac aactagcaga acagtccaga atgcattgca tactttctaa
360ttacctcaca ttctcttatt aaagagaaca gatagaaata aaacatgcac tccccacctt
420tagattacct gcacttggtt cgtcggagga cttctaggat cccgtttccc tgtaaattaa
480tttaggtaac taaatagtgt aattctgcct ttttttttcc aatgcaatat gactatgcta
540aacctgtttc atatttttga ccttctattg ttatatcact tcacatgttt tataactatt
600tgaaatgcgg acatgacttg ctattcagtg actgcatcgc tacttatttt ttatgtcttt
660tagacttggg tgtttagaat tatggatata aatatattgt agatctcaat ttctaaaaaa
720aatattttat ttaattgaac ttaaaaatgc ttgatctaga gaaagtcctc cagtggaaaa
780gtttctacta aactttgttt tggggaaaaa atgtttactt ggcagctctt ccactgtgca
840gttggtttct gccccttctc ttccccccta cattgggcca aatttataac tcttaatatt
900aacattaaaa gagctttggg tttgtcttta tgtaggttgc agtaatgaga gcagggttgt
960tccttttgaa tgattggccc tttgggctaa tcagaatgag gatttgttga aatctcaaat
1020atacatatat atatatatat atatatatat atatatatac acacacacac acacatacac
1080atatatatac acatatatgt atgtgtgtgt gtgtgtgtgt gtgtgtgtat atatatatat
1140cccatgttgt ctgcaaagtg acagttttta gtgcttaatc tccaaaattt tccccctact
1200gatacaatct aaagagcaca ggcccaaaga gaaaatgaca agaggctatt gttccttcat
1260tcaagcacat gaaaggatca tgtgtactgt atttgctgga ccattacaag ctcttgaagg
1320atctgcttgt gaagtactct gaggttattc ttggaacaca gagtgtggga gagtggggaa
1380gagccagctc cagcctgagc aaagctgaag ggtagaatga cagtccagtt tctgaaaaga
1440gatattttaa ttttagttca gctttatttt cctaagacga tatgaagcct acttttttat
1500ttttggctag gtctttgttt tctagctttg tttccaaaag gatgcttaag aaaacagagt
1560ttttttcccc tatccattta tctagggtag aaagtcactg ccctgtgaga agaattaccc
1620ttccaatcat gacagcttct attccaactt ttcatatacc tgactaacct gtaaacatat
1680cccacaaacc acatacacag gctacctatt aaaactctcc catataccta acacatacac
1740atgcacacac atacaaaata tatacatatc acacacttag agaatgacag atttcaggca
1800atggccaata gtcagtattg agtttttcta accccagaat ctggttgggc tttatgatct
1860aaaaaaatgt ggtttagaaa agaggataat tggtagagct gtagtttgaa aatatcaggt
1920caaagttgca aacatactta tgatggaatc atttcatttt tagttccttt cccagaaaag
1980ctgagagagc acactgttcc agctgacttt tttttccatt aatagccgtt ctattcaaca
2040aagagctgcc cacatgccag gaaagtgcta cagtccttct aagcttttaa aaatggggat
2100caagctacat gcagccagtt ggaatactta agatctttat gccaagacac aataacctga
2160cctgcagctg ttgtattgcc tccatagtat atctagccaa actgtagtct ttgacacaca
2220actttttagt cattcactat caattgcaaa gcttcatgct ccagccctgg ctattctagt
2280accagtccta tacctttaaa tgggacttaa ttcccagtta taatcctggc cgaattgatt
2340tgaaagtcaa gtggctgaag cagcttgcat ctcctttgtc attcccaatt tcctttggga
2400gaagtcaatg aaagagattt acttaagttt ttcctcctta tctcttgatc attaattatt
2460aagacatggg taaaattcat ttgtctcatg tcttttggtt gattgaataa tatagttact
2520ttttgaaagc ccatcatttc tttgtgataa aaccattttc tcaatgtgac agccacatgt
2580aaaatgtgaa actaatgttt cttagtatgt ttaaaatctt cacaatcata tatggccctg
2640caatactccc tgaattcctt ccctccaaaa aaaaactatg agttgcaaaa acactagtta
2700cgaattaatc aagaagcaaa gagagtatgt taagtggcta tgttctttaa aattttacat
2760tctaatagat gacttctagg caaaattttg gtatgattac cattaaacag gaaaacatac
2820catgatttac tttctataac ttgctaatca ctgtttcttt tatttcaatt tttgatattg
2880ttgcacaagt ttatgactct ttggtcttta tattcaaaat ttattaagta tcaattcttt
2940ctttcaacaa atatgtactg aacacatgct gtgttagaac aactgtcttg ggtagtaaaa
3000agaggggaac actacaaatt ataatggata atattaatga agggtggcat gaaataatca
3060agctttctca aatcctctta atatgtgatt ttatcaagtg acattagaaa aactgacatc
3120agttcttgtc taattatatg tgagctgcaa ctaaaagatg cttcctttac ctaaattata
3180gaactcactg gatcgccctt catctctctc cctccatctg aaatgtatga attaaatcag
3240gactcagcta ggactgaatc aaatgaaaaa gaaattttcg attaattgct tcttcaatgt
3300tagacctata taagtgatgc taaatttaca ccagcaaact cctatcagac agtttcaagg
3360aggatttgac tcatgcataa cccaggaaac atttcaggaa acttttaaga caacagtgtg
3420gaacacaatc actcttactt ctatttataa aaagcctatc aaaacagcaa aatcctgttg
3480caaattccaa agacatcact ttgcagcagg tggaaaacag agaggttgtt agactccatc
3540actttcagct ggagttacaa accatcaaat tagcaggtgg aaaatttata ttctcagaag
3600ccaacaggag actgcctttt taaaaggtgt catgaagcct tctatttctc caaacaactc
3660aatatttctc tttaaaatgg catctctgtt ccttattctc ttgatgacag tttgcagtgc
3720cattatagcc agtgtattaa ataccaggct ttaatgggaa gcacctttgt gctttcaaag
3780gcaagacttg tctgtaattt taaattagaa ctgttggttg tattcttacc acaaccaaaa
3840cccaaagttt aaaggcttga ttactctata ttaggtctaa tatgaatttt caccttctga
3900ttgcattact ttttctgaaa tctgctaact agtgctggaa taacaaaaat gcctaagcca
3960aaatgctgta gctctgcacc aacagcacag ctcatcagat gttttctata gtagtaaaga
4020atttgattga cttaattgaa tatcagcaat tttaataccc actagaatta tggaagtatc
4080agagtggaag tgaggacgca ataaaagctt aataagtggt ggtgtcttct aggaattatg
4140aaaaaaaaag ctcagcagag ctaagccaga tcttattaca tcataaagac tagagttaca
4200aatggcagcc ccaaacctag aaggggcagt tacaatgtgg acaccctttc agcccagttg
4260gtgctcacat ctgctgacca acattcatat caattacatt tttacagtct gtaagtcatt
4320tgatgcttta gaaaataaaa acacacacct acagcattct aaagaaacta ttttgttaaa
4380aaatttaaac acattttata ttaaaataag tgatcaatga ctttcatgat tgtcaaaagt
4440aaaaagtgca gacatttaaa aaagctcttc tttcacattt tacctaccaa atctagatat
4500tcaacgtgag tccttaaaac agtcacttct aaattttatt ttgactcctt tcttggcttt
4560ttcaaactgg accatgagac ccccttccta aacaatgcag ttgttttgtt ttattctttt
4620tttccagaaa ttcaagctga aacatttgat atgacctgtg ttttacctgg tgatcagttc
4680tcaggctagt ctaagtctgt gctgtcttgg catgccttgg gtttcctatc ctgagagtgg
4740taatgccagt agaatgatgg gaaccaggag agtgaatacg accagattca aagcctaaaa
4800caatatgttt cagtttactt ttatatgtag attttttaaa tattcacatc tagcctggaa
4860ttttagtaaa tacagatact tgttgtatcc atgggaaatg aggtcatttc aaagatgtaa
4920gtcacgttgg tgactacttt cttttacttg tgataaatat atcactatac agggtaattg
4980cttttataaa tttgatcctt tgcattggct tataattcaa gaaatgtcaa cttcactgtg
5040tcactttatg tcatctctca taaaagtttc tggcaggact aaagtttcaa aggaatgtga
5100tagaatttat tttggataac atttgtattc agcatgctaa tgatgtaact cctctcttta
5160tcattaccac ggtgatagtt aagttcattc gttaactcat tttagtgaca tgtgaggcca
5220tttaaatatt tttaagataa tgtatattga ttagaaaggg ttagaaggag acaaatttat
5280ggtagaaatt agccttgcca ttgtctaagt taatttatgt gacctgattg aacagtttta
5340gttgtaactt tagagtagct tctttgttag aggatctctt tcctttctct tttggttttc
5400tcccaatatt tttatctgtc ttgggattat cttaaaaggt atttattaat gagtatttgg
5460aagaaaattg acctagaaaa tgtctctttt aaatttatct aggtacttgt atcaccagag
5520accttcatca agtcctactg ccacactgga taaggccact acccccgacc cttcttgtca
5580gggacatggc ttcctaataa tggttttgct gttgtctcta cataggggac ctatggaaaa
5640ggagtttgaa atttctacct ttgtagtaca ttgtatccaa ttttgttttg ctcctccttt
5700tcatgtggtc accaagcttt tttccttagc cattaaaaaa gccacccctc tttaaaacaa
5760atttaaagat ctatatacat ttttgcaact ttatttaata aaaaagagaa gaaaaaataa
5820aatctaaatc tcaattaccc taaagtggaa aaacctaagt atctaaccta ggaacttgtt
5880gcctggtggt ggttgttgtt gttgttgttt tttaatttat ttagtgttga tccttgagtt
5940atttaatggt atccaaatga tatctgaaag tgattgattt taagatgtaa tagttggcaa
6000agtcatttca tttgtgtctc aaacaaataa agcccactgc catgggttac catctgaacc
6060ctgattcatt aggcactagt catgccaaat ggggatctca gaatagttta atcttttcaa
6120agatactcac aatggccaac tgtgtgtgaa tactggaaag tttgagattc ttctatattc
6180ttcaaattta tggtcctgag actaggaggg atctccagag gtcatatggc tcatcgatct
6240accaccaggg aagactgtca cttaggcatg cccaacagga gagatcgcca tctattctta
6300acatctctag ggaaggcagt ttccttggca acacaattca gctctcttag gttttctcac
6360aataagaaaa tatcttggcc agtctttgag ggttaggcac ctggcaacag ttgtggtgat
6420ttgggccatt gtactagggg attcaaggga agttgggttt ccccctacat ttggtgatta
6480ttacattaat atatgatcat tccataagtc tcctaagaaa tcagattttt aaagaaagac
6540acggattcag agacaatggt ctgtaacatg accaactttt gacaaatatg gctgtactag
6600gttttcattt ttgctgttgt tgttgtttgt ttttgtgatt gctcttatgt cggtaggtaa
6660tgtcaggaaa agtgactgtt tcagatcccc agtacaaaca tcagaactag gatcaaatga
6720cttgactttt aagctgttcc attttctaag aagttacaat tacaaaaagt aagcattttc
6780tacattctta cttctaagta atgaccttct cattaatctt tccttggctt aaaatgacaa
6840ataagaatcc aaagtttttc cactcctcct gtgggagata ggaatagttg tgaagagtct
6900gatggaataa aatgactcca gagatttaca cagccaccat ttgatcccag cctttgtcct
6960tgtgttattt aaacaatggc ttcctgcttt ctcaggggtc aggaaaatga tttaaaaatt
7020tgtcttcttg acaaggcctg aaaacgaagg gaggtgtcgt ggagaaatgg tgatttttct
7080ggttacatca cttgattcaa gcactccctg gagctttcaa agcctttttt atctgctcag
7140agttgacacc caactcagag tcagaactca ccagaagatg aggaacaccc ccgaaatcaa
7200tgtttccttt ctccatggga tttttttcaa tcttggtaca gctgcatatc tcccaaactc
7260ggatgctctt gtgaaatgac agccacatca gtgtggttac tccccacaaa taacactagg
7320gggaaatgac cttttttatt taaaaaaaaa aaaaaggcta atgctataga atgatcatgt
7380aaagaatgtt gaataaaatt tggcccatgt tttaattgac tctaggcacc tctattgaaa
7440taagtctgag gcatagtttg tgaaacatgt ttcaaaagtg tttgatgtat aataaatgct
7500agaaatttac agtacagaaa tagtaaaaaa ataaaataaa aataaacctg tagattacag
7560ctctgttcct aagaacctca gcctttattg tgtcttagta aagttgatct gctttatttt
7620tagtttattg aatttctggt gtactagcct cttaagatag aacttttaat acatatgtac
7680tggttaatgc ctgtttacgc agaaaatggc actttgttct ttcagtgtgt ttccctcagt
7740gtcacagggt atatcaattt gatagatcat ttgacatttg tcaaaaatac gtttcccatt
7800ttaaaaatat gtataattgg ttgcatagac atatgtcact ttttgcagtg tcttcatctt
7860gatcatagtt ttagacatca actatgtctc actttccaac tgccatagaa atttggtaca
7920tttgcaaatt ctggtggaat ctagtgagct gaatgtattg gtagatcaaa tgatccaaag
7980gacttgggtg ttatccttca gtctgtgttg aaccattcag atattgggaa tggtccttac
8040acaagggaag tcttttgaga atatttgttc tcttattttt tgtttgtttg ttgctggcaa
8100aaaaaaaagt aactttaaaa actcagaaaa gccactgaac tgtattttgt gaccttatga
8160ttagttttat ctttcaactg atttttattg ttacttttac tcaatatcaa cagtacttca
8220atagatattt attagttatg ttcaatctaa tgacttaaaa cagttgaaaa ggaggaaaat
8280caactccagt ccacacatac agtcatgtta cattaagatg tcatgttgta tgtcacacag
8340tttctagatt gatttctctt gtgtataagt tataacatca aaaatgccaa agggtatata
8400aaataagcta caataatatt caacagaact taacctggac cttatgttgt aataatttat
8460tcatattagc tgtagtcttc tgtatttata ttttcagtat ttattatgaa tgacatggaa
8520attcgtgtat ttattgtggc tttttattgc agtgtgtata tatatataac aaataagcat
8580ttttaagtct tatccttaag tttcttttat tagtggcact tgtattatgc tgtacttttt
8640attacatatt gtacaatatc ttgtccattt gtttgcagca gagtaaaacc ggtttctaag
8700ttgtatctac tgttgcattt ctgttgcctg ttgtgtaaaa cctttgttct tacatcctga
8760actgcaatcc gttacgctgt cccagcacac acaaggatac ctcccttcca ctgtgtaaca
8820gataatattc ttgaaaagtt gtgtgtgaag tcagttcctg tgaaatggtt cattacattg
8880tcatttcaaa tatgtgttaa ctttattggg atttatccct aagtgaagat gcttatagca
8940ctttactttc tcagctattc aaccccttca tcaagtgatg atgtgtaaac aaacacaagg
9000gccagaggag aactctttat gtaaatgaaa tttggaggct atttttacaa ggacaaattc
9060aatctcttgt aagtagtatc ctttcactta actgcttagt agtatttttg ttttgctttg
9120cttaaagcag aatctgtgtg atttgatgtt tagaatatat gcgtgtgcat ggtacgtata
9180tgtgcccaac tgtttaaagc tactggctta aggtaactaa aacttaactt ttattggcag
9240taggatgaag gaaaaaaaat tgcatttttt taaatctcct atcatatcca agacactttc
9300ttccaacaac ttgttttata tttagagact ggatctatat ttttaaaagc aaataataat
9360atgtgatttt gtgatcatcc taaaactcaa atttgaacat gttattcact gaattaaaca
9420tattttgtgg tgttccctcc tcactgccaa caaaacaaaa ttcagactcc ttggaatgcc
9480tcacaaggtc gcctgagatc tgggtacaag ctctcccttt acttcccacg tcttctgctg
9540cccacaagag cggttcctta gatttcacct gctagtccat tttccctgct tctgttcagc
9600ttgtctcttc tgccaagaat atacacccca taaacctatc ccgacagcac aattctagtg
9660accatctatt tatttatcaa gagaaaacac aaatattctt tcctttatgt tgcccttcca
9720atactcctga aaatcgtttg cctgttgttt tatgcaaagc agacatgtat tagagcccct
9780tcgagtctca ctgcactagc ttatttatgc atctactccg cctcagtact tcctgagaac
9840agaaaacact tctcattact ctgtcttctc agcattcagt ttagtgttta aaacatggta
9900gagacttaaa atacatttac tgatgtatgc aaagtaaaaa attgagtgat gcaaagtcca
9960ctcctgtata ggagttcaga tgagggaaag atgattggct gaaacagtgg aaaaggtgtc
10020agattcagag agagggagaa tttcagctag cagggttacc cccatagtcc acattatgga
10080acaatttgag gaaaggctga tcagaacaac atcactcttc tggtgggggg cagaaggacc
10140tggttaagaa cagataattt actatcaaga acaaatgaca aatcaggtta ggagagtgca
10200tttagacagt gcaaagcttt gaagaccaac ccaaggaatc tggctcatat ccttagggaa
10260ataaggatat ttccttcagg aaatcctgaa gattttagaa aaggaaaatc aatcaaataa
10320gagtgatttg cagtatggaa tatcagtgtg gctacagtgc acagacagtt tgggaggatg
10380gaaatgccag aggaaaaatc tttgcagtag ctattgcagt cagtattcat tagaaaaaat
10440aagagttaag aacctactct gatttaggat actgtgcaag aatgtgctac aatgaggagg
10500gtgtggggga aaagacaggc agagtccctc tagacaagca agtagaaaga tgtgggtaat
10560tacagcagag aaaaagtcag ctatttgaaa gcctattacg atgtgagaga ttaaaggtaa
10620caaaataatt atgagcctaa aataccatag gtggaatatg gggaaggaca taagaagata
10680aatatcatca acataaatta gaatgttata aggaggaaga gatgtgttca ttttggacag
10740cttgaattga tgctgggata ctcagtgaaa ataacaaatg gacggaagga aatgtaagtc
10800tagagcttag acaaagacgt aagatttgga gcgaatgaga attttaagaa gagaatatac
10860atatatatat atagaaggaa ctaaataaca gacaggactg aagctttaga aaggctagtg
10920tttcaggttg tgtttgctgt aagcacacaa ttctgagatg gagtttagca tgcaggattt
10980ccatgaggaa tcaattcttg tgggagctag cgggaggaag cagattagga aaagacagaa
11040atcaggctgt gatgcggacc gaccccactg gagtgctcta gaggataagg gatcctctga
11100gtcatcccct cgcgggctga aatggctggg cctttatagc cacgccacag tcagtctctg
11160gatgtcttcc accccagaaa gggagtgctc tcagatgagt tggcaccctg cagtgaggag
11220aaagcctgaa aagagctgag agctggagga tgtttttctg acagcgctct taggacctgg
11280gacaagtcct tccttgaaag ggaatttttg aaggtaaaac agctctgtgt cttctacatc
11340catccctcag gtcctcagat tcatgtcatc acttaaattt cagggaacag ctccttcagg
11400attctggtgg gcctctctcc ctgggggaaa acttaaagga gcaaggttag taaaataaat
11460tacagttcct gccactctgg ctggtcctaa agccacaact aaaactgacc atctccctct
11520ctcactatta attctacatt cttctcagct tcatcaaaca cagttgctag cctcaatggc
11580ttacttggtg gtgtgaccca gagccaaaag gcacctaagt gaatattact tcctgctcct
11640attatgaaac agtaggccca tctcttcctg aggatcaagg tcaaatacct ctaccaagat
11700ggtgaggtat tttttctcct tgcctaaaaa tcctaggatg tagcaatagc ttaagtccaa
11760tgggactctt tagtcccctg ataaaaatgt gacctctttg gggactagga cctttaagac
11820tgcaaagaca tttaacccta tagagttgtg gaataggaag cacagttttc tccagtgggt
11880catgttaagt agagacattc ctgcttccac ccagtagagt ttgcacccat gtgttcttcc
11940tatgggggac atattattat gtaaaggttt tttattcaat ctatatactg gatcctggag
12000gatagtaacg aattcatgcg gaacatcatc tccaaacttg cacttcaact gcaccttcta
12060caggccagtc aacgctctgt caggccggga gctttgaagt ctatgatatg atcagtgggt
12120ctcatggtca tgtgcccatg ctctgatatg atgttatgtg ggatcccatg tgagcagatc
12180aaatatactg ttagtccttg aataacggtg tttgccaagg ccctgcaggt aagaatggca
12240aacttatact tggaatatat gtctattttt atttaaaaca aacaacaaat aaaaacacaa
12300aaaatctctg gcccttttaa gatggaaggg gcccaatgta atcaatctgc caacaagtgt
12360ctggttaggc tccagaagaa tagtgccata tggagggctc agcatcagtc tctgttgctg
12420gcagactgta catttggcag cagcaggagc tttatcagtc ttggtaagtg gcaggccatg
12480ctcatggacc caagaagagc cttcgtggct cccactgcca cccaatccat gtacctatca
12540cagcagccct ggagtagcca ataactgagg ctggctgctt gctgccaacc ggccaagtta
12600ttctgtctat atcaatacta tcaaaaaata tatataatgt gagctacaaa tgtgagctgc
12660atatgtaatt ttaagtgttc tcatagccaa cttaaaaaga gttaacaaaa agataaaatt
12720aatttaatga tatatttcac ttaacccaat atataaaaaa tttcaacatg taatcaatat
12780aaaaattatt agtaatattt tacatacttt ttggaagact aagtctttga aatccagtgt
12840gtattttgca attacagcac atttcaattt ggagtagcca cgtgtggcta gtgcatacaa
12900tattggacaa cacaggtcca caccattgtt taatatgtct tctgcagtgg gtgctctcta
12960gttgataggt gttaacatgt gacacaaaga tcttcacact ttgtgtccac tctcacatgt
13020cattcacatg cttatactcc aagactccta ttccccaatc ttctgatatc tctgctttca
13080gcatcctgag ccaggctatt cacttctacc caccagtcca tatatattct aacagagctc
13140ctatttattc ttccacacaa agagaataac cgggtggacc ttaaagctct acctatttta
13200aggatttttc tctagcccca tgagctactc ctgaacaagg ctgcagtgta gctgccatgc
13260atttttggct tgtttggcat ccacctacta aaccagccca tctataaacc aaatctgaag
13320gctttttcct cctccagcag tgagccatgt ggcatcctcc atatggagtg agggatgagc
13380tgtggaagag gttcagggac aacagtggca aatgacatgg aagatgggac tgtcttgtca
13440tgcagctttt tttgtgtctt ccagtcctat ttatgctcaa cccaagtttt ctacttctag
13500aatccatctc ttaaagttct gctagccctg ccttaagata cgtggttcta caggtgcagt
13560gccttggtgt cccatagcag gcacataatc tccatgaggc ttcagtaata tatcaggagt
13620tgtttctcaa aaggcataca aataactgct acagatggca aagctttaat cccatagcct
13680atgttgggat tctaccactg gcacttgaca taacctccac acagcatctt ttcccaccac
13740tgctaccact aatatcacgg tgtctgctgg cctacatgac ccaacaacag ggcttggtgg
13800actgcaacca atacctccta ctgagatttt tcctgctcta cgtctcactc aaagctggca
13860gccttttaca ttgcacaaca tacgggtcag agcagtattc ctgaatgaga aatacgttat
13920caaatctaga acctgaagaa gcttaccaag tgttgtgctt ctcttttcat ggtgggagtt
13980gcagcatgta ataatgtatc tttactatgg aagagacaac ctgaactccc cacaccacag
14040aatccctgaa gactatatta gtgtggcagc ctccgacctc ttcaaagggt ttatctccta
14100tcctccattt tgtgtgtgtt tcaccaatgc caccggcatg ctgacaagtt attgctcatc
14160tggcctaatt agcatagtat cataaaggag tggtattctg ttggaaatct agagagttga
14220gatctctttg tgctatatta tgacagaata caaaactaaa tatatattgt tgcctattcc
14280acgcacatgc aaacaatttc tgatcctctt tttctaattg gaatagaaag aaagacattt
14340gccattcaca tgacctcatc catgttactg aggttggtta acccactcta gcaaagatac
14400tacatctggc acagcaactg cagttgaggc taccaattaa ttgaatttgc aataatctac
14460tctcatttcc caggatctct gtgttctgca agagcaagtc actgcattaa gacaacttaa
14520gtggggatat gatagcaacc accaccctta tatcttttag gttcctaaag gtactaataa
14580tttccactat tccccatgcg atacaacttg tattttattt actaccttga tttgggtggg
14640ggatatgaac agtttcagaa acatccactt ggctctcccc acttctatag cctctgtccc
14700actcactaaa ggttcagtgt tgggattgca tgtattccaa acatgagaac ttcaattacg
14760tacttggaac cagggaaaat gaccactgtg tgggtcctca gacctagtgg acttactgca
14820agccagatct ctgccaggag tctgtttttt tacctggttc ttatatgctc ccactttaga
14880acaggagggc cccatggtaa ggcttcagtt ctccaggtat caatgtccac tcagacccag
14940tgtcaaacag tcctaaaatg ttttgagtgt ttccctttcc cttgtacaaa gtcacccaag
15000taaacagttg taggtacttt taaagatgga ctgggggaat catcatcata atacttgcac
15060tgtgtagctg ggtctttctt cctagggacc tggccatttc tttagtcagt gggtcctggg
15120tctgaaaact aactcagatc tggaaagtgg gcaaaagact acaacatctt attggcatga
15180ctgccttatt ggagtgagtt ccttcaataa ttagggcttc taattattta ccctaatttc
15240tcgtgatggt ataaaccaaa gtatccttgt tggctgccca tctatttagt ccctagggtt
15300atgagtttct attaaccatc tccatgactg tctgtgggtc aggccccatt ggctgccact
15360ctggctctgc tgctcattat gataattgca tccacctgct tttcagcagt tatgtggcac
15420ctcctgttct ctgttgtttc agggtctttt catttccatt ggattatttg tgcctcagta
15480ccatcagtga tggagttcac attactagcc aagtgacaaa tgataaagct tcaaaattta
15540tcttatcaac ttcatttttg gactattctg ataccaaatg tcagattggg tttttaaaga
15600ctctaaaatg gagttggact tgcaagatat ttatgaaggt tcaacagctg tggaagccag
15660gaggaggaag caatcaaaca gagagacaag atgaattatg aagcaggcgt gacagagcct
15720cagcccaacc ctcagagaac tccagagcca tatgaccctt tggagttgtc ccctagtgag
15780ccaaaatttt cagatcttta tatgtcctcc atggtcagtc attggatatg gatagctaag
15840gaaggttggg cttttgaaca aggcatcact ctgcaactga ggtcatctct gaaaaagaag
15900taacaagtca gccctgaaag aaacctgagt ggtgcatctc catatccatc acagccagat
15960ttgggattca aaagaagaaa ataaagacat gattttgtat ccaagagaat ccagagtagg
16020agttaagaga gcaatggaaa gtcttgagct cgggttttaa aaaggaacat gtgtatttac
16080cagatcagga attttccttc ctgaaaatca gatcacacat ggaataccaa gttcaattat
16140ggctactgca gtttaaaagg aatatccact tactgaaatg tgttcagagg agaatgatca
16200aaacagtaaa ggagctaaaa aacacaacaa agggctagtt aaaggaatga atgcttacac
16260agaatacaag ttttctttag acagctaaag gggcttgaaa ttattccctg tggaatcaag
16320agacaaaagt ctaggaaaac agaagttaga agtctaggaa gacagaatat agggaagata
16380attttaatat taaaaccaaa agaaaaagta ctgtagatat aaattagcca gatgaagggg
16440aaaagaggca ttttggatca aagcagccat cacaggcaac cgtgaaaagt tcagactggc
16500caaatataag accaatagaa agtggccgga agggaaaatg actatgaagt caatgtcaga
16560tcatcaagtt ccttgtatct catgcttagg actttggcta ttatgcagtg aagtagtgcc
16620tggcttggca cttcaaaagg tgaatctgac agcaaggaag ataatggatt agggagaggc
16680atgagataga aagcaggtaa accaattaag ggtagctatg atcaatagaa ttcatgtctg
16740tctttctgct cattgtattt gcagtacctg gcattgaacc ttcatataaa agaccctcaa
16800taaatatcaa atagatgaat gttcgaggaa agtaccaacg gttgtctgaa ctaagccaat
16860ggctatggga atacagaggg gttaacattc aaaagatctg tagaaggtgg aatggaaatt
16920aaaggagagg aagaagtcaa ggataataga tttgttgatt tgttcctagt ggcatacctg
16980ctgtggacct gaaatactga aggaaaagtg gtcggtgggc aggtgaaggg aaggaaaaaa
17040agattgctaa aggtctcatt tatcatctat gattaggctc tatctgttgc tgaaatttct
17100caacaaagtc tctactaatg tgctttctca tcgatgtttt ctatgcacaa gttccccgtg
17160attaccagca ccctgtcagt atagaacagc ctgtttcttt gcttttctct ggtaaagcaa
17220agcagcaagc caattactga gatcattcag atgaaaattt tacttttgcc tgaattagcc
17280agagaattga agcataaatg gaccttttca cttgactgat tgagataatt gcctcacttt
17340tctaggggag acctatagct gggaggctgt atttggtcac caaaacatgt ttgtaatata
17400cattattgac tttcttgaaa caactctggt tggctaggtg ccacactcaa acacttgccc
17460tcatcttttt aggtatttga aacaatacag agaaatacca tgcctcggaa actcagtgtt
17520gaattatttc attcttgggc agcctttaaa cacatcagct caggcctcca gaagaggtaa
17580agtggcacct aggggagtct gggagaaggc ggcagggcag gagcttggtt cagtggataa
17640aatgcaagaa gtgagaatta gctccaggtg catgggaaca aatggctttt cctgaaagaa
17700ttgggtaggt agacttgaaa gaaacatgta atttacattt actattggga ggggtgagag
17760gggttaataa agagagctga gtaggggcag acaattaaag cagcagcagg gctcagatca
17820gagccttttg gcctggatct tgggtaatag aagcattttc taacagcatg agacatggga
17880aaagaaggct tctaaagaaa cagcacagca tctgagcttt tctgcagctt ttgctatctg
17940gtggcctgct atgacagcct gggtgtgtac agacaatgtg tggtcagtac tgacctacaa
18000aattcactga aaagcaagat gtgtattaga aaaactcgta caaacttcaa gtctatgagg
18060aaaggttgtc atgtaaatgt gtctgtaatt actttgtgat ggctcccctt actggccaac
18120gggcagaacg tcctagatcc tgaagagaag taggaggagg taacgctgag gcaacctggt
18180taatttctaa gtttcctgta ttaaaatata tatatatata tatatatatg tgtgtgtgtg
18240tgtgtgtgtg tgtgtgtgtg tgtgtgtgtt gctaaaataa attttattat aaacttattt
18300tattaaaata agttttattt tctgagtatc aaataaaaac agaaatccca gccatgatat
18360ttataaaatc caccagtatt atttttaaat tttttatatt tatttatctt cagtgggtgt
18420ttagagtctg cgtatggttt tgtgactata aaaaatatgc ctgctttaat ttttcaattt
18480ttaaactttt atattacata taatttcaaa agcaatttga ggtgatggat gagacatacc
18540cacacatcag gattgatata tccaaacaag ctgcattttg gggagagatt atatgcttac
18600aataaggagg ttttcattgt ttaaagcaac cttgattttt tttgcattgt ttcattgttt
18660ggaatattgc tttaactgtg gcaaatagat atacattaaa agtatattca acatcttgta
18720atatccatag atcattcaga atcaactatg ttgaactagt tgaactagaa aagttaaaat
18780gttgtatcca aaataataaa aatgatgcac ctatcttcta aacttttccc caggattccc
18840aaagaataat cttaaaaatg tcaaataatg ttcccacagg ctatgtatgt tgctgtttat
18900ttaaatataa gtactgggat attttatttt aaagatgttt tgtttgttaa tatctcttta
18960taggtgtcat aaatattttt atgtatataa aaattggtct aagaggacca attgaattgc
19020tagtatcaga ccaatttttt gctttaatat aaacccagac tgacatatct tgtttacaaa
19080caaataatat tggcatgaaa aaagtcacac cattacacag attttttatt taagctgatc
19140ctgcaataga taggatactc ttgaaattag cactttacat taaaataatt aattttcttc
19200agatttttaa aattctttct gtactttttt tcatatatgc acagtattag aagattctaa
19260atttgttgct gtagtaacaa aaacccctcc aaacctcaat ggcttaaaac atggtttcac
19320tccatgtctg tcttcagtgg gtgaggtacg ccatcatcct ttttcaggtg ctaagactga
19380tggagcctcc accatctgca acattgcagg cagggggagg aaatcagctt cttgtgccct
19440gcttcttaaa ggtttctaac tggaaataac acatgtcact tccgctcaca ttgcactggc
19500caaaataagt tacatggaca gaaatagaac tgcattttac ttccttttgt cttaattgat
19560cccatcaaag atgctttaat ctttcagaga aaaaaaaaga aatttgggtc atgctgtcct
19620acaatgtgtt tggaaggaga acagaaacat tgctgaacag caccaataac tcccacattc
19680accctaactg aggaacttta tcttaatgcc gggaaagggg acagtgcttt cacagctgag
19740aatccaaaat agtgacagga tgtagggaaa cttatatttt gatttctttt ttaacagaaa
19800gaggcctaac attatatttg ctggttaggt ttaatgtctg aacttctttt caagtcttac
19860tttttactac aataaattgt gcttaaattg agctctctgg ggattccaca gggattgatc
19920gatttgcctg gttagggctt ttcaggatgg atgtcaggag tggggcaatg gcatgagaac
19980agctgttttc agggctctgg cctcaatctt gagcgttgcc atagtgacag ctgcccgcag
20040gaacccaccg ggagcccagt aggagaactg gctgccagag ctttgtatca gtgtgccatc
20100cccagccttt cagaaacaag cgtaatgaac tctttagcca gggtgtttct gcctactaac
20160ctaagggaca gtttattctt gcttgaattg tcaactgcca gatgtcagtt caggcaaatc
20220aactctattc cctctttcct cctgtaccct ttctctcagc ttattttcat atcaaattcc
20280acttttcccc attttatagc tacataatta acagctatta atagagcaaa ttcaattcat
20340aaatttagat ataacaaaaa aatccaaaga tacaaagtcc tctaacacag cactgcccaa
20400tagaattttc tgtgatgata taaatgtttc atatctatgc tgttcaatac agtagccact
20460ggtcaccaag tggcaactca gcatttgaaa tgtggccagt gcaactgagt aaccaaatta
20520tttaactgat ttaatttgag ttcattttaa ttttaaatag ctacacgtgg ctaggactaa
20580tgtattgagc aacacggttc tagaatcgta ttcaaatcca gggagtggta ccagctagag
20640ctggccaggg aggagtcaca gttagcagca ttctctcatt caaagtagcg tgtttattat
20700ctgctctaga gcatattatt attattgctg tataaggtgt gccctggcct gccctaactg
20760gctgttgtaa atttaattat gtagctaaag aggaggaaac ggaggcatga ttatctgggc
20820cgttacaggc aatatcctct agaagcaaga ttgaaaggga aaaggtgtta ctatttctga
20880ataatatatc agccacatta cagtgattct acaaaagaac tggattctaa ctggaagtca
20940tcaattctag tcccaaattt tatgctagta aattgtatta cttaggaagg acactcagtg
21000ggaggcttgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtggcttt atttatttat
21060ttttgcctaa attcctcctc tgtataaaag ggagcgtaat aatattctct gtgtgcttat
21120taatatgaca ggggctttat gtaagtctca tttattcctt ttaagaaatc cataaattag
21180ggatttgtat gcagagtata tagatgagga aactgaagct tagcagagtt aagtgcctaa
21240gatttcccag ctaataattg gaagaatcaa aattcattca aactcaagtc tgtctgtctc
21300taaaacccaa tttctttcta ctaaatccca ttagaatgtt tctaaaatcc cttctaactc
21360ttaatcatca ctaagtgagt gccatgtaat aaatatattg aatgagtttt gcgtacaacc
21420caaaatagga ttcttaactg tatttcattt tttaaaacat agtaacaact gtaactacta
21480gttatttcct aacctggaac cacttaggat ttcctaatca gtggctttct cccctggctt
21540tgcattagaa ttatttggag agttttttaa aaataccgat acgaggtatt cttctcaaat
21600aacttaaatc acaatctgca caatggggcc tagagacagt ttttttcttg gttgtttttg
21660gtttgtttgt ttttgaggag ttttttgttt tgttttgttt tgtttgagac agggtctcac
21720tccatcaccc aggttggagt gcagtggcat gatcatagct cactgcagcc tcagcctgct
21780gggctcaagc aatcctccca tgtcagcctc ccgagtagct aggactacag gcatgcacca
21840ccatgctcag ctaattttaa aatttgctag cgatggcttc tcactctgct gctaaggctg
21900gtctcaaact tctggcctcc agacatcctt ccaccttggc ttcccaaagt cctggaatta
21960caaatgcgag ccaccatatc cagccaggat aatttgtttt taagtctctt atatgatctc
22020aaagtgcagg ttgtgctgag aaccgtttcc ctaatgtaag gtgtagaaat ctacttttcc
22080attgagccac ctgctaggac ttcaggacct ggcctccagg aggaagcccc ctgatagctt
22140ggttgatttt atcttatcag tttatgttag gggaaactgc caaattgaaa taagaaagcc
22200atctcccgca agcaagcctc tcctcctaaa gactaatcat aatgcaagag aaattttcca
22260tttctctcag gaatcagaat cctttgctac atgcacaact ggaaggaaag tagtacaaat
22320aggaggatcc ttacctgatg ggagaaggga gatcttgcca ttcaggagtt tgctttgaat
22380ctctttccac gtgcatgggt gctgtcacag tacatgtgac aggctgcaac agggtttgag
22440agggttcctt gctggaaggc aatgtcatag gaaaacaggt tttatgaagc agtgattccc
22500ccaaggagca aaggagttga ataaatagga tagttcctag actagaagag caggcttgcc
22560tacactctca cacccaaacc aactgccttc tgttgttctt tttttgaccc aaattctatg
22620cagaatcttc tcttatcaaa ataaataaat aaataaataa tgactgcaaa caaaaaactc
22680aatagctcct agtattactt aacttttgca tgattttaac gtgtcaatgc ttatcttaaa
22740gcactgtttt actaatttca ttcctatact tatcctaaaa gatctttagt gacgtgcctt
22800ttctcacagc aggacgcttc aaacttgggg gagtttattt aaaatgcctt tttctggact
22860ccattcctgg aggtctagaa tgggatgcat tcataatata tatgttaagt gttccaggaa
22920atcaataggt gattgggata ggctaaataa tggtccccca aaaatgtcct cattgctgtc
22980cccagaacct gtgaacatgt taccttcaac gacaaaaggg actttgcaga tgtgatgaag
23040gatcttgaga aggagggtta ttttggttat gcaggtggtc acaatgttat cataaggacc
23100cttatgagag ggaagcaaga ggatcaaaat cagaaaaaga tgtgatgaca aagccagagg
23160taagactgat gcactttgaa gatagaggag agagccatta gtcaaaggat gcaggcaaca
23220tctagaagat agaaaagcaa ggagacaaat tctcagatgg tttctaacac cctcaaaaag
23280aatgctgctc tgcaacatct tgattttaga cttctgacca ccagaactat aaaataatac
23340atttgtgttg ttttaagtca ctaagttggt ggtaatttgt tacagtaata attggaaact
23400tgtatagtta ttcacgaaaa gagtattacc ccaaataaaa cactttctat agacatcaac
23460ttcaatatct cttattattg taaaggatcc tggagcacta ggagcgaagt aaattgttca
23520acttcactga gggacttagg gaaaatccag gacaacagtg gaattatctt agtttggctc
23580tatctcttta tcctggcgtt caaggtatct tcccctagtc taggccaacc tttcccacat
23640tatcccatta tttctatatc cttacttcta taagcaagca aaatagtgga tatattttcc
23700ccctgaataa gccccattat ttctgtttca ctaccttgat acatagactt ccctcttcct
23760gaagtccttc cattctcgaa tgttaggttt cactaaaggt cgggtcctgc ctcttcctta
23820gctatccagc attaggtgag tactgacctt ggaactctta tggtgtcagc atggttgctc
23880tttaaagatc atgatgaaga atccctagtc tatttcttcc tcagtgtgat gatctggttg
23940gctctgaggc ttgtggatac ttcctaaagc aatattgaag aaagaatgaa tgactaaaga
24000ccttaatcca agtcaagtgc ctagattttc aagttccctc tctttatgaa ctatatttca
24060cttatttcca caaagtttta aaaataaata cccaatgaac ttaaaaaaat ttgtagagat
24120gagtgtcttg ctttgttgcc caggctggtc ttgaactcat ggctgcaatc ctcccatctc
24180agcctcccaa agtgctggga ttatagccat aagccaccac gccagctgca aaaaatttca
24240attacatgtt ctgttagtat acatagatat cttcctagtt taacagtaaa aatcttctca
24300gttacttgac tgatagatga caactcaata tttacatctc attggggtaa cacaacattt
24360taaaacatat ttcttattgg catatacttc acataacata aaattcagtt ttaaagtgtg
24420gtattcagtg gtttttagta tattcacaga gttgtctgac cattaccatt atctaattcc
24480aaatatttta attaccccca taaagaaacc tgttaaccat tagtagacat tcaccattct
24540ccctcctttc tctaagccac tggcaactac taagccactt tctcactcta tggattttcc
24600ttttctgaac acttcatgtc aattaatcat acaataatca agtggtcttt tatggctggc
24660ttctttcact tcacataatg ttttcaaggt tcattaatgt tgtagtatgt atcagaatct
24720catttctttt tacggctgaa taacattaca ttgaatgaat ataccacatt tgtaatccct
24780tcatcattga cagacatttg ggttgtttcc accttttagc taatatgaat aacgttattt
24840gtgcaacaat ttttgctttc tgttggtatt tgtgaaaata tgttttcaat tctataaggc
24900atatatatct ctaggagtag aattgttagg tcataaatca tttgtataac tttttgaggt
24960actgccagat tgcttgccac cttttcagtg gctgcatcat ttaacattcc cacaagtaat
25020gtataaggat tgcaatttct ccacaacctt gccaacagtt gttattttcc atttttatta
25080tagttatgct tgtaggcata aagtaagata tctcattgtg gtttttcgtt ttctaatgga
25140taatgatgct taccatcttt attggccatg tgcatatctt ttttggaaaa aatggctatt
25200taaatctttt gcccattttt taatagtttt ttttaaattg tagagaacat agcctttaaa
25260attaaaataa gagtccagaa gttgggctat atttcctagg tgttagctag tatcttgtcc
25320ccttttcata gatttatttg tgtatctcag gttatcagtg ccactataga tctcctccct
25380cacagcaatc aaacttagga cttcatccag aaatgggtgg agagaccact ctcccagctc
25440caaaaatgct taccgggatg gtcaatgact gcctccttct tgttgctcag ccctctgaga
25500gtccttggct ggctgtgcag tttgctgcac ccagttactt tctttctttc tctctctctc
25560tctctctttg tttttgagac agaatctgat tctgtcatcc aggctggagt acagtggcac
25620aaacatggct cactgtacct tggacctccc aggctcaagc gaccctccca ccttagcccc
25680ccaaatagct gggactacag gcgcatcacc acacgtggct aattttggta ttttggaaga
25740gatggagttt caccatgttg cccaggctgg tcttgagctc ctgagctcaa gagatcctct
25800ggcgtgggcc tcccaaagtt ctgggattac aggtgttggc catggcaccc ggcccactct
25860gttactttct attgcattgc ctgttacctt attttatagc tttcataata ctcatcatct
25920gatatttctc atttgtttgt ttgctgatta tctcttcctt ttggaagcgt gaattccatg
25980aggccaagac attgcctgtt atgactgact gtgtgactga tgctgacaca gtgcctgtca
26040catggtagac tggggctgag gttcagttcc caggggtctg gctcctcagg aagtagcatg
26100tgtcaaggga agatgtgtga catggggaag aactttctag aggagttacc aggtcctcca
26160gtattagact cctttatttt actttttaaa aatgttttga agaaaatctg gcaaactaca
26220agcatactca tcttaatagt aattatcaga agcaagtcca gattccggaa actaatttaa
26280ctcaatgaat gtgttaaact agttcaagat taatcagaaa cgcttagatg tctttattct
26340ttctgcaatc ctaaatgaaa cataataaac actttaggct aaatttttgc atagttttaa
26400gctcatcatt tgcatagtag tctcattgct accacagata gaaaagataa ggtctgaatc
26460tgtattgctc cccaggaaac tacattttag cagcacttac cgagtagtca cacccatcta
26520aattcaaatc ctagctctgc cacccacctt caggaaaaac ttggccaaga tgtttgactt
26580atctgagtct gtcccatgtc tcactgaggc tttcccaagt ggggaaaatg gagccagtaa
26640tacaggccta cagagctgtt tgaagattat aaacaaaaca ttgcccacaa taagggatca
26700ataaatgtta gctgattttt ctctctttaa atttctgttt cagctacaac tctgttatgg
26760gtaggtgtga gggaaagaaa ttaggtaaaa aaaaatctaa agaaaaaaac attggggaaa
26820ataaaataag aattctcatt tagtttgttt catgcgcgtc cgtgtgaaga gaccaccaaa
26880caggctttgt gtgagcaata aagtttttaa tcacctgggt gcaggcgggc tgagtccgaa
26940aagagagtca gtgaagggag ataagcgtgg ggccatttta taggatttgg ggaaggtaaa
27000ggaaaattac agtcaaagag ggtttgttct ctggcgggca ggagtggggg tctcaaggtg
27060ctcagtgggc aggagtggga gtcacaaggt gctcagtggg ggtgcttttt gagccaggat
27120gagccaggaa aaggactttc acaagataat gtcatcagtt aaggcaagga ccggccattt
27180acacttcttt tgtggtggaa tgtcatcagt taaggtgggg cagggcatat tcacttcttt
27240tgtgattctt tagttacttc aggccacctg ggcatatacg tgcaggtcac aggggatgcg
27300atggcttggc ttgggctcag aggcctgaca ttcctgcctt cttaataaga aaaataaaac
27360aaaatagtgt tgaagtgttg gggcggcaaa aatttttggg gggtggtatg gagagagaat
27420ggacgatgtt tctcagggct gtttcaagcg ggattagggg cggtgtggga acctagagcg
27480ggagagatta agctgaaggg aggtcttgtg gtaaggggtg atattgtggg gatgttagaa
27540gaaacatttg tcgtatagaa tgattggtga tggcctggat acggttttgg atgaattgag
27600aaactaaacg gaagatacaa ggtccaaata aaagaaggag aaaaatgggt attaaaggac
27660taacaattgg gaggacccag gacatccaat tagagagtgc ccaagggggt tcagcgtaat
27720tacttgcttg gttggcaagt ttttgggctc tatccttgag ttttttaatg ttgtcataca
27780ccaggccaga ttgatttagg taaaaacaac actcctcatt taagaatatg cagagtcctc
27840ctttttcagc agtgagtaag tcaaggcctc ggcggttttg gaggacaact gcagctaaag
27900agtcaacttg ggcctggagg actgataaag tttgtgatat gtctgtgatg ctagcagaga
27960agtcattaga caggctacgg aaggtcatga cagacgttga aatgcctgct attccagtac
28020cgagagcaac agtggaggca gaaagtccta aaccgaccat caagggaatt agtggaataa
28080ctcttttttg ttgtgtcggt gtcatgaggg gaacagggag ctcttcggtc ccatttgcaa
28140attgaatttt gggggtaagg aagactagtg tacatgtgcc tgtccaattg gcaggtaggc
28200acatgtaggt agaggatcca cagaggaaga agagaccttg tgcgaggcaa aactggagat
28260gtaaagtaaa aaggtgagaa ggagtgctga aaggggtgtc ttgtacccag actcctaggg
28320atccagctag ggcggcagct gtcagaggtt gtaatgggga ctgatggggt aactgcgtag
28380aggggaaggt tcgattttca tggtgtatga gaaaacgtcg agtatctacg agcaatcttt
28440cactgttatt ttcggggctg ggtataagta aacaagaaga gggcctggga ggagagtctg
28500atgagcaagg ggaagatagc caaggatgga gtgaaataca gggcaagtgt cttcctaagc
28560aataattact gctaatgttt ttaagtttgt cagtattgat agagggcttg tctgtaatat
28620ggagctggaa ggctccaatt gtttcattga tgtttgtagt tggacttcgg agatgaagag
28680taaaggaaca tcgagaaggt gaaagattac ctaggggaat tccagtgggt ctttgccaag
28740agatacacaa aggagcggcc acaggaatag tagtttgtgt tgtgagaggt ccaaatatgg
28800ggggagtaga gttgatataa ggagaaaggt tttttaaata agtgcgaagg agggcggcag
28860cttgctgatg tgaaatgtct ggggaagtct tgctggacct gtctagaaag taaatgagtt
28920cttcaggagg gtaaaggtga gggctgttaa aggaagttcg gaggtgtagg gagatgggag
28980atgttgccca gtctgtctgt aaggcgggga cagctgtgta ggcactggaa gaaagggaaa
29040tgcaaagcca gcagttgttc actaaggagg gattagaagc ggctaggaga gaatgggtaa
29100ggttgatagt gtggtggaga tagctgggga gaggtagagg atgacataag aatgggaatg
29160agaataagag tgagtataaa agtaaagaat agaacttcat cagggtggaa gtattggagg
29220gtgccttgcc agcaaagatc atctatccac tctaagacgg agttaagagt ggcagtttgg
29280ggatagcacc aagagatatc agctgtgatg gcttgaagaa acagtgtaaa ccggcggtgt
29340aaacaagagt agggcattta taagtagttg agaatggaga ataggagtat gaccggacag
29400aagataatag ggatgactag ttttttgggg cttggcctaa gtggtggggt gacttcgtaa
29460agccctgttg caaaaagtag ggtaaggatg aacagaccta atagaatgaa gggatgtatt
29520aggctcataa gggttattac tgttcttcag aaatatgagt gagtttaagg gaagtggggg
29580agagtacttg cgacttccag gaggaagagg agggattagg ctggctgtcc gatggacaca
29640gctttattct ggaatggtga acccagtggg gaggattctg caggcagacg gcagtcgggg
29700tactatagat gactaagtag ggtccggtcc atcgaggttg tagagtttga ggggtcagat
29760tcttaacaag aactgatcgt ccagctaggt tgtcttcata tggctgggga tctggagtag
29820gcaagagaag attagcagcc tggcgaattt cctgtctagc ctgctggagt actggaagat
29880agtcgcctag agggctggtg tctgggatga ggttggggcc aagcaagaaa gtgcgtccat
29940ataaaagttc aaatggactg tatcctgtag catctcgagg acaggctctg attttgagaa
30000gagcaagagg taaaagtgct gtccaatcct ttgtaagttg gaggctaaac ttggtgaggt
30060gtgcctttaa aagaccatta gtccgttcca cctttcctga agattgagga cggtaagggg
30120tatgaaggtt ccactgaata ccaagagcct gagaaactac ttgggtgatt tggctagtaa
30180aggctggtcc gttatcagac tgtatagagg tgggaaggcc aaaccgagga attatgtctg
30240acagaaggga agaaatgacc acagtggact tctcagaccc tgtggggaag gcctctaccc
30300atccagtgaa agtgtctacc cagactaaga gatattttag ttttctgact cgaggcatgt
30360gagtaaagtc aatttgccag tcctgggcag gggcaaatcc ccgagcttga tgtgtaggga
30420agggaggagg cctgaacaat ccctgagggg tagtagaata gcagatggaa cactgagaag
30480tgatctcctt gaggatagat ttccatgatg gaaaggaaat gagaggttct aagagacggg
30540ctagcggctt gtaacctaca tagaagaggt tatgaaatga tgacagaata gaatgggcct
30600gtgaggctgg aagaagatat tttccttggt ctaagaacca tttgccttgt gtgggaagag
30660attgataggt ggaagtttca gtgggggagt aggtgggagt gactgaagtg aaggagaaaa
30720actggccgtg agggacagaa gttggagagc tagctgcttg tctagccact ttatcaacat
30780aagcattgcc tagagcaatg ggatctgatg ccttttgatg ccccttgcag tgaatgaccc
30840cagcttcttt tggaagtaaa gcggctttga gcagagtttt tattaaagag gcattaatga
30900tggaggaccc ttgtgtagtg aggaaacctc tttcagccca tatgactgca tggtggtgca
30960ggatatggaa ggcatattta gaatcagtat agatattgac gcatagtcct tttgcaagag
31020tgagggcttg agttaaggca actagttcag cttgctgaga ggtagtggag ggaggcagag
31080cagtagcctc aatgatagat gtggaagata ctatagcata gcctgccttc gctggtgagt
31140ggcgattagg cctggtggaa ctgccatcaa taaaccaagt gtgatcaggg tgagaaacag
31200ggaagaagga aatgtgggga aatggggtga atgtcaggtg gatcagagag atgcagtcat
31260gagggtcagg tgtggtatcc ggaataatgt gggaggccgg attgaagtcc gggccaggaa
31320caatggtaat tgtgggagac tcaacaaagt gtaagtatag ctgaaggagt ccgggagcag
31380aaagtatatg tgtcaggtgt gaggaagaaa atagattttg gaaattatga gagctgtaga
31440gagtgagttg agcatagttt gtgattttga gggcctctaa aagtattagg gcagcagcag
31500cggctgcacg gagacatgat ggccagccta aaacagtaag atcaagttgt ttggacaaaa
31560aggctacagg acgcgatcct ggtccttgtg taagaattcc gactgcacag ccctgcactt
31620cggctgtgtg taatgaagca ggttgggatg agtcagggag agctagagtg ggggcagttt
31680ctaaagctgt cttcaaggaa cagaaagagg agtggggaaa ggatttagga tctatggggt
31740cagctaagtt tccttttgtg agtttatata atggttttgt taggatggca aaaccaggta
31800tccaaaggcg aaagtatcca accatgccca ggaaggaaag gagttgttgt tttgtagaag
31860gggttggggc ttgagagatt agtcagacac gatcggcagg gagagcacgt gtgttcttat
31920gaagaattat gccgaggtag gtaacggatg gagaagaaat ttgagctttg gagggggata
31980cccgatatcc tttggagaat aaatgctgaa ggagcagaag tgtgtcttat tgagaagatt
32040caaaggaggg gctacaaaga agaagctcat caatatattg aataaggtga gaagcggagg
32100ggtggaagga aagtagatca tgagaaagag cttggctgaa gtaacgaggg ctgtccctga
32160aaccttgcgg cagcacagtc caggtaagct gctgggactg atgggtgtca gggtcagtcc
32220aggtgaaagc aaagagaggc tgggacgagg agtgcaggag aatagtgaaa aaaccatctt
32280taagatcaag cacggaatag tgagttgtgg aggaaggtat tgaggacaaa agagtgtaca
32340ggttgggcac cacagggtgg ataggcaaaa caatttggtt gataagacgc agattctgaa
32400ctagtctgta agacttatcc ggtttttgga caggtaaaat gggggaattg aaaagagagt
32460ttataggttc tagaagccca tgctgtagca ggcgagtgat aacaggcttt aatcctttta
32520aagcatgctg tgggatggga tattggcatt gagcagggta agggtgatta ggttttaatg
32580ggatggtaat gggcatgtga tcagttgcca gggaaggagt agagatgtcc catacttgtg
32640ggttaaggtg gggggatatg agagaaagac gcgaaggagg ctttgggttg gggagaaggg
32700cggcaatgag atgtggctgt agtccaggaa tagtcaggga agcagataat ttagttaaag
32760tgtctcagcc taataaggga actgggcagg tggggataac taaaaaggag tgcttaaaag
32820agtattgtct aagttggcac cagagttggg gagttttaag aggtttagaa gcctggccgt
32880caatacccac aacagttatg gaggcaaggg aaacaggccc ttgaaaagaa ggtaatgtgg
32940agtgagtagt ctccgtattg attaagaagg ggatgggctt accttccact gtgagagtta
33000cctgaagctt gccgtccgtg atggtctagg gggcttccga ggcgatcggg cagtgtcagt
33060cttcagccgc taagccaaga aggagtcagt cagagagcct tgggccagag ttccaggagc
33120tctgggagtg gctgccaggt gagttgaaca gtctgatttt cagtggggtc ccgcacagat
33180gggacgcagc ttaggaggaa tcctgggctg cgtgagttcc ttggcccagt ggccagattt
33240ccggtatgtg tagcaagttc ctgggggagg aggttctgga ggaacgcctg gctgctacag
33300ttcaggcgtt tggaagttct tgtgtgctgg agatgtggct ggggtttgtc tcacagtgga
33360ggcaaggaat tgcaactttt ttctgttatt gcacaccttg aaggtgaggt taattaagtc
33420ctgttgtgtg atttgagggc cagatttcag tttttggagt tttatttaat gtcgggagca
33480gattgggtaa taaaatgtat attgagaata agatggcctt ttgacctttt agggtctagg
33540gctgtaaagt gtctcagggt tgctgccaaa tgagccatga actgggctgg gtttttatat
33600ttgatgaaaa agagcctaaa cgttatctga tttgggataa agaaaaagga gcattaacct
33660tgactatgcc tttggctcca gccacctttt taagagtaaa ttgctgggca ggtgggggca
33720ggctagtcat ggaatgaaac tgtaagccgg accaggtgtg aggaggggag gcgataaaaa
33780gattataggg tggaggagcg gaggctgagg aagaattggg acctagctcg gcctggcgac
33840gagcagcctg gggaggaggg gaaaggtcag atgggtctgt agaaaaggaa gaccggaaag
33900actcggcgac gcttggggtt gggactgagg ggacaggcgg gagggaaaga aggaggatct
33960gggaggcatc gcattgtgaa cagaggctag ggagggaacg aagtgtgaaa aatgcctgga
34020cataaggcac ctcagaccat ttgcccattt ttcgacaaaa attatttagg tcttgtagga
34080tggaaaaatc gaaagtgcca ttttctggcc atttagagcc attgtcaagt ttgtattggg
34140gccaagcagt gttgcagaag aaaataaggc atttaggttt taggtcaggt gtgagttgaa
34200gaggttttaa gttcttaagg acacaggcta aaggagaaga aggaggaatg gagggtggaa
34260ggttacccat agtgaaggag gcaagcccag agaaaagagt agagacacag agaaggggtg
34320gagggttctt gccctccaga aaagcagaga aggtgttggg gcacggaaat aagggattgg
34380ggcacagaga taagaggtca gggtgcgtaa ataagggatt ggtgcacaga gataagaggt
34440tggggtgtgg aaataagcaa ttggggggtt cttgccccct aggaaagcgg gacttgccac
34500taagggtgaa ggagaagggg ttgaggggta cttgcccctg ccccaggaaa gcgggacttg
34560ccactaaggg tgaaggacca aggcaggcgt ccctgcgtgg tctgacaccc ttgaaacgtg
34620agtgtagaat cagagaggcg tccctgcaat gatgaaacac caagggaagg ctgccttccc
34680agtctgtgac cggagccgga gttttgggtt cacggataaa acatgtctct tttgtcttta
34740ccagaaaatg aaaggaattg aaattaagag aagggagaga ttgaagtgtg gcaccaagat
34800tgaaaggaga aagaggttga gggatagtga gggaggttgg agaagagagt aaaaagaggc
34860cgcttaccgg atttgaaatt ggtgagatgt ttcttgggct ggtcggtctg aggacctgag
34920atcgtaggtg gatctttctc acggagcaaa gagcaggagg ataggggatt gatctcccaa
34980gggaggtccc ctgatccgag tcatggcacc aaatttcatg cgtgtctgtg cgaagagacc
35040accaaacagg gtttgtgtga gcaataaagc ttttaatcac ctgggtgcag gcgggctgag
35100tccgaaaaga gagtcagtga agggagataa gcgtggggcc attttatagg atttggggaa
35160ggtaaaggaa aattacagtc aaagagagtt tgttctctgg cgggcaggag tgggggttgc
35220aaggtgctca gtgggcagga gtgggggtcg caaggtgctc agtgggggtg ctttttgagc
35280caggatgagc caggaaaagg actttcacaa ggtaatgtca tcagttaagg caaggaccgg
35340ccatttacac ttcttttatg gtggaatgtc atcagttaag gtggggcagg gcatattcac
35400tttttttgtg attctttggt tacttcaggc catctgagca tataagtgca ggtcacaggg
35460gatgtgatgt cttggcttgg gctcagaggc ctgacagttg gataacagtt catgaaagtt
35520tttcactgca ttattgactt aaggattcag caccatttgg gatttcctct tggctagaag
35580ttggatttct atacaatttc ctttttctgt gggcagtgtt ccccaaactt agctggataa
35640agatgttatt tgagaataca gattctggaa tgcacttcag atctccaggt cagaaccttg
35700gaaactcggt cttcaaggat ctttgtgatt gcccaaatat agaaaaacaa tgctgtattt
35760tttttattct ttgccagaaa ttaggtaatt ttttatttct tttctacgaa gaccaaaaaa
35820tgaacatgaa agatagttag aatatatttg gcttgactac agagatagaa atgatgctaa
35880ataatttaac aggaaactta ggaccgtaag aataagctac taaagaacat tatgtcaagc
35940acaaacaagg taaggacttg cttgagatac atttggtcaa atgctgtagg cgtatgagag
36000atgaaactta agattctcaa tttcctcttg tgtcaagatc aactttcctc ttgtacggct
36060gtacctaatt tagtatgtgc actactgaaa gatgtgttga ttcaaagtga catgtgcaca
36120atgcatttct tagtttgcag tctttgttta taaaattatc tctaaagtag ttgcttagag
36180ccatataaat atattctagg agatgtagta agtcgttgaa atggttaata tgcttttaga
36240tttttatgat atgatttaaa aaatccattt gaaataacag caggacagtt caaatagtcc
36300tatcttaaat tctggaagta gaggccaggc ttggtggctc acccctgtaa tcccagcacg
36360atgggaggca gaggtgggcg gatcacgaag tcaagagatg gagaccatcc tggccaacat
36420ggtgaaacct catctctact aaaaatacaa aaattagccg ggcatggtgg cgggcgcctg
36480tagtcccagc tactcgggag gctgaggcag gagaatggca tgaacccggg aggtggaagt
36540tgcagtgagc cgagattgag ccactgcact ccagctcagt gacagagcaa gactccatct
36600taaaaaacaa aaaattctgg gaatagaaag tcatggggag aaggaaggaa gactgcgcca
36660aaagaagaaa caaacgggtc ctacatttcc tgagcgtaga gcagtctaag aaaaatgctt
36720ttgcacgttt cattgtcttt tttccaactc tcattgtttt ttccatcctt ctcctctgga
36780ctatagcacc ctcacagata tagtacaagt aggccagcca caccccaacc aaaactctcc
36840tccacttaac aaataaatct tttatttcct caaaacagtg caaaaatctc aactaagttt
36900agaaaaaagg ttgtatctca ctcataactt tctatcatct caatgatatc tatcttattt
36960tataatggaa gctataactc aagacacata taaaatttag ggaatgttag gcagtataaa
37020aaaactaaag aacaaatcaa gctatagata ggaatgccag agactctaat attatcccac
37080tttttaaaag aacttgaggg gcactacctc ttacagtatt catggttgtc tgaaacgtga
37140aattgaatat caagtcctaa aaaattcaaa ttccctttct tcatgcaatc ccaattcccc
37200taaataggaa gcacttaaaa ataatttgta accaaaaaaa tctcagtaat gcccaggttt
37260cagtaatatg ccaggtcaga gagaggtgga tccattcctt tatttaaatt aagttggtgt
37320catgatatct tcattatact aagggaaaag taaggtttaa aaatagaaat tcatggttgt
37380tttgtgttgt ttttagttgt agctgttgtt tgctctccat gttttatttt tacgtacttt
37440caaatttaca gaaaagttgt aagagtgctg ttatggatgg gatgtttgta tcccttccaa
37500attcatatgt tgaaatccta atccccaatg taatgtattg gggagtaggg atttggggag
37560gcattgaggt catcagggtg gagccctcat aagtaggatt agtgcagtta tatatgggac
37620cccagagagc tctcttgacc tcttttgcca agtgaggata caatgagaag atggcagcct
37680gcgccgtaga agaggatgct cgccacaacc caaccacgct ggcaccctga tcttaagctc
37740ccagactccg taactgtaag aaatacattt ctattattca taagccccct aatctatggt
37800gtgtgtatat gtattctttt aatatactag cccaaactga ctaagtagta cagaaaattt
37860tcaaaaaccc ttcactcaaa tcccccaaat attacatttt accttgtttg ctttatcttt
37920caaacatctt tttccctgaa tcatttaagc ctatggtctg gaaatgatgc ccttcaccac
37980taaatcctcc aaatcaccat ggatccaaaa cctgctagct gggtgagtct ccagaccctc
38040tgaaggataa aggcccataa catctctccc ttggctttca gatcaacatt tggttccagt
38100ttctaatagc tggaacatct ccttagaaat ttggagcagc ttctggactt ctctgaaagc
38160aagaacagag taactattgt ccactctacc agagcctttg ctttctaatg cttttcaaca
38220tttaaagcca ctccagtatt ttaaaaaata cagctcttct tttaccttag ctgctgacct
38280aatttagctg aacacatttc attaaatatt tattaaacat cagttcgtgg aagcactgta
38340tcaaatgggt acacaagggc aatgcccact gggcaggttt ccagctcatc aaaaagcaca
38400gctctgaatt aagagggtgg ctgaggctct gaaaggaata agggcaacag tgaacttgac
38460tatttgaaaa aatgttctgt gataatagta ggaaataaaa aaagatgagt taaagaagcg
38520gaaccaggaa catgataagc acagcataag atcagctata tttcatcttc ctgtgtaggt
38580actggaatat gatcttttca tttaatttgc aattgctcat tttcttgaca acaaaaggca
38640gatccaagtg ttgtggaggc tgaagattat ataatttggg agtttctcat taaaaaaaaa
38700atgagaacac aaaataaagt gtaaaaataa atacttaaaa tgatcatttt atctcaacaa
38760atacattttg aaatctgaaa aatgtcacaa aagttaataa aaataacaat ttgccagaca
38820cagtggctca tgcttataat cccatcactt tggtaggcac aggcaggagg atcacttgag
38880cccaggagtt caaaaccagc ctggtcaaca tagtgagacc ccatctctac aaaaataaag
38940caaaaattag ccaggcatag tgacatgtgc ctatagtccc agatacttgg gaggctgagg
39000caggaggatt gctaaagccc aggaggccga ggctgctgtg agccatgatt gcaccactat
39060acttcagctt ggccaacaca gcaagatcct gtctcaaaaa atgtaatagt aattttgtta
39120attaactgac atgtctctat aatatttttt tctgcattgt ttagctggat gctctcttca
39180tataacaagt actttccaca cagagaatat aaaaaataat tccatcttac ctctagcata
39240attggataca atttttatca ataataaata aattgactcc atcattagag ccaatttaaa
39300caattcttta aatttccccc tataattata tccttgatct tcttgacagt acaggcaaaa
39360ttttcctgta attaacaatg agagggtcaa gtcccctctt ggaggaattt atcagagtct
39420ttccaaacgt ggaacctttt gttagagtga gtgtaaaaaa aaagaacaag aggacctcag
39480gacatgggag agaaagaagg acaatgaaaa taaacaatag ccttaaaaat agaagcggaa
39540aacagagcta ggtgagaaga tgctgaaact atctttactg aacaccgact cttaaggctt
39600ttggtaactt tttctcttct cccatcacaa aataaatatg aatgccaaag ggcaggcaca
39660cttccactgt tgagagaaac agatctaaaa ataagtagga agcagacaag aacgagcaat
39720gaacatgatg agaaccatgg tgtctacttt taggttaact atggaggcat gtaggcacag
39780atggaggaag ttagtttatc tttgctgctg cacttaaaaa ataaaaacca aaaatataaa
39840tgccatcaaa acattgactc ttctctaacc aaaatttttg gttttatatt taaggccaag
39900cccatttaga agaaaagcac attcatttgg gttttgtggt ttggagaaag gactgccaga
39960gaggaggtga aagccacagt ggagcattcc tggacaatcc attgcaggct tgggagatca
40020ccatgcaggc agctcctgct ccataatagc caaaatagca acatgaccac aaataattca
40080aaggctgaaa cattttgttc ccaccagttc aaactctcct gcttctgtca aatggctggt
40140aatcctatct cctctactaa gcctttccta aatgaaacag tggcaactcc ttttgaattc
40200tactatttat tacataaaca cacaattccc gtggttccct tatcactcca ttaaatgcac
40260ttgccctctg attgaaacac aatgcaaatc cttttatgct acatttgttc aatgcttgtt
40320gccttgactg tatatagcct taatatccgg taggtgcttc taacttattg taagttccac
40380atctaattca caaagtattt tgcttagcaa tgcctcttat gatgcaaata aactagtatt
40440tatcacgagt gctttaggat acagacaaga agactatttt agagaaaata agaattaaat
40500aatggtaagg aaagcaaacc acccctgcga tgtgcatgct ctccaccacc accccttttc
40560cctccctgga tggtgtctca ctctccacct cctttttgtc acttgtcctt ggtcacacat
40620caatctgccc tcagccctcc agagaagact tattcttcct gcctgaaatg ttatttctcc
40680cttgttcatc taaccttgtt cctttttatt aagtgcttta tatatacgga agactacata
40740aattatgtct tcacgttaaa taatagtaat acaatgaaca cctgtgcatc cactttaaga
40800aatagaatgt atctttgaag tctttgtgtg tactgtcatg ctcgtgtcct tccacattcc
40860aaggtaacca tattcctagt ttagtgttaa tcactgtctt gtgggttagt ccaagtcttg
40920ctattcttta gagttttact acctatttct ccctaacaat acatatattt agtttgaaaa
40980aaaactcaga gcaatcgcaa gcctagtgga gtaagagaaa gatcaacatt gacactaaga
41040gaggacatgt cccagaagat gagctaacag aggctctccg ttacagcatg gcttcagttt
41100ccccaggagt ttacaaaagt catttgcagg gaagtcagaa ggagagagac tggggatgtc
41160gggctctcta agacagggca ttggggtggt ctgtgaatgt gagtctggag ctggaggtct
41220ccaaccataa ggacatgaga ggtggcagct ctgccttctc cacttctgag ttcaccacag
41280cccacctcct gctcttcaga gtaggtggaa gatcaccctc ttttgcccca tctgaagaag
41340aaactccaga ttactaatgg gaagctgaag ggcagtcagc caggctgact ctaagttgtc
41400cttcaaaatg tagctcaggg tttcctcttc cagcagaact tttctgacac ccacatgcaa
41460tgaccagggt caacctttta accccctgtg cactctgtgc taacccctat caacagctct
41520ccctcctact gctgtccctc ttccttatta aactggatgt tcttcgaggg aataaattat
41580cttatcaatt ttactaataa taacagtatc atctgatatt tgctttattt tataaatgtg
41640gacaaacctc attaacatta aataccaaga tttaaaaatt gtaaggcaga atatgtgttc
41700ttgttactcc gaatttattg gaaataaaga attttgagtg tctcttctat taaagaagtt
41760ctgtgagtgt tccttgcatt cttctttctt caggcatgcc tcatctcata acatttcagt
41820tactgtgtca tttgcacagt tgagaccttt ccacatggat acacgtgaaa taaaggtgtt
41880acaaaaagta gtatttttca ctttactttg tgcctaaaaa caaatacacg tgtatggtac
41940tacataaatt aaaaaatatg atttaattat cttctacttt ttataacacg cctggaaatt
42000gtttttactt ttataattag gaggtgaaca gcaggtctac aaaatattaa cagaaaaagc
42060taataccttt taattaaact tttctaacta taactttcaa gataagtacc tacactgttt
42120taaagtaagc tataaaatta aagttagtaa aaagaaaaaa aagatattaa tatatcagac
42180attacctcac catccagaat taaccaggtt ataatgctgc atatcctccc tgatatcatg
42240tatttttaaa taaaatcatt ctgaatattc tggtctttac ctgatttatt tcattcaaca
42300tatcccggtt atctccccat ttcaataaat gcacttctaa agccataatg tttaatgtct
42360gtatatgaat ctctaagcat gtagtaaaat cataatgcaa aataaataaa aataaacaca
42420aaattcagaa gactgggtgt ttttttaatg agagaattgg atttgttaag ggtttagcca
42480tgattccaaa cacacacata tttttcatta tttgtatacc gtacatgttt tatatctaaa
42540aatttccata tgaatattat attatttcat aatatatgaa aaaagcactc attccaataa
42600gtgtttgtat tttttcccat ctttcactaa acaaacccct gacaaatatt cttctattaa
42660aatgttgcaa atatcctctt tttaatccta ggataaattt ctagaaatag aattgctgag
42720caaaatggtt tgtacaattt taaggccatt aatatatatt actaaactgc cacccagcaa
42780agtaatgtca atttctattc ccatcagcaa tgaatgagag tcattttctc tcattctttg
42840ccaacactga acataatgaa gaagaaaaaa atctctgcca atttgatcag tgaaaatttg
42900tgtttcatat tttctctaac ttgcatttct ttaactacta gtgtgattaa acctttttaa
42960agtttttacc cattttttac tttttcttct attaattgct tattaatggt ttttattctg
43020ttagggtatt attattctta tgtatttata aggttatata attaaagatt tcgaactttt
43080gtctgtctaa tttgtggtag gcagaataag gcttccccca aatgtccaca tcttagtcct
43140cagaaagcct ccaaacatct tatgttacat gacaaaaggg aatggaggtt atagatggaa
43200ctgaggttgc taatcaactg acctaaaata gggagattat cttggattat ccagcggact
43260aaatggaatt acaaggttct tagaagtagg agagaggtgg aggagggatc agagtcagag
43320agaggcttga aagcgctgta ctgctggctt aaagatggag gaatggagcc acaaaccaag
43380aaatgctggg gggctctaga agctggaaat ggcaaagaaa tagatgcgct cttgaagctt
43440gcagaagaac acagctctgc cagcatcttg atttttgtca aatgagactc atttcagact
43500tctgatctcc agaactgtaa gataaataga tttgtgttgc tttaagccac ctattgtgtg
43560ataatttgtt gtaatggaaa tgggaaactg attcacaagt tgcaaatgtt ttcccgattt
43620ttatatacat aatctttaca aagcaatcaa aatgtccctt atatttagta ccattttaaa
43680atgtattcat tatacctctt ctatgttcaa ctacagtgaa gcagataaaa tttgaccaaa
43740aatacaattt tttttttctt taaaaaagga gggggtttcc tcttcaccct ttctttctcc
43800ttttcttttc cctttgcttg gaggtggctg ggagctgagg aagctagaag aacaaggaga
43860aaatggctga ggagatagtc ctggggtcag gagccaaaaa aagctgatga catagtcagt
43920agattggtta tatgcagggg gattaaggaa ataagcaaat attgaagata gtgagagcca
43980ggtttttcac tgtcagagaa gggatttata aaaatgcaaa tgagccagtg ctttggctca
44040tgcctataat cccagcactt cgggagggca aggcgggtgg atcacttgag gtcaggagtt
44100cgagaccagc ctggccaaca tagtgaaatg ccgtctctac taaaaataca aaaattagcc
44160tagtgtcatg gcacgtgcct gtaatcccag ctactcggga ggctgaggca ggaaaattgc
44220ttaaaccctg gaggcagagg ctacagtgag ccgagatctc attactgcac tccagcctgg
44280gtgacagagc aagactgtct cagaaaaaaa aaaaaaaaaa aaaaaaaaaa tatatatata
44340tatatatata tatatgaaaa tggagaaggc taaggtatta cgttgtcatt gagaaaaaga
44400aggagggagg gagagaggta cagatgaaac agatatatgt atatatatat acacacactt
44460ttataattag atggatatat atagagagat agatgatata taaatacagc tctgtcggcc
44520tggtagtaat gagtatacta agcacccaga tctggattta taaatgcctc taccaaaagg
44580aaccagactg cagagaaaaa tagttgattc taaggttgag acatggaaag tacaaaatta
44640gcccaaaagt atggaaatat tctaagaatg atggaaacat gcaaaataaa cccacggaac
44700ttagcttgaa gaggctctca tggccaaatc tgagacacat gaagtatcag aataaatgtt
44760gatagtaacc agattataac ccattgaata aagtaggaat ctatcagacc ataatgaatg
44820aataaatgaa taaaaggaaa aaattcttgc agtataatgc caacaaacta atgtggaaag
44880aatgatgaca catatcagag aggaagttga atcaggtagg agtcatcagt ggatgccaaa
44940gccagtgggt agaaatttga tgaataatta tatatttgca tagtcttgag tatctcccca
45000tgaaacactt ccactcacaa aggggaaaat ggtggaccta tggtccagga acctggcaga
45060caccaacttg aacaggttgt caatgttaac atctccaggg ttcagatgaa ttaacattga
45120aggattcctc atatactact ggaagtacaa tatcatttca gtgctattcc tgataagaat
45180gtatggtgtg agtgtgaaca tgaggaaata ttagatggtt ccaggttgat tgatgtggtt
45240tatactctaa aactgcaaat tacatgaatg tcaaggaaaa gactgtcaag ccgctccatg
45300ttgaaggaca ctaaaataca tgacaaccaa atgcaatgca tgatcctgga ttgagttcta
45360gaacaagaag gagaaagaaa cattatggga acaggaatat ttgaatgaga tctttggaat
45420aattggctgc tagtattata tcaatgttta ttacttgatt ttagatagtg gtatctctat
45480tacgtggtag agtgtttttt gtatgcaatg cactatgggg tgttaggaat cacgggacat
45540gctgcctaca actcctatga tcccaaaatg attcaggaaa aaaagaatga agtgtgcatg
45600tgtgtgtatg tgtgtgtgtg tgtatgtgtg tgaactttca tgccacaaat atttattcaa
45660gcctataaga gatgctaatc actgcaaact aaatattgat aatccaagaa tttaaaaaat
45720gtaatagtta atactttatc ttagtgggca attaaaaata gaaacaatgg ctagaaaaat
45780agttttacac aagaggtaat taaaatgggc ctagaagacc atagatttca aacattaagg
45840aaagtaataa gcaataggca aaatatggta gtgtaaaagt gaatggcatt tcagggaata
45900atgaggtcag tacgttagat gtaagtgacg agaaggaata tagcgagggg aagaaatccc
45960taaaaaaaaa gaagaaaaga aaaaaaccta gaggccaaat tatgacacta gagcctttgg
46020acagtatctg cccgaagatt agaagccgct gtagctgggc atggtggctc gtgtctgtaa
46080tcccagcacc ttgtgggcaa aggcaagagg attgcttgag accaggagtt tgagaccagc
46140ctgggccaca tagcgagacc tcatctctaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaat
46200taaaaattag ccagatgtga tggtgtgcac ctgtggtccc agctacttgg gaagctgagg
46260tgggaggatg aggctgcagt gagccgtggt tgcatccatg cactccagca tggatgacag
46320agaaaaactt tgtctcaaaa aagagaaagt cattgtaggc ttttagtggg caaatgaagt
46380attcaagtct gtaagttatt aatcaaatgc agtttaaata taaaagccca tataaaaata
46440tagtttaaga aataagcacc agaacaaaca atttctgaaa cattttcagt ggattatttc
46500caatggaaat agataaaaat gtgtccaatt aaagaatcca ttataaaaat aggttataag
46560ataaaaatgt aaacattctt catactcagc atctccaagt actcaaagat cccagagtca
46620tagcacttta aaactaatgt gatttttata aaggagtgga aaatatgctt agcatgcagc
46680ataaaaaagt aaaaatatta atctgtgctt tttaatatgg cagggaacat ctattctttc
46740atttaaactg gagtctcgcc ttctccacaa agacacaggg agaggaaaat aattgacaag
46800aataggaagt gaagaattta tgattctcaa taaaagtatg tattgaaaca aaatcagtac
46860tacacgtctt ctaaaaattg ctgctggatg gcctaataaa gtctaggtgc atgcaagaca
46920gctgaaaaat gtacttgtta ccacaagagg gaaggtgttt ctccaagtgc tttgcagcaa
46980tctaccaaag ttttgaagtt ggggcagtag agttgactga cataccctga agccatctca
47040gtatcagtct agtggtaggg caggaaagct gagagaagtc cctctgatat gcattagaac
47100ttcaccagct gccaactgaa agctgagtga aaggccaaga gaagtatccc aagttgcaga
47160aaagtggggg ccatactaca gagaaaatat aactcccaag tgacctaaag agttggacag
47220tttagttata aagcagaaag acctcttcca tgatttgaaa aacaggtgtc tgtgccataa
47280agtataaaga gatctgaaat ctcactggtg tataatcctc aagctctgct gaagacccta
47340attctgccct cagaatactt ttatacaata gtgagctgaa tcttattaaa gttataataa
47400aattcaaatg ttgcttaaga acagattaaa ttgacccaat tcccaactcc agcagcttga
47460cagaggagca tgcccttttc tgtagataaa tattgtcagt ctctactgtt ttttcatata
47520caatgctcag tataagacca ataacaaaaa agacaagcag gaaagtgtta cccccatgac
47580atgcaattta ctcatgtaac aaatctgcac atgtacccac tgaatctaaa acaaaagttg
47640gaaaataaaa acaacaatag gatatttaga atatcctcaa atttggaaat taagcaatac
47700acttgtaaag agttcatgag ttgaaaagat aaactgaaaa ataatttgag ctgagtgata
47760atgaaaacac catatatcat aatttgtggt gtgcagcagc taaagtatta caggagaatt
47820tatagctttg aacaagaaaa agggtttaaa atcaattact caagcttcca ccttaaggca
47880cgcaccacca tgcctggcta atttttgtgg ggattttttt gaagagatgg agtcccacta
47940tattgttgag gctggtcttg aactcctggc ctcaagtgat gctcctgccc cagcctccca
48000aagtgttggg atttcaggca tgaaccactg catgagccat gaaattatat ttatattaca
48060cattatcttt cagaaaataa aggtggagaa aacaactttc caaatggttc tatgtccagc
48120ataaccttga taataaaacc tgacacatta taagaaaatt cccaaataat atctttcaca
48180aacataaaca caaaaatccc ccacaaaata ttaacaaatt aaatcaatca aaatatagaa
48240agtatttgta atgcaggttg tagtcagggc aactaagtga gaacagaaat aaaaggtatc
48300cagattagaa agaaagaagt aaaacgattt ctacacacag atagcgtgat ctcgtataca
48360gaaagcccta aggaatccac taactataaa aactaacaat tcagcaaggt ttcagactac
48420aatatcaata tataaaaatc aattttatgt ctatacactt tcaatgaatt caaaataaac
48480tcaaaaacaa aactaagaaa atttataatt gcagcaaaaa gacaggaata aatttaacaa
48540aagaagtgca aaattaatag tctgcaaact ataaaacatt attgaaagaa attaggtatt
48600acaataaatg gaaaaacatc ccatgttcat ggatcagaag acttaatatt gttaaaatgg
48660caatctctat tagaatccca gatatcattg tggaaattga caagctgata ctaaagttca
48720tataaaatcg caaagggtac ataagagcta aaacaatctt gaaaacacag aaagcaaagt
48780agaaaaaaat cacacctctc aatttcaaaa tttactccaa aatgatggca accaaaatag
48840tgtggtgcta gcacaaggac agaatatagg tgaaaggaac tgaattgaga gaccatgttt
48900ctatggtcaa ctgattttga caagaatgcc acaaccattc agtgaggaga agaatagtct
48960tttcaacaaa tggggctgga actattggat atccacatgc aaaataatga ggctgaaccc
49020ttacctcata ccatatacaa aaataaactt aaaatgtatc aaagacctaa atttaaaacg
49080gaaaaccata aaaactcatt gaagaaaaca tagacataaa tcttcatgac ctcagatttg
49140gcaaaggatt cttacatatg gcatgaaacg tatgaacaac aaaagaaaaa aaattgataa
49200tttgaacttg ataaaaatta aaaccttgtc cttcaaagga aaccaccaat aaagtgaaat
49260aacaatttca cagaatggga gaaaatattt gcaaatcata tctcttatat gggactttcc
49320aaaatatata aataacacaa cttactaaga aaaaagacag cccaaattaa aaataggcaa
49380accacaataa acatttcttc aaggaaaata tacaaatggc caataagcac atgaaagatg
49440tttaacgtaa ttatttatca gagaaatgca aataaaaacc caaatgagat accaattcaa
49500acacacaagg atggctagaa tcaaaaagtt agataataaa aagtgttggt aaggatgagg
49560agaaatcaga accctcatat gttgctggtg gagttgtaaa atggtgcagc cactttggaa
49620aacaataggt aattcctaaa gtgattaaac atagagagac aatatgaccc agcaattcca
49680ctcctagata tgtactcaag agaattaaaa acatgttttc agtcagatgt ggtagcgcat
49740gcctataatc ccagtacttt gggaggaaag gcaggtggat tgcttgagct caggctttag
49800agaccagcat ggacaacaaa gtgagactcc atctctaaac aaaatacaaa aattagctgg
49860gtgtggtggc atgtgcctgt agtcccagct actcaggagg ctaaggaggg agaatggcaa
49920ggctgcagtg agccaagacc atgccactgc accctagact gggcaacaga gccacaccct
49980gtctcaaaaa acaaaacaaa acaaaaacag gcattcaaac aaaaacttat gcacaaatgt
50040ttttgtgtat acacagcaga attattcata aaagtcagaa agtgtcaaac gtccaacaag
50100tgatgaatga ataaacacat atggcttatc catataatga aatattatct ggtcataaga
50160aaaaatgctg atacttgctg aagtatgcat gaatcttgaa aacattgtga ctaaattaaa
50220taagccagtc acagaaaacc acatattata tgactccatt tatacaaaat gtcaagcata
50280ggagtatctc tacagacaga caagtagatt gttctttttt taatcctatg ttataaaagg
50340aatactttgt tatgaccaag tgtaggttat cccaagtatg caacgttggt ttcacacatg
50400aaaagtacaa catgagtagg ataaaggata agatcctatc atggactcaa tagaaaacat
50460atttgacaaa attttacact tatgcatgat aaaaactctc aaactaggaa tagaaggaaa
50520tttcctgaat ttaataagtg caaaatctat agcaaagacc acacttaagg ataaaacagt
50580gaatattttc ctcttaagaa cagaaaaaaa atgcaaggat gttcattatc acttctattc
50640aacattgtat tacaagtccc aataaatgta ataaggcaag aaaaagaact acaaggcata
50700aacattgtgg ggggaaagta aattaacaag tagattccaa aatttacaga aaaaatgcaa
50760ataatctaaa atagtcaaaa taatgttgaa aacagaataa gtaggacaat acacaatcta
50820taaaactaca gtaatcaaga tagtgtgtgt tgccataagc aaagacatgt agaccaataa
50880aatagaagag tgagttcaga aattgacaca cacaagcata gcatccattg attttttttt
50940ttttaccatt attatttttt cttttttttt tattattata ctttaagttt tagggtacat
51000gtgcacattg tgcaggttag ttacatatgt atacatgtgc gatgctggtg cgctgcaccc
51060actaactcgt catctagcat taggtatatc tcccaatgct atccctcccc cctcccccca
51120ccccaccaca gtccccagag tgtgatattc cccttcctgt gtccatgtga tctcattgtt
51180caattcccac ctatgagtga gaatatgcgg tgtttggttt tttgttcttg caatagttta
51240ctgagaatga tgatttccaa tttcatccat gtccctacaa aggacatgaa ctcatcattt
51300tttatggctg catagtattc catggtgtat atgtgccaca ttttcttaat ccagtctatc
51360attgttggac atttgggttg gttccaagtc tttgctattg tgaataatgc cgcaataaac
51420atacgtgtgc atgtgtcttt atagcagcat gatttatagt cctttgggta tatacccagt
51480aatgggatgg ctgggtcaaa tggtatttct agttctagat ccctgaggaa tcgccacact
51540gacttccaca atggttgaac tagtttacag tcccaccaac agtgtaaaag tgttcctatt
51600tctccacatc ctctccagca cctgttgttt cctgactttt taatgattcc cattctaact
51660ggtgtgagat ggtatctcat tgtggttttg atttgcattt ctctgatggc cagtgatgat
51720gagcattttt tcatgtgttt tttggctgca taaatgtctt cttttgagaa gtgtctgttc
51780atgtccttcg cccacttttt gatggggttg tttttttttt cttgtaaatt tggttgagtt
51840cattgtagat tctggatatt agccctttgt cagatgagta ggttgcaaaa attttctccc
51900atgttgtagg ttgcctgttc actctgatgg tagtttctct tgctgtgcag aagctcttta
51960gtttaattag atcccatttg tcaattttgg cttttgttgc cattgctttt ggtgttttgg
52020acatgaagtc cttgcccatg cctatgtcct gaatggtaat gcctaggttt tcttctaggg
52080tttttatggt tttaggtcta acgtttaaat ctttaatcca tcttgaattg atttttgtat
52140aaggtgtaag gaagggatcc agtttcagct ttctacatat ggctagccag ttttcccagc
52200accatttatt aaatagggaa tcctttcccc atttcttgtt tttctcagat ttgtcaaaga
52260tcagacagtt gtaggtatgt cgtgttattt ctgagggctc tgttctgttc cattgatcta
52320tatctctgtt ttggtaccag taccatgctg ttttggttac tgtagccttg tagtaaagtt
52380tgaagtcagg tagtgtgatg cctccagctt tgttcttttg gcttaggatt gacttggcga
52440tgcaggctct tttttggttc cacatgaact ttaaagtagt tttttccaat tctgtgaaga
52500aagtcattgg tagcttgatg gcgatggcat tgaatctgta aattaccttg ggcagtatgg
52560ccattttcac gatattgatt cttcctaccc atgagcatgg aatgttcttc catttgtttg
52620tatcctcttt tatttccttg agcagtggtt tgtagttctc ctcgaagagg tccttcacat
52680cccttgtaag ttggattcct aggtatttta ttctctttga agcaattgtg aatgggactt
52740cactcatgat ttggctctct gtttgtctgt tgttggtgta taagaatgct tgtgattttt
52800gtacattgat tttatatcct gagactttgc tgaagttgct tatcagctta aggagatttt
52860gggctgagac aatggggttt tctagatata caatcatgtc atctgcaaac agggacaatt
52920tgacttcctc ttttcctaat tgaataccct ttatttcctt ctcctgccta attgccctgg
52980ccagaacttc caacactatg ttgaatagga gtggtgagag agggcatccc tgtcttgtgc
53040cagttttcaa agggaatgct tccagttttt gcccattcag catgatattg gctgtgggtt
53100tgtcatagat agctcttatt attttgaaat acgtcccatc aatacctaag ttattgagag
53160tttttagcat gaagggttgt tgaattttgt caaagtcttt ttctgcatct attgagataa
53220tcatgtggtt tttgtctttg gctctgttta tatgctggat tacatttatt gatttgcgta
53280tattgaacca gccttgcatc ccagggatga agcccacttg atcatggtgg ataagctttt
53340tgatgtgctg ctggatttgg tttgccagta ttttattgag gatttttgca tcaatgttca
53400tcaaggatat tggtctaaaa ttctcttttt ttgttgtgtc tctgcctggc tttggtatca
53460gaatgatgct ggcctcataa aatgagttag ggaggattcc ctctttttct attgattgga
53520atagtttcag aaggaatggt accagttcct ccttgtacct ctggtagaat tcggctgtga
53580atccatctgg tcctggactc tttttggttg gtaagctatt gattattgcc acaatttcag
53640ctcctgttat tggtctatta agagattcaa cttcttcctg gtttagtctt gggagagtgt
53700atgtgtcgag gaatttatcc atttcttcta gattttctag tttatttgca tagaggtgtt
53760tgtagtattc tctgatggta gtttgtattt ctgtgggatc ggtggtgata tcccctttat
53820cattttttat tgtgtctatt tgattcttct ctcttttttt ctttattagt cttgctagcg
53880gtctatcaat tttgttgatc ctttcaaaaa accagctcct ggattcattg attttttgaa
53940gggttttttg tgtctctatt tccttcagtt ctgctctgat tttagttatt tcttgccttc
54000tgctagcttt tgaatgtgtt tgctcttgct tttctagttc ttttaattgt gatgttaggg
54060tgtcaatttt ggatctttcc tgctttctct tgtgggcatt tagtgctata aatttccctc
54120tacacactgc tttgaatgcg tcccagagat tctggtatgt tgtgtctttg ttcttgttgg
54180tttcaaagaa catctttatt tctgccttca ttttgttatg tacccagtag tcattcagga
54240gcaggttgtt cagtttccat gtagttgagc ggctttgagt gagtttctta atcctgagtt
54300ctagtttcat tgcactgtgg tctgagagat agtttgttat aatttctgtt cttttacatt
54360tgctgaggag agctttactt ccaactacgt ggtcaatttt ggaataggtg tggtgtggtg
54420ctgaaaaaaa tgtatattct gttgatttgg ggtggagagt tctgtagatg tctattaggt
54480ccacttggtg cagagctgag ttcaattcct gggtatcctt gttgactttc tgtctcgttg
54540atctgtctaa tgttgacagt ggggtgttaa agtctcccat tattattgtg tgggagtcta
54600agtctctttg taggtcactc aggacttgct ttatgaatct gggtgctcct gtattgggtg
54660catatatatt taggatagtt agctcttctt gttgaattga tccctttacc attatgtaat
54720ggccttcttt gtctcttttg atctttgttg gtttaaagtc tgttttatca gagactagga
54780ttgcaacccc tgcctttttt tgttttccat ttgcttggta gatcttcctc catcatttta
54840ttttgagcct atgtgtgtct ctgcacgtga gatgggtttc ctgaatacag cacactgatg
54900ggtcttgact ctttatccaa tttgccagtc tgtgtctttt aattggagaa tttagtccat
54960ttacatttaa agttaatatt gttatgtgtg aatttgatcc tgtcattatg atgttagctg
55020gtgattttgc tcgttagttg atgcagtttc ttcctagtct cgatggtctt tacattttgg
55080catgattttg cagcagctgg tactggttgt tcctttccat gtttagcgct tccttcagga
55140gctcttttag ggcaggcctg gtggtgacaa aatctctcag catttgcttt tctgtaaagt
55200attttatttc tccttcactt atgaagctta gcttggctgg atatgaaatt ctgggttgaa
55260aattcttttc tttaagaatg ttgaatattg gcccccactc tcttctggct tgtagggttt
55320ctgccgagag atccgctgtt agtctgatgg gcttcccttt gagggtaacc cgacctttct
55380ctctggctgc ccttaacatt ttttccttca tttcaacttt ggtgaatctg acaattatgt
55440gtcttggagt tgctcttctc aaggagtatc tttgtggcgt tctctgtatt tcctgaatct
55500gaacattggc ctgccttgct agattgggga agttctcctg gataatatcc tgcagagtgt
55560tttccaactt ggttccattc tccccatcac tttcaggtac accaatcaga ggtagatttg
55620gtcttttcac atagtcccat atttcttgga ggctttgctc atttcttttt attctttttt
55680ctctaaactt cccttctcac ttcatttcat tcatttcatc ttccattgct gacacccttt
55740cttccagttg atcgcatcag ctcctgaggc ttctgcattc ttcacgtagt tctcgagcct
55800tggttttcag ctccatcagc tcctttaagc acttctctgt attggttatt ctagttatac
55860attcttctaa atttttttcg aagttttcaa cttctttgcc tttggtttga atgtcctccc
55920gtagctcaga gtaatttgat cgtctgaagc cttcttctct cagctcgtca aagtcattct
55980ccatccagct ttgttctgtt gctggtgagg agctgcgttc ctttggagga ggagaggcgc
56040tctgattttt agagcttcca gtttttctgt tctgtttttt ccccatcttt gtggttttat
56100ctacttttgg tctttgatga tggtgatgta cagatgggtt tttggtgtgg atgtcctttc
56160tgtttgttag ttttccttct aacagacagg accctcagct gcaggtctgt tggaataccc
56220tgccatgtga ggtgtcagtg tgcccctgct ggggggtgcc tcccagttag gctgcttggg
56280ggtcaggggt cagggaccca cttgaagagg cagtctgccg gttctcagat ctccagctgc
56340gtgctgggag aaccactgct cccttcaaag ctgtcagaca gggacattta agtctgcaga
56400ggttactgct gtctttttgt ttgtctgtgc cctgccccca gaggtggagc ctacagaggc
56460aggcaggcct ccttgagctg tggtgggctc cacccagttc gagcttcctg gctgctttgt
56520ttacctaagc aagcctgggc aatggcgggc gcccctcccc cagcctcgct gccgccttgc
56580agtttgatct cagactgctg tgctagcaat cagcgagatt ccgtgggcgt aggaccctcc
56640gagccaggtg tgggatatag tctcgtggtg cgccgttttt taagccggtc tgaaaagcgc
56700aatgttcggg tgggagtgac ccgattttcc aggtgcgtcc atcacccctt tctttgactc
56760ggaaagggaa ctctctgacc ccttgcgctt cccaggtgag gcaatgcctc gccctgcttc
56820ggctcgcgca cggtgcgcgc acccactggc ctgcgcccac tgtctggcac tccctagtga
56880gatgaacctg gtacctcaga tggaaatgca gaaatcaccg gtcttctgca tcggtcacgc
56940tgggagctgt agaccggagc tgttcctatt cggccatctt ggctcctccc ccgcatccat
57000tgatttttta acaaaggtac cgacacaatt cctgtattag tccattctca cattgctata
57060aagaactgcc tgagattggg taatttataa aggaaagtag ttcaatcgca tggcagaagg
57120ggaagcaaag gcatctttct tcacatgatg acaggtagga gaagtgcaga gcaacagggg
57180aaaaaacccc ttataaaacc atcagatctc gtgagaatgc actcactatc ccgagaacag
57240gatggaggaa actgcccgca tgattcaatt atctccagga catgtggaga ttatgggaac
57300tcaaattcaa gatgagattt gggtagggac acagccaaac cataacaatt cctttgggaa
57360agcaagttcc caaatggtgt ttgaacaacg gaaaattaaa ttagtaaatc tcaacctcct
57420acatcactct atgtacaaaa attatttcaa gattaatcat tgacttaaaa taaaagttag
57480aaaaataaag catctagaaa aaacattgaa gtagatcttc atacgtttga gataggcaaa
57540gatttcttta gagcataaaa agcaccaaac ataaaacaaa acttgataaa ttacacaaca
57600tcaaaaataa gaacttctgc acattaaaaa atgttataag aaataaaatc aagcttcaga
57660cttggagaaa atatttacaa cacaattatt ttacaataca aatatctaac aaaggacttg
57720tatttagaat atataacgac ctgcaaatca ataatgaaga aatctactca ataaatatgc
57780atataatatg taaacatttt accaaaaaaa atgactgttc aacatgaaca taaaaaggtg
57840cttatcaggg aaataacaat taaaaccacc acaagataca tactagaatg tttaaaataa
57900aaaggactga aaataccaag ttttggcaag gaagtgaaac cactgggaac tctcatatat
57960tgctggtgga aatgtgaaat atcacaacca tttaaaaaaa cctgtggcag taccttttaa
58020agttggatat acatggtatc catgaatcca ttcctaggtg tctaccttaa aaaaaagtga
58080aaacattttt cactggaaaa tgttaaagat gtttcattgt agaacttttt ctccagtgaa
58140aaagtaaaac cttttaactt tttttttttt tttttttttt gagatggagt ctcgctctgt
58200cacccaggct ggagtgcagt ggcatgatct cggttcactg caagctctgc ctctcggatt
58260caccccattc tcctgcctca gtctcccgag tagctgggac tacaggagcc caccaccacg
58320cccggctaat tttttgtatt tttagcagag atgggttttc attgtgttag ccaggatggt
58380ctcgatctcc tgacctcgtc ctcgtgactt ccctgcctcg gcctcccaaa gtgctggaat
58440tataggcatg agcccggcca tgttttcact ttttttaaag ataaaaagac ttgtatatga
58500atgatcatag cccccttatt caaaatattc aaaaatttaa aatatctcaa atgtctgtca
58560tgaaagaatg gacaaagaaa ttgtggtata tctatacatg gaataatact cagcaattga
58620aaagacagtc tacaaatgga caaaacctca tggattcata tcaaaaaaca ttataatgaa
58680caaccaaaag cagatacaaa aatatgtaat gtattattcc atttagatga aacaaaagaa
58740taattgatgc taatgcatgc tgataaaaat caggatggtt gctttcaggg ttgaaagttt
58800gagttgaggg agatgcaaag cactttctgg tgtgaaggaa atattctaga tcttgttttg
58860ggcggcagtt ataaggatat ctacaagtca aaactcatta aactgaactc ttaaattctg
58920tgcattttat tatatacaaa ttattttgca ataagaatta atttttaaaa agcaataaaa
58980attgctgctc ggcgaaaaca aattatgaat atactatcta acacactttc tggtgtgaag
59040gaaatattct agatcttgtt ttgtgtggca gttacaagga tatctacaag tcaaaactca
59100ttaaactcaa cttttaaatt ctgtgcattt tattacatat aaattatttt gcaaaaataa
59160ttaattaaaa aacaataaaa attactgttc tgagaaaaca aattatgaat ataccatcta
59220actctacaga gtacagaaat aaaaaatata gtaatatctt tgacctattt gaaagtcaac
59280aaaagaaaag caagaaagtg cctacaatat gattaagtga atggcattta tatatatgta
59340taatcattta aatgtttaca gtttaaagga aaaaatagtg aggaaaagaa tgctatggtt
59400ttcaaatcct acggtttgta ctaaagttgt ttgtcttttt ttttttctta gctacctggc
59460atctgaactc cctttctgtg cctgtgaaat ttccaccttc aactgcagaa gctgcaaagc
59520cagatacttg ctttcatagc ctccattact ttgagggtag ggcacatgaa taggctcagt
59580tactctgact tcagcttgga tattgacttg ggaactagtg agaaaagaag cactcagtgg
59640agaattagtt ctggcagcaa tagtagcagt tacctcgagg atctggtcac agttatagca
59700ggggggtcat ccagtagcca atgtcagcag caaacaatgc caatgccaat gctagagaaa
59760tgagaggtac aaaccattca tctagcattt gctggcatca gaaaaaatat cctcactagt
59820caagttctgc agcataattt tagcttcaaa gctatgcact ttgttttatc ctgcctgccc
59880attttttaag ccttttctct agtgttccaa atgactctgt gaactactca atatcctttc
59940aacaaacttc ttttccactt gaatcatcca gagttaattt ctattgctgg caatactaac
60000tctgatggaa aaataaaata ataaatgcac tctaacacag aataacagaa ataatgctcc
60060acctagttaa caaggatttt acaaacaagt agaagttatt tggtcttctt tttaaaaata
60120cgttcttaaa aataacattt ttgacaccat gaattccacc tgtgttttca aactattttt
60180cactattaac attatgtttt aaaggagata cgttttcaaa agatccactg gaaaaaatta
60240gacttctcct ataataattt acacagtgag tggctgagat tctaaaggaa tatcataaaa
60300ttatgataat attaaataaa agcaaatact ctcataactt tcaaggaaac atgcgatgac
60360tgatgtagta tcaaagggat atgttttgga agatgctctc ttctatgtga ctgaaaatat
60420ggggacatgc tatacattct attttttctt attgattgtg aatttgttag tcatcaattt
60480tgctgagtct attcaaaatt tccacttaca taaactcttt gagtaatgag tttcatagat
60540atattgtgtg ctatgcgaac tagtacctct atttcttggt tccattattc ttgtactata
60600ggatattgta aataaaccta tgttttctgt aacatatctt ttgaaaaaat aatgcatctt
60660ctcagaaaac ttttcctatc aagaattttt gttgttgctc ttcatcagtc taagaaaaaa
60720ctgtcttttt tgtccttttt tatttttgcc catattttcc tcttcatcct ccattcctat
60780gctcatagcc catcctcact ggcaccaatt ctgtgtttaa tgtaaacgtc cttgaaaaat
60840agtgttgttt catttcgagg atatactgaa gttacctaaa tttgaacatg atatggattt
60900cattatttct tatcttgctt ttgtgaagat gacattttat ggagacaaaa gataagttca
60960cacataaaat ttcaggtagt ggtaaatgta acaaggaaat aaacacggtg attcagtagt
61020gattgggtta ctttataaaa gatggtctct tctcagaatg ttttatttga gctgagaata
61080gaaacatata aaagatactg ttactagggg catccagacc aagaaaatgt tggctctaaa
61140gcaagaatca gtttgataca cgagacaaat ccaggaggca actactctgt ctgaagcaca
61200ctcagcacag aaaaggtggt cctagatgag gcaggagaaa ttggcaagga gtgggtcata
61260tagggtcttg caggctacaa ccagaagcaa gaattttact ctaagaacaa tttgagagcc
61320attggaaggt tacaaagcag aggagcaatg ttgtttttta tttaaaaaga taggttgctg
61380tgaaaaaatt ggaaatcatc gaagaattaa tgggatttct tgtcaagagt atcagattct
61440atcttcattt tcttctgctg tcttccaaaa tcccattaaa atgacagtaa agatataggg
61500aaaaaaatat atatccatgg ggagaagagt aggagaagtt taatgacaac actgttttga
61560agcctggaaa gcaaattaat tagtagttag tccaaagcta tcattggaga aagcagagta
61620gtaatgcatt tcatactaca gaaccacaga aagaatcaag aactggtgga accaagcgcc
61680tccaaaagtg agaaagatac agttggggag gagaagtagt tgacgctcca tttaccaatc
61740atttagactc ctcgttcctc tcctacaccc aaacagaagc ttagacattc attcactgga
61800gaggataaga tagagtgtct ctagaacagg ggctaccagc acagttaaga atcttatact
61860gaaaattcat ccccagtcct cttctcccaa tgattctcag gtttacacct tccagggtga
61920ttggaagatt cttctctggg gatctaactt gtcattttgt tttccagcac aatggccgca
61980cttgggtcag cctacaggaa agaccatagt caataagccc tcctcagact caagagcttt
62040ttaataagta ttcaatcctc tgttcaatat gaacaattaa aaatcaccag actttagagg
62100aaagcatctg ttatgaaatg cagagagtaa atagggggaa aaaaagcagc ttggaggaaa
62160tagactatgc aagtagatga aacctttaaa aaaagaacta ccattaatat ttttattgat
62220aatctcagaa agacaaaaga aaaaaatcac attcacaaaa taatagtggt attcaagaaa
62280aattcagaga acaagaatgt gcttttggaa attacaaatg acagaaaaga aaaactcaat
62340agaaggctta aaggagtaca ttaagaatac atcctggata tgcaaagata cggaatatag
62400atcagaaaag ctgccctcct atatagacta gagaatcaat atgttaatgg tctgatataa
62460taagattccc agagagaaac tgagaataca aaggataaaa ttaataaata tttggaaaag
62520atttcataat ttaattacgt gagtatttaa agagcacagt aaagcatact gtaatataag
62580acttacagtg aaatttaaca cattgtggca aataggatat ctataagtat tcagagtgaa
62640aataataaaa tcactaacaa agaaccaaga atcaaaaata gactggagtt atcaatactg
62700aaatttagaa aaacaaacaa acaaaacttt taaaatattg aaaaaaaatc catccaagaa
62760atctatactc agctaacctt ctaattgtga gggtagaata caggtatttt ggatataagg
62820gtctcaaaaa ctgtatgcat cctaaaccgt tccccagaat attactgtgt aatgtactcc
62880tccaaaatga ggaagttaac caagaaagag gaagttatag tatacacaaa acaggagacc
62940taacatatga aagagagaga ggatacagga tttccccaga aagatggtga atgtgcccag
63000cctgacaatt gtttggatgc agaagtagtg gtagcaaata ttttggttta tccagggttt
63060ggggttttct aggtaagcac gatggagaaa cagacacatg aattgacagt gaactctttc
63120actgtcattg ttacaatgag gaaatattaa aaccattctg gataagaata aaagtataga
63180ctagaggaga atgatagtga acaagtagca aaatcaatgg tttagaagtc ctgaagagat
63240agaaaaagtt tctagagagc aagaactagc ataaatgagc taggaaagga ggtggtagtc
63300agagacgtta aggtgatcaa gatgttagag aagatatagt ttctagtgat gaccagggaa
63360tgtgtggttg agggaaggaa atgtggttgt caaatacaga gagctcagaa tgctggagtt
63420atctgcacca ataatgaagt cacccatagg gtgacacaag tagaggtata aagaaagaaa
63480ggccaacagg aactaaagta ttcaattaat gcagggaatg aattaagata aatgacctat
63540aaattcatgg ttgacagcag cacgaagggg tagagcataa tatatccaca tgacacggct
63600tcaaataagc tggggcattt ttgaagaagg acacacctat cccaaatcct agccttgaag
63660tgcaagagga atgatagaaa acaaaaacaa aaaacaataa ccctcactag aattggaaga
63720tttatcctta attaatgtcc agttcagtta aggcaagaca atacgtagaa ttttcagaga
63780caaaattgct gatataatag agttttctaa tcaaagaagt ttgcagagta gaaggttatc
63840agaaggtggg ggatttatca acttagagat atacaaagga tgtaggatct gccagggaag
63900aataatgatg ataatggttg atagagaggc attgacttct ggtcgtacta gaagtaatta
63960tggatgtcaa gcatggtcaa agtcatcatg gtagtctcga agaaccctgg gcattggttc
64020actgggtagc ctttctattc aaagcgcggt accagggcca gccgctttgg tatcacctga
64080aagtttgtca gaaatacaaa attacaagct ctaccccagg cttaatcaat cagaatctta
64140ttcaatctga ttgattctta atcagaatct tattcaatct gattgattct taatgaatca
64200aaatcttatt caaactgatt gattcttaat caatcagaat cttattattg tgtgtacaaa
64260aaggtttaag aagcattact tcaattattt acctctaaaa tatggggtgc ctgttttctc
64320ctctagctcc tgtcagtgga ctcaccagaa gttgaatatg ttgacttcat catgggacta
64380tttctcttta aaacatcatt atgagaccac attggtgacc acattactaa actataactt
64440tctccgcctt cagatgggga ctattttgta tattattagt gcctggtgcc taataaaatc
64500ccaaaaacag acacaaaaaa tttcctaggt gttgtggaag cctagagagc gagagggact
64560aggcgtgcag gaggaatctc ttttgcagtt gggctggtga tgtggaaaaa gtaggactgt
64620aggactggaa aagggaagat tgggtaggtt gataatttgg aaaaggtaat taaaatataa
64680attcataaaa agatgtcaaa tattttaatt taaaatatac acactacaca cacacacata
64740cacactacac acacacacac acaatgccac atttgtatgt gtattaattc cttcacagat
64800attcttttgt agggcccaag gccttttatc tgagtatgaa ctggtttctg agagttctgc
64860aaacatcttc cttgcacaaa gcatactaaa aatgcattgt ctaaagatat ttatttctgc
64920tgtctttaaa gtgaattgtg aagcaatctg agtacagaat ctgcctagtt ttttgttttc
64980cctatctttt acaattctca tctacacata acagcatttt aaaaatgatt tttttctagt
65040ccttccgata aagtcaacct tgtccttttt gcacttatat tttgatggta tataaagtac
65100ttaactaaat tgtccaatac tagtctctgg taatataggg gcttaaaaaa tccatcatca
65160ttggttatag taatgggcaa ttttagtgtg ttgcttattt tctcctatag ttaactctgt
65220gatcccctct cagatttcag tgaacttgcc catcaggtta accctagtgg atctggcagt
65280gcagcttacc aaccgagagt tcttaggact tggtgctctg gaatgaaggc agcatgccag
65340gtcttctgta gcaaccagac catttgccat ggcagcttca aattgagcaa gaattccaca
65400atttcacact tgagtgcagt taagtctcag gtggatggca gccaggttcc ataaataact
65460ctcggttatc attgttccaa aactcaagac ttttaaaagg cactacctac cctaaaggtc
65520cagtccttaa tgacatgatt tgtatgacac ttgagcaata gcctgaggct gccaaatcag
65580gtaagaagga ctttcccata ataatatcaa cctactctaa gtagatttca gttggatcat
65640gatatatcaa catatgagtg atacatgcag ggcactcact ttggtttttt tgaaaatatg
65700tttaagttca tttatgaaat atttttgaga aataattttg tgtttagaga tataataagc
65760tctaagagaa atatatgctc atagatattc atgcctagtg gtacttacct tctactttaa
65820aattatttgc ctttctttct gacccatgag acatagctca aaagtaataa caagtcttac
65880tctgcctacc atcacttctg atatggtttg gctgtgttcc caccaaatgt catcttgaat
65940tgtagctccc ataaatctca cgtgttgtga gaggggccca gtagtagata attgaataat
66000gggggtggtt ttccccacac tgttcttgtg gtagtgagta agtctcacga gacctaacgg
66060ttttataagg ggaaaccact ttcacttggt tctcattctg tcttgtctcc ctccacgtaa
66120gacatgcctt tcaccttccg ccatgattgt gaagcctccc cagccacgcg gacctgtgag
66180tccatcgaac ctatttttct ttataaatta cccagtctca ggtatgtctt taacagcagt
66240gtgaaaacgg gctaatacaa cttctgatag aaaattaggc acataataca cattagttga
66300aataatcaac tggagaatgt aggctttttt tttttttttt gaaagagtct tgctcagtca
66360cacagactga agtgcagtgg tgtaatcata gctcactgta gcctcgatct cccaagctca
66420agcaatgctt ctgcctcacc ttcctgtata gctggaacta cagatgcgtg ccaccacacc
66480taggtaattt tatttttaat ttcatttttt aatttttact ttttgtagag atgagatttt
66540gctatgttgc ccaggctggt ctggaactcc tgagctcaaa tgatcctccc acctttgcct
66600accaaagtgc tgggattaga agctgagcca ccatgtccag ctcagaatgc aggcttttta
66660atacaaattc aaacttacaa acaaacaatg agaagatagg gtcaataacc agtatatgct
66720ctttttcgta cagctgaggc tctgccagat ttaaaatatg aaaccgatcg tattttactt
66780cgggaacaag aatacaggag atctaaatat aaaatgacga aaaacaaaaa aaaaaagtgg
66840taaatctcct aagcaagcgt caaatttttt taaaacatca ttctatcaga tgcttaagga
66900actttatcag aaattaatac aaaattttct aaatattgaa cacaggaatt ttgctattcc
66960tcattttttg tgccataaga aatatctgat ggttttacaa acaataggcg cttagaaatt
67020cagcatacaa aattcatttt cccccaccta ttatcaatta cagattttac ccaaaagagg
67080aaaaaaactt aaccaaatga ttacaatagt gttgaggatt ttttattgtg ttttccacat
67140agataaaaaa ataaggcttt ttgatgaaaa gaatccatta caaagtcaaa aatccattac
67200aattataatt gaatcagtaa caaaatttag ctttaaatga gtcaagtatt ctgcatttga
67260aatttaatat cacaaacatt caagattagt gaattttggt aagaaaaaaa tactagaaga
67320aaggaaaagg acaccttttc aacagatagt aatttataaa aattttttta aaagtgcttt
67380gggaaaacac acagtatcat tacttaagaa aagtcattta aggaagactt aagtgcttca
67440agtggagtgt attacagact aaaaaatgtt ttaaaatttg ccaagaaatt taagtgttaa
67500aaatactctt ctccttattc agtttcatgt ttaaggaaac atttgacaga caagtaaacc
67560aaacgcaaaa aaaagttcac ctgcatttta aactaataaa ttctggatct gtaaaagctc
67620ttggtttgta cacagaggcc aatgctgaca tttattgatc tatttttatg tagttaaaaa
67680aatacagtaa caaatctttc ttcctatccc cccaaaaaac taggggaaat attttaaatt
67740gtgagtgtgt gaatgtagct atatatatat atccctaagt gtacaaaaca cacaaacatc
67800actttacttg gaaaattatt ttcatcatac tgtaaacatc tcttccccta catctggaca
67860ttttgaaata gtctttggta ttactagtta ttgtgctttg aaacagaaac ttgcagaatt
67920tctgtagtag tgctacataa agatataaat aagaaaaatg cacttggaat aagttacatt
67980tagctgcttt tgcataattt tcaaaaacta cagtgtatgc ctagtcacag ttttatgaga
68040aagaatattt cctttttcaa cttaatttta aggaacactt aatcattttg gctaagtatc
68100catttttgga gtggatctga tgggttgcat gacactaaac ttggatgctc tccatttgct
68160gaaaggcaca tttttaagaa tggattgtat agaagttgat cctagaataa aaggaaacaa
68220cagacattaa attccaagac tcatagttaa gtgacaaata tgaaaaagat tttatggttt
68280cattgaagca aacctaatag caaatctttc taaggtgagc catgtctgat aaactctttt
68340tggcttctag tgtaaatgcc accttttaag ctactactct tcaattagta agatattaca
68400taatgaagag aggcagacag aacaaggttt gaatcacatt ttcaaacaag aagtattccc
68460ttgatatatc cataagtgac aaactcttaa aaacatcata aattataaaa gccagtgtta
68520acgttttcta gtactgcaca aaacaatgtt caggtaagaa ttgctatgat acagctaagc
68580cttaaacatg tttaaaaaca gaagagacct gttatgtatt ctctcaattc taggtttgtg
68640aaaaagtgct aaaacagggt agaaaaaaat taatcacttc aagtgagaag atataataat
68700ttttcttctc tctgacagaa aaaaaatatg actgttgcta aaagcattat aagaaaccct
68760aaatagtaaa aaacaagcga ctaatattaa taattcacag aagtgctcat ctgcatatat
68820cacggttcaa ccaagcaact taattttttg ttactaagag tacttaattc atagatatat
68880tctcttatca catgggtaga aacacataat aacaaagtta ataaagggag agaaattctg
68940atagataata ttaatgtacc atgttccact cattttccca gataagcaag tagagctaag
69000aaaaatagtt aaatagatga attaattgca ctgaggtacc ttctggatct cccatatctg
69060ctctccagtg acaactgtct tgtgaccttt gtaggtacat gccttcagct gaacgagacc
69120ttgagcagca tgaagacata attccttctg gatgttgtgc cgaatttcat gttgagcaga
69180gtattcaaag tactatggaa ggaatagcac tgttactgac aaaagcaacc catgtgctca
69240gttgcaaaat aaataaataa atcagcaact cgttaaataa taaagtcatt tctatttcaa
69300caaaataata caataaaaat agttattaga aacataagct gagtttttgt gatagacgat
69360acaatagcct tccttcatca acaggtggtt acttcattaa caaggtgctg tcttcctccc
69420cataccagaa gttggaagtt atcaaggaga gtggaggcta tcccaggcat tcctcagaaa
69480agggaatgaa gagttataac aagaacatta attgcctctc tcccacaagt gttccatctc
69540tcttcttcat ctcaatcaaa ggtgttttca ctaaactagg gacttcaggg tgttcaccat
69600gcctcagttt tctcactaga aaatgggtaa tttaatagtt cctaccccac agggttatga
69660agataagtgg gttaatacgc ataatacatt tagaacaatg actggcacac ggtgagtttt
69720catacatttt agctattgtt actactgatg cttctctttc tccacatatc caagtaattc
69780gtcccttagc tgcactattc ctctcagatg ttaacatccc atccattctc acttccgtgt
69840actcacttct gttactcaga ctctcagaat ttttactgaa acacctacag ctacttccaa
69900tttctgttcc tatcatccag caatccaatc tgctgccaga tagtctttta aagcattggt
69960ttgatccctg actgcccttg cttaagtgaa tcactactgt tgctaagata aagtccacac
70020tccttaattg gtacatgaaa tctttctttt tgatcttgca tcaatcattg ccccagttat
70080atattatgct tctttcacac aattctacca atcagaacat atgtatctat aactctaaga
70140ctttgcttta cagtctgtcc tctgcctgga atacactttt gacccttttc tgcctggcta
70200actccttttt aatccttcaa gagccaactc aaatactacc ttccttcttt ggctttgcct
70260gaatttgccc tgctgcacaa aattacaatc ttttcaaggt tcccatagta catcagagat
70320aacttttcta gcactctatt agaattgtgt gtggtgtgta gacctggctt ccccatagac
70380tgaagtcagg caatttatcg agtcataaca cccatcacaa atccaatatt tcataatatt
70440gacattataa agataaaata agttaacata attgattttc aataatgaaa taattagtag
70500gagcactaat atttgttaag tccttattac atgttaggca ggcactgtgt taaactcttt
70560atgtgtatta cagtaggcac tctacaaaca ttcgctgaat tcacagagcc atatatcttg
70620agtttcccta ctcttagtct catgcttaat ttatctccgc ccacaagcaa aacatcagtc
70680atttttagaa ctagtagttc ttcagatgga aacacaggaa gaaactgagt ttagatgatg
70740aaaatagcat tctagctata cttggctaag aaagataagc cttaagatct caaaaatggt
70800aacaaaggct gtcgagtatt ttacccatta ttaaaaagtc attaaaataa aaattacaaa
70860aaatggaaaa tattgaaagt actttggtaa caaaaaagag cacataattg gataactgtg
70920aatgttggag agacccatta cttagaacga gtagagaaga ggcaaagcag taaatataat
70980caagttataa tgactcaaac tgcattacac tagtaatatt ggccagctta gtcacataac
71040caaaatattt atttttattt ggcatacatg aagttttatg ctataaaaat attatctgat
71100caaaagtaga aggaaacatg aaaaaatgat caagttatat aatatctaaa aattttagca
71160ctcagggaac taaaatttgc aaaacggttt aagtgcaatg cttgattaaa aataaaatgt
71220caaggaaata aagcaaaata aaatcaatag ctgtgttagg gtaacaaaag ctgaagtttc
71280tatttagtat atacaccaga taatatttat tcttctgctc aaaactcttc aattgtttct
71340gtcaaagtca gaattaagtc caagtttctt aatggggcca gttaagccct acatgatcag
71400gtccctatca atgccccgac ctcatttcct agtcctcttt gctagaccac tctgctctag
71460gtacactggc tatctgcttt ctccatgtat ctgcctctgg gcctccactc ttggaaatcc
71520ttcctccaag atgagcatct tctcctggaa cactctctca cagtattcag gcctccctct
71580aagatcgctt ttccaaaaag gtcttccctc acctcttaaa ataaccaccc taccccgaaa
71640cacactattc ccttacctgc tatatttttc ttcatgccca aaactacata tttttttctt
71700tgttcctgtg gaacataagc tccacaagat caggagcttt gtctttttac tacggtaaca
71760gtcagtacaa gctactttat aggttctaaa caaacactta ttgaaagaat atgtgaatgt
71820tttcccatcc cccaatttct aaaagatagc tatattctat ttaaaatgta aacatatttt
71880tgatacagag ctatcataaa gctgttatta tctttatgaa aatgataaca gaaaaatgga
71940aaaaagcaat gtgtacaata tatcttcagc ttttaagata aaataaatgt aatgctagaa
72000gaagcctaag agaaatttat agggatttac atttactagt aagattttaa caaatgcaga
72060aatatttccc tttgctcaaa atcttaattt ttacaagcat agatcagtgt tatttccaaa
72120gaatgcaatt atttatgtaa ctatttttct tctaatatat ggagggtaag aaaattctta
72180aaaagatgca tatagattta tgcacataca catatatgta cagatatata tgtatataga
72240tataggtata gatgtatgtc tgtctatatg catccatata tatctccatc ctatttcaga
72300attacacctt aaccatccag caatagatcc agttcaccag ctctggcttc tgggcatctg
72360tatcatctgc cctcagtaga ttgattggtt taggttaata aggattagaa ctagattaag
72420ctgagcctaa tgggtaggaa gaaatataga agaggatgga ccaggagata aactggctct
72480tttttcctaa aactgacatc tcaaagtttc gtcccattaa aataggacat catggatatt
72540aattattgca aaaatcctcc acacacatct tggactctac accaaaatac ctgcaatgcc
72600cagaagagat actccaggct cttctcttca tttcactttc cttcacactc ttcttattct
72660gttgccattc cccacagcca cctcactagc tcctacctac tttttttttt tttaatgttt
72720tcaaatatca tctttctcag gaaaatgtgt ggacacccag tctcattcaa tgcccttttc
72780tataccccca gaacactctg aatgctttat cctaaggaac tgctagttta tagatttcct
72840aaaggagtta gagtctaaat agcttgaact catcagagta tctggcatgc agggctcact
72900caatgaattt gttgaatgaa caaatgaggg aaatagtcta atctttccag gagttaagct
72960gcaaaaggta aggaaaggaa gcttaaaaaa acagaaactt tgtatataac atgttctcta
73020cccataggca agtaaatagt aatatttgac atgtgaatct taatcagtaa aatagtcaaa
73080aggtagtaaa gcaggtatgt cactaatcaa ggagaatctt cctcttttgt tggagaagga
73140ggaacatatt gaaataatat tgtggcccac aaaattaatt tataatgctc tattttgtta
73200ttaacacttg atgtaatttg tgattcaatc cagttatatg tggcctttat gtgtaaactt
73260gcaaaatcac catgccaata gtcatgcaaa agttaaagca tcaaagagct acatagaagt
73320ttctttttaa aaaaaatttc aaatggcatt attctattat tttctcattt tccttttttt
73380ttttttaatt tgtttgtttg tttttgagac agagtctcac tctgtcaccc aggctggacc
73440gcaattgtgt gatctcggct cactgcaacc tccacctccg tggttcaagc aattctccag
73500cctcagcctc ccaagtagct gggaacacag gcatgcacca ccacgcccga caaatttttg
73560tatttttagt agagatgagg tttcaccata ttcgccaggc tggccttgaa ctcccggcct
73620caagtgatcc acccacctca gcctcccaaa gtgctgggat tacaggcgtg agccactcag
73680ctcggcctcc tttttttttt tttttttttt tttttaatgg ttaagctact caaattgttt
73740tcagagccag agtaaaaacc aaaaaaaaat actcatcaat atcaattgcc aaactcagtc
73800tgaaaacatt tcacacacag cttacccaag tataaagctg ctgtgggact tctgaaaaat
73860aggcaacatc tcacttgtgc ttgtaaatgc tttacaagtg aaggattttt aatgcagtgc
73920tcacctggtt tcccccaagt ccatgacatg tatacataat taatggtttg cctccttgat
73980tgttttctcc aacatccaga catagaggct gaccaacgct tttaatctaa aggaaaattt
74040tcaagttatg aaaagttttt taatccatgt gatttatata tttttaaaat aatttagaaa
74100gtaatgttaa tgattataat gtaaaatctc aaaagcaata aagaaagtat ttcagcaatt
74160ttcttagaac cacataaagg ttggtggcaa ggagtatatt aattaaaaaa ggaacaggaa
74220aatactcacg tatccagata taacaggatt aaggtctggc acatacacct ctggataaat
74280gttgttcaga taccatgtaa aatttttaca ctgaaggcgg tgttttattt caaatctttt
74340tgaaagatca ccaaatgctt tctgtagaaa catgagaaat gaagggaatg cttaattaaa
74400acaacattga taaagcactt aaaatactct tatttgtatt tctatttagg tatttttaaa
74460aatccatgct aaaaattcaa gaggcaattc aacagccttc atcattcatt caagtaaaaa
74520ccatgcctgc ctgttcattt ctggctttaa aagagagtca acaccacttc agtgctgatt
74580aaaatactaa aatgatgcta ttttattctt catttgtgcc acaaacattc cctacaagac
74640aaagatttct agtaaaatgc tgtgggcaaa ggaatggtgg taggagcatc caaaattggt
74700cccaacttta tacctaaaaa tgatctgcaa agtgaggtgt cagaaatcag gtatagacaa
74760acagtgccaa tacacaatgc aacttctatc tcttctccat caagatcaca gaaaaccacc
74820agcaagaaat aacccactgg gagtaatttt tctagtttca ggatgttgct tgttcttaac
74880aatatcccca actgaacctt tagtagaaaa aggcaatttt caagttaaat gaattttgct
74940ttccttcatt ttaacgtact aagaaaataa cttttttaaa aaaccattat taaacttcaa
75000ggtatttgac ctacaggcaa aatattagag aaaaagtaca tgtaatatct aaatgtatgc
75060aaaagtaaat actcaatctt atcagttcaa ctaaagaaaa tattttctta tgccaacttt
75120gatgtcttga acccaggcgt tttagcaaaa agtatgagtc tttaggactt ctggctagct
75180tattacattt accatgtaga tctatgtgtc ctgtggtaac caacaggtat ttgctgattt
75240atgtgcccct ttgtggggga caagcagagc ataagcttca tttagtgccc acttgcctgc
75300ttagccaagt actattgtac agtacacaat ctgcccaatt tgtcttcatg gccttgggta
75360aaaagatagc aatctaactc agtagtgctc ttaatttgca tcctaacagg gagatactgg
75420agaccagaga aaaagggaaa gaggtaacca aatcgttctt caccgcaaca gccaaaatat
75480tgataccctt gactcccaat aaatactcca ccttccctca ccctgcaaat tagcactatc
75540tatttcctac aaagagatga gtactataca ctgaagcatg gtcttaaact atctagttaa
75600tttaccttat ctaatattat taagactatt ggaactcact tctaatatat taggtttcat
75660gattctgcag agcagaatct atttatggca ggttaataac aagagtacca tatatttgtt
75720agcacttatc actttcttat ttcacagcta ctctgggaga gagacagcat gcgtatttca
75780tctcttaaaa cagggaagga gagaaaagaa atatgttaag catgcactcc atgtccagcc
75840accttgcagc cactctatga agtgtcttcc ctattttaca ttctcaaagg ctttgccaaa
75900gtaacatctc cagtaagtag tgaaactaga gcttgaaaaa aaaaggcttg gcactcctgg
75960tttattctca tgccacatta agacactgtc tctatctcca gtgcaaaaag tcacttagaa
76020cctttattta ggtgttatat ctcctaaaac cagtaaaaac agttctactt aattttcttc
76080gagtaaaata ttgaaccagg gcagcaacaa aaatgaacta tctttcatat taaaaacatc
76140aagtagaatt cctgattttt aaaaagcagt ttacttagtt tttttcttaa aaaaggaaac
76200ttggattcat aagtcatgta ctttaagagt taaaaaccta ctttcaaatt ttttgatgtt
76260ttgtacttga gatgaatcga cgcaaaagga cgtgtgaact tgttatagat tttattgcaa
76320tttaactact tagctttaac ttacttgttt aacaattttt gctgcatctg tatttctcct
76380ataaaatatt tccttgtatt catccatcca gacttctgca aggcgaactt ggtttctagc
76440aatcacctga gtgccttttg gaaagctatg agggcttttg ctgcgaaaaa catgtccaac
76500aacagagcaa ggcataatct ccaactgccc accacattgc catacctgat aattaatgat
76560atttgtttaa atttacagta ttttagaaaa atcaaagtgt tgcttttaag ttcaccttta
76620aggtattatg aaaaacaaaa ttagagaaga gattacaata ttacaaatag tctctgtccc
76680ctgccatttc aacttaagtt cttaagtgta aggaattatt ttattaaatg gcatagacac
76740taaagacaac agaagtgcat tttcagtaag ttaacaaaat ggtgatttca gatttttcat
76800aaaaatgaat tccaggcttt tgtttttgca aataattatg taagttctct aatatgcaca
76860gaaaactaaa ggctcttatg catttgactt ctgtctgaac tactgacata gtcacttcac
76920ttaaactaga gtttctcttc aaagtttgga tataagtgga ctcagaccct atttatctct
76980tgtatattca attttgtaac caagaaaaca attccagatg gctattatta ccaatgatca
77040ttctactgaa cttgcaaagt agaggatgat tatttaaaaa ttcatgccaa tttagagaga
77100aaacgtcaaa aaaaatcctt attggcatta atgctaaata aaatggcaaa tcatgaaatc
77160aatgcctttt cacttaaata tcttaataat tgactctgcc atagttacca agcaaagaat
77220cagaaaataa gtcatattta atactatgtg cttgtagtag agcaagtgat tacaaatctc
77280tttctctcct ttctttcttg aacaaatgag aagactcagt gatatgttca attcaacaaa
77340ctgtgcttgt gacccaacac gtgctagggg ttggaaatac tgaaatgaat aaacaagacc
77400ccgccataag cagttcaaac ttggctaaga tcatcactgc cagtatgtga tggagccaga
77460gccaaaactt aaatcttcta actcctagtt cagttctttt tccactacat tccagcctaa
77520gagatgaaaa atgaatgata aagttactgc agaaaatgaa actcatgaaa tcaaatacat
77580aaggtacata cggagtctac ccaaagcctt tttttatatt aacttataat atgtatagaa
77640aaaatcaagc tcattcattc caaacagtta agtccaatgt cctttaaaat tattcataaa
77700tgtttttcca ttctaactcc cttgatgatc aactcatttc ctgaagcact agatttaatt
77760atatttatct tggctggaat tcagtagcta caagtgcatt aattggtgtt actgcctgga
77820cacaggagtt taactgattc ctcttgttac actggcgaca atgaataaat atattaagtt
77880acatattaaa agaaaatatt cttattccaa aatttccagc aattaagttt cagaaagcac
77940aaaaagtaga gtgcacacat ctgtaatcat atgtaccagc cgattagaac acaatatatt
78000tcattgttta atatatctgc tatatttaat ttccataaac ttactctgaa agacatttct
78060atattttcac ctccccagat ttccatttct tcatcatagc ttccaatata ctcaaaatat
78120tcttttgata tggaaaaaag tcctcctgca aaagtgggtg ttctgaaaaa taatccattg
78180tcaaattttg ttataaaaac taaacaagat aaaacagcat attttaagaa ctgaagttaa
78240aaaattaaag ccatatcaca ttttcaaagc ttttattcag attaataaaa actagaaatg
78300ctatcttcaa agtcaagatt tttggggtcg gggggagtac ttcgtgtcct ctcccattgg
78360agtaaactta aatgtcttct cctaaccatg ctttacgatt tttaagtaat gattgatgag
78420gaagacataa gagctcactc actgctacct cttactcagc aacatcatgt ataagcaagt
78480acacaaatga atgactttta gattctcttt atatagaata tgtgtgttct ttatcaatta
78540cctcagaggc aattgctata aagcaaacag tgtgtacata ttcaatgtat ggtatagggt
78600gtaaatataa gactctgaga gcaatcatct tacttaattg ggtaggtttc atctttcctt
78660ctttgcttct catgatcagg aagcgactcc cagccaaatg aaagactcca gtcaaaattt
78720ccacggttat ggttacttcc ataaggagaa ggtttgttga attcaaacgt gttcagatct
78780atggatgcaa tatctggact tacgacagcc gtgtagttct cagctattct ggccaacaga
78840ggttctagcc aaccatagaa acactcacct ggagggaaca gaattttaat taattcaata
78900aaaacattga agtttttttc tttagttatt taggtgaagg tttacagaga aatgagaata
78960acagagtcaa tttgtgtttt gaacatgtat ataatggaga tttctttctt cctcagattt
79020attgagatat aacaaataaa aattctatac atttgcagtg tatatcatag tgtttatata
79080tgtatacatt gtaaaatgat tattgcaatc aagctaatta acatatccat caccttatcg
79140gcttatggag cgggtggtaa gaacatctaa gatctactct tagcaatttt tgagtagaca
79200atatattatt attaattaca gtcaccacgt tgtacaatag atctccagaa ggggagattt
79260tttaaaggtt tattgtgatg agatgagggt aggatgaaaa aggtgaaaaa agattcacta
79320cagttagaaa aattccccta cagatgatga ccttctttct tctacaaaag atgcaaaata
79380acaccagtgt atttaaagtt tagatgatgt gttaaaatgg cctatgagtc aaaggtagct
79440ttttgctgtt ccacattacc tggccctaaa agtgttctga tttatttaaa ctcgtatctc
79500cctgaataag gtacagtaat tgaaattttc atttatctga ctgggtctta ggagaaaata
79560aggagtcagt ataaattccg attttagcaa ctggagaaga aggaccattc aaagctgccc
79620cagacctaat ataaagggag ggccaaggag tttcatgtca tgataggtct aagccctggt
79680cacaggccag gctgttttat gtaacacaca gcaagttcag ccaatgcatg tgctgtccta
79740ttctgggctc cccacaaagc agtatttggc agtcatggtc cgggaggacc gtcagacata
79800tccacccagt gggtagtggg taagaatgtc cagtatgatc ttattgtaat tttgaacaac
79860gagaagaaat accagaggat ggtattttcc tttttaaagc acctctctag cattaccttt
79920agtgttctct tcattttcta aatgtctggt attaaaagag aactgaatgt gatggctcta
79980agcaacacaa gcacatccaa aagcccttct gtaataatcg gtgtgtgctc aaaactagta
80040attgggtgaa acataaccta agcactggcc aattcaaagc tagacactca tacaaagtat
80100agcccactaa actcagagct caagggacaa aatgacagtt tagtaataac tagcatgcat
80160atgacacatt gactctaaaa atgctttcta aatacttcaa aaactttgac tctgaaaata
80220attaaagtac aaagaaaaac caacagcagc aaagtcaaac aaaaattgac aatgtttatg
80280taagggatac caaagaagat gggatggggc acccctaacc tggcataaaa agtatatata
80340taaacatatt ttggttttgt ggtttttatt gaaaattcgc agactccttt ttatgccaag
80400gagtctgcaa atttaaaaaa aaaaacacac acacaaacta aaatataaaa aaagcagcag
80460tactaggacg aagtttaatg atagggtgga ttatattcac tcactcattc attcattcat
80520tcattttgaa acggagtgtc actctgctgc ccaggctaga gtgcagtggc acaatctcag
80580ctcactgcaa cctctgcctc ccaggttcaa gcgattccca ttcctcagtc tccctagtag
80640ctggaattat aggcacatgt caccatgcct ggctaatttt tgtattttta gtagagacag
80700ggtttcacct tgttggccag gctgatcttg aactcctgac ctcaaatgat tcaccaacgc
80760cggcctccca aagtgctggg attacaggaa tgagccacca cgcccggcta catttaaata
80820gtaaataagc tttcattcac tttaaagtgt gggaatcaag aaaacaaaca aacaaacaaa
80880caaacacttt tttttttaac agaaaagaaa catggaaaaa aaaaaaagag gcaaggataa
80940ctggctggaa cagctagtaa ctggagaaca ctgaagttga ggaaacttaa acacaaaagg
81000agataagaat tctgcataag tgaaaggaat tttctccaca gagctgttac ctgcttgggc
81060tgtcattgct ataatcgcca gttgaaaacg ctgttaaaga aaatgcaatt aggcatttag
81120aaaagaaaag acttccttac cttttaaatt agagggagag ggagggaaaa tgttacaacc
81180atatccatac atatatactt acagtgagca tctaaaaatg tgagcgtttc agctgttgcg
81240actgttgctc ctagcaaccg agcagtgatc agaccttttc tttctctttg tctgactatt
81300tttactatag aaaattgttt tacatattca tctagtttat catgtaagta ctctgtaagg
81360aaaaaaaatc agggttaatt ctttctaaat gcattttcat atatagctta tgaaagctaa
81420tgaaaccaca gagcaatgat ccttccaaat tcataatctt aacagttgca tgtgtccttc
81480cagcaaaatg aaaattttat ctttaactcc ttactgggga aaaaggctag ctatgcaaat
81540tatcactact gtaacacaaa acactgtaat gcatacttat tctgtacact tcctatcaat
81600aaatataatg tgtttggagg gaagtgagaa acttcaaatg gagtcaaccc tagccgttca
81660aaagactcat ttagaggtct gagaagactg actacaagag tgaaagatat ggaatttggc
81720caaaaagata ttcaatgctt tgaaattggt atttaaatct ggaaatctta aagaaaaatg
81780tcaaatatct aaagacctct gatatttaag caaggatcaa aataatttaa caccaaaatg
81840ttgagccacg tcagaaagtc cacagctata gcagtttggg acgattccta cacagagtca
81900gcagaaaaat ttgcgaaagc tttggagtct cctgacagtt ttatttagta aaatatgaaa
81960caggcaaatt ttagtattgg gtatatactt taagatattc aatataccaa attggataag
82020gtttgtaata agcaaaattt gagatcatag gttttaaaaa agttattaac aaatgtcaat
82080ccagtgttgt ttttttgtct ggttttgttt tgagacaagg tcttgctctg tcgcccaggg
82140tggagtgcag tggcacaact cactgcaacc tccgcctctg ggctgcaagc agttttcctc
82200agcttcccaa gtagctggga ccacaggcat gctccaacag gcccagctaa tttttctttt
82260cttttctttt tttttttttt tagagacggg gtttcatcat gttgcccagg ctggtctgga
82320actcctgaac tcaagctatt tgcaggcctc agcctcccaa aatgctggga ttacaggttg
82380agccactgcg cccagctgtt catattctga aacaacttct taagcctgtt tttctcaatt
82440aaatttttga tttcttattg agtacaaaat gcagttcttc aagcagcctt cttgggccca
82500aaacatagcc tcaaaaaaat ttgggaatgt taagaaaatc aataaatgtc ctggggcaaa
82560agtttgcttc tgccagattt tagaaggaaa tgcagatgac tttgacttta tctgtgataa
82620tataaccttt ttttcaattc tccagtggtt cagttaagag agctgcttat ttaaagctta
82680aacattcata agagaggaaa gtgcaggaac aacccttgta gaaaaataaa tctatctaaa
82740cactgttgtc tcagagtaat agattaactt tttaacacct tctaactacc tcaaaataat
82800tctcagatta agcttttaac accttctaac tacctcagaa ataattctca gaaagacaag
82860cagaattgta actcatctta aatatataat cataaacatt ttctgtttta agaggcttgt
82920aaaaatttca acattcttgg ttagaattat tttaaatgga gttactacag attgcttcaa
82980tataccctct cactgttgga atttgttaaa aaaaaattta cttacccaca acatatgaac
83040ttcccgtaaa tttatatctg caccctaaat ttagctccca tgcacaccct ttatttggga
83100ggttaagaac agaaagctac atgaaatcag atgccctgat ttcactttat ggcacaatta
83160cttttagctg tgtgaccttg ggcaaggaat tttacctttt ctgcgcctca gtttctttaa
83220gatgaggata atcggactca ccatcacaag acattgctgg gaggataaaa tgagatatct
83280gaccttcagg aatctaactc caaacttgga tttattaagg aataaatttt gtaatcattg
83340aaataatatg tatctgtttg acacaatatt taaaatactt atgatgtttg aaaatttaca
83400agttagagta tttgttaaat tagccttgtg attccaattt aaaatgtgta atttagtatt
83460tgacttccaa ttttaagtgg aaatgtacta aagaatttga ctaacattgt aagtaatatt
83520ggaatattta agaacaccga aataaaatag aacatggtga ctctttttaa ctattcattt
83580atattattta ttgatattta tattaactca ttcagtcttt aaaacaactc tagaaattat
83640gtactagttc tgtatttcct caattttaag ctacacaatt ggattcttct tataatcaat
83700ttcttataat caatggcatg ttaagtttaa ttagctgtgg gttttctcta gtgatttata
83760aattaacaac acataatata tttaatgaaa tatggcatta ttcccatttt gctgataaat
83820cacagcagag aggttgagta acttgcccaa gtcatatagt tggttaggaa aagagctggg
83880attcaaatca aggcagcctg actcaaaggc tcaagtctgt gctcttaacc tctatgcctt
83940actgtaacag tctcacattg aggactacat agttatgaat aatggtgttt catacatttc
84000tgcatttgaa aatatttgaa tatgtccatc cttggattct aatttcattc accataccca
84060ctatccaaac tattatattc aagctctgag atggcataca gagagtacca cataaccaag
84120tagactgtag ataccacaat atggatttgg gaaacaatct aatttgcatg tgctttctta
84180ccatctacac tagcatcatc caccaaaatg atttccttca gcagtattgc aggtgaagaa
84240tagagcacac tgtggacagt tctaagcaac gtggaccacg cttcattatg aaaaactatt
84300atgacactgg tggtgggcag gggagggcag cgcttaaatt tttgttcaat acatctagaa
84360gaagtcagag aagtagaaaa acaattacaa attttattat tttatttcac agctatacca
84420ttgctaacat tatcattatt gaatctttat cactgaaaaa ggatagtttt cttgtatgtg
84480atataatctc aatgctatat aaaatgtaaa taaatcaaaa caattttttt acaagcaaat
84540cactcaccca gagatgatgt ctaccaacag agatgacaac atatcaagga ttacaacatg
84600ttattatagc tttctcaaca atgtaatttt aaatgcaaat aacattttaa ataatgaaaa
84660taacctacaa tgaggcagca tgtaaaaatg acaaaccctt ataattgctc aaataaaaac
84720caaatttttc ttatttcatg cagaaatcat tcatttttca tatttgcaac tttaaaaatt
84780gtaccacttt attaattttt ttttttttat ggagtctctc tctgtcgccc aggctggagt
84840gcagtggcac gatctcggct cactgcaagc ttcacctccc aggttcacgc atttctcctg
84900cctcagctcc cgagtagctg ggactacagg cgcccaccac cacacccggc taatttttgg
84960tatttttagt agagacgggg ttttaccatg ttagccagga tggtctcgat cacctgacct
85020tgtgatccac ccgcctaggc ctcccaaagt gctgggatta cacacgtgtg ccactgcgcc
85080cacaatatta aatcattttt ataaagatta tctttaatta gatatttacc taagtatgta
85140atataggttt cttaagtatt tcctaggttt attgaatcaa tttacttaat ctatacacag
85200aatcaaaagg gaaaaaatag ctttattgta aaacctatgt tcttcttatt tttatttaaa
85260atagagacag tctgtgatgt tgcccaggct ggtcttgaac tcctgacctc aagcaatctt
85320cctgcctaag cctcccatag tggtgggatt ataggggtga gccatcatgt ccagcctaaa
85380cctatctttt tatgatgttc atcattcagc aaatacttat taagtactgc catttgccat
85440gtgttaaaga ttttgctcca ataatttgat ctactatcaa ataagactag cgtttaaaaa
85500aaccctttaa aaatacgtag aaataaagat ctaaataaag acctaaatcc atgttaaaag
85560tcctgtgctc atgtcaccat ctaaaacatt gattgggcac agggagagag accctaagtg
85620cccttgactg attagtggta ctactatcca ttagttaaaa acattactta taaattgatc
85680tcaaattcat ttgtatagga atgtctttcc tcaatgaaaa atatcttcat attaagccac
85740tggggtgcag gagagtctgc atagtctcca gcctttgagg ctagagatgt agagataacc
85800acagtgccaa gcctgctgaa tggaggtgag gattaaatga aagaacatat aggagagggc
85860ttcaagtcac tgggacataa aataaaactg tgtgaggttt atgggaaaga taagcaaata
85920aagcaactat atctttaagg tcacttaaaa accataaatt ggagataaaa gcccatcacc
85980cttcagttcc tctgccaggg ccaatgggga aactgtagcc tccagtgtga tattaggggc
86040ctgagaaaac tgccctaaaa aaagatacac aaattatccc attaattact ttcatagtca
86100tacctcaata ggaatgtctc tgataccctt ttttaacagc aagtctttag aatggattat
86160ttgtagtgat taaaaaaagg agttggattg gggtaaaata tcaagagaag atgtcaacaa
86220cagattagaa aaaggagagg atggattctg agtgaaagtg gcattgctct tcagtttctt
86280catgatttca aagcatgata caggtgcatg ggcggaaaaa ataagcacct tcttcatcat
86340tcataaataa aaacaaatta tttattctaa attgaatttt ggataaacta ccaattaata
86400aaaaagctca aacacaaact attaatatac acacttgaac cacaactaca cagaaataaa
86460tgactaaata taagcttata ttttgaaccc ataaaagttt ataaccaaca taaactagaa
86520gtaaaaatta tgaaaaattc catattgtgg tattttattt ttaaagaaaa aaaaaagagg
86580aaacctatga tttgttataa gcattttgag gagaaagtga aattctgagt ataagatttt
86640tttcctacct ttatttgcta tgtggttaat taatatagta ttaatcgata attataaaat
86700ggttttgaag atttaaagta taaaatttca caaaaattca tactagtaaa atgttatcac
86760tttaatcttg caagattttc cgtcagttta aactaggatt tatttgctta actgataatt
86820accaatatca agtacaaatg tcatatcaat acaagtaata aaatttcaca atataatttc
86880ataaacccca ttcattttga aacaacatgt taaatgtatg ctttgaaaat tgttttacat
86940atttaaaata gtgcaattaa aataacttgc caaagtgact gtatcattcc agtcaaacca
87000tttgaaaaac ctggcacata gaaacatgct taaattatca gtcacatgac catctggctt
87060tgggagaaac taggtctata tttctaatga gcatggaaaa cattagcagt tcttcagaaa
87120atgtgttatt tgctgatact ggttacattc aaagtaaaaa acaaatcttt tttttttttt
87180tttttttttt gagacagagt cttgctctgt caccaggctg gagtgcaatg gtgcaatctc
87240ggctcactgc aacctccgct tcccgggttc aagtgattct cctgcctcag cctcccaagt
87300agctgggact acaggcgcac atcaccatgc ccacctaatt tatgtatttt tagtagaaat
87360gaggtttcac catgttggcc aggccaggca cagtagctca tgcctttaat cccagcactt
87420tgggaggcca aggcgggcag atcacgaagt caaaaaacaa atctttacca cctgtacaaa
87480accctgtcca aagtaagaaa taattaaatc ttcactccaa ccaaaaaatg ttgttaatgt
87540gcttacttgt ttttcactta cttagcgatt aacattattg aaaattattg cagcttctta
87600atgctatatt gggagaaaaa agctcatttt gagctgtcac tgattgtcat gtcaaaagaa
87660taaaaatcat gattattgat ttttaatgta gattactttt caatcaaaac ctctcttcaa
87720gtcagtaaag tgcaatactg cattctaatc tattactgta tgaaaggaac tataatgtgc
87780cttgttatat cagctagaat attttataga tattagtttc tttacacatt catttaaaaa
87840ttatgagcaa cctaaacttg aatcaatcag attagaatag ctcaacagaa atctaagcta
87900cacagtttca atgttaaggg actcatatac ttggatccag tattgataat cctaaatcta
87960gaaaaacctg agtgaaacaa tagaccaaaa gttatcttca agttcactgc caaatgtaat
88020tctctaatat ccagaaagca aagcaaaata cacaggcatt gtttgcttcc agtttgctta
88080tcttgtactc tttttttttt ttttttttgg agatggagtt tcgttcttgt cgcccaggct
88140ggagtgcaac ggcacagatc tccgctcact tcaacctctg cccccaggtt caagcaattc
88200tccagcgtca ggctccccag tagctgggac tacaggcaca cggcaccctg tccggctaat
88260ttttgtattt ttaatagaga cggggtttca ccatgttggc caggctgctc tcaaactcct
88320gacctcagct gatctgccct cctcggcctc ccaaagtgct gggattacag gtgtgagcca
88380ccaaacctgg cctatcttgt acacttaaaa acttactgag gggctgggtg cagtggctca
88440cgcctgtaat cccagcactt tgggaggctg aggtgggcgg atcacttgag gtcaggagtt
88500cgagaccagc ctggccaaca tggtgaaacc ctgtctctat taaaaataca aaaattagcc
88560ggacgtggtg ccacatgcct gtagtcccag ctgctcggga gcctgacact ggagaatcgc
88620ttgaacccag gaggcagagg ttgcagtgag ccgagatcac actactgcac tccagcctca
88680gtgaaagaga aagactccat ctcaaaataa taataataat aataataata ataataataa
88740ataaataaat aaataatctg ttgagggtgg gccaggtgca atggctcatg cctgtactcc
88800cagcactttg ggaggccaag gcagatggat cacctgaggt caggagttca agaccagcct
88860gggcaacatg gtgaaacccc gtttctacta aaaatacaca aaattagcca gctgtggtgg
88920cgggcgcctg taatcccagc tacttgggat tgtttacaaa aaatagggca ttctgaaata
88980taattaggta ctcaaaaact ttgagtcaaa gtaatcacaa agtctgtgaa ttgctttaca
89040catatataaa atattatcca acagttccaa aatcttggca tgtttttaaa catttttatt
89100ctaaagaaat acagagaaat ggaaacaatt cctctacctt ctacttccta tacaaattgt
89160ccaataattc cccctaaata gatgagaaaa ccacagtatt taggaataaa gaatttcctt
89220gtggatagca gtcagatcta gaattcagtt ttctaatttc caagctaata acttttcata
89280tctgcactgc ctcaccaagc ataaataggg atatgtagcc tcaatcatta taagcaaaaa
89340ataaatacaa caggttaaat aaacagtatt tgctgatttt ctgccttaga gtagctaaaa
89400aacagataac ttccttagct cacccctctc tcccctgcaa agcctggtga taaagaataa
89460acaataaata ttcttcaaca tactcaggag gtcgagtgtc tggtccaaga tctcggtgca
89520aagaaatcct gtcacttgcg aaagcattaa agcagtgttt agcttcccca cgttcctttt
89580ccttttgctc ttcaacactt aaattggttg tcttgaatgc tttaccagaa gcaccaggtg
89640catttgaatc ctgaggtgga cggtcaagga caggcttcaa ttctgctgct gtataatatc
89700cttgcaaaca aggtctctca ccagcatcaa tgttttgcct gacaggtgct cctatttgca
89760tttttggcat ggcatcctta atattgttta cagcttctag cattaaatcc aacatcttgt
89820ttttgttttt catgttcctt tccatccttg attcctcttt ggaatattga acacttactt
89880ctctttgcat taaaaccaaa actattataa agaaaaaaat tactgcacca agcttccaga
89940actttttatg gtaatgtctt ttaatgtgta attttactag tcgctttagg tgagccattc
90000tgacattaaa agcttgtcac ttgacaaata acagttattt cttcttctgt tacttatatt
90060ttttatcata gatttgctga gaagaaggta tctttaatgg tagtacctat aaacagaaat
90120gatcgatggt attagtagct attcagtcct acaggcatgg ctcaattcat tcatttaacc
90180aacaactgaa cataagctac caatgtactc ttatggttaa tgagaataca aagatatctt
90240gtcctcaaga aaattacact tttggaagaa aataattgtt acaatttact acaatttatt
90300ggggttgcaa aaatattgat attcctctcc attaaaaaat acattctttt taaactctaa
90360tatccactat atactatgta taaaattaat tttataaata tattttgagt taaaaaagaa
90420tatcttcact tgataaccaa ggaatcaaag gacctcatta aataaattac aaaaaagaga
90480gctccataag gaagtaaggg tatactgata agtactatta ggcatccatc taaaatagtg
90540gccttcattt tatgtagttt accctcaaac tgtgatctgc tccagcttaa tgaatgataa
90600tacagcagtt tcatcagtaa gtaaactatt attctcccag attttccaac ccaatagtac
90660ttgtgactcc tgaaggaacc cctaaaaaca agaaaatcaa cagcatttaa tttgttgtta
90720tataacagct agttttaata gctcacagaa atacaagatc taatagtaaa aagagaattc
90780attagcttta cttgaagtgt ctgtagggag cctatagaag tcataaagtt aaaaaagcag
90840aatttggttt gataaaatat tgcttgcaat tgaccctaag aacaggacag caagtttatt
90900aaaccctaat atgggacaac aggagcaaag gaaaagaaaa ataggacttt ttaggaaacg
90960attacatact atgagccgcg atagccagtc aagaaatttg cagggacgaa ggggagggaa
91020aatgcctgtt cttcctaagg tctaaacata ttttaaagaa aatatcattt atagcaatga
91080aaattaactt ttgacacctt ctttaacttc tcttccaaag ttctccctaa atttaaaata
91140agaaaaccag gttctaagat ggattctctg gcttttggca ggaaatttta agctgaataa
91200acaaaatagg cagaacttag ttctatatac tatttctaat tggagtgctc tatattcttt
91260tcagtatgga gtggatactt ctgctccaaa agataacccc aattctgaca tatattgctc
91320ataaaagtga gattccaatc tttctgaggt ttggttgctt taagaacttc aggtaaaaaa
91380tgaaatgacc tcttaagctt ttgaaaagaa tataagaagt tatagaacca cactatgaca
91440gttgtgtaat taattatgtg tcaaatgttg tggtaggcta tggttaagaa attaaagata
91500gcccttacct tcaagaagca gaattgaaat ataggaagcc agaggacagt cacaaagcag
91560tgtgatatgt gctacgacag gagactaaga aagctacaca ggcttggaac agtggctcat
91620gcctataatc ccaacacttt aggaagctga ggccagagga tcacttgaga ccaggagttc
91680gagaccagcc tgggcaacat agcaagaccc catcctacaa aaaaatacaa aaattagtca
91740aatgtggtgg tgcacgcctg tagtcccagc tactcaggtg gctgaggtgg gaggatggct
91800tgagctcaag aggttaaggc tgcattgggc tatgattgtg ccactgcact ccagcctggg
91860tagcaggaca agactctgtc tcaaaaaaaa aaaaaaaaaa aaaaaaaggg aaaacaatgt
91920agaaggtcag gagaaatgtt aaggaaagtt tcctagagat gacactgttg agcaaaatct
91980tgaaatacaa gtacatttta ggcagttaga caggagggaa aatagggaat gtcagatgca
92040aaaccataaa agcaagaaaa ggcatggtca gtccttgagt acagcataag tggaaggcac
92100agggagagaa tgacaactga tgtggcttga aggtcagtta aagatcagtt cataaaaacc
92160cttgtgcatc tttcagtgaa tttggaaatt accaattttt tccaaaatag atggaatgga
92220gaggactgtt tttaagaaaa gaccacaatc aaatttatgt tttagaaaaa tcacaagttt
92280ttcagcccca gtaaaggtga tagatattaa tgagatgagg tgggtcaaag gatggagtgg
92340aatgatttca agagagtcca ggaagtggaa tcaaacaaca ggacttggtg agagaaaggg
92400aagaatggga gatgaatggt gatatggttt ggctgtgtcc ccacccaact ctcaccttaa
92460attgtaataa tccccatgtg tcaagggcag gggcagatgg agataatgaa tcatgggggt
92520ggtttccccc atactatttt tcatggtcat ggataagtct cacgagacct gatggtttta
92580taaatgggag ttcccctgca catgttctct tgcctgccat catgtaagac gtgcctttgc
92640tcttctttca ccttctgcca ttattgtgag gtctccccag ccatgttaaa ttgtgagtcc
92700attaaacctc tttttcttta taaattatcc agtctctggt aagtctttat tagcagcatg
92760agaacaaact aatacaaatg agtaaagtga taacattaac aaaggcaaga aacacaggaa
92820ggatagattc agggtaggaa ttaaggagat aatttgttca gttttggaca tagcaaatgt
92880aatatgctaa tggaacttcc aaagtgagtt tccaagagca aaaagatata tgggactgga
92940aattaggaaa aagttctgca gtagagatga aaatttgagt cagtcttggg gtaaatgtaa
93000aacaaagagg atagagaaat acctacattt aagagtagaa aaaaagagac tgactggaaa
93060gtccaatttt tccaatccta tgaaggaggg gtggtaaaaa aaaaaaaaag acaaatgaaa
93120acacagataa attatccacg tcaagtggaa gtaattttac aatcttatct cactcttgag
93180agatgttaaa tgtgcaacag ttggttgacc tttaaaactg aacttcatta gacctggcct
93240aatgggcttt agtgaaaaat gagaagagaa ccttggcaga gagagtagtt gtagtcatgc
93300cttcaagatc ctaaggctta ggattcaggg cacttccaga ataatagggc agcaacctca
93360cctatggggt tccggaagat aagaatggtc aaaatgttgg ggtctcacag gaaagtggtt
93420cccagttgat gaactacaac ctcatcccct ctaactgtat cttgacaaaa ggtagatatt
93480catcaacctg tgttcacctc ctttctgttc ctgaggggac taccaaaaat ccccttcccc
93540agggctatcg agtgacagtc aatttttctg cagctagaag taggctaaat taggaatttg
93600agaagaattg agctggactc cctattttga cagaacatgg aaaaagagtt cagagaaagc
93660acagaatagg aagatatgac agaactagat tcttctacag tagtgatagt tttcccaaaa
93720cattttatgt cagaaatcat acttgtggat agggtgggaa ttgaaaagaa ggatagaatt
93780aatgacgcag atagaagata gttcttttta gaataaatca taaactagag gaattgttca
93840tcagaatgac agtaatccta tcattgtgag tgcatgtgta tctatgtaaa gacccattca
93900attagtaggt tttagtttgg gatgcttctg ataaaggagt caaggaaagc tcatgagaga
93960catggatagc atcttattca tggctgtttt tctggccctt accacaatgt tgataacata
94020gtaggcaccc agaaaaattt attgactgga aaaaggaaga aagaggacca agatttcatc
94080agaaacaaca acaaaaacaa aacttaactt gtgttaaatg ttgactaggt ctctggtatt
94140aaataacatt taatacataa gttctcattc tcccagcaat attgtgaagt attcttcttc
94200ttctctcttt tttttttttt tttttttttg tagagatgag gtctcactat gctgcccagc
94260caggctgatc ttgaactccc aaactcaagg gatcctcctg tctcagcctc ccaacgtgct
94320gggattacag ccatgagcta ctgcgcctag cctttgtaat tccaatttta ctgacagaca
94380gggagagtca aagagattaa gtggcttggc catgccagtt cagtttaaga ccagcatttt
94440tctaactctg ttcaaactca agttcaatgt aaaatcctga gcttttaact gaaattatct
94500aaaataaaat gtttaagagt caagaactga tatcctaaat aaaaggcgat aagaaaaaaa
94560actgaggaaa aaccataaaa actacaaata gcttcctatc attgggtaat attttacaaa
94620taaatgattt ttaaaaactt ttcaaagcac catttcagta ataaagattg aaaataagat
94680tgtagatctt aaaatggaag attaagaggt tgagaacctt acagaggcag agcctagaag
94740taggaagatg atgttttgat cttttcagac tatttctgaa gggattaatc ttctcttatc
94800aaccctgatc acttaaagat tccaacattg atgggatttt cattatgact tttctgtact
94860ttgctgttaa tgtgcaatat taggcttcca ttaatgctga atgacactca acactaagca
94920tctccatcag ctgtcttcaa ttctggatat acattagtga aaagctctta aaaaaataaa
94980gtgccttagg cttcacctct gatttaattg atgtgggggc agggcatata ctctgctttt
95040gttgttactt taagctctcc agataattct aacaagcagc aagggttaag aattacttgt
95100ctagatcaca atattttggg caaatcaaaa ctagcatctg ataagggttg tatctgtcca
95160aaggcaacca cattactaat ttctaataag ccaaagatgg gtgtttcaaa atagattctt
95220gatgtaaatg aatagaagtt atttagacat ggagatacac gataaaatcc aaggcaagaa
95280tgacccacag ttttccaaaa agactcaggg atttacgctg atataaatga ggcactgagg
95340ttttactgca aattatgttt ctgtaaaata attttataaa tattttattc caatcataaa
95400aatgggaaaa ttgatacctg aacaataaaa gacataagcc aaaagataca caataaataa
95460atgagtaaaa tgaaaccagg atttttaatg cctttttccg ggaataatgc catgttcctt
95520gtcagtacta cctgccaaat tggaagccct acgtaaaaac agctcgtgaa acaaaacaaa
95580tctctcctac agtcaacttc cagttggact cagaagtttg agaattgata agaaaaaaat
95640cagcacaaca gcaaactcag gatataacat gtcatatttg cacattagaa atgattagca
95700gataaagggt ttgttcttct tgtactcttt ctaaaacctt taaacacacc tatccaagat
95760ggctatttgc taattaccaa agatcagatt cacagaggaa gagaagtcaa aaatcatgaa
95820atagttttta aaaactaaaa aaaaagaaaa aaaagaaaag aaaaattcag agatcttcta
95880aattctaaat tcttatgaat tcctaagagt gaaattcaga gacctggttt taaaaagtta
95940ttaatctgta acctggtata tatgtagcca ttgcaaaaag tcattaataa aaatattcat
96000aaagtgaaaa gctaaagcaa tatacacaat tttaactggt tgtgacactc gctatgttaa
96060aatcaatgga ttattctcag gggtaaaaat atgcttccat ctgaaatctt acaaatttaa
96120aataatctaa ctaacaagtg gtagcttcca gttatctaga ctacagcttc tcaaacttta
96180aagtgactat gaatcacctg gggatcttgt ttaaacacaa attgagaagg gaaaatatca
96240aggtaagatc catcatgatt gtccctctag gtgaagaagc ataggttgca gaaatattag
96300ttgttcattt aacctaaaaa ttccaactgt agcaattttc tgaagtagtg gaaagcagaa
96360cattataatt ttacatgaaa gtaacattta atgaaataag aagatagtgg ttcctctcac
96420ttcttgtttc catgtttgtg acaataaacc attacaactc cagaagttta aagtctggtt
96480cctgatgaat ttaattgctg tgatgtctaa tttggtatac aaaacttcct tactactgag
96540ttaaggcatg ctgtaaagag ttaggggtaa ccacactaag cagaaaaaaa acaaaattac
96600atcccaatat cctaatatag attatagatt aagaaaagta cactattgtg aattattata
96660aaactcattg gatctttgcc acacattatc tatcatctgt atttcctaat tgaattcaaa
96720acaaaacaaa gaaaaacaaa atttaaagac cttgccctga taataaacct ttaagtgctt
96780gggtagaagt tttattatgt gcccctcatc tactggttaa agtgttaggg aacagaggag
96840tggcagaaag tcatttctgc ttgcatttat tggtgggtca aatctcatgg tgttttgctg
96900ctgtagtttg ttagttttga ttatgtttaa gttcatgttt acattcttgg cagcatttct
96960tctgagcagg aagtggcatg tggatgtatg acaagagaca aacactgatg caaagcaaac
97020tgcatgaaga cagccacatt ctgtctcaga gggggcagca gtagacatac ctcaaagatc
97080tcagctaaag gcaaagtaga gttactctgg gtggctgggc aatgccacca gtggatgact
97140aaactacttg caattgttat aaactaagaa cagctttctg aaccaagata tctgcaggct
97200gtgagacaaa aagctcagtg acagagttaa ccatcagcaa ccgtgaggca caggtttcaa
97260tactgagaag gacagggtca gacatccaac ttctcagcta cccctggtca ggtagacatc
97320tccctcatcg tcatctctga atgtgaaagg ttgctgatag aatcttgatt tgtgagctag
97380ccaactctat cttgacagtt tgacacaaac aagtaattat gatactacat tatctgtcaa
97440gggagtcaca aactcagggg ctcacatgct ctttcctgcc acatctacac ctgtgaatga
97500cttttacatt acatatgaaa gatatgcatt gattacttat ggcatactac agagtagagg
97560aattctgagg agtgatttag gtggatccag agattattca tgtaactggt attccctgcc
97620ccccaggtag aggcactgca ttttaaaaca tttgtccatc tatttacctt gtaaacatag
97680ttgttggctt ttggattagt aaggagatag aaaccacatg ctaatttgaa cagagaaagt
97740ttaatacaat ctggtattaa aagaggattg gagtaacagg aaattgccta gtgggagtta
97800aggagaattc taaagaatac aggagttgta aatacagtat aaggggcaac cactccccct
97860atggctgaaa taagtgttca aggaagggct ctcctctcac cagggctgag atccagagat
97920gtctatggag atgccctagc tttgactacc tggtaaaaag tcatagaagt acttcattca
97980tgggacttac cagaaatctg cccctgaagt tccagaagag gccattcaca gccgggggac
98040agaaccctag aactagctag gtgctgagga aaactgctgg ctgctgaggg aatctggaat
98100cacagaggtt gtcccctgca ggagtttagt gaactttcat gtgccacaca ggtaacaacg
98160gctctgggct ggctgcctgg gacaaaggtt gggcagctgg ctccctagac acccagtctt
98220gtggggcccc gagtttggca tgtgtggatc tgaggagaga gtttggggag cagtgactta
98280ggtgacagag caggtccatg gtgtctgtgt ctcaagggta caccactgct gcctaaatca
98340agctctaaag aaagtgcagt gtgtggggaa caccaactct agaaaaagct gtgttctgca
98400gatacctagt actggagaaa atcatactgg ggagtgagca gtggaggaaa actatgctca
98460aaaggagctt agccagtgag catcacagaa accagaaaga gaagcctcag ccacttcagt
98520gtccctccag tgccctctac tgacaaggct taacattgta cgagccagca aaaaagaaat
98580ttacaaggcc cagctccatt attacagagc aggctaatac agtaaatttg gaactaagag
98640acagtaaact gataagcagc aaagtctaca cttttcacta ctcagcttcc acatgcaccc
98700tctcccacac actgaaactc ctgtacaata acaacacaat tctatatttc acctaacaaa
98760gtctaactgt ccttctttat aagtgaagaa gttctatctc ctcaaaaacg agagacatag
98820agtcctaaca gctattgtag tcattgctgt gactctgagt tactccttaa attctgtcac
98880agtcccacta aatattctgt tacctaaatt ggaaagttga tctccaacgg cttctattta
98940acatttttaa aatgaaaatg agagaagaaa attgttaaca tatacaaaaa aaacctagat
99000ataagaggca aagaaaaaat gtgcattgaa cctgagaaag ctgattgaaa aaaaattttt
99060aaagaaaaaa tacgcaaacc tacaataatc ctcacttaca taattggctg cgaggccata
99120atgaatatct atggcttctt tcttacacta actactactc tgtattcacc atgcccttaa
99180gcattctagg agtccacaga tgttggtgat gtcaaggaag ggcaaatcca tatgtagaat
99240acttgtctat tctagtgagg agaagtctct gccccctcaa tgaaggaaga aatccagtat
99300aatgaacctg ccacccagag gctggctgat ccccgcaggg gctcaatgct ggattctgca
99360gattaggaac tcagcagcag cgctatccca gtcaacttta caaaggggaa gccatgttgc
99420tgagcccatg catacacagc tccagtcttg ccaccaagac tcctgtttat gggcccactg
99480agacagctgg agagataggc tgactgacat gcagaatgtg tcattttgtc cacttgatta
99540ttaagagcct cctctgcaat ggatgtgctt ttgtgggcac tcagagggga tacaaattct
99600ttcagggtct tttcattttg aaaggttcat tcacatacct cttcctcaaa ctaactacct
99660tgtcaccaat cttccaatcc tgttccttcc aactctgacc ataaaaacca ttaacaacta
99720cccatcgatg accaatatag atctgtattt ctaggcatgt ctcattccca acatggtgga
99780caaccatatt tactgttcaa atctctgcct acagaaaagt tttccttatc cagtattatt
99840cacagttact cacgagtagg gctatagtgc tacacagtca ttaatcttgg gtagtactag
99900tgttttgttc agattcatct gcaaatcagg tttgaggggt ttttttgttg ttgttgttgt
99960tgttgttgtt gttgtttttt cttctaacaa atggactaag ggaatcgcca agaagccaca
100020ggcatgagtt gaaagagaca aggcaatgcc attagagttg ctaccaaaga agtctgagtc
100080cctggttcat gcaacttatt tgtcctatcc agatctgctc tagttctgtc tcatgtacta
100140tttccactca atgatatatt gctgttgtac atcgctaacc ttatggaaag ataggtcaga
100200ttttacccag ataatgatgg gaaacttggt ccacatgata acctgacact ccataattat
100260gcattcaata tctacctagt agcaagtcag aaggtgttta tcaaaaagta aacagttatc
100320tgaagaaaat atttatttca aaaccctaga tgtctgtatt gtgattctgc tattggcact
100380ttccagaggc tccatagagc attcctcttt ggcacagata ccaagagagt cgttgagttt
100440gctagatcat aaagctgaag tggtagcata gcctgtactg ctgggcttgc tgcagagcct
100500tcttctactt tgatcatcat taaaaactgg cagccttatg agttactctg taaacaggtt
100560gaggtagcat attcaaatgt tgtaaatgtt gcccccaaat tcaaaaatga ctattgaggc
100620tgggtgcagt ggctcatgcc tgtaattaca gcactactga ggtaggcgaa ttgcttgagc
100680ccaggagtgc aagaccagcc tgggtaatat ggcaaaaccc catctccaca aaaaaaatac
100740ataaattagc cgggtgtggt ggagctcacc tatggtccca gttactcaag aggctgaggt
100800aagaacattg cttgagccca ggaggcagag gttgcagtga cccccagatc ccaccactgc
100860gttccatcct gggcaacaga atgatactcc ctctcaaaaa agaaaaaaaa aaaaagccta
100920tcgagcatag tggctactga atgcccagaa gttttgctga atttttgtgg ggctgattta
100980tcaccttcta attcttatat gctctacaag gatatctaaa gtatttgcta ctttcttctc
101040atcagagaca atcagcatat tatcatcaat gtagtggatc agtgtgatga cttgtgaaat
101100gttacgatga ttaagattaa gaattgatct agccctgagg caagactgtg aagccatatt
101160gttggctctg ctagataaaa acaaattgtt tttggtggtc cttgtaaatt ggcagaaaaa
101220gatattaact aagtcaatag ctacatacca aatgccaggg gccatgtggt ttgataaaga
101280tattacatct gggtcagcaa ctacatttgg agtcacccaa taattaagtt taggggagtc
101340tacagccatt ctctaagatc catttaactt ctgccgaggc aaaataagtg agctaagtgg
101400ggatgtgata gatatcacaa gtcttatttc aaatctttga tagtgacatc aatcttgaga
101460gtttcctcag ggatatgtta ttacttctga tttactatct ttgaagaaag gggaaattct
101520aaagccttct actcagctct cctatcacat ggtcctctct ctatgggtta gcaggggtgt
101580ccaatctttg ggtttccctg ggccacatta gaagaagaag gattgtcttg ggccacacat
101640aaaacacact aacacgatag ctgatgagct gggggaaaaa aaagaattac aaaaaaaaaa
101700aaaatctcat aatgttttaa gacagtttac aaatttgcgt tgggccacat tcaaagccat
101760ccagggtcgc atgtggccca tgggccacag gttaaacaag cttggtttag agagtcaata
101820tagagattct gccagttacc cagtatgtct attccaatta ttcattaaag aactggggaa
101880ataagcacag gctagatctg ctgaccccct ggacccacta tgagtacaac tggagctaag
101940actccatttg ttacctgagc acttttaagt ctccactttg aacagttggc cacagtattt
102000ttgggtcccc ctaggaatta gtatgaaagg tctgattatt tccttttccc caaagcatag
102060tctctggtaa atggctacag gtccctttgg ggaaaagctc tgggagtgtg aattgactta
102120ggtctgaaaa ctgagtgaga tgctgtgact cttgattgca gcaactaaag ttaacttttt
102180tgctaccaga gctagagatt ttcctattat atagatgaag ggatgctata gtactgctta
102240cgtatttcat tcattagcta ccactaggca tataccaaag acattatctc agctgtcaga
102300aacatctgca agccaatgtt ttctgatacc tctatggccc agcttttcat tgtgggagat
102360gtatatactt tgtatttggt gtataagggc ttactgccac ttggcttttg ctactatgga
102420atttcatcat ctccagtaaa atcagagagc caatttcaat ggtctacaaa ggagagcagg
102480cacagatttt ttcatggatg cagggaatcc cttcactaat gtacttctta atactttagt
102540gaaaggagtg tctactgggc cttttcatgg catgtatttg gaggatgggc aggttgcatt
102600caattaattc aaccaaccat tccttttttg aaggagtcca aacacacaca agcagtaggc
102660aatttctctc cattcattct tcctttgagg ccccaaaaat attaaggaat agggggtgaa
102720ggaataaaga agattgcttg ctcaattgtt atcattttgc attcaccttt ctacaggttt
102780accaatgctg atgtgataga cactcctcct ccatctctca gcttccatta ccctctagaa
102840aaagtgtcaa caaacttttt ataaaggact agataacaaa tttttttttg ctttgctggc
102900catatggtct ctactgcaac tattcaattc tgttgtagta caaaaaagac aatttgtaaa
102960tgaataatca tagctgcatt ccaattaaac tttgttttca aaaacaggta gtggatcaaa
103020tttggtctaa gaactatagt ttactaaccc ctgctctaga aattctaaaa gacttcaatc
103080tcaatatttg taagtctccc catatttacc ccttccccat tagctgccac ctccccagat
103140ataaaacaga atttttttca agtcttccat aacatcattt tttattatac ataaaatctg
103200tgtctacctt taagattagg ctatatatgt gcctaatttg aagcgaacaa tatccctcca
103260cttctccaac taccatctga aattcctgca ttatccactc ttcatggtta aggcacaaga
103320aaagctcctt taatgtaaaa tttgggttgg aaataaaccg gtctcagcct aaactttaat
103380tcttctatca acagctggcc tcaagtaggg taattttacc cagaaccatg catagtcaat
103440taccatacac agtgaggcct actaaacaca cactggatgc caagctccaa ataggggagg
103500gtggggtgct taattcagtg ggatttatat ttaattcttt aggcaaaggg agaggccctg
103560atgagttttc agcaaggtga tgacctaatg agaacggaag tttagaatga ttgtcgttga
103620agttgtgctg aatatggaat ttttggggag gtagaaaaac ctgataggag gctaaaggca
103680attaggatac ccttaaaagt agtccaggca agagataaat ataaggggca tggaagctta
103740gaaacagatt ttggaagtag agttagctga actcagcaaa tgagtcaatg tggaggctac
103800agaggttgtt aagttgtgac gggtgttaag gatgtgttct ggacttgttg aaaggtgcta
103860gctagctagg tgcttgtaat cccagctact cgggaggctg aggcaagagg attgcttgag
103920cccaggagtt caagaccagc ctaggaaaca catcaagacc ccatcctgaa aaagaaaggt
103980gctagcagat ggaatgctct ctcttctgca ttatcttcag tggtagacct agaactcaaa
104040aatatgagac aagaggccag tgaaactaac agaggcaatg atagagggta gagtacaaga
104100aggagactga ggtaacaaca gaaacaagtt ttatccagtt caaaatgatg ggtcacatct
104160gaaaaaacaa aggtcatgtt caaaaagctc aaagaaaaca ataatttcta tgagaaaccc
104220tgtctgcttt attaattgtc ctctagaaat gagccccctc tgccattaag tatttactat
104280caggtgtttt tccccctttc tccttatctc atttgactca gaattatcaa acttgcaggg
104340ctgcttacta ctgtagtctc tataaaagca tcaatcagat gtggggatga cttctagaaa
104400tcccatagat acgctaatgc actagtgaaa tggcacacct gtaacttgca cattaaagca
104460ttgttctaat atcaaagggt tgaaacaacc gagaactcta ccaataggga aagagataaa
104520ttatgatgga gtaatatgat taaatattat gcaaagtaaa aaacaaggcc aatttatatg
104580ctgatatgaa atgatcccaa tatagttaaa tgggaacaaa aaaagcaaga attatttgta
104640ttttaaaaag agggaaaaaa catacatatt gtttttatat ttttaaaaaa tagcatcggt
104700tacctctggg gaagaaaact ggtgtcagag ggcaacagtg aaaggaagat ttatcatata
104760cctctcctaa cttttgaaaa ttgtgaactt caaacaactg gagttcacaa acaactgcaa
104820ctactaacac atttttaaga attaaataaa acagaaagga aaatgtatga aggtagaata
104880aagagtttta gacattcaat atctcacatt tcttcattcc atgcaccatt ttccagcaag
104940ctagtggtgg atatgtgttt catccaaatg agggggtgaa taaaaaacaa aagtcatagt
105000atctaaggaa cagaggctct atcagagatg tcagcaaaaa gaaattccga acaaccacca
105060cggaacaggc ctagagagca acagtccgaa caggggcaag aggacagatt gctccagtaa
105120gggacaaata aatctgttat gttagactac atggaaaacc aagctgaaaa gatgttagag
105180tacatagaaa ttgtactcca atagatatat ggaaacacta agccaagtgt tttcttaaat
105240gagttcatta ttacttctag gattaaaaaa aaattctaca agtaaggaat cctactcgta
105300atatgcttgc ttgactctgt agtaaacaat atctatgtaa tcacagtaaa taaatactaa
105360aatgcaaaca aacaatgtga gaattatgtt gggaggtcaa ggagggagag tagaggtggc
105420tgtgtatgaa agagctaaat atttatctac cataatagga agtcagtaga tattgccaaa
105480tattgataca ttaagaaaca gcagtataag catattattt gaaaaggtaa gggctagaag
105540aaacatttaa ataaatgaaa gtagttgctt ttaggcaaca ggactgggga gacgagaaag
105600gaggggcaga gttgactgga tttttattgt tgttggaagt cttttaataa ccctatttta
105660ctttatttac atgtaagtag gataaatgta cagtcatgtc ctggtatctg caggagattg
105720gttccacaac ccctgtggac accagcatct gcagatgttc atgtcgcttt tataaaatga
105780tgcagtactt ggatttaacc tatgcacatc atcccaatta ctttaaatca tctctagatt
105840acttttaatg cctaattcaa tgtaaatatt gtgtaaatag ttgttatact gtgctttttt
105900atattatttt ttattgttgt gttacttttt tcctgaatat tttcaatcta aggttgtttg
105960aatctatgga cacagaactc atggaaacag aaggcgaact gtgcaggatt tggaattgta
106020ctgcttcgac ttgagtcttg gctctaatct ttcactaacc ttgtaaatag aggcaaaata
106080cttaatctct caattttcct atctttaaaa tagggaggaa aatagtatgc acctcatagg
106140gttgctttga agactaaata agtaaactca tgtaaattgc ttaaaatatg cctgccatgt
106200aagtgttcaa tagtaaataa ggaccagtac ttattatgag aataaatata aactattatt
106260acttgataaa aatataaagg cattaataac taaaatctat ttttaaaaat atgggccata
106320tattaatgtc tgcctccttc gtggggaaac ttttgttttt ataagttaat gctcccaggt
106380actaaatagg actttatttc tttagcctac agccttcact ctaacccata gttaggggga
106440aggatgtggt gagactgttg aaaaattcca agttacagtt agtgatgaaa aagtgggagc
106500tcaaactgga atttgaagac tgcatgttaa gtagttatgc caaaatggga gggaagagcc
106560ttccaggcca ggaagcccac ttgcaaagac ctagaggcag aaggaacaga gcaatataaa
106620aagattggtg gagctggagc aatgatgagt gtgctatatg tttgatggga gccaaatcat
106680gccaggcctt tgggcatgtt aaggagtttg ccttatctta agggccatga agggctttat
106740gcagggtata acataaccac atattttgaa aagatcactc tgggtgcagg gtgaggaact
106800aacaagcaag aagccagaat gagctcagat agacaaacag cgaggccatg gcaaaagtcc
106860aggcaagaga tgatgataga gataggaatg aataaaagta gatggatttg aggaatattt
106920agaaggtaaa acttacagaa cttggcatag gactagccaa aaagagatga aaggactgct
106980aattccaaga aatcttttgc ctcattttat ttttgttttt ctttgtttgg tctagtctaa
107040ctcaattata ttttttaagt aaaggctgct ccctttaagc tcctcaacaa taataatata
107100ctcaattgtg gaatatgtat aagcaaaagc tgcaagacat ttcaattaca aagttaactt
107160ttcactttta aatgataaaa ccccaagatc ttggaacaga accaaaaggt cccaaaataa
107220aatagaattc aaagtaaaaa gatagaagta acagcaatcc aaattaaata gactaattaa
107280gtcagtgaaa caagttcgct actccccagt ttctttggtt acttcccatt aaaaaggcag
107340attgggccag gcgcggtggc tcacacctat aatcccagca ctttgggagg ccgaggcggg
107400agattcacaa ggtcaggaga tcaagaccaa cctggctaac acggtgaaac cccgtctcta
107460ttaaaaatac aaaaaaatta gccaggcacc tgtagtcccg gctacttggg aggctgaggc
107520aggagaatgg catgaaccta ggaggcagag cttgcagtga gccaagatca cgccactgca
107580ctccagcctg ggcgatagag caagacatca cctcaaaaaa aaaaaaaaaa aaaaaaggca
107640gattgactta tattgcctgc cgatgcctct cccctttctg tgacatttct accccaaaat
107700ttttattcca caaaaggctt aatggaaaga gaaattttgc atgttttgca aataatccaa
107760aagggtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtatacag acaagagcaa
107820aagcagacta tatttatatc gtatcaagca aataatagaa gtatacacag gacacctcac
107880cctgctcagg tatatgtgga ggtgtgggtg aggggacaca gtttcccaaa tgaagcaaga
107940ccttctttga gttttaagag ccattaagag ctatagatag atgtaaagat agaaggaaag
108000gcactctctg cagtgggaag tgcaaaagaa aagcacacaa gaaaaaaaaa acaataagaa
108060gtctattgct gggaaataaa atttcggctt atggagtatc actccagagt taagcaagtc
108120tcagatccca tgggaccatt ttaagggctg tgggttctat ccggtaagca acagaaaatt
108180gttgaaggat tttaagtgag agagagaatt tgcatttcta aaaagatcat cctcatcctc
108240cagatggtgg acttgaggtt gacaacacta tctgctgaga taccaggtag gaggcagcta
108300tgataccttg gttgagagat gttaagacct taaactagat cagtagaagt gaaagaattt
108360atggctggga gaaaggtata aaatgttaag tggattcagt gttctaatgg ttgttgtctt
108420agttcatgct ataacaaaat accttagact gggtaattta aacaacacaa atataccact
108480cacagccaga aagctgggaa gtccaatatc aagctgctgg cagattcaat gtctggcgag
108540ggcacatccc tcatagatgg cctcctaggt atcctcacat ggaggaaggg caaaaggcta
108600acaagctttc tcagtcctct tttataagga cattatcttt tacaaaaaca cacgaggcca
108660aggccctcgt gacctgatga cctcccaaag accctactcc taataccaat acattggatt
108720tgatttcaac atatgaattt tggaaaaaca caaacattca gaccatagca gttgtagagg
108780ctaagaaaaa gagtttaaaa ttacctctgg atttctggta tggcacctgg atggtagtac
108840acatttatga ttcactctcc caaatctctg cagcaaaata tatttcaaaa ttcaggattt
108900gacagcctgt agtctcagct acttggaagg ctgacatgga aggatcactt gagtccagga
108960gttcaagatc agcctgggca acatagtgag accttgtctc ataaaaaaaa aaaaaaatct
109020gatttttttg gatgttagaa aggtgatatg gtttacataa tcaatgtaaa agtgtacccc
109080agcagggcct gggcagtatt atgtaatcaa acagcactat ttctgcagct ggacataatc
109140cactaagttg aatacacaat cacaatagcc tcctgacagt tcactcaggc tttgctccaa
109200ctgagttcaa gttttgctaa caaatgagtt aaattttgtt ttgctttgtt ttcagaggat
109260gatttttgaa ttatagataa gaaatggtac aatgtactac tatctataaa tagaatacag
109320agagtgagca ggttgtttgt tcatttgttt tttgatgaga tgaaggttct ggtttgaaca
109380tatataatct ccagtgatga acaaatgtta aagatgtcca ataggcaatc atatagaaaa
109440gaaaattagt ttggacagtt tgaaactgaa acaattatag ggaaaaggtg tacttggggg
109500aatattctct ctttagtgcc aaaaataaga gcccaactct gagaatgacc caaggaagtg
109560tcatataatt gtcagtagtc tctaatgacc tagaattcag agtcctactt gtctttgctt
109620tgcaataagt gaatgtactt ttgtctcttg ccactgctgg aattgatttg aattcagttt
109680tcccccttag tgtacatgag ttgtgttaaa tttcacccaa attacatagt atgaattatt
109740ttgatactaa ctaaatgcat aattgaacag ctaactgaaa acaccttcct tttcaatagg
109800attttatatc tataaagtat ctatcattcc aaatgtattt ttgcacttga tggactaaag
109860aactgaatac atgtgaagat gattcaggca ctgatcattt ctaaaatcct aaatatgata
109920atattttcct taggagaaat atttcaccca ccaagaatgt aaaggcaggt taaaattgaa
109980agtgtctacg gaaggcttaa ttatccaatc agagcttcca agactacatt cctacaaatc
110040attgagggat ctattttaat ctctcaatgt agtctttttg tggagagggg gagtgtaatt
110100cctacaattt gtctcatact tgtttcatag taaaaaagaa aagcattaaa tattaacaga
110160atataaacca aataataaac tggctatgtt ttctctaata aattttcaga ctccacccag
110220aactctaact cagtcataaa actttaagag ttcagaatag agctggacag aaggtttctc
110280cctcaaaatt tcactgaatt cttatcatca aaatcccctc agaaaaaaat atttctagtt
110340tttcctgttc ccaaatacac tacagcatga tgagaaccca tttaacacaa aatatgtata
110400atgaacaagg tttcatgtag ttcttaatat ttgaaagatt taatctagca ccaaggtcct
110460ggagcctcaa gccctacaat acatgtttaa ttaggattac agtaaagttt ttaagaaata
110520atttattttc tttcatgtga gtgggtgtgt gcctgtgtgt gtgtgtgtgt gtgtgtgtat
110580acgcatgtat gagacagaga aaaagaaagc tggtagaaaa atcagccttt aaaagaacaa
110640aaattcaaca gaatagtgct attttcacat tgagggctcc agcactttcg tggtcataaa
110700gcagtctaat tgtttagacc aacgttttta aaaataaaat aataaagaac atacatgttg
110760taaggttaag cactgtttta tattaataaa acttgtttca tttatataca ctcacattgg
110820agtttacaga gttgaggcag agaataaatt agttattgag gattgcagta aaaggaaaaa
110880aaagtttaaa gaaacttagt gttaaggaag tgtgcttgaa ataaaaaatc tggagtgttg
110940caaatggatg aaaggcactg ggtagctgcc tcataaaata tattttaagc caatataata
111000aaaagatcag aagactaaat aatgttatcc acattgactt aaaaaattaa ctgtggcttg
111060gaaataccaa gaaggaacta aaagtctcaa ctgacatggc atcatcaata tgcatgctat
111120tccacaatga gaggagcatg atgccttcac tgggtagttt ccacgaagtc aggcaatatt
111180tggacaagta gaaatgaggg taaatcagag tgaagaaaga gcaatggaca attctgtaat
111240tacaaaggcc ttacaaatta tatgaaagaa aaaagataaa catcttatgg taagtgagga
111300tgtgggtggt gaaaagtaag atacaggagt catgagtcaa gccactggaa tgatcatgcc
111360atctctgagt gaaatgaagt tagaatacct ctcttggctt caatttttca taaatggatg
111420gtttgagtat tgaatctcca aagtgagaaa gggatctcga gaatgctagg gctggaagat
111480actcctgatt gtttttaagg ttttgttatc ttaatctagg aaagtattat ttttcctatt
111540taacagatag attaagttac actgttactc tagctaatga aaacatgtta catttctcac
111600tagaggttaa gaggaaagac agtctggtaa catctgccta gtaagtaatg aagactaata
111660ttcaaaattc agggaacaac tcctatatct catatttaaa cagtatttag cacatttgtt
111720tctctgtatg gagacaagat taagaggtca ggtgagatcc ttttaagaaa tgtagaaaag
111780tggtgattga attaataatt acatcatgtt atgctgtggt tggcttttag atttccatct
111840aatcttaaaa agaagaagaa aaataacccc actacatgtt gcatcaggca ttttaagtaa
111900aacgtgcaat ttaaaaaata tttttaaaat gtgtggagat agaacaaaac aatggaattg
111960ttattattat cagaaacttc tacgtaaaaa tgttggttaa agcaatatat aaagattgat
112020tctcggtaca aattgcttgg aaagtttaac atgtgcacta gatttcagat attaaaagtt
112080aaattaaaaa aaagaatcta atggcagcgt gtgactttaa cctcatggat tttagccaca
112140aacgcaaaga aaaatatata caataaaaga agcaacagat tttcccacaa gcagagattt
112200tctaattaaa atgctgtcac tttaatattt tttagctata gaatttgact gtaaatgtct
112260tcattatgta gtgtaacttc tatagcaagt tatgttaacg ttttcaagaa tcggaaatat
112320tggtaactcg atggggttct taaaacagaa atagtttctt tgatgagttg gaaagagaag
112380gaaataaaag tctaaggtaa gcgccagttt caccctcaat tcttaggctt cagaaaagcc
112440taattaggca gggaaaggaa tctgggcctg gcacacccaa gcttagacgt ggcaggatcc
112500tgaaacaatg tttttttgaa cctttgagtc atatgccaca ggcaaaccat agccgcgata
112560tgcaggtcct taagtcaaca ggtcctctaa cgagatggtg agtggttggt gcgtggcagt
112620tggatttctt cccgccacgg aagaaaagaa ctcttctcca ttgatcagga aagtgggccc
112680aactctggcc tggagagcgc acccggaaaa gtgcagagtc gaaacagaga gagaggggtg
112740tccgcgtccc ggccacagag ccctttccaa accactcgga aaccatttaa atggctcggc
112800ggccgcggtt tgcagcctag aactgtttgg ggtccccggc taggaaagtt gcctggcaaa
112860gccatgtctt tcgctgtcgc gggatgtcct ctcgggcaga gaggctggag gagacaggca
112920acctgctagc cggagtggaa gcgcgcccgc ctcccgccgg gccctccctc tcagggtccc
112980cacgctgcga ccctcccaag ggcaagtccg gggccaccag gtctttggtc tccgtccagc
113040tcctcagggg ccgccccata gggactggcg ggggcaccgt gggcgagagg gtgtgaccgg
113100gctccccccc gaggcgccac ctgccccttg gcccagggcg cccctttcgg gatctctcca
113160ggggcaaccg acacactgct ctcgagaccg tctccaaccg gccacctgga ccgtagccct
113220gtccccaaca cgtgaacccc tgcggccgtc ctcccgcctg ctctaacgca gcccaggggt
113280accgcgtctc cctccgcctg ccgccggctt acctggcggg tgggcagggc agggtggcgg
113340gaagcggcgg ccgggcaggc gctggacgtg ggctaggcgc caggtgcagg tggcggcggc
113400tgcgactccg gttgctgtcg ccacagttgc ggctcagtag agctcctcct ccgccgccgc
113460ctcctgcctt cccgctgggc ctcccgcgtt gcctggagag gcagaaccga ggctcggctt
113520ccacttggag tctcccaggt gagctccagc ctgcgacgtc ggcaggggcg aggccccact
113580tccgcgcctg cgcgccagcc tcccgccccg ccccagccct acctgagcgc tccaggtgag
113640aaccttggat cgcgcgcgca gggtgggggc gccgtccggg ccaagcctgg ctgtcgcgcg
113700gcttctctct gagtggtcgg cgaggctgct gctccgcgca agttgtggct cccggcccat
113760ctacattgga ggaatcctgc actgacctgg tggcagtgat caccttgtag ccagaacaca
113820gtctgctggg tccttgggga accagaagtt ctagatttcc cccacacggt tcctcccttc
113880ctcctcggtt cgccaaaatg aaggggtgcg ctgcctccga ggaccacttc gggagggcag
113940caactgctgg ctcatgtggt ttcttcgggc agggttcagc agcttctgtc accagttagg
114000tttcgtgagc ttccttcccg tgtagtcttg atttcatcac ttgactcact tcagcgcagc
114060tagtggtttc cgtttctgta cgcgggcgct ctggtagggg cgcctcacgt ccaaaggagg
114120agtcttcttc caaagaagag cctttggtat ttggcagagc ctcgccaatt tggggcgcac
114180ctccccttcc tgggatgctc tcagaaggca aaccaattcc cagaagagag ggaaaaacag
114240ccacaacaga gagaaccttt gctgtccctg tttggaaaga gattgggatt tcctggagag
114300ggcttgcttc tactgcaggt ttgtttaaaa gctcaacatc tattctggaa gagaacaacg
114360cagactccag gaagatggtg ggttttgctg tttttaagac aaaaaaaaat tgttaaacag
114420tatttgttcc atttatgaca caactttctg tttcagcctt ccttctgctg ctgcaacaaa
114480cattgacaca cccaaaacca ggacttaaaa tggcctttat acctttctat gaactcattt
114540ctttggccct atcccaaaga tatctaggga tataaataga aaactagcta aaaacttttt
114600ggtgtcgtgt ttaatcacat ggtggtaggt tttactatga agtttagttg tggccttcct
114660cctacatttt gacatgctct tatggtgctt attcactgtt ttattagtgc tatgcgtgac
114720ctaaaaataa ggtttaaaat acagtttagt atagtatcaa caagtgtatc tttagtattt
114780gactactcta agacattggt tttagcatcc atagtgctat tttatactag gacatctatg
114840agttagggtt taagacttta gtaaactagg tagttctttg cttgttttac atgtcatggt
114900tattttgcga gcaccaagga acctccatgt atttgcaaca ttttaatatc agtacctaca
114960agcaattgcc aagctccctt tccctccaaa aacaaacaaa caaaaaaata gttcaggaga
115020tacttgtgtg tattgatata ctaacttggc agtgtttttc cttaggtcca ggtccagaac
115080tgaaaatact aactactgaa attctcttct ccttctcctt cttcttcgtt gtgccacttt
115140gtcgtttacc cctaaccaga ggttaatgtt agtctttgga aaatttgtaa ggtatcctaa
115200atgtataacc aagaaacctg tacccttttg cacgcaccac ccatctctga atgaagagcc
115260aaagattgtc agagttttga tgtcccacca ggcgaagagc aacacttttc actgacaatc
115320aggacagagc agatggatat agaataacaa tctggaatcc aaggactgag atgcattgac
115380agcaagacta gtaacgtcaa ataacaactg aaaagagaag ctagggtaga cctattaaaa
115440gagagagcca gcaaacagga ccaaaatgct ggatgaggag tctaaaaaga acagaagaaa
115500caaaaaagat caggagttac taaaatttga ctgtcagttt tacaatggag acagacaatt
115560taaagaagga agcagttttt ctgagtgtgg cattacttgt gcacgttgag tgtcatatat
115620atagagagag agaaagagag agagtgagag agagtgagag agagagagag agagatatct
115680tggggatgag acctgtctaa acacaaaatt catttatgtt tcatatacac cttatacaca
115740tagccttaag gtaattttat aaaacattgt aaataatttt atgcatgaaa caaagtttgt
115800gtaaagtact tatgtgtgga attttcaact tgtggtgtca tgttggtgct caaaaagttt
115860tgaattttgg aacatttcag attttggatc tgtggattag ggatgctcca cctgtactat
115920tttacacaac gttgcttgta ctatttcaaa gcattctgaa agcaaagtct taatattaat
115980aaaaaatttt ctatgtgaca ataaaattaa aatgctgcag gcccaagaaa aaaatattat
116040catattgaaa tatttaatgc cccaaatcta atcagatgca ggtttttgaa agtaccatcc
116100tattcaaaat tatgaaataa agaatttgtt tcataaagag aaattagcat ttcatcataa
116160ataaaatcca ggaaccttag tgtagacatg cttcagaaat aagctaattt taatagtata
116220tgtttgacat ataatgacag ttaagataga aaactgggca gattttacac aggaaggaaa
116280caaacattct ctctctctct ctctcacaca cacacacgta cttattacac acacacacac
116340acacacacac acacacacac gactagccac aaaatgctca tttgcatgga tttgaaacaa
116400tactcactcc ttaatacttt ctgttaacat ttaaattaat gccaagacca gcacctggca
116460cttagtaggt acttaataaa tatgtgttaa atgaataaat tgacccatca agtaccataa
116520aaaaaatgcc tagtgtcttg agcaatttac aggtctaaag aatggtttta cccaacagta
116580atgacaaata atccaaggca gataagaaca ctagactggt aatctgaatt gcaaagttct
116640aagaacaata gctaccagct ttcacatacc tacatttcaa aagacactac gcaaaacaac
116700ttacactcct acaaattact ttacatctgg cttaaatatt ctcttccaca aaacaaaata
116760gtggtacaag atgatctaca ggcttcttta cattctgatt catataaact ctatgccaag
116820agaattgggc aaataaataa ataaataaat gaaaccttct ggatatagca ataataatta
116880atgggacagc aaagatgaag aaactgaaat tatttagctg agatcgaaat tttaattcat
116940aaaaaatagt tgtaatatat aatgtaacaa gtaaatgtgt aaagttacta agcctaactt
117000taaactattt tagtttgtta attgtttaat tgttggttac acaactttgc tttgaaacat
117060actctagaaa cagttttgcc tcatgaggga actagagctt ttagtgagga tctcaaagtc
117120agactgagag atttggctca aagactgaac agactggaat aatttcttct caaagcagta
117180aaagtgttga aatgctattg attcaagcag agaatagagg tggtatcaca gtgaagagcc
117240acaaacacgt gggtattttt taaataatga tccaacataa ttctacattg tgtttaaaat
117300atagcaagtt aaaacaggga aaatataaat aggttatttt agtatgaact tttttggaca
117360ttgttttata tgattcttcc ccctgtagaa tctcagaaca atcatctcag attttcaaag
117420tggttcacat actatgaggg gttgacttag attacaacaa aactatgtct tgctaatctt
117480gtattcttag tacttggcac atagtaggca ctcaataaat atttgatgaa atgtattgaa
117540tagagtgcat tgtttcaagc ttttaagcat tagcataaga gcaaaaagaa atgtcatgct
117600gagtcagaat aatattcaat ctaactggct ctaaattctg acagtggcac caaagaatat
117660tttgtggaaa actatggtga tagtcttgca tgatgttgac ctcagtgatg tactcgagta
117720tccctcactc actcttttcc taaattgatt tctctttttc tgtaacatgt ttctttaaat
117780tgaaaagaaa ttaatattca ttacagagaa atcagaaaat acacataact gcaaagaaac
117840tagctttgaa atactcagaa ttccttatac caagatgaat cagtgctatc agaccctttc
117900tatcatgtat ctttgcagat tttttttatt gaaaaagttg gcaggaggca cggcatggaa
117960tggggaacat agagggatac agagacaggt ttgtaaaata aaaactagat cattctgggg
118020acttgttttg tagccagagt tttctacttg acatataatt aaatgtcaat aaatttattt
118080tacagtatta ttttaatggc tgcctgatat ccctttgtaa ggatttaatc tgtcaattac
118140tgttggacaa tgaagattgc tcccaaattt tgctattgca accagtgcta taatgatcat
118200actcttcatc tgttgaaaca tttattacga ttttcttcta attgactttt attttgtttt
118260tgttttatat agagatgggg gtttcactat gttgcctagg ctgcaattga actcctgaac
118320tcaagcaatc ctcctgtctc ggcctcccaa agtgctggga ttacacgcat gagccaccag
118380gccagggata tttttgtctt ttgagacagg gtcttgttct gttgtccagg ctggatcaca
118440gtggtgcaat catggctcac tgcagcctca acctcccagg ctcaagtgat ccttccacct
118500cagcctcctg aatatctcgg actacaggca tgtttcatca tgtctggctt ttattttatt
118560ttattttatt ttttggagag atgaggtctt actatgttgc ctaggctgtt ctcgaactcc
118620tagactcaag caatcctcct tcttcagcct actgtagtgc tgggcttata ggcaggaacc
118680accatgccca gccctgactt ttattaagcc acattgtgtc aattatccag atcctaaact
118740atgacaatgt cattagaggc atcatatcac aaaaacaaga ggtagaatac ctattaacaa
118800tttacaattt tctattggta agaaaaataa taagaatatt aattctagct accagacctt
118860catacaaaga acgttctatt caatgaacac cttataattt tagaaatatg gaaatagaat
118920acgtcactta ttttaaaaaa aaactcagat ctcttatttt cctctgatca taccacagtc
118980agtttcccac ctgtgaaagt ctttgtatct tctaatatga ttttcttccc cctctccaaa
119040tcccctccag cccaactgaa gaccctagtc tcccatggag tcttgatatg gtttggcggt
119100gtccccaccc aaatctcacc ttgaattata ataatcccca catgtcaagg attggaccag
119160gtggagataa ttgaatcatg gtggtggttt tcctcatact gctcttgtgg tagtgaatac
119220gtctcatgag atctgatggt tttataaatt ggagttcccc tacacaagtt cttcttacct
119280gccaccatgt aagacgtgac tttgctcctc attcgccttc tgctataatt gtgaggcctc
119340cccagccatg tggaactgtg agtcaattaa acctttttcc tttataagtt acccagtctt
119400gggtatgtct ttattagcag cataagaaca agactaatac aagcctcctg gatcagttca
119460aatcaccttg atatttctta attccaatgc attggcataa agtaaatttc cttcctccct
119520tggccctcac ctctctcttg actccaataa gggagagttg tggagagatg gtacctgggt
119580ttgatataat ttgttgccag ataagttata ataatttgtt aaggaattca agccgtagag
119640gatgaggatg ctcttttagc atattaggtt tgaggtaaca ggccaaaagg aatagcattt
119700ggttattaag gtaaaaagag aaataatttt tatgcatgtt agaaggggaa ttttgaagga
119760aattacactt tttctgtata tttatatatt ttacttttta tttaccagga gaaaaggcat
119820ttgctgccca aggtagaatc ccatcaccat actcctttcc tacatccaat cacaaaatca
119880gaatatgcca attctctatt cccaagtaag tcaacagacc ctaagattac attccctttc
119940tcccctctgc aaagcttcag ggacaggagt ccctgacagg cagctgacat ttggggttgc
120000atacaaaaag tcaagtcatt gcaagagaag gaagtttgtt tctattggaa gagttagtgt
120060acaacatata aaaacggagt gaggatgggg atattgtagt agggaggggg ttaaagggct
120120tagaaaatga tttttttaaa ggtggacagg ggaagagtag aaggcaaaaa aatgtcatcc
120180atgtgggaca tcttctgaac gcccaaggag aatctttggg gatacattcc ccccaagatt
120240taaagaagtg gcttcactcc tgagaatgta cagggagcgg agaagctaaa ccaggagcaa
120300catccaattt ctttgtctct ccttctcttc tcactgcaaa ctctgtacag gtttggcata
120360actatctaaa ctttctagta aaaatcatag ccttcatgaa gatccattgt gtatttgatt
120420cacagaaagt tggtattgaa ttgtaaaaca cggctacaaa tttctccctc tccaacaggc
120480gtgtcatttt ggaatgtgtc ttgctgctct tctcatcaaa ggtaaagtcc tcttcttcac
120540cttttaacct agagtagctg tgcaactttc tttgaccaat caaatatggt ggaagtgatg
120600ttgcgtgact tccagatata agtcttaaag agatttttca ctttctgctt tcactctctt
120660ggaatgttgc cctgagatca ccacaccatg aagaagtctg gggcgaaagg ccatgtggag
120720tgagaggctc agcttcccag atgatctcac tgagcacagc ccccagccac ctgccagctc
120780aatgcaacct ggtgatggac ccaagcaaat accagcagaa gaatcacctg gtcaacccac
120840agaatcatga gaaatactaa tttgttgttg tcttaagcca gtaagttttg agagagtgtt
120900ttaactgttt cacctaagtt tggtctctta acatgaacat aagcactttt ttgcatttgt
120960acatcctttg tactcttcct gttcataccg agaaagcttt tgcaatgcaa aagctcaaat
121020gtattggaat tttagaagtt taagccttga aggactctgt acaaaaccat gccaaaaacc
121080atatattttg aagaatatgt tctcagtctg agccagtttt ctatttccac ctcaaatctt
121140gattggtgca gactggagaa aggagaagag agtcaggggt aagaccgtgg gtcggaggtg
121200gatatcgaca atgtacctga gtaaaagccc tgtagcctct caccatgttg gaataccata
121260tggcacaact acatggaccg ccatttacat agagtaggat gtttagtact gcgtcttgac
121320ctttgaaacc agactggtga gaggactcac caacaaaagt gaagctaggg tcataggccc
121380taccttggag attggtaaga ccccagagtt attccttgcc caatgttaag taagaacaga
121440aaacatttct aaagagggtc ctgaggcaga caggtcttaa gcagtcccac agccctcatg
121500tgccctagaa agatgcagag cgcccagaag cggtttttca gtgcaggtga gaatggttta
121560gatccacatg tttttgtaaa ttagttttta ttggaagaga ggcaaaccca tacatttacc
121620tgttgtctat ggctgctttg acactacaat agcagagttg ggtagttgtg acagagatca
121680tatggtacac aaagcctaaa acatttacta tctggtcctt tatttttaaa aagtccttat
121740attagatgaa acacaaagaa ctggtaaaac ttaagccttg tgtaataata caaaacaaaa
121800atataccatg tagaccataa tcaaagggcc cattgtcttc agccgctgac attgggacca
121860gggtgtagtg tcagaacatg aaagcctgga tacctacaat ggcctaattt tctcatacaa
121920atgcatccta ccactcccat cccatacctt atgctagata ggatccatgg aattagctgc
121980atctccagtg aaacaggaaa agtccctgat tttactgaga tttagctatt gtcacatggt
122040gtaacaggga tttaagagaa aaaataacct caggtataga cataggaaaa taatgttaga
122100ttaagtttct tatacacctg gttctgtctt cagaaactgg cacccactat aaagccttcc
122160atagtgataa ttatcgcaca aaatcgatac aaaggaaata cctgctgaat gttctgcctc
122220agtaggtttt cctttggtct tttaccttag aaaactgcat gatagcattt ttctattatt
122280ttgacatttc atgtaggaag catgttattc acaacttatg ttttataaaa tctttttctg
122340atgatgatga tgatgacact tgttaatttc catctccttc cttcactgta agtttttcta
122400ttccctgaga caactatttc tctcacttgt cctaaacccc aaggatacaa gagatgcata
122460ggaaaatatc tatgaagtgt gtgggaggag acaacagggc agggttccag aggtctgtgg
122520tttattgcac gtaaaacact ggaaagtgct ttggatgata aggaagtagc ttgttgtcct
122580ttgcaatggc ctgacattct tacactgaga aaatcttact ccccactttg agctgggatg
122640tgggagctgg gaggacattt actgagtatt aactctgtgc caggaattat tcctacatta
122700tctcatttag tctccggaaa accctacgta acaggtgaga agacctcagt gacaagctca
122760aagatgcata gctaaagagc tgaaatgtaa aagcaggtct gccagactca aaaacccatg
122820ctctataaac tacatcatag aagttgtaaa ataattttac cttgaatcca agggtgaata
122880atttgtcatg gttagcttgc cctttggcat tcataaacaa tttctactgt cccagttttt
122940gttccattca tataggagta acatgtggaa gtattaagca atagaaggct ttgttttcat
123000gtataaattt ttacgatatt ttcaaatata ataaagccaa aattgttaat ttcacttcat
123060gtagagattc cttcaaatat aagatgctat gaaacagtgt cataccaaaa agcaaacaca
123120ctgatttcta taatcaaaac attgaggaac acaggaaact ctagataact cagaggaaaa
123180agggagtaca aaatcgggtt gacacaatgc ttcatttaag aataggaaca ttgtttgttc
123240atttgggtac tttataactt taagtataat gaggtttctt taaaaaattg taacaatttt
123300ggtataattg tataataact tggtatgtgg tatttttatt tctatttatt tatttattta
123360ttttgagaca gagtctcact ctgttgccca ggctggagtg cagtgatgtg atcttggctc
123420acagcaacct ctgccttctg ggttcaagca attctcctgc ctcccaagtg gcctccacgc
123480ccagctaatt tttgtatttt tagtacagac agtgtttcta ctaaatgttg cccaggctag
123540tctcgaactc ctggactcaa gcaatctgct ggcctcggcc tcccaaagtg ctggaattac
123600atgcatgagc ctatcacact cagccgtagg tgttattttt aaaacatatc caaaatgatt
123660tggatcattt ttaaaatgat cctaagtaat tctttataat attaatagta taatttggtc
123720cattaaatta taactagaaa aaaataaata aaattcttcc aaattgatat attgatgtaa
123780aaataagggt ctctccttta agtctctcca taaactttag ttgagagtga aagacttttg
123840gaccaatgat tcttaactaa agcaatttct tgacagaagc aaaagtgtga aatgagaaga
123900tacaataaat ttaacatcaa aacaagaact gtaagtttta tcatttaatt gtattgaaat
123960acatacaaac cactagatat gtggaatttc agaggcattt aaagagtcat tctaaaagta
124020tatgaaatta tttcatgaaa aggtcgatca aaataagaag aggatcctca taaataaaaa
124080tgttttataa tatctcgatt ataaaagtaa tttcacagta acttttcctt tctgcatctt
124140gtgaaacaca atagacctga gacagcctgt cagtttgctt tatggaattt gactttttgc
124200agcctcacag accacgatga actggtatta ccagttttgc catcttatct ttttatttaa
124260acagcaatta aacactgcca taatatcata taaatcatta aataatgttc cagaccacac
124320acaagtggat gagttatgaa agactgaagg attatattac ttttaaaata ttttgcttag
124380atagaataat ggcaataaat attagcaccc tttgttttat acttttacca aatattgagt
124440agtttcaaag gaacacttac taaatacatg caatacataa ggaatcataa tacaataaac
124500aactatgtac ctactactta cttaaagaaa aataatattt ttgtatctgt catggaaccc
124560tctcagatcc tgtcccctcc taattttgaa ttttgtgttc aatcatttcc ttgtttttct
124620ttagaggttt actgcatatg cctaaacaaa acactggtta atttcatggt ttttacagct
124680ttgaacatta atgaagtgta tcgatattat tttgctactt gatttttttg cccaaaattg
124740gatacatgag attcgtacat gttgttgcat gtagctgtaa tcaattaact ttcacaccgt
124800attatgttaa tccattctgc tcttgatgaa catttgaaaa atctccaggt gcatgtttat
124860gtgtgagaga gattacaaat agtgctgcta agaacatttt tatactaccc tggttaaaga
124920gcatgtgaaa gatcttctct tggaaaaata actaacagtg aaattgctgg gtagtagcaa
124980atttaccaga taatgccaat tattttccga agtgcttata taacacaccc ttatggtttt
125040aatggcatgc ttctttacta atttatctct ctctcacaca cacacaggca cacacacata
125100cacgatatag agatatatcc ttttaatata cattactcct tttctgaaaa tgattttata
125160tactaaaaat ctaaataata gtattagtgc tggcttttca gaaaacctag aattgtgaaa
125220gatgtggttg attgggaatt tttgtcattt gttgccagga aacaaaatat taaggaaaaa
125280atgggaagaa tgttttagtt gtattttttt tttttttttt tttttttttt gggacagagt
125340ctcgctctgt agcccaggct ggagtgcagt ggcgtgatat cggctcactg caggctcctc
125400ctcccgggtt cacgccattc tcctgcctca gcctcccgag tagctgggac tacaggcgcc
125460tgccaccacg cccggctaat ttttttgtat ttttagtaca gacggggttt caccgtgtta
125520gccaggatgg tctcaatctc ctgacctcgt gatccgcccg tcttggcctc ccaaagtgct
125580gggattacag gcgtgagcca ccgcgcctgg ccagttttat cttaacagat ggaaaaacaa
125640atgtatttat ctttcatatg ccttctatta cacttctgat gtcttcattt atagggctaa
125700aatatgcatt cactctattt tcaatcttgg tctttggtct tttaaagaag cccaagggct
125760gactggtgac attcaaaata atgtgttatg caagaaacca attttaggga tgaacgatac
125820ttcttcagct ataactccta aaaatcatca gaattggaac actggaaaag aatttaagat
125880atttttgtct actctgtaac tctttttgta cattttttct cttataacaa gcatctactt
125940taagttgaag aggatccgtt aactatccaa ctaagtatat ttcaaatgct ctaaaagttc
126000agacttgact taactaaaat cctctaggta caatactacc agaagcacat gcttctcatt
126060tttatctaat aaagtttctt taatcagtga aggcctataa gaatgtgatg tcccttaaaa
126120gaaatgcatt gcaaaattcc aaaaaattat ttcttttata ctctctttgt gtaaatatcc
126180tacttttcaa aatttaccaa tgttataaga actccaaatt atttaaatta gaatcaagca
126240aatggtaata ttattttaaa atgttcctag agtggtaccc tcccagctct tgcactctgc
126300tgtttttaat gtgtgggccc ctgatggtct tccatgagaa ccagaaatct gtaagactct
126360atcttgcatt taagataaag tggtttaggt acttcattac gtgttcattt taatggtgca
126420gtgcactcat taatatgctt tatcaacact aaataagtaa cctagtgtat gtatgagcaa
126480tcattgatat tagcctaatg agaaattact gggcagtaag tactatgata agaaacatat
126540cataggttta tattacaaat taaatagaga attgcaaaaa ttttctgagt tttctaagca
126600aaatatattg ggccctaaat tatttggcta ttgttattgc tttgttgatg gagaaatact
126660cagcacagag atgcttttct aatctataat ccagtatcac aaaggaagca tccaccatta
126720aaaaaatcaa aaatcatacc tcaaataata ataaaaatat caatggaata ttaatgattg
126780cttattatat gtaccaataa ttttacatac tctaatttgt acaaaagctt ttcaggtagg
126840taatattatt atcatgttgc agatgaggag attgagtttt agaagatttg tgtgaattac
126900ccaaggcaac aaagctagtg agcagcagag tgaggattca gttcatgcca gctgatgtag
126960aagctatgtg cttactatac tattcaactc taataagata tgtctttcat tagtaccttt
127020cttctcatat gtatgaactc tgatggtcca aacatatata agcaccaggt acatacactc
127080atgcctgaat gaaatgcctg ctgtatacac tatgccatgg gaagtaaagt aaggactcat
127140tcaccctttc ctcaaggagc ttacaatgta gatcgagggg ataagacgtt aacaaagata
127200cgtataatca caagcaaaat atgtcaatag ccataagaaa catattctgg caatgcacgg
127260aagaaagaaa tcctgttggc taagtgggac aaaaagagtc tttataaaga aggtaaggtg
127320aatcttttgg ggaacagtga agtggaaaat caggattttg cacaaagcat gctttatgcc
127380aaaaagctta aggtacgttg taggcatgac ttgttttatt ctggctgggc tgggacaggg
127440catgtataaa aactgcctag agactgggca ctctttatac tctttgatga atttgtacca
127500aattattaaa cagtgggaaa ctattgaagt tttttttttt tttttttttt tttttttttt
127560ttttttttga gacggagtct cgctctgtcg cccaggccgg actgcggact gcagtggcgc
127620aatctcggct cactgcaagc tccgcttccc ggcttcacgc cattctcctg cctcagcctc
127680ccgagtagct gggactacag gcgcccgcca ccgcgcctgg ctaatttttt gtatttttag
127740tagagacggg gtttcacctt gttagccagg atggtctcga tctcctgacc tcatgatcca
127800cccgcctccg cctcccaaag tgctgggatt acaggcgtga gccaccgcgc ccggccacta
127860ttgaagtttt gaagtaagga atgatatcat tcattcaaaa actatttgtg gggtgtcagt
127920atgtgcctgg cactgtgaca gatgatggga atattgaggt gaacaagacc agtccatttc
127980ctgccctcat agatcctaca agttgatggg gagaaagagg ctaattttca ctatgactaa
128040gaatgtgatg gccctgcatg aaacagaagc aaagcctctg agtagaaaaa actttgatgg
128100tttggtttac atttcctcta ctaccttttc ttaaagtaaa tattttattt tagaataatt
128160ttagattcta ttactattag agtctaatat catttaaagg taatttagaa aatttatttc
128220aattactatt aaggttgcta tttgatcatt taatgcgtat ccttctatgt cataactctg
128280tgggacaaat ttcagaaaga ctaacacatt taaacatcag ttagttgcac aatctaaact
128340gctactgcaa ctaggtagat aaacatgtta aagaaatgtc tttggtacat ctgtacttgg
128400ctctgatccc tctgagcata gcaagttaca aagtgttgag ttggaaaact ttgtaatgac
128460caaagaggta ttacaaatac agaaaggcca attttaaaag aagtcaacga gaaaggtata
128520caaactgccc aactataatc aaacacttgc atgaaaaaaa aattgtaggt aagacctgct
128580tgtattaaag gtttttcaca gaaacataaa actcagagat ttatgaaact aaaaagtatg
128640gaagattact gaattaatcc tgaggtaaat aatgtcccag tagaaattag cagtaaggta
128700agcactccaa aacaatcaaa ttagcagggg gacataaact gatatgaggg taaagggacg
128760actagtgaga aataagaaat cagactacag ccaaattaaa aaaaaattta aatatattga
128820tgcttagttt cctttaagca ttgcaacata tgatattgac aaatgtggtg acagctggta
128880aaaactgtat atgttaattg ccaacatcac ataactaata tttactataa aaagtgtccc
128940taaaataact ttctttcatc atcagatagc catatccatc tagcattccc tgtttatgtt
129000ttgaaatgta tgtattacat tttttcttct ccttttagga ttaaaatata aagttttatg
129060ttccctaata tatctacttt aagaatattc taaatattga aaataaaaaa ttttttaata
129120aaataccact gacagctgtt ctctacttat catttcttag cagggtgaag actatatttg
129180ctcacataaa ttgtgtcctt ataacagaaa aagcttctta gggctggtgc ggtggcttat
129240tcctgtaatc ccagcacttt gggaggcgga cgcaagcaga tcacttgagt tcaggagttc
129300tagaccagcc tggcctacat ggtgaaaccc cgtctccact aaaaatacaa aaattagaca
129360ggcatggtgg tgcatgcctg taatcccagc tacctggaag gctgaggcag gaaaattgct
129420tgaacttggg aggcagaggc tgcagcgaac caagatggga ccactacact ccaaactggg
129480tgacacagtg agactccatc taaaaaacaa acaacaacaa caaaagaaaa agcatctcag
129540aaaaacgttc ccctttttca ttgattccat gcaaatgata cttcacccta ttgtttactt
129600gtcctgaaat ttcaaagcag cagaaattct gctatacatt atccttttat ctggtattta
129660ttgaaaatgt atttggctat gatttgaaat atataacata gtgtcaggtt tattttctca
129720tgtaacgagt caagaagtag gtggttgctg cctttctttc agttattcaa cactataatc
129780aatcactgtc ttgtcttttt atatttctgt tcgctatctt ctatgtgttg ggtttccttc
129840ccggtgggtg tcatatcagg gtcacaaaat ggctgctatt gcccagtaat actcgaatgc
129900tcgagcgagg gcaggatgga gggaggggct gctagctgta tctgtctcct tttgtcagaa
129960aagcaaaagc tttccgggaa gctctcctag cggcgactcc ttacatctca aggccacaaa
130020tttcaaagag tggaaaagca gaaaacaaga ttctcatttt tggcaccatt gtgagacatt
130080gcctaggaaa ggcatattga caattcaaac aaaatcttaa attctagtag caaaggacac
130140agaggaatga atatttggta cgggacagaa aggatctgcc taaatgccct agttcatttg
130200aatgtaaaat tcttaaaggc tgggattgtg cctgttctct tctttgtgta taggactcag
130260atcataagaa aaatctttga cactacaaat tctatacact aaggcagtat ccccaaattt
130320cattcaattt ttctaatatg ggggtgctac tgagcacaaa actagaaaga gattataaat
130380atacatattc ttggtcagtt tagactacta tagcaaaata tgatgcacta ggtggtttaa
130440atgatctcct cttagggccc ttgctgtgtc cttgtatggt ggagagacag ttctctggcc
130500tctcttctct tcttataagg ccactaattc catcatgggg acttcaccct cacgtcctga
130560tctaaacctg agcacccccc aaaggttcca cattcaaata ccatcacact agaggttagg
130620gcttcaacat ataaatttgg gggaagataa gcgtttagac cattatttat ttatatgtta
130680aaaataatat agaatattcc cagaagtcaa tttttgctcc atgtattaga atacttatta
130740aaccttccta tgcccagcaa aattgtagac actgaaaaaa taccaacaga aaatggtcca
130800gcttttaatg catttaagct gaaataagaa gtggtgaaca cagatccaaa ttgaactaaa
130860atgttatgta atctataata gcaacctttt atctggtgtc tattatgtga taatggtata
130920acgtacttta tctaatcttc acaacaacac tgctaggtag ttattagcct catcttaagt
130980gttagaaaac agagaacact gagaggctaa gaaattcatt caaggccacc cagggcataa
131040gcagtggagc tgaagtttgg acaagatttc tttttctccc caagccatgc ttgcacataa
131100tgcacatacc actacatgct gattaattca ttttatgaat aaaatcttcc ctcttataat
131160tctgctttgt ttttatgctt tttcattcct agctttctca ctggtgactt atgtggatga
131220atcaatggga acaaggtcag atcatggaca gatgagaaaa taatgagaca ttagattatt
131280aagagagatg aagttaagag tgagaggtga atgaaaggga agaaggggtg aaagctcagg
131340attatgaagg gaataaacag gaaagaggag agagaagaag agaatgaata ctacccagat
131400atttctctgg gcactgtgga tctgatggaa tggtggtatc ccaccctgta accctgtctt
131460ctctgatctc tggccaagtg tgtggcttgg cactgcacaa ctaacagtta gcagcattct
131520ggggagaggc tcatggtagt gaccaaattg cactttccct agctattctt gtgttctaat
131580cctcccctga aggtctttta tggttaatct caaaccatat aaatatttaa ctctcctttt
131640agttacattg cagaagcctg ttctcaaaca agtataagtt tcagtaacca caaattgata
131700ctaatctacc ctaatgtcac tgtgttcctc ttcttcaagt gagttgaaag catggatcca
131760tacaatttgt cttgttcagt ttgtacctac ttgtatcttc cattaacaga gcatagactc
131820tcaggtatgg agaacagatt aagatccaat tactcagcca aaatttcaat ccacctctct
131880ccaggacatt tttttgtttt tgtttttgtt tttctttaac atcctccaaa ctggtagtat
131940agctatatcg agtacttcca gtccatggaa cctaacactt cctcaagtag tcctttccat
132000gcttagacaa cttcgacttt agagtgtggt ttcttatttc accagtcagg atagctaagc
132060tgttctgcag taacaaacag ccctaaagtg tcagctctaa tacaacagag gctttctttt
132120tttttttttt tttttggcta acattatgtg cctatcaaat cagcttggct ctggtatata
132180ttgatatcca tctgggatcc tggctgaggg agaagcccct ctctgggaca tttctgatct
132240catggcagag gaaaagaggt cctggaggat cgtatactgg ccattagagc ttccacatgt
132300cattagccaa catgcatcac atggagtcat cccacagaga aggacagggc caggagcaag
132360acaacccaga gaacagccgt aaatatctgg aataacagtg caatgtacca catagttatt
132420gagtcaaatc cttttccaag tcatatccct ccttccccat tgccttttct ctttaccagc
132480tctacaaatg ttaggaatcc ttagccatgt tttcttcgag ctgtctttct tctagtctaa
132540ttggctccaa ttcctccact gacctgttcc ctgtatctag tggtcttatc tgacattctc
132600ttgagccttt tcagctgtct caagtcaact ggtgagagaa ttgttggtct attctccaaa
132660ggaagtggtc ccttgacata ctttccttta ggatgatggg tttggcgatg tggctgagcc
132720ttctagacaa aaaaagcttt ccttaaacaa tatctcaact aatcagtctc caaatatttt
132780agtagcttaa atctttaggc ttttcacaaa tgcccagaca ctttgcctgt tatatattag
132840aatgtagaga tgtttgagca cttccagagg gcaaatgtca catccctgac tatactttag
132900gtaacttagc aatatatata catggttgta ttgacaaaac tcctcaagcc tactattttt
132960tcataggact ggctcagaat gtggactttg gaaataacca gtcttggatc aaaccctgat
133020tccactcagt actacctata cgacctttga caagttaatt aatcctgagc tcttatttcc
133080tcatctaatg agatacagac aataagagtt atctttctca tgtggctgtt gagaggatta
133140aatgacaaca gtggggctca atcagtgtct tcttttgtta ccatactggg gtcctagcac
133200tcagagctat ttctggtctt actcaaggac caggtcctct agcatccctg ggtttaccat
133260ggttatgcat agctccctcc tctctcccag gtgtccttct ctcctcctga aatgtttcag
133320cctcaagtca gcatcatccg agcttgacca gtatctctca aggtgttgtc caagaacaag
133380ctgcactgga atcacctgga ggcccacgtg gctccatttc agactaactg acagagttag
133440aagatgaaac tagtaaatct gtattttaac aagcttcccc cagctagagg tgccagataa
133500attataggac atccagttaa atttgaattt cagataaaca atgaacaatt ttcagtataa
133560gtatgtctgt cccatgcaat atttggaaca tacttatact aaaaaaaaat ttattattta
133620tctgaaattc aaattaaatg gagtatcctg tacttttaat tgctaaatct ggcaaacaaa
133680tctcagggat tccaattctt actaaatttg aagaccactg gtttaagata tttggcaaaa
133740atctcaaatt caacttcatg agcttccaag gagggaggga gggaaagaac gaaggaagga
133800aggaaggaaa cgaaaggaaa gggaagggaa ggaaggaagg aaggaaggaa atgaaaggaa
133860agggaaggga agggaaggga gggaagggag ggaaggaagg gaagggaggg aagggaagga
133920agggaaggaa gggaaggaag ggaaggaagg gaaggaaggg aaggagagaa ggagggagga
133980aggagaagag aggaaagaaa gaaaggaaga aagagacaaa gagaggaaag aaaagcaaag
134040caagcgaagc ttatagacgg aacacagatg gcccatgcat ccacaaaaca aatatgcaca
134100agaccaagta ggttagcatt tgtttcagca gccacagtgg caaacataga caatccagca
134160gccagagaaa aatccacttc cttgtttctc tagtttggaa ttgggcttta ggtgtctctg
134220tctccttctg ctgctttctc cttttttttt tttttttttt tttttttttt tgtctaaaca
134280tctccatgct atgctagcca acacctactc atcctcattt ggtatttcct cttttgcttt
134340tttgctttcc tacaaagcat cttgggccca tcctcctctg tctgccccaa acatcagaat
134400acctaatcaa atagtcatat agccatcttt ctccctctaa tattaagtgt ggactccctc
134460aattttagtt ttctgatggg attttcctaa ttctcaaacc attctgtctt atgtgtacta
134520gagtaatgca ttgtgttttt ggttcgtgta catttacaga ggccttaaaa gagacattag
134580ctgatttttc acaataaccc cataatcgag gcagatgaaa aacggagccc ttttattcct
134640aggccaagac ccttcttact cagccattgt ctcatctcag gttattatac tcacatcatt
134700catttgtcat tttagaatag tgattaaagc acagctgtgt gccttggtta gttttatttt
134760tttcttaacc aatctaagct tccatttcct catttgtaaa cttaggttaa caatagtgcc
134820tacatcatga gcactgggtt tagtcagatg atcaaagtct tagcactgtg actgcaaatt
134880gtaagcactc aaaatattag ccattattaa aactatcttg tgggccaatt cttttccagg
134940cccagctttt ctcttgttgt ctttcagcaa atagagatat aaaatggaaa acttagacta
135000aaaccagcag aacaagaagt atcaatggct gcacaaggaa gtaatgcaga cctatgctaa
135060attttctctt ggtaaacact tcctttttca tacccctctc ccttgaactt tttcacctcc
135120actcctacaa ttttatgtta tgcagagtca aaaatataat catcctaaac taggctcagc
135180agtgtggaca gtaactgatt gaaatatgag gttctttaat tagaagcagg aactagagta
135240tagtactaag aataatggta atataattat ttgcatcttg tctaacttcc tcatttacac
135300tggccttctt catttctatc tcattccctg ataaatatgg tatctcaaag ccaaatgttt
135360gggactccaa tattatcatt gtgttcacct tctttaccca ttagggcctt tatatgataa
135420taagtgtaat atgatatccc caatgaatga gatagagtgg cccatacagg agcgacctca
135480aagcactttt tctgtactgc agatgcagtg caaaattagg aatattagga aaattacgac
135540tgctttcttc agaaaatact cagggcaata ttgttgtgga taatgggaaa taactggtgg
135600tttttgaaca aggaaagtga atgtcggtta tagtattgaa tagaatagtg ttctagaaat
135660agacaatcta tgtagaccag taagtctgaa gtcagggtca ctgtgcagca acagaggaca
135720taatctgaga ggctaccttt tcatgactgt gtcagcaaat acaccaccat tgcttttcat
135780cttagaggca gcacaaaact atgcaaaaga aaaatcaatc aagaacatat ttaggacaca
135840gcagggaagg ctcctataaa catttttttg ggaagaacat agatatatga tgctttttta
135900aaaagttatc agtggaaaaa attggaattt cacaggactt tgttatattt gtttttggct
135960tagagtttgt tttgtttggt ttctttgttt aactatattt ttggaagggt agaaggtgga
136020tggacattgt aatcttcttt actatttaga aactctaaat gtcttaatct gattcagtcc
136080aggactgcat aggaattact tgatttttgc catcgacaca ggtatcataa tccctttact
136140gctctatccg gagaatgttg tgttttttcc cccctttaag atgaggaatc tgtcagtatg
136200gttgctccct tatgactaac aggttagatt atgcttaagt attctacgtg gtccctccca
136260taaaaactgc ctcagaaacc tccagtgcct caaatactct tgtccttgaa tggtacaagg
136320aagtagagga ttcctggggc atatatgata cttaatctct ctctctgcct actgacattc
136380taacattttg gtgagcccca ttttatttgc tatggaattt gacaaacagt tcaatcccac
136440aaacatcgag tgagcaagta ccatgtgcaa gcaattcatt caatgaatat tttcaactcc
136500tactacgtat catgcataag attcacccta atgaaggaga agaaggagga gaagaaggaa
136560ggaaggagga caaggaggaa gttgggagga atccctgttc tcaggagctt caaatcctgc
136620tggctagaag atatgtaaac ataagggcaa tagaaatctt atgctgcctc tgtggactga
136680gagtgacagg ggatagatcc ttgaagaaat agcagtgtgc ttgggggaaa cagatgctgg
136740gcaggcaaaa acttctcctg aaaaatacaa actgtgactt gagccatgcc tgagggagaa
136800gtagaacttt gattccagtt gaagcatagg cagtagaaaa tgtatggatt tgagagttaa
136860aaataatttt gaacccatct tcaccaatga gctgtggtga ctgtgaataa gttattttgc
136920ttctctaagc ctccactcta aaataggaat tgtgatgttt agcccaaagt gttgaggtat
136980acatctcctc aggcctcaat gcagtcttgt ataatgactt agagcatgga ctctgaagcc
137040aaactgcctc agtttgaagt ctggtgctaa cacttagcta ttaaacttgg gcaaattatt
137100gaatttatca gtacctcagt ttccacatct gaaaatagaa atgataatag caaactgacc
137160tctcagtagt tgtaaattaa acagacaggt taatgatata aagtacttta ataagtgact
137220acacatggta agccctataa ccatttgcta ttattaccaa gtgtttagtg atacaatata
137280agccattaaa ttaccatatt acctggttag tagatgctat tttttattat agtgcactgt
137340gataaacatt gtaagaaata taaatgttcc ttgtattttt agggaagaca tgaaagaaat
137400tatagcctag tcataagtga cactcacaag taatcataat aaagatggta tgtaataaat
137460attgcaagaa agatcagata tgtccaactg atagttcaga agaataaggg atcaggaaga
137520aaaaaaaact ataatagaga catggccttc tcatcatggc ctgggcatga tagcaagacc
137580ccatctcttc aaaaaatgaa gagatatccc ctaggagcat gcaatcctag aggaaggcat
137640gggtgtacct tccagattgg aaggagaggt aatgattgtt gagtcagcaa tgcaagatta
137700ggtagactat tgctgggtgg attagtgcag ggaatgcctt ccctgatgaa accaacaatg
137760gttgcaaagg catagaagca tgaaagggca aaaagtgtgt gtgtgtgtgc atgttacaga
137820aaaagcaaag cagtcaagag agatggaaga tgtgtacttg gggacatcac atgagcgatg
137880gagacacaaa gatgaataag acatgaagga aatatttttg gatgatgcat gctggagact
137940gtttttgttt tttgtgtcct tttgtcctta acttcctcct tccttttttc cttcaaaata
138000catttactga tacctacaat gtgctagaga aatatgactt tccttatatc acctgttata
138060taacagagtt ttgtcccatg ataagtgata taatagaaat cgcctcatat gaaacaaagt
138120tatgttatat cagaaatgtg ataaataatt ttccaagtta caccgaaact aggccccaaa
138180tagggctctt tcattaaact ctttgttcag ttgctttctc tcactcccat gtccctaaag
138240tacatggata gtagagaatc cttttttttt ttttcagttt atgtgggctt agcaaagagt
138300gggcaggcaa tagagacttg cagatctcaa ctgaattact ggagaattta attttaaatg
138360ctctagaagg ttatcttctt cacaacttgg tggcaaattc tttcaggaac tgaaagttgc
138420atgtgtttta aaggggcata agcttgtgga gaacctttct ctttggttta gctgcatcga
138480tatgctgatt ttttaaaatg cactgaacat gcaaaaatga gtatattatt gacctcaaat
138540caatgcccat cactgtttct atcttataca gtgagcttct tggcttttta atattttcat
138600tgtgaagtta ttccaatgaa gacaaaattt tgtcaaattt attacccagg gagctttcaa
138660aatgaagcag agagactttg accgagcaat ttcacttctg gaattttatt ctacttatgc
138720atacttatac atatgtgcaa agacatatat atctgaaagt ttattatttt ttcgaagcat
138780tgtttatagg gagaaaaagc aggtaacagc ccaactgtcc ttcaaaagag aactagttaa
138840tttatgatat atccatataa ttaatattat tcagtcatta aaaagaataa tatagatcta
138900tgtgagctta tttggaatga tctccaggac atgtttttaa attaaatcac aaatacaaac
138960aaaagaaaga gagaaagaaa agtacagaat aggtataagg aagatttcca tttgtgagac
139020ataaatattt atctgtgctt tgtatattta tttagtacac atgaatgcta tgtaggcatg
139080gaacatttct gaaaagatca gcaagaaagt acaaatagtg attatttctg gatgggaatc
139140tggactacgc agcaggcaca cttttcattg cctgtccttt gtattatgtg atttttacca
139200tgctaatata tttttattaa aaattaatta aagaaagaat aactactgga gataagccat
139260ttttggtcaa ttccatagtg gctagttaga ggctaagtaa tatgcaatca tgaaattaat
139320gctcaaatta ataaaacagt atttcccaat cttgatttta ttacaagttc atttctgagt
139380agttcaagaa cttgtagaat tattttaaag atctcaaaat aaaagaaagt ggtaatctaa
139440ttttgaaagg aaatatgttt cacacagttt gggaaagaga gaagtattgt tttcaaatag
139500ggagcagtgc cttagtgagt aatttcattt gcattcctgt gtatctttaa gctgagtaca
139560aactaagaaa aactgttctt gcagaataca ctgtaaaatt actttaatca gtaattactt
139620tgacctttcc ctatcaaagg tcaaaacacc ggacagcctg ttgctcttaa catcagctct
139680tctgcgtaat atttctcttt gctctggaac acaattagct cttgggaaaa gaggccaact
139740tttcagtttc tgcctttcat ggtttctgtc ttcccctctg cttcagtgca ggtcagtcac
139800acagataagc attatgccca gaataaaact ggctctgttt atctggacta ataaatgaat
139860ggaaccagtt attttcattg gcttggtctg gaaggaaacc aaactaattt ccacagacct
139920taaattggac ctaatccaag ctacttgtcc tcactaataa gagcaataac aattagtata
139980aaagcaaaaa tttttacaaa agcaatccat atttactgct gggcaagaaa attggataaa
140040actaggaact aacatgaggg actctgaaag ctctgagaaa tccacttcat ttgtatttca
140100atgagtctga gctttgcagg tgaatacaac ctttctttag gttctagata ctccagtttc
140160tctctggtca atttttctaa tcaagttgtg ctaacttccg ctgatgagca gaaagtatgt
140220gtatggtaca gtcaaaactt ttgcactttt gtcttactta agaaacttaa aactcacatt
140280tgtattccta gggtttctga caagtcggca tactttattt cacaatgtgt tcatggaaca
140340gtgagatttt gacttccatt gtgcacatgt aataaaatgt tctcattagt gatattcact
140400cagtgtatga aagcagttac tgcctctgcc caaaaggcat tgctgtttcc atttaagatt
140460tatggttggc cttttaatta tactcccaac actgccccta aatatatccc catggaaaaa
140520tatataaaag tatttcactt aattattatc ttgctcaaag atcagggcaa tatcttggag
140580ctgcttccaa gactattgct ttatgatttg aaagctgttt agctattttt accaaataat
140640taattcagat atatcaagaa tagggtaaac tctttattga aacgatttgt agggaactta
140700aagagtaatt aattttccat atgtattttt aaatgaaaat aacttattct ttctttctga
140760tttaaaaaaa tgcctacttg tagcaaaaca aaatagggga attcattaaa aaattgttta
140820aacccaatgc aacccaccta tatttctagg taccaagaat accactatga aaatgttcat
140880gaacatcctt tcagacattt atatggatag acattaggca tacacatttt ttaaaacaaa
140940tgagataata ctgaaggagt tcaggacatt ccaccccaaa atatgtccct ttggcgtatt
141000gattattttg atttaaagac acctggggct gggcatggtg gttcacacct ttaatcccag
141060cactctggga ggccaagata ggcgatgact tgaggtcagg agttcagatc agcctgacta
141120acatggtgaa accccatctc taccaaaaaa atacaaaaat tggccaggag tggtggcact
141180cacctgtagt cccagctact tgggaggctg aggctggaga attgcttgaa cccaggaggt
141240ggaggctcca gtgaactaag attacacctc tgcactccag cctgggtgac agagtgagat
141300cctgtctcaa aaaaaaaaaa aaaaaagaca cttagaagac aataggtatt agaagggctt
141360tctgaccacc ccttttctat ttaaaaacag tccataaaat ttcctatgaa aaagctgcct
141420tccttgtatc aaaaagtgaa gaacatcctt atcagcagag actgggaatc aacactaaaa
141480tgaatccata tacacaaatt tactaaaata actcttattg tccaccagtt ttccccactt
141540aatgttttat cacttccccg caattaactg cccctagccc aaaccacttt gtcttgtcat
141600ttcttcacaa attcatcact tctttgtcca aaaggtacat atgctttctg ctctggtcct
141660ttcttcatgg tttcattttc ctgtgaggat tcccatgcac atgtgccatt ctaataaaac
141720atatgtgctt ttctcctgtt catctatcta tgtcaatttg acttttaggt ccagtcagag
141780accacaagaa cgcagaagac acattttatt tcctctataa taccatactg ttttaaaatt
141840aacaatatat tctttaaaat atgtaaatat aagtagatga ttagatagag agatctccaa
141900ctcaccactt aaagtgctgc atcatattag attctgtttt catattttac caatccacta
141960ttgatgaaca tttaagtaat ttcaattcaa ttttaaataa cactatagca aataccaatg
142020tgtgtttttc tgattatctt tctaggtttt attcccaaag gttactgact atggatattt
142080aaaatgtttg aaatgtattc ttaacttccc cttcaacaat gttaaaacca atggttttaa
142140tatccttaaa ggtagtttct actttacaca aaagttattt ggaaggagat ttggggtaaa
142200ggaagtgatt aggaggaata gaggattaag agatgaggat gatactggat atatttctta
142260aaaaagtcta tgagaagata gaaaaatgag tacagcttag agatgctgaa atatcataac
142320agtaaaaaag gtatgtgtta catgaagtta ggcaccaatc caaagaagtt gctgagagaa
142380caatcttgtg aacctacaag ttaattgtag gagagaaact cagaagtttt agtatgaatg
142440agctggaagg ttactgtttc actcaggaga ttagaaagat aacaagagac attgcttaaa
142500tctattttca aatataatgc taataatttt ttcagtaggc tataaaaaca agtcttccct
142560ttcctactgc agttaaaaaa aatgcattgt ccttttcccc cctcacactt tgtttgtatc
142620aggaccttct tgtttagatc aggttactaa atatctttag aagacactac atttagaaaa
142680agaaattatt gttttgaagt ttttccaaag caaataaaga taattcttat acttctatag
142740atatttaaat ccaaatgtgt ctgttcaatg gataaaacca tagaggtctt taattttgca
142800aaacaagccc atggcaaacc catgaaatag aaaaatgtat tattttgaat actactatcc
142860aatcttcccc cacgctttct ctggtgccca gtttttccag gtgacactgc tatacctcag
142920cctgaagcct ctacccctct tgttcagctc tgtccctgca gacagaactt ctaggtgggt
142980ccccaaggac cagatttgcc tcactctata ccctctgaat gctttagcct ttgacttgtt
143040tcctcaaggc actcctaaat gatgctcttt taactgatga actattttga aaattagtga
143100ctgagggtag accagtgtgt tactactcca agtaacaaga gcaatttaaa agtgtccttc
143160agtgcttcag tggaaagaaa tctgttccat aaagggaatt caggcttcct gcaacaggat
143220gttttctgtt caatccatag tcataccaag cagggcaaca gctcttagtt tctagtcaca
143280atcatagata caggtaggag atactgataa atatttgata aatattattt cattggagct
143340tcaattctat ttaattttta attttatcag agtaatgcac agatacatgc ttttataaag
143400taaaatagca cttagagagc ttttaataaa attagcttgc ctgccgcatt gccccacttc
143460aaaagcagga tcttccacac ttgatttgtt tatgtgtact tctgaatttc taaactacct
143520gcccacttta ttgacttttc aattttagat atcattattg aatgatatcc ctgtaagaga
143580aatgaagatt aagcagtctg atgcacgctt ctcaccacac accacaaata catgtccctc
143640cttccatctt cttattctgt aacagacttt ggggttaaat taatatttaa tatttccatt
143700gatgtcactg tgtctatcac tcacatctga aagatgccat gttttgtaat ttacatttct
143760tttctcatat taattttcat tattcctaaa ctttaaaata gccatgcact gttttgtttt
143820cttcatttcc tatgtgccta tcatttacgc aatctcaaat tttctgatac tgctgctaaa
143880ctctcagtaa aatcagacac attaggtgaa ctatcagtag tacttctcct ttcatggaga
143940cattactcct ggggctgtct gtccttttgc ttctctggtc tattgcagat aatctgttat
144000tcctaactgt atctcttcat tcaaatgaca ataaagctca atatttagtg aagagcaatc
144060ttgacatgtg attcgaagac ataactttat tttcattttg attatttttc ctatgtctca
144120aataaggttt cttgtgtgag tgagggataa ccaggaatga gcaaatgttt ctgtgttttg
144180cctataaaac agacacagag tcccaagact gtctctctag aagaattttt ccagagattc
144240cttagttcat cctactctca aatggctaat tttattcccc ttgcaagtct ttaccaaaaa
144300tgattttgta gcctgaactg ctgtggtact agaaatttca tctgcttttt tctgagtatt
144360cccattcctt ctcattttta atcagccctg tatagtttag atttcatgtc ctcctctttt
144420ttatttaccc tctcattttg aagaagcaaa tagttcaaca gcctcttgag aaggaggact
144480tgagaggtaa atatattgag gtattgcatg tcaacaatat ctttattctg ttctcatgct
144540tgattgtggc ttggcacagt gccaaactca aggttggaaa tcattttccc tcagaatgtt
144600gaaggcagtg ctccgactgg aatggagccc atgctgctgt tgagaggttc agtggaatct
144660gatccggatt ctgaatcctt gtaatgagaa ctgatttttc tctctgggag acttttaagg
144720tcttttcttt atctctggct tcctgatgat aagccttgct tcatgtgggt ctttcttcat
144780tcattgtgtt aggttcttaa tcagactttt caatctgaga atgtgtcctt cagtaccaaa
144840aatgtttatt ctatgatttc cttgataatg ctatcccctc tgttatatct ctttcctgtt
144900tgtgaaactc ctattaatcc atttatgcct agtgttccat tattggaaca ctaagcttgt
144960gagagttatt tatatcttac tgcccaaggt catcaccaag gtctgatttt tcacacaaaa
145020aaatttgcaa cctcctgcat aaatgggtta attggatgtg aaacctctga ttcttgtctt
145080ttctatactg tttttcatct ctttgatttt tatttcagtt tctgattttc tcaaattttg
145140tgtttaaatt cttctgtttt tatttgtgct actgtatttt tttttaattt aaagagcttt
145200taaaaaattt atttctttta gatagcattc tgttcttttt catgggtgca aaatctactt
145260ttgtttctgg ggatattgct tacagaaact tttcctgtta ctccatgaat ggtttccagt
145320atatggtgta ctggctggaa atttagactc tgcatccagc ttaaatgtca atcaacacca
145380ccattgactg tgcctatgac cttgggcaag ttatctaaac tctgctcatc agttgtctca
145440gtaaaataga aacaataaga gtatacagta acgcatggca ttattgtgag gtacaaatga
145500gagcaaaatg tattcggcac tcagaacagt aactggaaaa gaggaagccc atatgatatg
145560ttattatatc catttcctcc aagttccttt ttttcctgtt ttattttact ctttatcatt
145620tgtgttagac atttttctca aatgcctgat gatccttggc tgtgtgttca tatttaataa
145680tgaggaacta agaagtaaat tggaaacctt ctgtgtgcct ttgaactcct gaagagccat
145740gtgggaatat aacaccccac agcttcagtg ctggaaagac ctcatgatag agagtggtga
145800tcaaggagca ccagatgttg caatcactgc ctgcagccat atcagatacc ccaataagaa
145860ccgcctcgct gagtccagtc agcacccaaa accatgagaa gtcataataa caacaagtga
145920ttgctgttga tttgttccac caagtcatgt aacaatagac aaatgatgca gagaagaata
145980ggccctagaa tgagcaagag ccccacctag tatcagatct tgtgacttga aagtttccct
146040tcccatctag gaacagcctc tcaatctcta ttcctctaaa atctactaga caatatcctt
146100gtttgattct ctatagatat tttagtgtta atctttcctt gaagtttaag acaggtcatc
146160caaacacagc tgaaggtaaa tcggagtgtt ggagatgatg ccactctaag gcatagtaag
146220ccagttttct aagagttgag ataacacata atgataaatt acctacgaca aacacaatat
146280aactgttact tggattaata accctctttt tctcttcaca tctcatcagt tccctctgtc
146340atctaaatac attcctatgc caggaataga atttcagaaa acagtttact ttgcaaattc
146400aaaagtatct caaatttagg gaaaagaaag tgagatgaaa gtttcagtga agtcttctcc
146460aaagctaaaa caatcatttg aaaatagatg cccacaaatg ttgcagcttg atttatagag
146520gcaaataatt cagcattaaa tgaggagcaa catgattgct aagagtatat atgtttaaac
146580tgtgtcaaca gctattacac tgtgaagagg gatttggatt ttgaacatct tgtcaagtca
146640agagacgttg tttacatttc actgctcact ttatttctgc taccgagaat gagatttgac
146700ttcttccaaa gaactcattg ctacaaggaa gggtaaatac agagagtagt caaggtgatc
146760tcacagggga gaaggtaact aagcatccaa attgaaacac ttaaatttaa acagctgtaa
146820aacatgtaat cagccattca aaatgtaatt gtcaaaactg gctacactta ccgaacattt
146880cactctgcat ttgtttgaaa tgttttgaaa ctctaagttt aggttgaaca ttatatttgg
146940ttagcaggga aaattttgcc tattctgatt tacagtgaac tacaggaagc taataagttg
147000cctgggagat actgactgaa actatgatct gcataggtca cttagttact aattggttat
147060gggaaaggca tggctaagcc tactgcactg aagatttgaa actatgaaca ccacacacag
147120gaggaacctt aacccttagc agtcaaatgc tgcatgtaat aatatgcaga agaggaaaat
147180ctatccttta agtttgttaa gaaccagcag ttttaggagg aatcatttgc cattctctga
147240cagtttacat gtgtttccct gaaaaatgag ataaagtaaa ataaaacaaa aataagcaca
147300agtttactca gagatgatga aaccccaaac tagattggtg gttgagaaac atgatattca
147360tggccttcat agtaaaagca ttgttgttaa gcactctact ctcattacct cttttagccc
147420ttgtaactgc tgtgatggat atgtctatat ctccccctct atatttgagg aaactgatgc
147480ttaaagagac taagaaatag gttcagcaac catcctgatt tgcccgagac tgagaggttt
147540cccaggttgc agaattttca ctcttaacac caggagactc ctgggcaaac cagaatggtt
147600ggtcacctta tgcagcagca ggtcacttac ttactaactg gttatggaaa aggcatggct
147660aggcctactg cactaaagct ttgaaagcaa ctccacatgg cagcaagcat ttctttctgt
147720gttatagggt atcttacttt tttggagaca gggtcttgct ctgtcaccca ggctggagtg
147780cagtgataca actatagctc actgcagctt cgaactcccg ggctcaaagg agcctcccac
147840cttagcctcc caagtagcta ggatgacagg tgcctgccac catgtctggc taatttttta
147900atttaaaaaa ttttaaattt tggggtctca ctatgtttcc agggtgatct caaactcctg
147960gactcaggcg accctccttc ctcaccttcc caaagtgctg ggattacagg catgagttgc
148020catgtccagc cttatagggt atttttcatt gtactgattt agatattcaa ccctctccac
148080tcatccttag acctgagaga tactcactcc atatgatctg ctaaaccatc caaatgtgaa
148140catcctcccg tgcccctgag gctgaaatgg cagcattcac atgtatctga tacaaaatct
148200tatttttatg atagagaaaa ctaaagctca gagaggttta agtagttaac caaggtgcca
148260tttaacttga cttctgtaaa tcaccttgag acttaaagga acaaattgtt tttcctttaa
148320attagaaatc tttaatacct ggattaggtt caaagggtaa actgtcaagg gtaaatttca
148380agagagataa tatttcaaat tggtggtatc gaatgataaa aatacataat ctgtaatgtg
148440tatttgcaag taattaattc ggctgtagaa ggcaagaaaa caaacaggat gggtgaagct
148500cagaagatgc attaagcata gataagtaaa ttagtaatct gtaagacgtg ttcaatataa
148560ataaggtaag tggagcatat gcgtgtgaaa atagaaatgg ttgaaaggac gaagtgactt
148620taactattca tgatatacta attatatatc aagcatgtgt gttcccaact agtcacatta
148680catgaagtat gtaatgtaac gtgtaagtac tcagaagagg aactcttgaa ctatatatga
148740aaggtaagtt ttctagaaga ggtgatacct aaaccaaatt cttaaggctc tgagaaggta
148800agggtgggct ggggaggggc attcagatgg agacaaccat aggtagttgg gtatatggat
148860ggatacaggg taaggaatca ctgtaggtta attctatgaa aatgattgaa gtcaaaacgg
148920aagcacagtc taaaaagtat gcaggatgga actcaaggag ctcacagact gtcaaggaaa
148980gaggacgata tgtagcatct gacttactta tattgcaaat aactcaaagc atgtattaat
149040atctttagta ataatagatc caggttgggc cttgggaata ggtatagatt tggacttatc
149100atgtcaaaag acattaatgt tcaaaaattt tattaccatg tcatcaaaaa tgtctttact
149160taggaaccta agaggttttc ttttaatact aatacagcaa ctgaaaagag taaaaccttg
149220acatgcatct gttaaagtcc catctatagt catcattatc atcattgtta cctttattat
149280ttaagtaaat ctagactgtg ttaaataacc accacacatt atcttactta atatgtaata
149340ctataaagta ctatacaata tgctatacta atattataaa ataggtacaa tagtccccca
149400cttatccatg ggggataagt tccaagaccc cagtggatac ctgaaaccta gaagagtacc
149460acacctgatg gccatcaatt ggaatacact tctgttcatg tcttccattc acaagtttaa
149520tgccttttcc atttaaccaa gcacttagca tgcactgtgg ctgtaatttc tgcaatttgc
149580gatgtgacaa ccaaactggc agaaatttct tttccttctt cacaatttca cagatagaac
149640atcttagcaa tctcagcata tgattctttt ccttcctaaa gttgacaact tttatctttc
149700acttaaagga agcaactgac agcttctttt tggcatattc aaattgctag tatccttcct
149760cttgcacttt ggagccatta ttaagtaaaa taaaggttac atgagtccaa gcgctgggct
149820gccaggacag tcaatctgat aaccaagaca gatactaagt gactaatagg tgagtagtgt
149880gtacagcgtg caggctctgg acaaagggag gattcatgtc tcagggagga cagagcagga
149940cagcacgaga tttcatcatg ctactcagaa gggtacaaaa tttaaaactt aagaattgtt
150000tatttctgga attttccatt taatattatc aggttgacca gggctaactg aaactgcaga
150060ttgtgaaact gtggataatg ggagactact cttgacttaa atagacttta actgaaaatg
150120aggctctgag agttgaagtt tatacaatca agaagtggca aagttggcct aaaaagccta
150180atgtatatga ttccaaaaca ataaggtttg tatctagaat cagatacatt aggcttctta
150240ggccaacttt gccacttctt gattgcataa acattatcag aaatacattc tgataataaa
150300tattttaaca attagacttt agttaacata actcaagtca gtacaaattt caacaaagct
150360gatagtacca ggagtcgttc caagtgcttt tcatgattat ataacacttt tctattcttc
150420catagccatg agcaaaaagc aagaagcaca atctgagtgc caaccctgcc cacaatgttc
150480agtagacaaa aatttcccat gatactcaaa attgcctggt gcagtctcac ctaccaccaa
150540taccactgtg aggatttatt tagcagattc atgatctatg cattcttcac catcaacatg
150600aaaggctaaa tagcatcaca acattaacct ttctccagag taatttatat accattaaga
150660gatgcagtct gtcaaaggaa aaatgccact gatggtagaa ttttaatcat tgacaaacat
150720ctttaggaga cattttgtgg ttttcttttt tttagattag ggaagggaga tagagatagg
150780atgtcagggg gtatgataga gagtagtttg gggaaaagca aaccctctct tgccataggt
150840ccctcacttt atttgtaatg actctacctc tggatcataa atattgggaa aaaaatcatt
150900aatgagtgca aattgttgta aatgacaaaa tagaaagtta ttagtcttta gctacacata
150960tgtatgtgtg tgttatcaat acactgctct aaacactaca tggtgtgtta tgaatttacc
151020actaaaaaca ttatgcataa attggaacaa ttatctgtat cacgaggtaa gatctacctg
151080tcaaaacaag gggcttatag tgccaggaaa tggagtggga ggattttatg gcatacccca
151140ggagaggcac tcgtgattag cggtccactt cccataagac aaccaaagtc agttgactgg
151200tgaaaaccaa aattcagtgg cttccagggc cacttctact tagaatacca agaaaaatgc
151260ttctgctgtc cactcacctg caaagaatgg aaggaccttc tccaactcac aggaaatgca
151320tttctttcca acctgaagaa ttaagagttc gctagtcatt ctccctactt tttgttttct
151380ttcctgtatc ttggtcaaaa gtgtgggaaa atcgaggcat gctaaagata ttttgagctc
151440tcctaatcct cagttaaaat tgtctgctct gctgaggcag gagaatggcg tgaacccggc
151500aggcagagct tgcagtgagc cgagatcgcg ccactcttgg acgactgagt gagactctgt
151560ctcaaaaaaa aaaaaaaaaa aaaaagaagt ctgctctgga gaatggtaat gaaatgtaat
151620aagagctcta gaatgaaacc ccatacgtaa aaacttttca tcacaccaac tggttattct
151680ttctgagatg ggcctgaaat taaaccaatc tttaaagaca gaagttcata gcaatctcta
151740ggatgagaaa acctgggctc attcattcat tcatgcatcc attcattctt tctttcaaca
151800atatttacca agtgaattct aggtgatgct gcacttccat gtcttcttgt tgagcgttga
151860agcagaaaac gagtgggagc caaaccatga caattcaatg cctgacactg agaaactaga
151920aaattcaaat gcagatgggg cttcattttt gtctactgat aatgtttttg aaagccttga
151980tttcctcccc acaactgaat tcccaataaa ggtatggaaa gttcattgtg caatggccgg
152040ggaattcctg ggtcacttgc accgcttgaa gaagcgatta gggatggggt cactgtgttc
152100cccagtgact gtagtaaaga gctgggaagt caaggaccca gtcactttct gagcttttgt
152160ttcctcagat ttcaaatagg cattaaaaaa acaaaacaaa acaaacctat ttcaagaaga
152220attaaagaga gaacttccac aaagaacctg atcatggtat gtgagataac atacaaaatt
152280gctaagtcag gaacttgaac caaagacctt ctggttcaat ttgcaagaga tctacatagc
152340cacttgagag ggtcagacaa aaattttact attaaaggag ggcttgactg tttttctctg
152400gactcctgac cactcagggg ccatgtgatg tgttctgaga aatggtgtca cggcagtaaa
152460agatagtggc cttcttcaga gtcatgcctt acagagcctc cttagtgttt cacctggaca
152520aaaagaaaaa cagaatttca cagaaaatta aagccttact ctctttcttt gacaaagata
152580tttggcaaga ttcatcatac aattccacaa gcattagtca ttgaaggatc caaagcacgg
152640actgaagaac agaaaaacct gctgatacaa gtcccttctc aggaatggct tggactgacc
152700aggctggttc ttccacccaa tttggccaat ggatttcaaa gacaaagttt tggacttgaa
152760tttcggtaac tttaagtcta cggaagaaac ttatcgtttt gacaaacttg aaatatgatt
152820tgtgtgtact tgtctattct atctaatgag agatggcaaa ctccttaaga acaagaattg
152880tgttatgtct ggcacattgt aggtgctcaa gaaatatatt cactacatga ataaataaat
152940aaaggtcaac aaaagcctgc aaaatagttt gtgtgattat tttttggaaa gttttaatga
153000taaagagtgt ggtaaaaatt aaatagaata ataagaagaa atgaatccac gaattcatgt
153060tatttaaaaa aatttgaaat tttaaaaata acaaataaaa ataaaaataa aaaaataaaa
153120aataacttga aaagaaagcc aatatatcca gaaaagcaga gcctgagaaa ctgcagaaaa
153180atggagcaga agcctacttt atctctggac ttagtgttga gacaatatgt ttcctcattg
153240cttaagccag tttttctatt atttgcagtg aaaaagtgct ataattgatt caaaatcaat
153300gcatttgctt ccagtggggc atattctttc caataaccac aaggcattgg ttgcttcccc
153360tagacactcc ttcctctatt ggaagatctg ggaaaccaaa ttagttattg gaaatttctc
153420tgcctcaact cctcccttcc ctcctgaggg tgagttcttt aaaagagaat gggatgaatt
153480ttcagagagc ctgaaataca tttttagaaa tcctgttgaa gtactgtgac ccagaaacaa
153540ggtcccctct ccagtgactg agagattcaa gacactccac agtgagaact ttttttttct
153600tcttgaacaa tggtgaagac cctctgtcct tgtcttctcc tttgcttctc tttgtgtaga
153660ccatcattgt tcaaaaagtg tacatcacat gagcttctca tgcaaactac atatatgtaa
153720tttaaatttt tctagtagtt acatttaaaa agtaaaaaga aataggtgaa aataaagtta
153780aatttattat gttttattta acccaatata tctaaaatat cacttcaacg tgtaattaac
153840ataaaaacta tagagttatt ttacattttt ttatactaag tcttcaaaat cctgtgtgta
153900ttttacactt agagcacact gcaatttgga ccagccacat ttcaagtgct caatagacag
153960cacagttgta gccacacaca aatctccttt acttctgtta cagcataaaa ctcttagaca
154020cacagcgttt agcatctaat taattatgta actgtattac actgttattg caataatcat
154080taaacaagtg tcattccaac cccctatctg ttttaatgca atcttacatg aaacagtttc
154140ttatattgag atataggcca gtcttgatgg gtatagagat gtcattcctc tttatccaaa
154200gatttcctta ttattcttta ctgaatgtca catttacagc tattagatgg gaagattata
154260atttattcta gaaacttcta tttttaaact atgtattaac caaaattaca catgtatggg
154320gtacctgctt atttctagta agcccactag ctgatgtagc aaataggtag ccaggccaaa
154380agcaaaattt tgatagttgg ccttttgttt aaaaagtgtt ttatatctga ttagctgaat
154440tctaactatc ttaggaaata aatcaactaa cagtgtaaga gacagtctac tatggtggaa
154500agagataaac ttcagagtca aacaaactta accctatcac ttactagctt tgtaatggtg
154560gatatattca ttagcttctc acttgtagaa tgagaataat aatatattat gtctgagact
154620gctagtttta tcccaatatc cattctcccc ttcttcatca gtaaataacc accaaaagtt
154680agctagacac agatgcccag aaaaaagtcc atgttttcag ccctttcttg cagctagatg
154740tgtttatgtg gttaaatact ggacactgag gtcaaaataa aagcggtgtc tgtaacttcc
154800aggaagtatc cttttaaaaa gcagggagaa tgctattttc ttccttatgg ctagaattca
154860gacatgatgg gtggagctag agaagtcatc atgaaccatg aaatgtaaac tgtgttgaga
154920acagtagaac aagagcagag ccctgggtct ctcctgatca tggaatcgcc accaaagccc
154980agggttttag aaaagaaaga aatgcatttc tatttttttg agccattgtt gttacttatt
155040ttctctaact tgctgctgaa cctaacacat gcttaatccg tagcatcttt ttgaggatta
155100aatgggatgg catatgtagt ccactttgta cagggcttgg cacctgggaa tgcaggtgct
155160taatatagga ctgtttttat tactagcata aaatatctcc ttaaaagggt cactataagg
155220gtcagatgaa acctataagt gagcttgctt ttcaatctgt aaatctgaga atcactgtat
155280aaatgttggt cataattatc atcatcacca ttaaattatt acaaggaact ggctaactgc
155340cagcatgctc tactatgatc aatctatatt taggtcaatt aaaaatagat agatacagac
155400ttctttgctt ttgttaggtt tacttaactt tagtcaactg tgaatgattg attataggct
155460tgccattaca ttgttgcttc agagcttaga tgaaacactc atttgggtag tataaagaaa
155520gcttagaact tgatgaggct atgacatctt ttattcattt acttttacct taaaaagttc
155580tctattacaa aaatcaatat agtgtttgtt gttaaaataa attatagaga catgagtaaa
155640gtgaaagaag agagttcacc cccttccttc ttttccatcc cattctttag aattaaccat
155700attacaatgt aaccaattat tccttgtatg taaatatcat ttttatataa tgggatatac
155760acatgtataa tatacatttt ctatttccaa atgaaaaaca aaatcttttt tttgcagaaa
155820gttcaaagtt gttgaaaaga aaatcttttg caacagcttt tttgatttgt tcgtttccca
155880atttaaaatg tctcctgggc atctttccat gtaaatacat attcatcaat cccattttaa
155940aaaccatata cagagtcatt taaacatttt tactaattaa ttattggaaa attgtttgta
156000aaataaagga gactgccctt aaaataaaat agcaacttag ctaggattaa attaggcaat
156060tcagaaaaat gcttagcaaa tgtttggcag attgtaaatg attgataaat aaatattagc
156120cattatgata ttgatgatag tgatgattaa agatgactat ctctctaagc tcactctcaa
156180taaatttctg ttgtcagaga ggtggtttat tttatttaat tgcccaatat gtacatttat
156240atataatatt cccattttat attttcattt taaggacgta cttggctgtg ttatatggtg
156300gaggtgggtg ccgtgttctg gaaggactgt cagactcaag ttggttctct tcaggatctg
156360cccaacccat ggcgacctag tacagtgcca aaattgaaca aaaaaaatac aatccatcca
156420tctgtcagtt agtgaggcag caaagaaggt atttagcttc taggacattt aaaaacctaa
156480gttaattcta tctaaaagaa agacatctgt ggggtgggac tcgcctcctc tgagcctttg
156540agaccagtgg ggatttaaac aggtataaat aatgactctt gaatagccac cagacagcca
156600gtggtgcaac ctactctagg gagatgagtc acaggctgag ccgtgggtag taagtcccta
156660ggagaaggga agaggggtgt ctgggatccc aggaagagca ataaatgcag tctgtcagct
156720ccgcagcaga ggaggggctg tgacagcagc cctcacccag aaggggtgaa tggtaaatct
156780acctcaatag acagtggcct gtgcattgta aatctgatca ttttgctgtg cccaaaacat
156840cttaagtata attataccaa agcctgaaaa aagactggct aaagagaaaa ctaaaataga
156900ttttttaaaa tgcaaacaca caattctgcc tacaaaaatg ttaagtgcca aggaaaatat
156960tgttaaaatc caagaggacc tcctttccta agctgcagtg ttcaggaatt tatatgtata
157020taaggttggt agtgaccatg gacaaatcat tgcctacacc tctgccagaa catctgtcat
157080ttctcatgaa gccggaactt taaatgtcac aactagatca gaaataacat tgatatcaga
157140accatcatgt actttttttc tgctctgcat gtgagcatct ggggtgccta aaatccaacc
157200ctcacagctc ttgaaaagtc ctgtgtcttg cagaagggat gcctttcagt gatgcatccg
157260tgaggaggcg gcgccttttg ataattcaca caaaggtctt gctagtagag ggaggcctta
157320ccttgactgg aggagagacc tacgtttgga tctcggctaa agtatcttcc aagagcaaca
157380ttcttgttag cataacatgc cattttcatg tcctacagat tcatagattt atggagtttt
157440gaactagtta aatgccttag aaatgaccaa tacccaccca ttttgttgat aagggacctg
157500aagcatagag aagtcagata gtttgaatat ttgtccctac ccaaatctca tgttgaattg
157560taatctccag tgttagaggt ggagcctggt gggaggtgtt tggatcatgg gggcaaattc
157620ctcatgaatg gcttgggcca tccccttggt gataagtgag ctctgagtta acactgagat
157680cacatagttt aaaagtgtgt ggcacctccc tcacacctca actctctttc ttttgcttct
157740gtttttgcct catgatgtgc ctactctccc ttgac
157775
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