Isolated genomic polynucleotide fragments that encode human lipoprotein-associated phospholipase A2

Abstract

The invention is directed to isolated genomic polynucleotide fragments that encode human lipoprotein-associated phospholipase A2, vectors and hosts containing the fragment and fragments hybridizing to noncoding regions as well as antisense oligonucleotides to these fragments. The invention is further directed to methods of using these fragments to obtain human lipoprotein-associated phospholipase A2 and to diagnose, treat, prevent and/or ameliorate a pathological disorder.

Description

PRIORITY CLAIM

[0001] This application is a divisional application of application Ser. No. 12/323,364, filed Nov. 25, 2008, which is a continuation application of application Ser. No. 10/161,127, filed May 30, 2002, the contents of which are incorporated herein by reference. application Ser. no. 10/161,127 is claims priority under 35 USC 119(e) to provisional application no. 60/294,404, filed May 30, 2001, the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention is directed to isolated genomic polynucleotide fragments that encode human human lipoprotein-associated phospholipase A2, vectors and hosts containing these fragments and fragments hybridizing to noncoding regions as well as antisense oligonucleotides to these fragments. The invention is further directed to methods of using these fragments to obtain human lipoprotein-associated phospholipase A2 and to diagnose, treat, prevent and/or ameliorate a pathological and/or medical disorders.

BACKGROUND OF THE INVENTION

[0003] Chromosome 18 contains genes encoding, for example, GATA-binding protein-6, cadherin 2, retinoblastoma-binding protein 8, aquaporin-4, H+transporting ATP synthase and lipoprotein-associated phospholipase A2; the last of which will be discussed in more detail below.

[0004] Human Lipoprotein-Associated Phospholipase A2

[0005] Human lipoprotein-associated phospholipase A2, also known in the art as Platelet Activating Factor Acetyl Hydrolase (PAF acetyl hydrolase), is one of a family of enzymes that catalyze release of fatty acids from membrane phospholipids and can thereby initiate synthesis of proinflammatory mediators. During the conversion of LDL to its oxidised form, lipoprotein-associated phospholipase A2 is responsible for hydrolysing the sn-2 ester of oxidatively modified phosphatidylcholine to give lyso-phosphatidylcholine and an oxidatively modified fatty acid. Both of these products of human lipoprotein-associated phospholipase A2 action are potent chemoattractants for circulating monocytes. The enzyme appears to play a central role in the development of atherosclerosis and is regarded as an independent risk factor for coronary artery disease (Caslake et al., Atherosclerosis 150: 413-19, 2000). Specifically, this enzyme is thought to be responsible for the accumulation of cells loaded with cholesterol ester in the arteries, causing the characteristic `fatty streak` associated with the early stages of atherosclerosis. Recently, medicinal chemists have begun to design and prepare lipoprotein-associated phospholipase A2 inhibitors for use in preventing or inhibiting progression of atherosclerotic diseases (See, for example, U.S. Pat. Nos. 5,981,252 and 5,968,818 Boyd et al., Bioorg. Med. Chem. 10: 2557-61, 2000).

[0006] The level of PAF acetylhydrolase has been found to be altered in several disease states. For example, acquired deficiency of PAF acetylhydrolase activity has been reported in patients with systemic lupus erythematosus, stroke and asthma, and increased levels of PAF have been reported in children with acute asthmatic attacks (see, for example, Hiramoto et al., Stroke 28: 2417-2420, 1997; Kruse et al., Am. J. Hum. Genet. 66: 1522-1530, 2000; Stafforini et al., J. Clin. Invest. 97: 2784-2791, 1996). Miwa et al. (1988, J. Clin. Invest. 82:1983-1991) has also described an autosomal recessive form of PAF acetylhydrolase deficiency which has been observed only in the Japanese population. PAF acetylhydrolase activity was absent in 4% of the Japanese population. This inherited deficiency is the result of a point mutation in exon 9 and completely abolishes enzymatic activity. These patients suffer from severe asthma. Results from further studies indicated that the variant allele thr198 was found to be highly associated with total IgE concentrations in an atopic population and with `asthma` in an asthmatic population (Kruse et al., 2000, Am. J. Hum. Genet. 66:1522-1530). The variant allele val379 was found to be highly associated with `specific sensitization` in the atopic population and with `asthma` in the asthmatic population.

[0007] The full length cDNA clone has been isolated (see U.S. Pat. Nos. 5,981,252 and 5,968,818) and the DNA sequence has been determined The complete amino acid sequence has been deduced from the DNA sequence. It was originally thought that the gene encoding the human lipoprotein-associated phospholipase A2 polypeptide was located at gene map locus 6p21.2-p12.

OBJECTS OF THE INVENTION

[0008] Although cDNA encoding the above-disclosed protein, lipoprotein associated phospholipase A2, has been isolated (e.g. see accession no. AX006795 and NM_--005084), its exact location on chromosome 18 and exon/intron/regulatory organization have not been determined Furthermore, genomic DNA encoding the polypeptide has not been isolated. Noncoding sequences play a significant role in regulating the expression of polypeptides as well as the processing of RNA encoding these polypeptides.

[0009] There is clearly a need for obtaining genomic polynucleotide sequences encoding the lipoprotein-associated phospholipase A2 polypeptide. Therefore, it is an object of the invention to isolate such genomic polynucleotide sequences.

[0010] There is also a need to develop means for identifying mutations, duplications, translocations, polysomies and mosaicism as may affect the lipoprotein associated phopholipase A2 gene.

SUMMARY OF THE INVENTION

[0011] The invention is directed to an isolated genomic polynucleotide, said polynucleotide obtainable from human chromosome 18 or chromosome 6 having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of: [0012] (a) a polynucleotide encoding human lipoprotein-associated phospholipase A2 depicted in SEQ ID NO:1. [0013] (b) a polynucleotide consisting of SEQ ID NO:2 and 3, which encodes human lipoprotein-associated phospholipase A2 depicted in SEQ ID NO:1 [0014] (c) a polynucleotide which is a variant of SEQ ID NO:2 or 3; [0015] (d) a polynucleotide which is an allelic variant of SEQ ID NO:2 or 3; [0016] (e) a polynucleotide which encodes a variant of SEQ ID NO:1; [0017] (f) a polynucleotide which hybridizes to any one of the polynucleotides specified in (a)-(e) and [0018] (g) a polynucleotide that is a reverse complement to the polynucleotides specified in (a) to (f) and [0019] (h) containing at least 10 transcription factor binding sites selected from the group consisting

of AP1-C, AP1_Q4, AP4-Q5, DELTAEF1-01, GATA1_--04, GATA1-06, GATA2-02, GATA3_--02, GATA-C, LMO2COM-02, LYF1-01, MYOD_Q6, MZF_--01, NFAT_Q6, NKX25-01, S8-01, SOX5-01, TATA-C, and TCF11-01

[0020] as well as nucleic acid constructs, expression vectors and host cells containing these polynucleotide sequences.

[0021] The polynucleotides of the present invention may be used for the manufacture of a gene therapy for the prevention, treatment or amelioration of a medical condition such as asthma or systemic lupus erythematosus by adding an amount of a composition comprising said polynucleotide effective to prevent, treat or ameliorate said medical condition.

[0022] The invention is further directed to obtaining these polypeptides by [0023] (a) culturing host cells comprising these sequences under conditions that provide for the expression of said polypeptide and [0024] (b) recovering said expressed polypeptide.

[0025] The polypeptides obtained may be used to produce antibodies by [0026] (a) optionally conjugating said polypeptide to a carrier protein; [0027] (b) immunizing a host animal with said polypeptide or peptide-carrier protein conjugate of step (b) with an adjuvant and [0028] (c) obtaining antibody from said immunized host animal.

[0029] The invention is further directed to polynucleotides that hybridize to noncoding regions of said polynucleotide sequences as well as antisense oligonucleotides to these polynucleotides as well as antisense mimetics. The antisense oligonucleotides or mimetics may be used for the manufacture of a medicament for prevention, treatment or amelioration of a medical condition, such as atherosclerosis. The invention is further directed to kits comprising these polynucleotides and kits comprising these antisense oligonucleotides or mimetics.

[0030] In a specific embodiment, the noncoding regions are transcription regulatory regions. The transcription regulatory regions may be used to produce a heterologous peptide by expressing in a host cell, said transcription regulatory region operably linked to a polynucleotide encoding the heterologous polypeptide and recovering the expressed heterologous polypeptide.

[0031] The polynucleotides of the present invention may be used to diagnose a pathological condition such as asthma in a subject comprising [0032] (a) determining the presence or absence of a mutation in the polynucleotides of the present invention and [0033] (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of said mutation.

DETAILED DESCRIPTION OF THE INVENTION

[0034] The invention is directed to isolated genomic polynucleotide fragments that encode human lipoprotein-associated phospholipase A2, which in a specific embodiment is the human lipoprotein-associated phospholipase A2 gene, as well as vectors and hosts containing these fragments and polynucleotide fragments hybridizing to noncoding regions, as well as antisense oligonucleotides to these fragments.

[0035] As defined herein, a "gene" is the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region, as well as intervening sequences (introns) between individual coding segments (exons).

[0036] As defined herein "isolated" refers to material removed from its original environment and is thus altered "by the hand of man" from its natural state. An isolated polynucleotide can be part of a vector, a composition of matter or can be contained within a cell as long as the cell is not the original environment of the polynucleotide.

[0037] The polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA includes genomic DNA and synthetic DNA. The DNA may be double-stranded or single-stranded and if single stranded may be the coding strand or non-coding strand.

[0038] The portion of the human lipoprotein-associated phospholipase A2 gene that contains exons 3 to 11 is 24696 base pairs in length (SEQ ID NO:2) (see Table 1 below for location of exons). Exons 1 and 2 are encoded in SEQ ID NO:3 which is 17889 base pairs in length. The gene is situated in chromosome 18 genomic clone AC008104; contigs 21 (SEQ ID NO:2) and 20 (SEQ ID NO: 3) of gi 8072415.

[0039] The polynucleotides of the invention have at least a 95% identity and may have a 96%, 97%, 98% or 99% identity to the polynucleotides depicted in SEQ ID NO:2 & 3 as well as the polynucleotides in reverse sense orientation, or the polynucleotide sequences encoding the human lipoprotein-associated phospholipase A2 polypeptide depicted in SEQ ID NO:1.

[0040] A polynucleotide having 95% "identity" to a reference nucleotide sequence of the present invention, is identical to the reference sequence except that the polynucleotide sequence may include, on average, up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence, the ORF (open reading frame), or any fragment specified as described herein.

[0041] As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the presence invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.

[0042] If the subject sequence is shorter than the query sequence because of 5' or 3' deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5' and 3' truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5' or 3' ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identify, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence are calculated for the purposes of manually adjusting the percent identity score.

[0043] For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5' end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5' and 3' ends not matched/total numbers of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are made for purposes of the present invention.

[0044] A polypeptide that has an amino acid sequence at least, for example, 95% "identical" to a query amino acid sequence is identical to the query sequence except that the subject polypeptide sequence may include on average, up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the referenced sequence or in one or more contiguous groups within the reference sequence.

[0045] A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Com. App. Biosci. (1990) 6:237-245). In a sequence alignment, the query and subject sequence are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.

[0046] If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.

[0047] The invention also encompasses polynucleotides that hybridize to the polynucleotides depicted in SEQ ID NO: 2 or 3. A polynucleotide "hybridizes" to another polynucleotide, when a single-stranded form of the polynucleotide can anneal to the other polynucleotide under

the appropriate conditions of temperature and solution ionic strength (see Sambrook et al., supra). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a temperature of 42 C, can be used, e.g., 5X SSC, 0.1% SDS, 0.25% milk, and no formamide; or 40% formamide, 5X SSC, 0.5% SDS). Moderate stringency hybridization conditions correspond to a higher temperature of 55 C, e.g., 40% formamide, with 5X or 6X SCC. High stringency hybridization conditions correspond to the highest temperature of 65 C, e.g., 50% formamide, 5X or 6X SCC. Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T_m for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher T_m) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA.

[0048] Polynucleotide and Polypeptide Variants

[0049] The invention is directed to both polynucleotide and polypeptide variants. A "variant" refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar and in many regions, identical to the polynucleotide or polypeptide of the present invention.

[0050] The variants may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred.

[0051] The invention also encompasses allelic variants of said polynucleotides. An allelic variant denotes any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

[0052] The amino acid sequences of the variant polypeptides may differ from the amino acid sequences depicted in SEQ ID NO:1 by an insertion or deletion of one or more amino acid residues and/or the substitution of one or more amino acid residues by different amino acid residues. Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

[0053] Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine) Amino acid substitutions which do not generally alter the specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, as well as these in reverse.

[0054] Noncoding Regions

[0055] The invention is further directed to polynucleotide fragments containing or hybridizing to noncoding regions of the human lipoprotein-associated phospholipase A2 gene. These include but are not limited to an intron, a 5'-non-coding region, a 3'-non-coding region and splice junctions (see Table 1), as well as transcription factor binding sites (see Table 2). The polynucleotide fragments may be a short polynucleotide fragment which is between about 8 nucleotides to about 40 nucleotides in length. Such shorter fragments may be useful for diagnostic purposes. Such short polynucleotide fragments are also preferred with respect to polynucleotides containing or hybridizing to polynucleotides containing splice junctions. Alternatively larger fragments, e.g., of about 50, 150, 500, 600 or about 2000 nucleotides in length may be used.

TABLE-US-00001 TABLE 1 Exon/Intron Regions of the Liporotein Related Phospholipase A2 Gene in Contigs 20 (17889 base pairs) and 21 (24696 base pairs) of AC008104, gi 8072415 (reverse strand coding). Exon Nucleotide no. Peptide Amino Acid no. Contig (stop codon) 12311-12313 21 11 12314-12448 441-397 21 10 12905-13054 396-347 21 9 15744-15914 346-290 21 8 17080-17169 289-260 21 7 18296-18409 259-222 21 6 19247-19372 221-180 21 5 20022-20090 179-157 21 4 22213-22305 156-126 21 3 24136-24279 125-78 21 2 4-129 77-36 20 1 5819-5923 35-1 20

TABLE-US-00002 TABLE 2 Transcription Factor Binding Sites on the Lipoprotein Realated Phospholipase A2 Gene. Transcription Factor No. of Binding Sites AP1_C 14 AP1_Q4 4 AP4_Q5 4 DELTAEF1_01 5 GATA1_04 7 GATA1_06 8 GATA2_02 5 GATA3_02 4 GATA_C 7 LMO2COM_02 4 LYF1_01 9 MYOD_Q6 9 MZF1_01 14 NFAT_Q6 10 NKX25_01 16 S8_01 7 SOX5_01 2 TATA_C 7 TCF11_01 46

[0056] In a specific embodiment, such noncoding sequences are expression control sequences. These include but are not limited to DNA regulatory sequences, such as promoters, enhancers, repressors, terminators, and the like, that provide for the regulation of expression of a coding sequence in a host cell. In eukaryotic cells, polyadenylation signals are also control sequences.

[0057] In a more specific embodiment of the invention, the expression control sequences may be operatively linked to a polynucleotide encoding a heterologous polypeptide. Such expression control sequences may be about 50-200 nucleotides in length and specifically about 50, 100, 200, 500, 600, 1000 or 2000 nucleotides in length. A transcriptional control sequence is "operatively linked" to a polynucleotide encoding a heterologous polypeptide sequence when the expression control sequence controls and regulates the transcription and translation of that polynucleotide sequence. The term "operatively linked" includes having an appropriate start signal (e.g., ATG) in front of the polynucleotide sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the polynucleotide sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted upstream (5') of and in reading frame with the gene.

[0058] The invention is further directed to antisense oligonucleotides and mimetics to these polynucleotide sequences. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription or RNA processing (triple helix (see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)), thereby preventing transcription and the production of said polypeptides.

[0059] Expression of Polypeptides

[0060] Isolated Polynucleotide Sequences

[0061] The human chromosome 18 genomic clone of accession number AC008104 has been discovered to contain the human lipoprotein-associated phospholipase A2 gene by Genscan analysis (Burge et al., 1997, J. Mol. Biol. 268:78-94), BLAST2 and TBLASTN analysis (Altschul et al., 1997, Nucl. Acids Res. 25:3389-3402), in which the sequences of AC008104 were compared to the human lipoprotein-associated phospholipase A2 cDNA sequence, accession number NM_--005084 or AX006795.

[0062] The cloning of the nucleic acid sequences of the present invention from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleic acid sequence-based amplification (NASBA) or long range PCR may be used. In a specific embodiment, 5'- or 3'-non-coding portions of the gene may be identified by methods including but are not limited to, filter probing, clone enrichment using specific probes and protocols similar or identical to 5'- and 3'-"RACE" protocols which are well known in the art. For instance, a method similar to 5'-RACE is available for generating the missing 5'end of a desired full-length transcript. (Fromont-Racine et al., 1993, Nucl. Acids Res. 21:1683-1684).

[0063] Once the DNA fragments are generated, identification of the specific DNA fragment containing the desired human lipoprotein-associated phospholipase A2 gene may be accomplished in a number of ways. For example, if an amount of a portion of a human lipoprotein-associated phospholipase A2 gene or its specific RNA, or a fragment thereof, is available and can be purified and labeled, the generated DNA fragments may be screened by nucleic acid hybridization to the labeled probe (Benton and Davis, 1977, Science 196:180; Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). The present invention provides such nucleic acid probes, which can be conveniently prepared from the specific sequences disclosed herein, e.g., a hybridizable probe having a nucleotide sequence corresponding to at least a 10, and preferably a 15, nucleotide fragment of the sequences depicted in SEQ ID NO:2 or b. Preferably, a fragment is selected that is highly unique to the polypeptides of the invention. Those DNA fragments with substantial homology to the probe will hybridize. As noted above, the greater the degree of homology, the more stringent hybridization conditions can be used. In one embodiment, low stringency hybridization conditions are used to identify a homologous human lipoprotein-associated phospholipase A2 polynucleotide. However, in a preferred aspect, and as demonstrated experimentally herein, a nucleic acid encoding a polypeptide of the invention will hybridize to a nucleic acid derived from the polynucleotide sequence depicted in SEQ ID NO:2 or b or a hybridizable fragment thereof, under moderately stringent conditions; more preferably, it will hybridize under high stringency conditions.

[0064] Alternatively, the presence of the gene may be detected by assays based on the physical, chemical, or immunological properties of its expressed product. For example, cDNA clones, or DNA clones which hybrid-select the proper mRNAs, can be selected which produce a protein that, e.g., has similar or identical electrophoretic migration, isoelectric focusing behavior, proteolytic digestion maps, or antigenic properties as known for the human lipoprotein-associated phospholipase A2 polynucleotide.

[0065] A gene encoding human lipoprotein-associated phospholipase A2 polypeptide can also be identified by mRNA selection, i.e., by nucleic acid hybridization followed by in vitro translation. In this procedure, fragments are used to isolate complementary mRNAs by hybridization Immunoprecipitation analysis or functional assays of the in vitro translation products of the products of the isolated mRNAs identifies the mRNA and, therefore, the complementary DNA fragments, that contain the desired sequences.

[0066] Nucleic Acid Constructs

[0067] The present invention also relates to nucleic acid constructs comprising a polynucleotide sequence containing the exon/intron segments of the human lipoprotein-associated phospholipase A2 gene operably linked to one or more control sequences which direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. Expression will be understood to include any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0068] The invention is further directed to a nucleic acid construct comprising expression control sequences derived from SEQ ID NO: 2 or 3 and a heterologous polynucleotide sequence.

[0069] "Nucleic acid construct" is defined herein as a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature. The term nucleic acid construct is synonymous with the term expression cassette when the nucleic acid construct contains all the control sequences required for expression of a coding sequence of the present invention. The term "coding sequence" is defined herein as a portion of a nucleic acid sequence which directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by a ribosome binding site (prokaryotes) or by the ATG start codon (eukaryotes) located just upstream of the open reading frame at the 5'-end of the mRNA and a transcription terminator sequence located just downstream of the open reading frame at the 3'-end of the mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

[0070] The isolated polynucleotide of the present invention may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the nucleic acid sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying nucleic acid sequences utilizing recombinant DNA methods are well known in the art.

[0071] The control sequence may be an appropriate promoter sequence, a nucleic acid sequence which is recognized by a host cell for expression of the nucleic acid sequence. The promoter sequence contains transcriptional control sequences which regulate the expression of the polynucleotide. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0072] Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention, especially in a bacterial host cell, are the promoters obtained from the E. coli lac operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and the prokaryotic beta-lactamase gene (Villa-Komaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proceedings of the National Academy of Sciences USA 80: 21-25). Further promoters are described in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

[0073] Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium oxysporum trypsin-like protease (WO 96/00787), NA2-tpi (a hybrid of the promoters from the genes encoding Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof.

[0074] In a yeast host, useful promoters are obtained from the Saccharomyces cerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiae galactokinase gene (GAL1), the Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP), and the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.

[0075] The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3'-terminus of the nucleic acid sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

[0076] Preferred terminators for filamentous fungal host cells are obtained from the genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

[0077] Preferred terminators for yeast host cells are obtained from the genes encoding Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), or Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.

[0078] The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5'-terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used in the present invention.

[0079] Preferred leaders for filamentous fungal host cells are obtained from the genes encoding Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0080] Suitable leaders for yeast host cells are obtained from the Saccharomyces cerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene, the Saccharomyces cerevisiae alpha-factor, and the Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP).

[0081] The control sequence may also be a polyadenylation sequence, a sequence which is operably linked to the 3'-terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the present invention.

[0082] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, and Aspergillus niger alpha-glucosidase.

[0083] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

[0084] The control sequence may also be a signal peptide coding region, which codes for an amino acid sequence linked to the amino terminus of the polypeptide which can direct the encoded polypeptide into the cell's secretory pathway. The 5'-end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide. Alternatively, the 5'-end of the coding sequence may contain a signal peptide coding region which is foreign to the coding sequence. The foreign signal peptide coding region may be required where the coding sequence does not normally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to obtain enhanced secretion of the polypeptide. However, any signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice may be used in the present invention.

[0085] An effective signal peptide coding region for bacterial host cells is the signal peptide coding region obtained from the maltogenic amylase gene from Bacillus NCIB 11837, the Bacillus stearothermophilus alpha-amylase gene, the Bacillus licheniformis subtilisin gene, the Bacillus licheniformis beta-lactamase gene, the Bacillus stearothermophilus neutral proteases genes (nprT, nprS, nprM), or the Bacillus subtilis prsA gene. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

[0086] An effective signal peptide coding region for filamentous fungal host cells is the signal peptide coding region obtained from the Aspergillus oryzae TAKA amylase gene, Aspergillus niger neutral amylase gene, Aspergillus niger glucoamylase gene, Rhizomucor miehei aspartic proteinase gene, Humicola lanuginosa cellulase gene, or Humicola lanuginosa lipase gene.

[0087] Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding regions are described by Romanos et al., 1992, supra.

[0088] The control sequence may also be a propeptide coding region, which codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from the Bacillus subtilis alkaline protease gene (aprE), the Bacillus subtilis neutral protease gene (nprT), the Saccharomyces cerevisiae alpha-factor gene, the Rhizomucor miehei aspartic proteinase gene, or the Myceliophthora thermophila laccase gene (WO 95/33836).

[0089] Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.

[0090] It may also be desirable to add regulatory sequences which allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems would include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences. Other examples of regulatory sequences are those which allow for gene amplification. In eukaryotic systems, these include the dihydrofolate reductase gene which is amplified in the presence of methotrexate, and the metallothionein genes which are amplified with heavy metals. In these cases, the nucleic acid sequence encoding the polypeptide would be operably linked with the regulatory sequence.

[0091] Expression Vectors

[0092] The present invention also relates to recombinant expression vectors comprising a nucleic acid sequence of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the polypeptide at such sites. Alternatively, the polynucleotide of the present invention may be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

[0093] The recombinant expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleic acid sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.

[0094] The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.

[0095] The vectors of the present invention preferably contain one or more selectable markers which permit easy selection of transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. A selectable marker for use in a filamentous fungal host cell may be selected from the group including, but not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5O-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), as well as equivalents from other species. Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.

[0096] The vectors of the present invention preferably contain an element(s) that permits stable integration of the vector into the host cell genome or autonomous replication of the vector in the cell independent of the genome of the cell.

[0097] For integration into the host cell genome, the vector may rely on the polynucleotide sequence encoding the polypeptide or any other element of the vector for stable integration of the vector into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell. The additional polynucleotide sequences enable the vector to be integrated into the host cell genome at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleic acid sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

[0098] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB 110, pE194, pTA1060, and pAM§1 permitting replication in Bacillus. Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin of replication may be one having a mutation which makes its functioning temperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75: 1433).

[0099] More than one copy of a polynucleotide sequence of the present invention may be inserted into the host cell to increase production of the gene product. An increase in the copy number of the polynucleotide sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

[0100] The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).

[0101] Host Cells

[0102] The present invention also relates to recombinant host cells, comprising a nucleic acid sequence of the invention, which are advantageously used in the recombinant production of the polypeptides. A vector comprising a nucleic acid sequence of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.

[0103] The host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-unicellular microorganism, e.g., a eukaryote. Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans or Streptomyces murinus, or gram negative bacteria such as E. coli and Pseudomonas sp. In a preferred embodiment, the bacterial host cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cell. In another preferred embodiment, the Bacillus cell is an alkalophilic Bacillus.

[0104] The introduction of a vector into a bacterial host cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), using competent cells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169: 5771-5278).

[0105] The host cell may be a eukaryote, such as a mammalian cell (e.g., human cell), an insect cell, a plant cell or a fungal cell. Mammalian host cells that could be used include but are not limited to human Hela, 293, H9 and Jurkat cells, mouse NIH3t3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells and Chinese Hamster ovary (CHO) cells. These cells may be transfected with a vector containing a transcriptional regulatory sequence, a protein coding sequence and transcriptional termination sequences. Alternatively, the polypeptide can be expressed in stable cell lines containing the polynucleotide integrated into a chromosome. The co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells.

[0106] The host cell may be a fungal cell. "Fungi" as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra). The fungal host cell may also be a yeast cell. "Yeast" as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980). The fungal host cell may also be a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.

[0107] Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81: 1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.

[0108] Methods of Production

[0109] The present invention also relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a host cell under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

[0110] In the production methods of the present invention, the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.

[0111] The polypeptides may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. In a specific embodiment, an enzyme assay may be used to determine the activity of the polypeptide. For example, human lipoprotein-associated phospholipase A2 can be assayed by its ability to release fatty acids from phospholipids. Caslake et al (Atherosclerosis 150: 413-19, 2000) have described a specific immunoassay for the enzyme.

[0112] The resulting polypeptide may be recovered by methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

[0113] The polypeptides of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).

[0114] Antibodies

[0115] According to the invention, the human lipoprotein-associated phospholipase A2 polypeptide produced according to the method of the present invention may be used as an immunogen to generate any of these antibodies. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library.

[0116] Various procedures known in the art may be used for the production of antibodies. For the production of antibody, various host animals can be immunized by injection with the polypeptide thereof, including but not limited to rabbits, mice, rats, sheep, goats, etc. In one embodiment, the polypeptide or fragment thereof can optionally be conjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

[0117] For preparation of monoclonal antibodies directed toward the human lipoprotein-associated phospholipase A2 polypeptide, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals utilizing recent technology (PCT/US90/02545). According to the invention, human antibodies may be used and can be obtained by using human hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or by transforming human B cells with EBV virus in vitro (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96). In fact, according to the invention, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, J. Bacteriol. 159-870; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing the genes from a mouse antibody molecule specific for the human lipoprotein-associated phospholipase A2 polypeptide together with genes from a human antibody molecule of appropriate biological activity can be used; such antibodies are within the scope of this invention.

[0118] According to the invention, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce polypeptide-specific single chain antibodies. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al., 1989, Science 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for the human lipoprotein-associated phospholipase A2 polypeptide.

[0119] Antibody fragments which contain the idiotype of the antibody molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')₂ fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab')₂, fragment,

and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.

[0120] In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitin reactions,

immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. For example, to select antibodies which recognize a specific epitope of a particular polypeptide, one may assay generated hybridomas for a product which binds to a particular polypeptide fragment containing such epitope. For selection of an antibody specific to a particular polypeptide from a particular species of animal, one can select on the basis of positive binding with the polypeptide expressed by or isolated from cells of that species of animal.

[0121] Immortal, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al., "Hybridoma Techniques" (1980); Hammerling et al.,

"Monoclonal Antibodies And T-cell Hybridomas" (1981); Kennett et al.,"Monoclonal Antibodies" (1980); see also U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500; 4,491,632; 4,493,890.

[0122] Uses of Polynucleotides

[0123] Diagnostics

[0124] Polynucleotides containing noncoding regions of SEQ ID NO:2 or 3 may be used as probes for detecting mutations from samples from a patient. Genomic DNA may be isolated from the patient. A mutation(s) may be detected by Southern blot analysis, specifically by hybridizing restriction digested genomic DNA to various probes and subjecting to agarose electrophoresis. Alternatively, these polynucleotides may be used as PCR primers and be used to amplify the genomic DNA isolated from the patients. Additionally, primers may be obtained by routine or long range PCR that yield products containing contiguous intron/exon sequence and products containing more than one exon with intervening intron. The sequence of the amplified genomic DNA from the patient may be determined using methods known in the art. Such probes may be between 10-100 nucleotides in length and may preferably be between 20-50 nucleotides in length.

[0125] Thus the invention is thus directed to kits comprising these polynucleotide probes. In a specific embodiment, these probes are labeled with a detectable substance.

[0126] Antisense Oligonucleotides and Mimetics

[0127] The antisense oligonucleotides or mimetics of the present invention may be used to decrease levels of a polypeptide. For example, human lipoprotein-associated phospholipase A2 has been found to be associated with atherosclerosis and diabetes. Therefore, the human lipoprotein-associated phospholipase A2 antisense oligonucleotides of the present invention could be used to inhibit progression of atherosclerosis, including coronary artery disease.

[0128] The antisense oligonucleotides of the present invention may be formulated into pharmaceutical compositions. These compositions may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal

delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.

[0129] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0130] Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers ,dispersing aids or binders may be desirable.

[0131] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0132] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

[0133] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active

ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0134] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0135] In one embodiment of the present invention, the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.

[0136] The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to

several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀ as found to be effective in in vitro and in vivo animal models.

[0137] In general, dosage is from 0.01 ug to 10 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and

concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 10 g per kg of body weight, once or more daily, to once every 20 years.

[0138] Gene Therapy

[0139] Human lipoprotein-associated phospholipase A2 deficiency states have been described (Yamada et al., Metabolism 47: 177-81, 1998). Therefore, the human lipoprotein-associated phospholipase A2 gene may be used to via gene therapy to correct any such deficiency state or disorders associated with such deficiency states (e.g., asthma).

[0140] As described herein, the polynucleotide of the present invention may be introduced into a patient's cells for therapeutic uses. As will be discussed in further detail below, cells can be transfected using any appropriate means, including viral vectors, as shown by the example, chemical transfectants, or physico-mechanical methods such as electroporation and direct diffusion of DNA. See, for example, Wolff, Jon A, et al., "Direct gene transfer into mouse muscle in vivo," Science, 247, 1465-1468, 1990; and Wolff, Jon A, "Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs," Nature, 352, 815-818, 1991. As used herein, vectors are agents that transport the gene into the cell without degradation and include a promoter yielding expression of the gene in the cells into which it is delivered. As will be discussed in further detail below, promoters can be general promoters, yielding expression in a variety of mammalian cells, or cell specific, or even nuclear versus cytoplasmic specific. These are known to those skilled in the art and can be constructed using standard molecular biology protocols. Vectors have been divided into two classes:

[0141] a) Biological agents derived from viral, bacterial or other sources.

[0142] b) Chemical physical methods that increase the potential for gene uptake, directly introduce the gene into the nucleus or target the gene to a cell receptor.

[0143] Biological Vectors

[0144] Viral vectors have higher transaction (ability to introduce genes) abilities than do most chemical or physical methods to introduce genes into cells. Vectors that may be used in the present invention include viruses, such as adenoviruses, adeno associated virus (AAV), vaccinia, herpesviruses, baculoviruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression. Polynucleotides are inserted into vector genomes using methods well known in the art.

[0145] Retroviral vectors are the vectors most commonly used in clinical trials, since they carry a larger genetic payload than other viral vectors. However, they are not useful in non-proliferating cells. Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation. Pox viral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature.

[0146] Examples of promoters are SP6, T4, T7, SV40 early promoter, cytomegalovirus (CMV) promoter, mouse mammary tumor virus (MMTV) steroid-inducible promoter, Moloney murine leukemia virus (MMLV) promoter, phosphoglycerate kinase (PGK) promoter, and the like. Alternatively, the promoter may be an endogenous adenovirus promoter, for example the E1 a promoter or the Ad2 major late promoter (MLP). Similarly, those of ordinary skill in the art can construct adenoviral vectors utilizing endogenous or heterologous poly A addition signals.

[0147] Plasmids are not integrated into the genome and the vast majority of them are present only from a few weeks to several months, so they are typically very safe. However, they have lower expression levels than retroviruses and since cells have the ability to identify and eventually shut down foreign gene expression, the continuous release of DNA from the polymer to the target cells substantially increases the duration of functional expression while maintaining the benefit of the safety associated with non-viral transfections.

[0148] Chemical/Physical Vectors

[0149] Other methods to directly introduce genes into cells or exploit receptors on the surface of cells include the use of liposomes and lipids, ligands for specific cell surface receptors, cell receptors, and calcium phosphate and other chemical mediators, microinjections directly to single cells, electroporation and homologous recombination. Liposomes are commercially available from Gibco BRL, for example, as LIPOFECTIN.sup... and LIPOFECTACE.sup..., which are formed of cationic lipids such as N-[1-(2,3 dioleyloxy)-propyl]-n,n,n-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB). Numerous methods are also published for making liposomes, known to those skilled in the art. For example, Nucleic acid-Lipid Complexes--Lipid carriers can be associated with naked nucleic acids (e.g., plasmid DNA) to facilitate passage through cellular membranes. Cationic, anionic, or neutral lipids can be used for this purpose. However, cationic lipids are preferred because they have been shown to associate better with DNA which, generally, has a negative charge. Cationic lipids have also been shown to mediate intracellular delivery of plasmid DNA (Felgner and Ringold, Nature 337:387 (1989)). Intravenous injection of cationic lipid-plasmid complexes into mice has been shown to result in expression of the DNA in lung (Brigham et al., Am. J. Med. Sci.298:278 (1989)). See also, Osaka et al., J. Pharm. Sci. 85(6):612-618 (1996); San et al., Human Gene Therapy 4:781-788 (1993); Senior et al., Biochemica et Biophysica Acta 1070:173-179 (1991); Kabanov and Kabanov, Bioconjugate Chem. 6:7-20 (1995); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Behr, J-P., Bioconjugate Chem 5:382-389 (1994); Behr et al., Proc. Natl. Acad. Sci., USA 86:6982-6986 (1989); and Wyman et al., Biochem. 36:3008-3017 (1997).

[0150] Cationic lipids are known to those of ordinary skill in the art. Representative cationic lipids include those disclosed, for example, in U.S. Pat. No. 5,283,185; and e.g., U.S. Pat. No. 5,767,099. In a preferred embodiment, the cationic lipid is N⁴ -spermine cholesteryl carbamate (GL-67) disclosed in U.S. Pat. No. 5,767,099. Additional preferred lipids include N4 Dspermidine cholestryl carbamate (GL-53) and 1-(N4 -spermind) -2,3-dilaurylglycerol carbamate (GL-89).

[0151] The vectors of the invention may be targeted to specific cells by linking a targeting molecule to the vector. A targeting molecule is any agent that is specific for a cell or tissue type of interest, including for example, a ligand, antibody, sugar, receptor, or other binding molecule.

[0152] Invention vectors may be delivered to the target cells in a suitable composition, either alone, or complexed, as provided above, comprising the vector and a suitably acceptable carrier. The vector may be delivered to target cells by methods known in the art, for example, intravenous, intramuscular, intranasal, subcutaneous, intubation, lavage, and the like. The vectors may be delivered via in vivo or ex vivo applications. In vivo applications involve the direct administration of an adenoviral vector of the invention formulated into a composition to the cells of an individual. Ex vivo applications involve the transfer of the adenoviral vector directly to harvested autologous cells which are maintained in vitro, followed by readministration of the transduced cells to a recipient.

[0153] In a specific embodiment, the vector is transfected into antigen-presenting cells. Suitable sources of antigen-presenting cells (APCs) include, but are not limited to, whole cells such as dendritic cells or macrophages; purified MHC class I molecule complexed to beta2-microglobulin and foster antigen-presenting cells. In a specific embodiment, the vectors of the present invention may be introduced into T cells or B cells using methods known in the art (see, for example, Tsokos and Nepom, 2000, J. Clin. Invest. 106:181-183).

[0154] The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0155] Various references are cited herein, the disclosure of which are incorporated by reference in their entireties.

Sequence CWU 1

21441PRTHomo sapiens 1Met Val Pro Pro Lys Leu His Val Leu Phe Cys Leu Cys Gly Cys Leu1 5 10 15Ala Val Val Tyr Pro Phe Asp Trp Gln Tyr Ile Asn Pro Val Ala His 20 25 30Met Lys Ser Ser Ala Trp Val Asn Lys Ile Gln Val Leu Met Ala Ala 35 40 45Ala Ser Phe Gly Gln Thr Lys Ile Pro Arg Gly Asn Gly Pro Tyr Ser 50 55 60Val Gly Cys Thr Asp Leu Met Phe Asp His Thr Asn Lys Gly Thr Phe65 70 75 80Leu Arg Leu Tyr Tyr Pro Ser Gln Asp Asn Asp Arg Leu Asp Thr Leu 85 90 95Trp Ile Pro Asn Lys Glu Tyr Phe Trp Gly Leu Ser Lys Phe Leu Gly 100 105 110Thr His Trp Leu Met Gly Asn Ile Leu Arg Leu Leu Phe Gly Ser Met 115 120 125Thr Thr Pro Ala Asn Trp Asn Ser Pro Leu Arg Pro Gly Glu Lys Tyr 130 135 140Pro Leu Val Val Phe Ser His Gly Leu Gly Ala Phe Arg Thr Leu Tyr145 150 155 160Ser Ala Ile Gly Ile Asp Leu Ala Ser His Gly Phe Ile Val Ala Ala 165 170 175Val Glu His Arg Asp Arg Ser Ala Ser Ala Thr Tyr Tyr Phe Lys Asp 180 185 190Gln Ser Ala Ala Glu Ile Gly Asp Lys Ser Trp Leu Tyr Leu Arg Thr 195 200 205Leu Lys Gln Glu Glu Glu Thr His Ile Arg Asn Glu Gln Val Arg Gln 210 215 220Arg Ala Lys Glu Cys Ser Gln Ala Leu Ser Leu Ile Leu Asp Ile Asp225 230 235 240His Gly Lys Pro Val Lys Asn Ala Leu Asp Leu Lys Phe Asp Met Glu 245 250 255Gln Leu Lys Asp Ser Ile Asp Arg Glu Lys Ile Ala Val Ile Gly His 260 265 270Ser Phe Gly Gly Ala Thr Val Ile Gln Thr Leu Ser Glu Asp Gln Arg 275 280 285Phe Arg Cys Gly Ile Ala Leu Asp Ala Trp Met Phe Pro Leu Gly Asp 290 295 300Glu Val Tyr Ser Arg Ile Pro Gln Pro Leu Phe Phe Ile Asn Ser Glu305 310 315 320Tyr Phe Gln Tyr Pro Ala Asn Ile Ile Lys Met Lys Lys Cys Tyr Ser 325 330 335Pro Asp Lys Glu Arg Lys Met Ile Thr Ile Arg Gly Ser Val His Gln 340 345 350Asn Phe Ala Asp Phe Thr Phe Ala Thr Gly Lys Ile Ile Gly His Met 355 360 365Leu Lys Leu Lys Gly Asp Ile Asp Ser Asn Val Ala Ile Asp Leu Ser 370 375 380Asn Lys Ala Ser Leu Ala Phe Leu Gln Lys His Leu Gly Leu His Lys385 390 395 400Asp Phe Asp Gln Trp Asp Cys Leu Ile Glu Gly Asp Asp Glu Asn Leu 405 410 415Ile Pro Gly Thr Asn Ile Asn Thr Thr Asn Gln His Ile Met Leu Gln 420 425 430Asn Ser Ser Gly Ile Glu Lys Tyr Asn 435 440242611DNAHomo sapiens 2cttttctttt ttttctctct ctctctcttt ctttcgttcc ttctttcttt cttttgaggc 60ggagtctcat tttgtcgcca ggctggagtg cagaggcgcg atctcgactc actgcaacct 120ctgtctcccg ggttcaagca attctcctgc ctcaacttcc cgagtagctg ggactgctgg 180cgtgccgtat cacgctcggc taattttttt gaatttttag tagagacggg gtttcgctgt 240gttcgccaca atggtctcga tctcctgacc tcgtgattcg cccgcctccg cctcccaaaa 300tgctgggatt acaggcatga gccaccgcgc ccgccccgga gttttcttat ctgtagatct 360cctacaggga caatctctct ctccgtctct ctctctctct ctctctctct ctctctctct 420ctctctctct ctctctctct ctctcacaca cacacacacc actacacacc tctcactctg 480cctcctctct ctctctccct ccgctctctc tctcacacac ccttctccct ctctccctct 540ctctcactct ccctttgagg agtattgcgt ttttggagcc cagttatgcg tggctgctgt 600aagaacaaaa caaagcactc tttgtcctac ctggagcatc cccctgccgg ccagataaga 660tcacagctgt gtcaccaagt caagggagag gctcattttt aaaaaattaa gcaaaatcag 720aaaacagaga acactgtctt cctcatcatc tcctggagct tgtaagagaa atttctgtca 780ccaaaaacga aaagttcggg cttttattca tgcctgtggt tcttgggatt actgcagtgg 840tgatgaggaa ggagaggtgc gtatggctca taaatggcag cttgccacct ttaaaaggca 900cacggattca gatttgggat gtgttaacaa atcaggcttg cacaaattac ttttgatcca 960aatgagaatg ctgagctggg agactggtat atgggtagga gctggaaaat gtatcccctg 1020aggtgttggc aatcccaaga ggctcagcac gaaaccttga gcttacattg aattcagaga 1080tctgagcatt aagcactggc ctgatcaaga agaagcagat ggccttctgg tcacgaggca 1140ctggtgagcc tccatcaccc aggcagattc cctcacagag gaattctgct ggccctgcag 1200aaagtggtga gacagcgcac caccttggca ttttccagca ggcctacttc agctccttgc 1260acttgggcac tgaaagtgct ttcccctccc cagatatttg tgcaccagaa gctgctgctg 1320gccatgtggt gatgtctcaa tgtctttctt cattgaactg ggcccttcat acccaaggct 1380ggcaccatag tctgtgactg cctactgtac agccagcaag actgaaggag ttgcagcatt 1440aggagtgttg tgaatctggt tcagccactc attttacagt tgagaaatcc cttctgaggt 1500gtggaagggc ttgcttagca ccgcaggact tttccctcag acagaacctg ggacagctgg 1560gtcctcattt cctctccatt gtgtctctgt cccctttgca cttttgttgt gaggaacata 1620catgggccta aacacattcc ctcactctca cctttgcaca tgaatatgac ttaccacact 1680ctttgaagct ctgtcagcac agaacattaa gaaaagtgct aactgggtac ctgagcagct 1740ggagtgggag cccctgggaa agtcagttta ggagtcccct ttatcttcct tttcctgata 1800cccaagaaga tggctatttc ctggaaagga aggtggatat tggattattt acactctgat 1860tcctcttaag cctaattttt tttcaagatt cgtgataata agcctgactg acgtctcaag 1920atgttgagaa atttccataa aataaaagca ttgaagagaa agtactgagt gaagggaaat 1980tgaagagaaa cattcagttc taaaattgta ttgtttcaga atatgattct gggttcttac 2040tgactttctt catgacgcca gccacctgtt agacacatta tgtgccctca ctgtcatttt 2100gctcaatttt atgggcgtga ctcaacacta acagaaacag gctaatacat acaaacatgt 2160aggtttttgg tttttaaaga aacttagcag ttgaggagaa ataaaaagtc tattgtaaga 2220gtagatgctc attgaattaa gtgggctagt tgattcctag tagaacttgt attcattcat 2280tcattcactt caaaaaatat ttattgatca gatcctaagt gccaggtact gagtacagca 2340cagttagtaa gatagatatg ctctctgctc tcacagtgct cacattctag aattgaggga 2400tgaacagggt gaattcatat aatgagaaga aaagcaagca gattgctgca gtatagtggt 2460gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtatgtgtgt gttggatata tgggaggggc 2520ctagcttctt ggattggatt gtgaggagag acttctctgc aagaaggcct ggagatgaaa 2580cctcaggaag atgggcagac gtccctgcag agggaaccac aagcacaaat gtcctgctcc 2640tgggaacagg tgtgatgtgt tggaggcaca gttggaatgc cagtgtggct ggagttaggt 2700gagggagaag gagagttgga ggagtgagct ttaggggcag gagccaggtc aggaagaccc 2760tcataggcca agggaaacat ttggatttta ctctttacaa tgagagaagc caaaagagag 2820tttgaagcag agatgtgaca taattgattt actttataaa gagatgactc tgggtgctat 2880gtggagaatg gattacaggg gaaggagagg ggacagaaga aggccagaag cgagtttact 2940gatggggggt ggtagcagta gatgggcgta ggctattttt tggagttaca gtgggaagtt 3000aatgtttctc tgtctcgaat ttcccatcta caaaatggga atgagattgc tatacctggt 3060ccacttgtaa aaactaataa acaaatgaat atgccctgaa gcataatgta agacattatt 3120accattatta tttgctcata ttgtattact ttgaattccc tgtgctttat cttaaaattt 3180attttaataa atttgatgtc ttattatatt attttcagta aacctaattt attatacata 3240aaagtatctt tctctgttcg gagaactgga aactcttaac taagagttac caataaaaac 3300aagtcgtcac aaaatgcagg gaaattattg ctaaaataat agctggctta atttcaaaag 3360ttgaatatgg tcagataaaa attcaaaaat agaataatta aagctgggaa tgggaaaaaa 3420atattttgga cagatttgcc tttgtcctgg ctgtggtcat gcagatatct gtgagtgcaa 3480agatttgtag ataactgaaa atatctatat ggcagcagaa tttctggaga agtctgtgga 3540ccagagttgg gtagggttgt ttcccttagt gaagcattct ctctcagagg aagctcttga 3600attttctctc gtttatcact aagttttaga gtgtcaggaa gttttaaagt tgtttaccaa 3660ctaggatagt gacgttcttc gcagagacag gacaagtaac tgccacaata aggcaactct 3720atttctcttt cctgactcac tctgatgtgc aagttactag aaggagatat gcctgcttag 3780aggaaaaaaa gctcttcctc atttgtattg tgggcagcag aaatgtcagc tcgtgactta 3840atagtcccta ggtcatgtta acttgccaga tccccttaat tatttattgg tcagtgtgtc 3900tgaatttcag cttgaagtgg ctagtctggt gattcaaatt gcagacaaaa tgtgatagta 3960attctgggat ctgtatgatt tatctcttgc catatcaacc accatatttt tcttagggct 4020ggagagtggg ggtgtttggg tatcttaaag ggagaggtta catctctcct tgaagccaga 4080aatctatttg tttcactcac gttttgaaag gttgaattag ttgctccttt aaaaatcagt 4140ggatattaca tacaaatgta attttccagt ttttcttgga agcaatcaga tgatctagtg 4200acatggagct gccattccta catggaagca attaactgga atagagtggt agttgccttc 4260tttggatggt ccagtttgca ccatccaaat agaataagga cagttcacgc cagtccttat 4320tctattgcat gaacctgttt gattcattta aattacctgt ctggcccttg tagacttttg 4380atttttaact tcaggtttag ggcaatcaag tgtaacctgt ctgttatact ttcaccttca 4440tgtaatgcag gaatctatat tccagtatcc ctgctggtag tccccaaacc tgtactttaa 4500ctgctcctct gataaagaag ataatgctaa ttgttgaaaa gctactgttg atggagctgt 4560acagtctggg cctacataga acatgtttag gaactttgca caggactgtg gctgtcacat 4620gcaacctagg ttttccacat taaaagtaaa cattcctcat tccttcaaca gctcctcatg 4680tgacttgctg ccttgttccc tcatcattct ggttattttt gggttactgt cccttgtcct 4740gaacctttat ttgttaaaat ccttaaatcc ccaccaattc aataagatgt ggtctaactg 4800gtgcagaatg cagtgggagg gtggcctccc tttctctatg atgtcatact tttgtcatgc 4860aactgaagat gtctttagat ttttcctggc ctatccattg tgtttttaaa tttactttca 4920gggcttattt tccagttaga ttgttcctga gagtttacag gagaactgct caatgtacag 4980cttgagagca atggttttca gctgaggatg atttatgccc ccactccagg ggacatctgg 5040caatgtctgg agacgtaata tttagttgcc acaactgggg tgtgctattg gcatctagtg 5100ggtagaggcc agggatgttg ctaaacaccc tgcaatgcac aggacagccc ccacagccaa 5160gacttatcag cccctagtgt cagcagtgct gagcttaaga attcctactt tagggtccgg 5220gtctaggttc agatcctggt ttagctactt cctatccttg tgaacttggg cacgttattt 5280aacctctttg tcttcattat ctcaactgta tgtcaaagaa ctctgaggac taaatgagtt 5340catttatatg aaatgcttag aagggtaact gtatgtacaa acactatctt atttttctac 5400catattttat tcattttggt gtgtacatat gacatcagat aagtgggtgg gtaaagtttg 5460agggatagca cttcatttct aatcttgcct atgggatggg cactagtgca tttggatgtt 5520ttagggagct aacagctttt ctcagaaatg ccttctctct atggtcccca ttatcatttt 5580tccggcttct tttagtattt tcctccatat tggggcatat atgtcagttt aatactgcat 5640cctcctggag aatatggaca ctgagttcct ttagcgggag agcatctgtg cctgttgctt 5700gtacatccca tttccggtgt gccccaggct cctccacacc ccagtcaccg tggcttgcac 5760ctgcacaaca gattcctggg gctgtgtagc aattggtaga ctttggataa taataatagc 5820atttctccaa gggctcacat ccccataaga accccattat cagggttcct ctttgacaga 5880tgaggagaga aaagcagaaa gtaggtaaga acttaactga agtcaccaaa ctagtaagta 5940gcagattcaa gattcaaacc ttggcagcca gaaactggag ctaaatttct aaaccacttt 6000attactctga tacaggtaag ttttctcaag aagtataccc atgaccttaa cctaaccatc 6060atgtagccat ttatgcatac aacactcaac agatccagtt actcttactc ttactattgt 6120gagtaatata gttgggggac tgagtctgag agtaagatca ggaacttaca gtatagcagc 6180aggctgccca ttgatcttag acaactttta ccttttatga cctttaagcc atatagaaca 6240ccaaacattt ggacatttgc acatgggatg cagattaatt cccatttcca aaattgtgag 6300tagaatcaac taattatatg ttacttgcag gttttgcatc tcatgcatct gcctccctag 6360aatgaaataa aatttcttcc ttattaaaca aggatagagt tgccccagct tttcttagac 6420cagggtttct caacctcagc agtattgaca ttttgagatt cctctatgtg tagggctgtc 6480ctgtgcacta cagatgctta acagcatccc ctgactgcta ctcactagat gccagtagca 6540gccccctcct ggttgtgcaa cgcaaaaata tctccagaca ttaataagtg tcttcttggg 6600ggccaaaaat gccttagttg agagacactg ctgtagagtg atgttctagc tgcattctcc 6660agccttcatc ctattgtaaa atatccaaag aagattttat cttcttcagc aatcatttgg 6720tgtggtaatg ggtcagtttt gaggtattta agccactttc tttgacttta gttgaaactg 6780aattgggcta ttatttgctt tttgtctcat tcagttactt tatatgctcc atattagttt 6840tctattgctg ctgtaaaaat tttctacaaa tttagtcact taaagcaaca taatttatta 6900tctcatggtt ctgaaagtca gaagtctgac atggccttac taggctaaaa taacaggttc 6960agtgaacctt cagaggtcag ctagtctgtg ttgctttcct gaggctctag agagaatctg 7020tctccttacc tttccaagat tctagaggcc atccacatgt tatagctcat gcccgcttcc 7080ttcattttca aaaccagcca tggtgggttg agttctcaca tcacatcatt cagatctctt 7140cttctgccat cctcttatac ttttaaggac tcttgtgatt acattgggtc agataatcca 7200tgataatctc cctattttaa ggtcaactga ttaacattaa ttccatttgc atcttaattc 7260ttttttgcca tggaacatta catattcaca tgttctgggg tttagaatat ggacatttgg 7320gtgggagtgc attattcaac ctgccacatg cccccattcc aaaggtcaat caggaagaat 7380ttggaaagag agtactgata cttaacaaat gagcattttc ttttctgact ctttactaat 7440aacacaagag ctaaggccca cctgccctaa tctcttgcca ggactgggtt ttacttggat 7500ggtttatctg tcagcttttc acagagattt caaactcgag attggacatt ttatctattg 7560ttttgggttg tcttttgttt cgaaaaggga aatgatgcct gatgagaatt taacagtgac 7620aaatagggga aatcgggatt tgtggcagtt atggttgtgt atggagacta caactgagat 7680ctgaagaaca ggtagggcac tgtggtttca gtgggagaaa tgaagtctgc aggagagagg 7740cagaaaggta gaatccgttt aaatggaatg tattatctga gtgctcattg aaagtcttct 7800tgattgccag gatgatattt tatctagtgg tcaaggggcc aaattgaaga tagttatttt 7860caggccttac tatttgaaga aaatcattga tttttaaagt gaacattccc attgatttag 7920aatgttggtg gctcatgcct gtaatcccag cactttgaga ggtagaggtg agcggatcac 7980atgaggtcag gagttcataa gcagcctggc caacatggag aaaccttgtc tctactaaaa 8040atacaaaaat tagccaggca ttatggtggg tgcctgtaat cccagctact cagaaggctg 8100aggcaggaga atcacttgaa cctggaaggc ggaggttgca gtgagctgag gtggtgctat 8160tgcattccta ggtgacagag tgagactcca tctccaaaaa aaaaaagttt gtgtttttgt 8220attgaccagt gattggggtt atggtaatag attttcatac aatatgatga tgaagacggc 8280tgtatcaagg gttttagtgt agtcctgttg gattctctag ttctaagaga atgtctaaag 8340ttaccattaa gtttttcagg gtcctagttc ttaaatataa atacatttct agctaactca 8400tatcccatgg ttgagaaaaa ggcacttatc tgtgtaaaga gaactttaaa agttaaaaga 8460cttcttatga attattcctg tgtgttcata ttcagaagcc tctgtggtca ttgtgattat 8520tacatgttaa tatggttcct ggctcataac tcccgttgcc cttgctacag tctttggtta 8580taatattgga gtgctttaga ccccagaagc aggtctcaga aagcagaatc tttctctctg 8640accttttcct gccttccttt ctactgccca aggcaggact ctaatctgat tgtaggttat 8700aagaccctta ttcagggaag ggtcctctta ccttggaaaa aggaatgctg cacagacaga 8760ccaagaagaa tctgaataga cagggcttgc tgggttgccc ctctgagtct attagcatta 8820gagcttaccc ttactgtcca gtcatatttc tacacagctg ttcatacttt gttgaaccta 8880tgcataaaaa tagacagttt cccctgtatc tttgggtctt cattctgaag gctccttgtg 8940aatacacatt aaataaataa tctgcctttt acaagttgat ttttcagtga cccttcagag 9000gtccaagggg aatgccaccc gtggccccct tggccccttc agtgtgcatg tattcgagcc 9060tagaaatgcc tctggaaaaa aatctgttag caagtcatta tttgcgaaga gataactcaa 9120tttacctttt tctgttatgt ttggattttt gaaatcatgt attaacttta taatataatt 9180aaatgtaata ctgaaataag atcaaataaa atcaaccaaa attataaata atcttaaaga 9240gttgcacata tatacctttt ttttctgatc aaaagaaaga tgttgaataa ggaaatacag 9300tccctctgca agtagtttaa gaagttttgg ctggacacgg cggctgatga ctgtaatccc 9360agcactttgg gaggccaagg tgggcagatc atgaggtcag gagatcaaga ccatcctggc 9420caacatggtg aaaccccatc tctactaaaa atacaaaaat tagccgggca tggaggtggg 9480cacctatagt cccagctact cgagaggctg aggcaggaga attgcctgaa cccaggaggc 9540agaggttgca atgagccgag atcacactac tgcattccag cctgggtgac agagtaaaac 9600tctgtcaaag aaaaaaaaaa atttagagct ccagcaacac atttaccagc tgcctatctg 9660ggtctttcta gcctgtacaa taggagctgc ccttacacga aagccccatc tctttgaaat 9720ctttctgtct acattcctgt tgctctcaga acttgtacca tatcatctaa gaattataaa 9780atgcccctgt aatttgcttt cttcctgtat tatattgtta atatatgcta gtcttgtctc 9840tgaaacacaa ttgtaaactc tctgagagta gggattgtgt caaattctct ggtacttacc 9900acagttgttg ccataggaga gggcatatag tatttgctta agaaataccc tttgattgaa 9960tgactgcaga tctgataatc aggataggcc atgctgtgct ccattaaggc aataacagct 10020tcacctggaa atttcagtgg cttaacacag caaaggttta tttcccattt gtgaaaggtc 10080atttgaattg gcaggggctc ttctctgttc agtgactagg tatccaggcg tctttcactt 10140caagactgca ctacatcagc acatgatttc taagtttgct gtgtcaggga aaggaaggca 10200tgaaggagat acagtgactc tgttgcagac gacttctgtt caaatttttg tttctcagga 10260attagcccca tgggcccagc ccaaccacag gggcggctaa gaaatgtagg gaagcacata 10320gatgttcagg agcactcatt gtctgtgcca cacatctata ttacatcttc ccgcaaaata 10380gctatactaa cgagtgcttc attagtgtca gtgggacacc ggatcattaa acttggctcg 10440gtttgtatta gtaaccagat gtgctagatt tattcactca gcaggcattt actgagtgcc 10500tctgtacacc aggcactatt ctaggtcttg ggaatacagc agggggaaaa accatcaaaa 10560atccttacac tcacagaact aagtttttag tggaaggtga taaacaacta acaaataaca 10620tacaaggtac gttagttggt gataaatatt ggagaaaaat aaagtaggga gggaaatagg 10680gaatgtattg gtgtgaagaa ggaggttaga ttgcaatttt aaatagattg aaccgggaaa 10740gtgtcactga gaaagtatca ctggagcaaa aacccggagg tgagaggacc agtcatttgg 10800gtgtcagagc aaagagcatt cccataaaag ggaacacatt caaagccctc aaagtgggga 10860tggaaggtgg gtgtggagta cttggcatgt ttggtgaatg gcaaagaagc aagtgtgatt 10920gcagcaggtg tccaagaggg gctaggggaa ggattgggag tgtgttgagt aaaggttatg 10980tagatcatta taagctattt tgggtgaatg gcttctcccc cgtaacatat aacaccattt 11040gggggttttg tgcagaaggg tgacacaatc actctggctg ctgtgttggg gatagatttt 11100agggtcaagg gcaaaagcag ggagactcgt tagaggtgat ggcaaatttt aggtaagaga 11160tgatggtggc ttggaccttt tctatccccc acaaaagttg aagggccaaa ttttaggggg 11220gaaaatggat gcacaaatgc aagatagttt ttgctaataa gatgttagca tacagactaa 11280ttataattta ttattaccac acatctcata ttctcttgtt tcatttactg attcaacaag 11340catctatgca atactacttc ttccaggccc tctattaggc actgagacaa caaataagac 11400ctgacttctg ctttttggaa acttatatca tagtaggatt atagaaatta gcaagaatat 11460aacatcatat taacacccaa aacaaccaca gcagcagaaa tatagtcact atataattaa 11520atgatggaaa tagccaaaat ttatgaatta tttactatgt gctagacata cctatggata 11580ttattatgat gatgacagat ggttgttata tttgttatat tttgttggct atttttatag 11640ctgaccatac tgttataaag ttagaaaact agtgaccaaa aacttaatct aactgtctga 11700aagtattatt tctctgctct acaatccaat gtaatcctta gtaatgtagg taatggtaat 11760cctacattac caaggatttt accattgtag taccaatcta aaacccagca cagaaaatac 11820atgttttatt ttttccaagt gttactagta cctcagcctt tcttgatttg tcagcttatt 11880taaggcctct tcattgcata cttctttttt cttttaatca tctgcttcga aggagactaa 11940gctgaaactg ctgctcagct cccaagatgg tgccacccaa attgcatgtg cttttctgcc 12000tctgcggctg cctggctgtg gtttatcctt ttgactggca atacataaat cctgttgccc 12060atatgaaatc atcaggtaag aggtgtattt gttcaaggtc ttgagcaact gatctgtcgc 12120catacttcaa gtgggcccca agaagttgca catctgcaca tctaaacaag tcctatttaa 12180aggcttatgg agatcctgta ttctcctgga gtaggggaga ggttctcact ttttcctcat 12240ttcaactgaa attgaagaag cacttgtgtt cttcagtatc aggactgcag gacagaacca 12300ggatgggaca ttttaagata tactggacgc tcagcttgca

atcaccacgg agaacctcct 12360taatgccacc ccttctctct ctgactgccc taacgcatgc ccacacaagc atagtaatgc 12420aactggtctg gagtccactc caagacagcc tcaaaacctg cttccatgag gtgaaaaaga 12480agcaggtgtc ttgttcaaca ggcatactcc tggttaatag tggaaagtca tgtgagataa 12540caatttcagg tcttgggatc caaaaggaaa caatctttcc tacctgataa tagagagaag 12600tagggacaag taggtgctag ggagttccag atgagaatgg atgtgatcac tcattctttc 12660ttgccttgtg gtaagtgggg aaagaaagag gtggaaggca tagactaaac ccaggaggag 12720tggccaaaac gggacagcca tctttagacg gctacattta atgtcactaa gagagacctt 12780aattggaaaa agattgtttc tggagctttg caaattcctg ctcatcccag aggattgtga 12840tgcataaact tctctttcca gtctcctatg gagagcagtg taggtggctt ctgaatggtg 12900tcatactgac tatacctaca gagctgtgca cctggatagt tgtgggtcac tctggatcta 12960attgttcaag cttgatgggt gactccttac gatagcattt gcaaaagagc tttcataatt 13020cagtacctta aattttgccc tactacataa gcaggaaaat gaatgcatta ggagaggttt 13080tatatcttat atctacattt gcataggaaa tagaataata tctcacaggt ccattcatcc 13140tataacgcat caggaagtta agtcatcaag gatacactgg tgactaagac aataaatgtg 13200tagtcaaatt caagggtcct gcatcctaga tgacattaag ttcatttttc ctactacctt 13260tttcatggat tgagaacagg taatatctta gatggtggga aaatcatcat tcgggaaaca 13320tttatatcct tcacttttaa ttggtacagg gtaaaaattt aaacattttt aaaatccctt 13380tgtctttaaa aataattgga aggctgttgc tggactgaca gaaacttgag gacagggacg 13440ttgctttatc tttatagtcc cagcattgag cccaatgcaa tgcccattct aaatgttcaa 13500aaatgaatgc aggccgggca tggtggctcg tgcctgtaat ctgagcactt tgggaggccg 13560aggtgggtgg atcacttgag gtcaggagtt cgagaccagc ctggccaaca tggtgaaacc 13620ccgtctatat taaaaaaaaa aatacataaa attagctggg tgtggtggtg cacacctgta 13680gtcctagcta cttgggagac tgaggcagga gaattgcttg aacccagaag aaggaggttg 13740cagtaagctg agattgcacc actgcactcc agcctgggct acagagcgag actctgtctc 13800aaaacaaaca aaaaaagaat gcatatatat tggtttaaag tattagaaac agtttattaa 13860ataagattaa aataataggt gattctttat aatttcttac taaatgaata cacagaaaat 13920atggctatgc aggttaaaag gtttgtagga atgggctaga gatatcctct ttactaatgg 13980aggacataaa gccatataag agtgtaattg tcctagcatg tatacagttc atctccaaac 14040ccaagactga acatcaggaa tatttgacaa atgaaaacca ccaaaaatat gtaataaaat 14100taatagcatg ccataggttc caatcctcct attaaaataa agttgaagaa aactaagaag 14160agacatttat aaattctgaa ccacaaaact gtgtgagtgg tcatttgatc tgatgagtct 14220cctccttcca cttccttaat gatgactcct gacagatggt tagagaatgg gaaatccagg 14280cccataaccg ggtcagctgt aggctatgga gcctgttcca cagccacccc caggcaccct 14340ggcactggtg cataggaccc catagaccta taccaaaacc tggatcagcc tgatgattcc 14400tgaggtcttc tcagaggtca ggtgtcctgg aacaggcaga cctcaccttg acctgaatgc 14460tcctgaatgc tgagtgagga aacttcccag ggagaacagg tgttggacaa ccagggcact 14520cctaacttat tggaaaatgt tcccctgtcc tttggaattg agatctcaca agcggaggct 14580tgtgtttggg tgaaaccctc ccatgcctcc agaactgagc ctccaattct agaggtagtg 14640tctccaacag cagctgtggc cctggggctc cttagcgagt tgtgtgagcc agaggtctcc 14700ctgcagcaag ccctgcacag gcacaccatg cctgcctagg ggtggggttg cagtagcacc 14760aggctgctta ccacagaaga agaagcaggt gtccctacct taaggtcctc ctccttggga 14820cttcacatct ccctgttaca ccagaagcat ggctcagtag atactctttt aatggaagaa 14880gagccttttg taattcaaaa tagaccctga aagttcacct tgcaggtaaa gtttaaaaaa 14940aaagtttctg gggtttgaat ctacctgggg agaggttaga cctgctaaaa cttccctaag 15000gactcagcac agatttacct agcaagagta aatggactct ggggaaggag taaacagcat 15060tatttgtcta ccccttatgt gtcatgtttt gtactaagaa cttgacctac attctcacct 15120catctttaca actgtactgt gggctggtca agtgctttta tgtcatttta cgaatgtgga 15180tcagagaggg gagcaactag gccagaggat tatagcttgt aagaggtaca cctgaaatct 15240gaaatagaac catgtccctc caattcacag ggtttgttca tctccatccc ctctcccctt 15300gcctttcttg aaactacctc ctaccatttg accatggatt gctgacctgt gtgtcaggca 15360ggtggtctgg aacttcagcc cagtgtctct taaactttaa tgggcactcc ttttctgatt 15420gagtggaatt gtggtgaagt ccaggagcct gcatttccaa gctccagctg gtgccagtga 15480tgctggtcca tggatcagct ttgagtagct gagccttatg tattgaaagg cttcatcatg 15540ggcaggtgtt tcacacatga gtcaccagtg tggaggctgt aaattggttt cttcccatga 15600ctcaaaactt ttctgggcaa tgttccatgg tattgatgca gacgctttat ctaacatagc 15660tcttgatagc tggctagcag agagtaatca actcccacca tccaatggaa ggaatgggtg 15720gacaagctga atatgtcaag aaaagtgaga gtctgaatac actgatattt tgtaattaaa 15780tatggatttg attgaggttt agaaaggaga acggatgaag ctgctggtgt gtggaagatt 15840gcaccagaga gagaaaagta gttttaggag tggatgagta aaatactgca tgtgataaag 15900ggtttaaagg aaaacaagag agaccagtgg ccattcaagt taactgagct tattttcaca 15960agtatttgtt gagcattgac tatgtgccag acactgtgtt aagaggatgc aaagacatgg 16020atatggctct gaagaaagag cctagtgtag catgcagaca cataagttta atatgtgaca 16080ggaagaatga ttacatgtgt gtatacacac atgcacatag aatctatgta atattgagga 16140tgaaatgatt aagtctgtct gggaaatcag ggaaggtttc agagaagaca ggacatctga 16200ccaaggtatt aaagtgtgaa tgggagtttg ctaaggggat ttaagcatag gcagtagcat 16260ttacaaaggt gcagtggcat caaccagccc agtgtgttgc tcaggagtac tgtagcacaa 16320ggaaggggta taacttaagg ggaagaagaa gaggaagagt atgaggcaat gggcctccag 16380tggagggttt ctttgcagtg cccagaaact tgggatttaa cttgtgcacc tgtgaacctc 16440agtatgtttc aaccccagag cactggaagg ataggccaca tttttgcaag tagcagagta 16500ttggcttagg aaagcatgtg tagatttaaa ttcctcctac aaactgcctt ccaaaaagat 16560tacaatacat tcctccttca aggaaagaac acttgctgat agggaggcat tgaaggattt 16620taagaattgt ctggtgtgat ttgtgcttta aatccctcca ggagctgtgt aaaggatgca 16680tttgaagggg tcaagatcca acgtgggtag aacagttagg agagagagca acagtctggt 16740caagagacag tgagggcata aatgagacag ttgagctaga gggtcagcga tatattgtgg 16800agatcattct aagctaaaaa ctaatgttag caatagaggt ttggggctgg gcgtggtggt 16860tcacgcctgt aaacccagca ctttgggagg ctgaggtggg cggatcacga ggtcaggaga 16920tcaagaccat tctggccaac gtggtgaaac cccgtctcta ctaaaaatac aaaaattagc 16980tgggcatggt ggcacatgcc tacacctgta atcccagcta ctcaggaggc tgaggcagga 17040gaatcacttg aaccagggag tcggaggttg cagtgagcca agattgcacc actgcactcc 17100agcctggcaa cacggtgaga ctctgtctca aaaaaaaaaa aaaagaaaaa gaaaaaagaa 17160agaaaataga ggctccgttg aatgtagggt ggttggtgag aaagaggtag gagaatagga 17220tgattcccag gtttctggct tgtggtgcag gagatgagga gttcttgagt tgcaggctgt 17280gtggttggaa agtgagaagg gagagttgaa agatgatcat ttagtttcat tttggaaact 17340agtttcattt tgacattctt atgggaagga catcaggtag agatgctcaa ggagaagtta 17400taggcacaaa tctgaaatgt gaaataaagt tcatgtttag gaaaaacaga tttgtgaagt 17460catctttaat ttactctgga tagaagtaga atgagagggc aaggctgcaa acattaggag 17520gtaacagtcc aaggcagctt gagagaaagg ctatgtctac tttcatctct ttaccctcca 17580aaacccctac acagtgtttc aaacagagca gaccctcaat aattgcatat cttacttgtt 17640aggttgagaa agaaagaagg ccagataact atgggaagta acttgattcc gttggaattc 17700ttttgcataa taaaatctga tatgtaatgg atgacaaatg agataatatt tacctgtttt 17760tcagcatggg tcaacaaaat acaagtactg atggctgctg caagctttgg ccaaactaaa 17820atcccccggg gaaatgggcc ttattccgtt ggttgtacag acttaatgtt tgatcacact 17880aataaggtaa tgctttgatt tatacaactt atcctgatac tctaatattg tctgtcgcta 17940tggaccacta gaaggtgttc aaatgtgacc ttgccctcac ctgagaatga ctcattttgc 18000aatttgtatt gtttcatatg aaggctttta ctagtttggc cattcctcaa ttctttgtta 18060ttgtctgatt aatttctcta taaaccttat ttttcacttc cttaatacct gaagccaggc 18120tgcttgtatt ttcctttcac tgagatagaa tattgttttt ctgtttctct ttcatgacta 18180tcttcaatca ccacagcagc ctaaaaagtt ctttagacct ttttgtgaac acagaggtat 18240ttgagtcccc actaattaaa tatgcaaaat agctgctgga atatgtttga gacacaactt 18300ctctaaaagt gcattaattt ctttcttaac agggcacctt cttgcgttta tattatccat 18360cccaagataa tgatcgcctt gacacccttt ggatcccaaa taaagaatat ttttggggtc 18420ttagcaaatt tcttggaaca cactggctta tgggcaacat tttgaggtta ctctttggta 18480agatttctgt tgatccttct ttgtaggctc ttgcatgtat gaaaaccttg aaaacaacaa 18540gaacttcaag tagttaagac caaagtagat ttttcttcag tccaaatagc tcctaaaatg 18600ataaggaaag tatttcttta aagcccaggc aactacgaca gaatcaaggt tctcattttg 18660tccattctga gttggatggg agtggccgag agtatcagac tgactctgac atctttctgt 18720ggctgctctt tagttttcat ctgacatacc atggagaagg caataccgtg gtgagaatag 18780caggttattt ggtgtggcca ctatcccgca tgctctgtgc taaagttaga caaaaagaga 18840aagaaagtag aagccatcaa actctccaga tccaaaacaa aggagtcaaa gctaatgcct 18900ttcttgtcat acatgaagag actctactct cattcccatc acctaccccc atcgctaaga 18960caatctgatg tgtattcttc acttactgtc tacattctgc taatatagac ttttccttcc 19020ttatctgttt gactagacac tactgttgga ctgatgttaa ctgtgtcttc acatttaccc 19080ctcaaaacga tctacaaatt ttgggattca tcttaaacat cggcttttaa ttttcttaaa 19140aaccacttat aacagacatg aaaatagcat aactctgcat atttatgttg caaaataatt 19200tataaatcac ttccatagca atcatcacat tcagtcttta tgactgcatc tgcattttat 19260agatgagaaa atgaaacaca aatgattgct gataataatg gtgacaataa aaattgagaa 19320gtgaaagaag acaatatttc cattttttga gccccttact atgtgatgga ttgcaccact 19380gctttacata tattacctaa ttaaataaaa ttctcacaaa aaccctatga aagagatatc 19440attttctgaa gtttacagaa gaggaatctg aagtacagaa agtttaagga acttgcccaa 19500gatccatagt taggaagtgg cagagctgca tccatgactc agtatgaagt cagtggtttc 19560tctacctttt cctgcatgat tatgaccaaa tcataatgaa cagagtctct gtttcttaga 19620ttctggctct tagtgtaatg tatccaaggt tttatgggta gtttggtttc agaacattcc 19680ctttaaaatt tttccaaaac ctgggaatgg cagtaagtag cagcctttat gaatacacct 19740ataagtggga aaatctctca ccttaagtcc agagctgtca gtaagaactc atcgttaatg 19800atcctatggt ctgaggaaag aacttacgcc gggtgcggtg gctcacgcct gtaatcccag 19860cactttggga ggctgaggca ggtggatcac ctgaggtcgg gagttcacga ccagcctgac 19920caacatggag aaaccccatc tctactaaaa atataaaatt agccaggcgt ggtggcgcat 19980gcttgtaatc tcagctaatc gggaggctga ggcaggagaa tcgcttgaac ccgggaggca 20040gagtttgcag tgagccgaga ttgtgccatt gcactccagc ctgggcaaca agagcaaaac 20100tccatctcaa aaaaaaaaaa aaaacttaca gttcagctct ttggttggtg ggtatctagt 20160agcagtcttt ttaatgaatc tactattcat ccataaaaaa gtagatataa atcagatggg 20220tctgcatttt atgctaatga gatatgaatt aaattcacta gcaacactca gagaaaacct 20280taactataac cttccattgt tgtctaggtt caatgacaac tcctgcaaac tggaattccc 20340ctctgaggcc tggtgaaaaa tatccacttg ttgttttttc tcatggtctt ggggcattca 20400ggtaatgttt gagaggttga acaattttgg cttccaggaa taaatgacaa tttttttatt 20460caagaaagaa atagcagagt ttggaatgtc atgcaggccc ttgtctggag gagttgggtt 20520tcctcaataa ttggctgtgg gtctattgat cagtcctaga cctgtctggt caagtagttt 20580tttccctact atcagctcat tgggattagc ctcacagcag agaagaaagg gtgttgcatt 20640ttctatagtt gtccttcatt attgtaatat ttacactctt aaaattatcc tctgtaaagt 20700ttagaacttt tgagaaacca attttagatt agctgggaaa gactgttatt aagagaaata 20760tctgtgaagg caagagaagg agcaggaaaa gatggggaga acctttggac catgacacag 20820gttggacacc tgaaaaggtg atggggaagg gagaaggatt ggataagaag agcttcagaa 20880tgaagcatgg ttctaagaaa tattgagcca ggccaatgga aggagtgctg gactatattt 20940gcttatcaag agtcctgtat ctcataggaa tgggctaatg ttagtaccca tgctgtgcca 21000agtctctggg agcagctcac tggatgctta gcttccactt gaccatggtg atggatacag 21060aggggcagga gctggatagg gtctttagta agctatgcag tctgtagcat gatagctgag 21120tggcacattt tcataacaac aaccaccctc ttcactttct tccaccactt tctcacctgt 21180cacccctctt ctacttcaag ggattgtaaa actcagaaaa taagagagat ggcttttagc 21240tgggaagaag ttgggtagca cttgcaataa agaattctct tcagcactgt aaatgtagtt 21300cagctagcta tttgatttct cctacattag tgactggcaa attagaaccc aagggccaaa 21360tccagcccac tgcctatttc tataaagttt tattagaaca cagccacacc cattcatttt 21420tgtacactgt ctatggctac ttttgtccta caacagcaga gttcagtagt ggtgacaaag 21480actatggccc acaaacctgg aaatatttac tctctggcct tcaacagaaa aagtttgcta 21540atccctgtgc tatatcattc ccccctcttg aaaaggtcct gtatcttcag caataaataa 21600atagtttcta gctacctcct ttttattctc tgtggacatt tcctattgaa tgcatggtat 21660gaggaaaagg ttgtgcaaat gaagctgtgg aagagggcat aaatgcagtg cttagcacat 21720gatttttcaa ctatttcacc cagtccaatc atttaaataa tagctaacat gtgttaagta 21780tttacctgtt tctaatataa gtactgaaaa tgtattaatt caattgatcc tcactgatga 21840ggtagcatac tattatagtg tcaccatttt atagataagg aaccaaggca gcaaaaatat 21900tgtgacttat taaaggtaca cagctaacaa gtggtagaac cagggttcaa actcagtggt 21960cagactgcag gtctcattct tgtaactaca atactgtact gctttcctat atacattatc 22020ttagcaaaac cttacaacta ccctaaaaca aatgctttac tcttaggccc attttacagg 22080tgaggacaaa aatgcagtaa ttacctaaaa tcatatagct gggaagtggc agagccaaga 22140ttaaaaaccc aaaactctca aaggcaatat ttataatcac tgccctgggt tttctcaact 22200ttagtagtct tgacattttg gaccagataa tctcttgttg tgaagggttg ccctgtgcat 22260tgtaggatgc ttgacagcat ccctttaccc actagacacc agaaataccc tcgcccacat 22320tcatgataat gaaaatacct ccagacattg tcaaatatcc cctaaggaac aaaatcaccc 22380tcctttaaga accgtagctc tccactccac cccagggcac tactacaggg gtgtaatggc 22440ctccatgttc ccagttttat tagtggactc agccttgtaa tcatgactgg tagttgtaat 22500tcttccctct ttttgttttg aaggacactt tattctgcta ttggcattga cctggcatct 22560catgggttta tagttgctgc tgtagaacac aggtatgtta cctgatataa ttgggctctt 22620tggccaacta cagggaatgt caatgctcat aactatgttt ctaattttca taaaagttta 22680tttaaaatgt tgatggaact ttcaagtatg gtaacatcat gagcaaaaaa ggagattgag 22740tttatcgact taaaagactt aaaagcacct aacatttcta gagtgtttat tgcctgccat 22800gtcctatgcg aaatgttttt aatacgccat ttcattcagt cttcataaca atcctatgag 22860atggttatta ttattattgt tgttgtttta aggaagagga atatagaggt gataaagggt 22920aatttgccca aagtcacaca actagtaagt ggcagtgccg ggatttgatt cctgagactg 22980agtttcaaca tggctaacat tgcctcccag aattaggaag atagaatgga ttgagtttac 23040ctgcaacttg atgatagaca ataaggtttt tttcctggaa ctcttttaca gtcttcttta 23100atttaagaga aaatatagag tggaagaaga aagggaagtc aaaagatcag aggaagttga 23160gtcaaggatg gaactgagaa acatgggtca gatgaggaag ggaaggagca tgcataaata 23220ataattttgc ttgtattata gagatagatc tgcatctgca acttactatt tcaaggacca 23280atctgctgca gaaatagggg acaagtcttg gctctacctt agaaccctga aacaagagga 23340ggagacacat atacgaaatg agcaggtaca ttgcagtgaa aggagaggtg gttggtgacc 23400taaaagcatg tacaaaagga tgacatttgt taatttaatt ttacacctgg caagttatgc 23460tcctagctct cctatttccc attcccaaaa gatctgtcaa tagattcctg gagcagtaaa 23520attcccttaa tggaatatct agttcatagt aaaaacaaag gcaaatacaa aaatttggga 23580gatgacagtg aatattcaga attcccttga attaaaactt ctaattttag aatctaaaaa 23640gtgctagaaa ataaattaat agattcttcc tcacagacaa tataaaaaga ctacttttca 23700gagaggcagt atacaataga attaagagta tgggccttag aacccaactt tctatgtttg 23760aatcctggct ccaccactta attttgttat agaataatga atagaaacat tatttgtctt 23820acagagctat tagggcagtt aagttaagtg aagcatattg gcttcctctt tatagtgggt 23880gttttacatg tgttattatg tttgagtcca cccacatttc ctttaggtag gaattattat 23940acaatcaatg gaaactcaga gaggttaagt aaatcgtctg aagtcacata gtaggtaagc 24000aacagagcca ggatttggac taagctatac ctatgtgcaa agctggggcc tgtgtcatta 24060tggtagcaag taatagtcac taatcagatt tccagtttat aactgaccaa cgatttttcc 24120caaatacagc ttctacctaa actttaaaat aagtgttata actttttact ttgtcatttc 24180cttcttctaa taattatatt aggtacggca aagagcaaaa gaatgttccc aagctctcag 24240tctgattctt gacattgatc atggaaagcc agtgaagaat gcattagatt taaagtttga 24300tatggaacaa ctgaaggtaa gctataaaaa gtaatttttc tcttgtccta cagttcttta 24360ttgttttttg tcatttaatt ttctgctata ttgcaaggta caatatgata aagggctgca 24420accagccccc tccccaatgc gcacacacag acacacaaag cagtacaggt aaagtattgc 24480agcaatgaag aatgcattat cttggactag atatgaaatt gccaaaagtt agtcagtttt 24540gatctacaaa aacagcaatg tcatatggtt caactcaact cctgtggaag tattattatt 24600tcatggacag ctcctgcatg tttttaagct tgatcttgaa aacttgacac aagtttggga 24660gcgggtggaa aaatatcaga cagagtgggc agcatgaaca aagactcaaa agtgtgtgtg 24720gcatgtgaag tgcagggatt agggggaact agagaataag atgatggtct gttctccaga 24780tacaatgaag aaatttgtta tccctcaagc agccactctc ttctgtatcc ttgcctttgt 24840acatgttgtc cccttggcct gacacaccct tccccttgcc taactcctac ctaatttcaa 24900gactccagtt gagcatcacc tcctctaaga agctttcttg gaccccaata cccacttctg 24960gactgggctc gctgtctgtc atgtgtgctc ctttgtacca ctgtactgta ttgcatcatg 25020cctctgtata actttcttcc ctgatggact gcaaactcac tgaaatgaga ctgcagtacc 25080tggcacagag taggtactca ataaatactc atggaatgaa caaacaaata aacatggggt 25140gaggagaggc agaagtcaga actgatgttg aagtttccag tgtgggtgac tacaaagaac 25200attaagttta ctttcaaacc tttacatatg ttatatatat gtgtaaatgt gttttatatg 25260tgtatataga tgtatatgtg tgtatggtat gtataaatgt atgtgtgtat atgtatattc 25320tattttataa gaaatcaatg tatttaacca tccccatgaa atgaacaatt atatgattga 25380caaaatcatt tcttctaaca ccacgaaata gctataaatt tatatcatgc tttttcaaat 25440aggactctat tgatagggaa aaaatagcag taattggaca ttcttttggt ggagcaacgg 25500ttattcagac tcttagtgaa gatcagagat tcaggtaaga aaataagata gtaaagcaag 25560agaatagtaa attattggaa gaaattatat tgtgagatat aattttttat tcaaattctt 25620agtgaagaag ggatctcttg gagtttataa ggctattctt ttgcccccat aaaatactct 25680atatacattt tcctaggcta aaacatctac ctctcctgct attaaaatct ccccctactc 25740ccataagttt tccctcatta ttcttgttta cccaaggggt taacactttt cactgaaaaa 25800tttatcttta tataattttt tgtgacataa tgattgtgat aataatattg tcatcttaac 25860agtattgcca gacttctcca gtgggcagga cattttttaa ttaggttttt cccccttcca 25920cctttaagat ggagctggat ctaaaattac aattatttgg gtaagagttg ggacaagacc 25980ccagttttct agcttgcagg aacattttct ttcttaaacc ttcattccag actgatgaga 26040tcacatagaa ccaaccgtac agagagtggg caggatcatt tttgaaactt cacctcaaat 26100ttcttaaaag tccatttcag tgtaagaatt ctcttctttg atccatttac caaaagatca 26160ggaaacatag gataaactta cttttaaaaa ggaagttagt ttcatcttca aatgatatgc 26220tccatatgtt tttcttcaac cctcagacag tttttcagct gaagtgcctc taaatataaa 26280gtaatgggcc caattgttac tctaataaag ctataataaa tacactacaa ggtgcataaa 26340agctctgaaa acaaaggtgt atttattata ctttttcaga tactccacag tgttgaagaa 26400attattatgt attctaaaat ttattttgaa taaaaagttt taaggatata tcactcaaat 26460ggttgcaatt cctaaaggca tataaagaaa atgattatat aaaattccag gtctgctact 26520ttacaaagtt aatcatatcc ctttcccaca ttgaagtatg atacctcttt attccaatga 26580gataacccat aataaactgg tatggtgcgt gtccaccaat cctagcatta ttaggatgtc 26640ctcaatgttg gctagtatgt aaccagttta atttcatcat tgtcaacaaa tatctacaga 26700tgtggtattg ccctggatgc atggatgttt ccactgggtg atgaagtata ttccagaatt 26760cctcagcccc tcttttttat caactctgaa tatttccaat atcctgctaa tatcataaaa 26820atgaaaaaat gctactcacc tgataaagaa agaaagatga ttacaatcag gtaagtatta 26880gtgacttatt tcattatgtg aaacaaactt gaagcttggg taaatatcaa tcgatatcat 26940ttggtaacta ttaaagaatt gctgaattgg ttgtttagac tttcaataag gagagaatta 27000gataatctca gtttctaagt acatttagtc ttactctttt taaaatggga atgttaacgt 27060atatagtata tatactggtt atattagtct gttcttgcat tgctatatgg aaatacctga 27120gactgggtaa tttataaaga aaagaggttt cattggctca cagttctgca gcctgtacag 27180gaagcatgat gctggcatct gctcggcttc tggggaggcc tcaggaaact tacaattatg 27240gtggaaggca accggggcat gagcacttca catggccaga gccggaggaa gagagggatg 27300ggtgaaggta ctacacactt ttaaataacc aggtctcaca agaattcact gtcacaatga 27360cagcaccaag ggggatggtg tgaaaccatg agaaactgct

cccatgatcc aatcacctcc 27420caccagtccc tgcctctgac actggggatt ccaatttgac atgtgatttg gtggggacac 27480agatccaaac tatatcactg gtaattaaaa ttagcattat actacatgct acttcaatct 27540aaacaccaga atatgcctac agatttttgg gggtagagct agggggaagt attccatcat 27600taggctgggg taggaactct ttaaagaaaa agtcagatta tcgactgaga cctgcaatat 27660actaacctgt tgagaaagaa tatgagttta taaattcccc aaagctataa tggggtacca 27720tgacgtgttg gcaattcttg tatcctggag gtgaaaaaga actcctgata aggttttgaa 27780ctctcgatga gatattacaa agcaaagatg gacctgaact cacccctttc ctatctgaaa 27840tctggtttat ttaggagaag agaagaggca ggaaggaaat atatggagta taggcaataa 27900acactatggg ctcagagaat agaggggtct cttccaacag aaaaggattc atagatgaca 27960ctaaaattgg attttgtaag atcaagagag tttagatagt aaggaagggt ttaggtattt 28020caggcaagga cttcatttag cttatgtgta tgtgcaactc tttctaattc taaaacggaa 28080ataatacaga acacctaacc ttccctcaac cctatttcct tactaagctc tcacctttgc 28140ttttgtccct tctctgctag aaaccttcaa agaatagcca atattagtca ttgcatgtgc 28200ttacttctat tcactctttg actcttgtgg tctagcttcc tgggccccca cgggcatgcc 28260aaaacaaaga cactaaaggc ctgctgcctg ccacaccagc agcctcttca gagtctcaac 28320cttgctctct gcatctgttc aacattgctg accacccctc ttcctgctcc cttggctact 28380atgacccacc tctttgctgc ttctcctgtc atttatacca ctccttccat ttcttctcct 28440ctggcagttc tctacagtca gatcatccaa gcttctgctc gcaattctct tgtattcctg 28500ctgtttatct tctttatgtt tctgacattc aagtttccta ctacaatgtg gggaagagtg 28560agagggagtt aagtggccct gttttgagct tagatgacta aaagaatgat gggttgctta 28620taagaaccag cagagtcaag aggcagagca gctttgggca ggactgggaa gatgtctttt 28680aacttcacaa tttcttagct tgagaactga ggaatctgta cacagatgtc tggccacaca 28740ttggatgatt acataattta catgataaca cagatgaaac atttgaagta gaagagattg 28800caaagtgaga aaagagttgg tctgccagta gaatcctaag gaatgcctac attcagccaa 28860tgggaaatgg gtgaagagcc agtgaagaac atagagtggt tagcgtcaga agggaaggtg 28920caatgttaag gaaacgtaaa gaaaaaaagt gcaagaaggc catcaagtgt caaatgtccc 28980agtggtggta gaaaacgagg acttaaaaaa ggccattcta tttgacgtta ggctattgat 29040gaatttacag agcagtgcaa atgatattta taaagtgctt acagtttaat actgggaaaa 29100gtggtgtttg aaatctgttt cctctaaggc ttaaatctaa agtgatttaa atttaaagtg 29160actagcatca aatacatacc acgttcagtg gtgagggcag gtagcaggct ctggctctga 29220gttcagggac ccttcaatac agaacacatt ccagtattca aactggaagt attccaattc 29280actaaaaagc aagaatcatt tcttctaaaa tcaagatacc aagcaagaac aagattcttt 29340gagttgtatt tctagaggga agaagaatat actctgggat ccctaaacaa acagcctgtg 29400acccttgaaa cacatctaag tagatcaaat tacaagtttt atttcttctt tggttttcag 29460taaacagacc aacaagacca gtacctttct tacactctaa ctaaaaaaat aataatttta 29520tcaaacaatg tgacttttaa atgtcttgtt ctcttttagg ggttcagtcc accagaattt 29580tgctgacttc acttttgcaa ctggcaaaat aattggacac atgctcaaat taaagggaga 29640catagattca aatgtagcta ttgatcttag caacaaagct tcattagcat tcttacaaaa 29700gcatttaggt aagaaactat ttttttcatg acctaaacca gatgaatctc aggacaaagc 29760tgtctatctt aatacagctt tagtactatt taaactattt ccagttggtt tacaatggaa 29820caaagcagta tatcaatttg aaaacagaaa tttgagaaag tcaattttgc tgctttacat 29880cctctatatc atagaaagca aatccaactg ttaaaggtaa tattctttgt atgaagccta 29940gagtggactt ccatgttgag gatactgaca gcaggttgcc tcactcctat cccgtttgca 30000ttcagctgct aaagcagcca tgaggcagct gatacagagc acatcgtctc taccatccta 30060acggaacttg tgtaatttgt aaatctttat tgccacctag gggcaccaaa ctgtttaatg 30120ctctcaaaag tttaatatgt tgattaacac tttatatttt ataggacttc ataaagattt 30180tgatcagtgg gactgcttga ttgaaggaga tgatgagaat cttattccag ggaccaacat 30240taacacaacc aatcaacaca tcatgttaca gaactcttca ggaatagaga aatacaatta 30300ggattaaaat aggtttttta aaagtcttgt ttcaaaactg tctaaaatta tgtgtgtgtg 30360tgtgtgtgtg tgtgtgtgtg tgagagagag agagagagag agagagagag agagagagaa 30420ttttaatgta ttttcccaaa ggactcatat tttaaaatgt aggctatact gtaatcgtga 30480ttgaagcttg gactaagaat tttttccctt tagatgtaaa gaaagaatac agtatacaat 30540attcatatca gcctaaattt taattttaaa gatgattcct tttcagtgtc gaagttaaaa 30600actgttttta cattactttg acagacaagt agattaaaac aggcaaaatc ccagtgaaaa 30660cctgttgcaa tgatacaaga ctccctaaac atagagtaaa aaacaatttc ttgcctttta 30720ttatctacta tgggcagtgg agtttaatta tagcaacatg atattctagg tagaatttgg 30780cagctcttct ctttgacttt tggtcacggt aagaaaaatt agaacaagca aaagccattg 30840tttcagcaca ggattggtgg tactacacaa tttcaaatga tgactaaaag gagtagagaa 30900gggttaagaa tattagagat gaggcaaagg cagtggatta aattggtaat tttttgacac 30960tttctttaat tctttaaggc attctacttg ctaaagaaag gttcctcagg tattacagca 31020tatagcaggg attccagtta gaatacatta gttactcaca caaaggctgt gaaggataat 31080aaaacattca gaattacatt tagcttcttc atattccagt taattttatt taatgttttc 31140aattctatat gtagtagtaa aacagaataa cacgattaaa tggaggtaaa aatatagctt 31200aagaatgtat acttataaat atgggaatca tggaaaatga ttttatttcc caggggacat 31260aaaaatttat accactcatt tggtacccta gtcctatctt agtggtgtca ggtgcaaagt 31320cactcattct gagaaatttt acttctgtag ccattcagat taaatttttt aaaataggga 31380gttaaactca gcttgaaatt gaaggcttcc tataatctga tcctaatgca tcttctctga 31440gctttgcttc tggcaattca cattatatta tctatatgag taagccaaac ttgtctattc 31500attattccag gaataagtaa tatactcatc tacctcaatt acttttaatg caaacctctc 31560tcccttctac ttacggaaat tataatgggt ttttaaggtt cataattcta tttccattag 31620gcttcagagt ctgataatgc ttcacagaga tacaccttta ccccagtctc atttagcaaa 31680ccttttccct attcatttta gaaataagca ttccaactgt atggatgtcc cattcaacac 31740ttttttgttt gtttgttttt tacagtcttt tgttggtggc gcatgctgat gctgaaagga 31800gaaaataact attgaaaata tctaataagt taaggttaaa aagaaataac ctaacatagt 31860gcacactgag tatttttcag ggtaaagaaa gacacattta tcaaatgcct actccttttc 31920agggacttta tatcaattat ttctcatctt aaaaaaacac tgcaagaaag gttttattat 31980caccacttta cagatgaaga tcaaagttca aaatattaca taatttgccc atgtcacata 32040tctgttaagc agcatatagc tcagatctgt ttctaaaagc taagatctat gctttgcaaa 32100atcatgttta aaaagtcatg ggcttcaaag ttaggtaggt ctgggtttga tcaagcctct 32160gagactcatc agctatctga ccttaggcaa gttatttaac ctctttatga agcttcaatt 32220ttctcaccta taaaatatgg ataatattga tatgcacttc aaagttgatg aataaagtga 32280aaaaatgcat ttaaagtata agctaagtga ctaatacata agagctaaca tttaatatta 32340ctatgctttc cccaatcctc cattacagag gagagatgga accaagtata tttaagagtt 32400ctatttcctt tttaataaaa tgagaccatc atattaagga taaactttat ttaaggataa 32460actttattca aatcttgtat tagcatactt caaattcaaa cagaatgtta gtcaaatgtt 32520tagacaaaca ggcaaagatt cagagtcaag acaaaaattc tgaaggagga tctgctaata 32580agctatctaa gtagaatcaa ataatgtact tgtcatgagc ccttattttt ctattttaat 32640ttaaacactg tatttgaaag gtaccaaaat agtttttgaa atgtaatgtg ttagtaaact 32700gtttagcaaa ataaatcctc ccaactaatt agaaacgtaa gacaataaag acgaaagatc 32760ccagatctgt atatataaca ggatcactat aaacacaccc caccatcaac ctgagaccta 32820atttgttttc cacctatata gaagtttttg aaaatgacaa cttcttgtgg ttacatgttt 32880tcaattttct caaatactct gttggtctgc gtaagatgcc acttgttcaa gggcagcttc 32940tgactgattg gccacagcta tagatccacg gttaaatctc catcacctcc aaaccccttt 33000tctgaaacaa aaagagaaaa acttcaaact gagaaagaaa catacaaaca aaaaaattta 33060tttccatggg tgttaccatg acaaatgtaa tgttctctaa aagggagagg ggacagatga 33120gtattctaat tactataggg cattatttct atagaaacaa tgttttattt tcttattaat 33180attttggttc tcacactgta ccatcaaatc aagtttctct aaaaattatg aaattggcat 33240attttatatt ttgctagaga atggtgtgaa caaggctgat aatcaattag ttctttttcc 33300tttttgatta gtatatattt ttcacatgat attaaagcta ccattagctg attttattct 33360caatgtgcct caattctaag aaccactgca ttatttatga acaagaatgt gcaataccaa 33420gaaccagcca ataatttgaa tcttatataa actaaagatt cagaaattcc aagagattat 33480ttaacaaata tcatttgaac atatgagcta attaacagag aaaattcttt aaaatgtgaa 33540tttatcacat tcataattta actttcaaga ttaattcaat taataaattt cctatatttc 33600aagaaatcct catcttgggt tatttatctc cataagtata tttaaatttt tttacctaaa 33660gtggtttttc ttccctgcta cctatgtcca aagaaattga taaaccattt ttgtttgagg 33720gattcagcat aacaatattt ttaagaaatt gtaattctgg gtgataaaag tattttgttt 33780taaaaacaca agttaccttt aatagtggta tcacccactg acatgaagta aagatctttc 33840cctactcttt gggatacagc ctaacaaaag gaacctttga attgctattg attctttatc 33900acgagtacaa aataattcaa atcagaaatg gaaccacaga taagccagaa aagatgtaaa 33960caacaaaatg tatttctatc aaaattatga caatctgatt ttataatgag gaatcatgtc 34020aagtactttt tctattcttc aaatactcat ttctctagaa catatccctg gagcactcta 34080cacagggagt aaagtaccct tttccaatgt ttagctctca aatccccaac aactctggta 34140attggtgaag gagggaggga catcattgtt caaggggctg ttattcaaac acagtatcca 34200tgtagagcca cagatgcttt attaaggaca atgaaaaggg aaatatttct cctatattcc 34260ttttaacaac caatagcatc atgtgcatgc attatgtgat acagtaacta acaaaatact 34320cttatggtaa aataaaataa ctcagataag aaaccatttt tttaaaaaaa gtaattagat 34380tcctagatca tagtgacttg atgtatcatt aaaaaaaagt catgcataat acaacactca 34440ctttgtaagg aaagctagct catttgtaga caggtatcca gagttaagag cttcctcgaa 34500tttgagggaa ggatatttaa atctcattag taaccaaatc aaaattttta gagtcttact 34560cataagtagt ttctatatgt ctgctaactg gcctagtcaa agccagaggt agttatctat 34620ttacttatct agatatataa ttttatgtct actttaaata gttcttttct atgtagcagg 34680catagagaac tttatccaaa ctgaatcttg tatgtacata tgtatgcatt tggctgtgga 34740ttcttcataa ttaatggatg gagacaaccc aaatgtaaaa agttttagta aatactgatg 34800caaaaagtgt agtaggctgg tatttttgac ctaagcaaaa aagtaattaa cataaagaga 34860gatattcatt gattttttat agtaaaaatt tcaaatgacc taaatatcca acactatggg 34920aaatggttta atacattatg gctcactagt gtcatgcagc attatttagc tattaaatat 34980tgtttatggc cttctgggtg cagccaagat ggaataagct gactataacc tctttcgctg 35040attacaatga aaaattctag acagaatata aaaagcaact actcaaagac tcaaaagtga 35100acagaaatag gtagattaag aattaaagta aaaacgtgaa taatgactca caacaggagt 35160gagtttcata ggccttttct tttctttttt tttacttctt tgtctcctga ttttgatctg 35220agggtgggcc aaatcaaaac tgcctagcta gcgtagacag caaaactctg agagaaattt 35280cttatttata gccagtggac tagggataaa ggaaccctga atgctagaga gtaagaaata 35340ggtaggaaat ctctttgctg tattgtggtg ttatgtttat ttctattttt ttttctggcc 35400ctgccctgag ggtggctaac cccagttact gcctggtgac agctatggca acataggcac 35460ctacaaatgt gagagatgac ccgtctctct agccagtgga accaggaaaa gagctttctg 35520tcatctgaag aatagtaata ggagattgtt tttgttatct ctttatctgc ttcgctctta 35580cgggcaaaca gaactgtgtg gtggagcaga ggctgagaga aaaatactgt ctggctaggt 35640gaccagaaaa aggggccatt ggaggctgga taagagagga tcctgggagc cagaaaatgt 35700ggaggacatc tcagagagga aaggagttga aaatgtttaa ctcctaatgc tacatgtgaa 35760ctctaagtct caggattgcc cctgagctat gcacatgcag aacagagcca aataagcaca 35820gcaaagtctt tgacacttaa ctactatata aaccactgac caaagtccct gactaccctg 35880ggtggcttat gtacctggaa gacctgaaca acaaaggctt tgaaaactaa actgacagga 35940caatcaccaa ccacagaagg agaaagagaa catgtaggat gaattgaaac cagctgatca 36000tctgctgtaa caaaaacttc aacattctcc agattttaac aggacccaat gtcttaaaac 36060ataatatgca aaatgttcag gatataatcc aaaattactc aacatacaaa gaaccaagaa 36120aagctgacca tttctctagg gaaaagaaaa tcaacagcta caaaccccaa gatgaccaga 36180tattaaaact atcagacaaa gattgtgaag tgagaagttc aagaagtcca accaacccca 36240aacaggataa actcaaatgc ccagacacat cataatgtca tggcaaattg cccaccatat 36300agtcttaaat gaagaaaaac agaatataaa actatacata ggatgaaatc aattgtgtct 36360aaaatatgta gatgaaatac ttatattaca tataaacaga aaaattataa aagaaaacca 36420aactggtcaa cctatttgtc tctggatggg tatattatcg gtgattgtta ctttctcatt 36480tttaatattg ttatatttct caggttttct gcatctaata tgtattttat tagagtcatg 36540aagaccaaaa cattttctaa tcaaggagta aaacattaat aactagtttt ctcagaagaa 36600tacttggcat aaggactaaa atcactgatt ttgttattgc tgtttttaac tattgtcaca 36660ggatttttct cccatttttc caaacacaag gactttggaa atagtttgtt aaatcacctt 36720tcaaaatgaa gaacaatgct ctcactaatt actatttata gaattccata cctcaggtgg 36780actcttatcc aatttgggta tgccattcca gacattctca gggttcacta tctcctccat 36840accatttgaa aggttcaaaa cctacaggag aaaacaaatt tttttaaagt gtccaattca 36900aaaaaataac tgatttatgt accagagcaa ttattttaaa aatgaaatac tattattatt 36960attattattt tgagataggg tcttgctcta ttgcccagga tggagggcag tggcaggatc 37020agagctcact gtgaccttga cctcctgggc tcaggcaatc tgcccatctc agcctcctga 37080ataactggga ctacaggcgc acaccaccat gcccagctaa ttttttgtat tttggataaa 37140gacagggttt cactatgctg cccagtctgg tctcaaactc cttggctcaa gcgatccacc 37200tgccttggcc tcccaaagtg ctgggattac aggcataagc cactgcacct ggccctaaaa 37260tgaaacactt taaaagacaa tgaaaatgaa tttattgagc ttcaacctta tgtttcaaac 37320tccatgctac ttcagtttcc caaacatatg tagagtatct atgactggtg atcaaagatg 37380gataaaattt caacatcaaa tagttcaaaa tctagtggag aaaagacctc taagcagata 37440attacaatac accataggaa gttcagactt ctgtagtagg aggtatagga ttgtaataca 37500gaaataaggc tttttttggg atgggggaag gttgaggaca agaataaatt tagaaaataa 37560catcagtgaa tgctccctgg tagaggcaat gggtaagtaa attggaatta accagtttgg 37620taacaggatg gaatataggt aaaatagaac tgcaatatta aatatatcga aagagcaagc 37680agatggtata tgtaagagaa gtcaagaatt ttgatgtcca tttaatataa agtacaaaat 37740gtatagaatt agggccaaag agagtacata aacacttaac cctagaggat ttcctatgcc 37800atgttaatga gcttgacttt tatgttccag gccaggggtc tccaatttta atggaatatg 37860aatgcgtatt ctggaatcct aaccccagaa attctgaatt agcagtccag gcgcaggcgt 37920aggaatctat aatacaattt aaaaaacagc cgggcgcagt ggctcatgcc tgtaatccca 37980gcactttggg aggctgaggt gggcggatca caaggtcagg agatcgagac catcctcgct 38040aacaagggga aaccccgtct ctacaaaaaa tacaaaaaat tagctgggcg tcgtggtggg 38100cgcctgtagt cccagctact tgggaggctg aggcaagaga atggtgtgaa ctgggaggca 38160gagcttgcag tgagccaaga tggcgccact gcactccagc ctgggtgaca gagtttgacc 38220ctgccccaaa aaaaaaaaaa aaaaaaagag ggaaatatat atatatatat atatacacac 38280acacacacac acacacacac acacatatat acatatatat acacatatat acacacatat 38340atatacatat atattacata tacatatata tatatatata tacacacata tacatatata 38400tatatatacc aagtaactct cgaacaggtg gttttcaaat cacgggagaa acacgatcaa 38460caggctccct aataagtttt acacaataat gaagtaatca aatttacatt ttacaaactt 38520caccctgaca ccatggacaa tcaatttaag gaagactaga agaagcaaac aaaatagaag 38580gttattaatg tagttgtgaa atgactcaac taaggtagtg gcattagata taaaaaattc 38640caactaagag ataagatgtg taaagtgtgg aactaggtga ctaatagcat ttagaaagag 38700taagggagat agatgataca gcctaggatg atttcttgct acgattctta tatatggaga 38760tttaaaatat atgtgcatta catcaagtta tcagggggaa caagcaagta aacatttgtt 38820caatcgcgtt ctctagtctg acagagtctg gtaaactttc cttaatagtc tgcaaataca 38880tttggagata aagtacttaa acatcactta cccttgttcc ttcttcagct gtttctaaaa 38940tctcacattt agaccatgtg tttcttagac ttgaccacac aacacattta gatccaacag 39000taaaggcttt cagagtgtag gtgttctgta gttgagctgc caaattgaat tgagtaagac 39060tcaatgcata tctcttaatt ccttaatgta acatacacaa gcaaagcatt ttcctaatct 39120gcattatttc tatatcatca atatcatgta attagtacat tggtatgact gtctccctca 39180attttgttta aaattcttac agtgcccgcc cccccatata atctcttatt gcttttctaa 39240accaacaaac caaacaaaaa atcaccacaa ggatgtgaat ttgatttcta acagtgaaaa 39300tcgctaataa aatatggtac tattaagaca agtggtgttt tttatactta caacagccat 39360ttctataaga aagcagcatg ttacaaatga gcagacattc ttatcacaga gttaatacta 39420ttattaaata tgacaatgag gaataaatat ctaatgcaag ggtaatactt aaataactta 39480tgtgttaccc ataggatggc ataccatgca attaaaatat tttgaaagca tttttaaggg 39540actaaaatgc aggattcaat actgtatgat agtatgatct aaattgtttt taaaactata 39600tagtagacat acacaaagac agtaaaagac aacaaaaaga aaatacatca tggtgttaac 39660attgatcact tcttgttgat tttcattctc actattccat tattttcctt atttttacaa 39720tggttcaaat ttaataatat aaaaattgac atttaaaaag tatagcattg attataaaaa 39780gctaaaagtt acccatttaa caattctttt taagtagcaa acagtagtaa aaataattta 39840taaaaaagtt aataaatgtt ctccaagatt tacacatgaa acagtttttg attaatatag 39900gctacctaaa tctatttttt ttctttaagg gtttctaatg aattgcaaga tcaatgcatg 39960atacagttaa gtctaaaaga ccctgttgaa ataatcctta gtgtaaatta atgcctgtag 40020tagtgccata aatttggcaa gggaatacca tgttttataa aacttaccaa cacatgccct 40080cttgattata aatgtgatta cattttaaat gtgattacct tttggagact aatagatttc 40140aagcattcaa cttttaaaca caaaactaat atttcagtgt ttcttccagc acatattttg 40200cactgatttc acaatatatt ttccccaagt ttaaaatgcc cttcataaaa tccaatctga 40260ttcactttcc aggtttttaa tctcttacaa aaaattattt tttagaaatc catataagaa 40320ttattctctt ttctttaaaa caaataacaa ctctggaaag tcactatgtt aaacttaaaa 40380gtaaaagcac actttgggag gctgaggcag aaggactgct tgagcccagg agtttgagac 40440caacctggat aacatgacaa accctgtctc tacaaaaaac agaaaaataa gctgggtgtg 40500gtggcacaca cctgtagtcc cagctacttg ggaggctgag gtgggaaaat cactagagcc 40560tgggaggcag aggctgcagt gagccaagat cacaccactg cactccagcc taggtgacag 40620agcaagaccc tgtctcaaaa aaaaaaaaaa aaaaaagtaa aaaggaaaaa aaattcacat 40680acctgagata tgatctggta aaccattatt gtgcttgctt ccatcactgc tttcttcctc 40740agagaacaaa ggcatcaatg ccgaaatggc atccctgttt ttatactcta caaattcttc 40800ttcacatata ttcatttcat tctgtgtttt aggatcacaa tctgctccat gtagatgcaa 40860tgacatcctg cgctggtcat caaaagattc tacacaactt gactttctca tgtcctcagt 40920acacatggat tcctgtgctt tctgactaac tcctaaagac aaaggatcca tcttgtcatc 40980taaaggcagc tgggctgtag gtagttctag ttctggctgt ttctcagctt cacagctgag 41040aggaagctgc agtgtaaata gctccatggc gccttttgtg acacactctt gggagagtgg 41100cacattcggt ggcaccgggc ttagctcccc tttttcttca tccaccacca gagaattctg 41160taactcaata gattccagtt ctaagaattc ttttgattca tcatcaggag aaagcggcac 41220ctcaagtgaa ttcagttcta gtatctcctt tgtttcatta gcatgtggta gtaatgtgtt 41280aaatcctgta atcaggtatt tatcatcaaa ctctgctttg tcaaccagat ggcactctaa 41340ttcaccttcc agttcctcag tatcaatttt atctttgcag gggttttcaa acccttcaca 41400aatgttatca gttttagggt cacggaagtt acttggtttg gttccaataa tgtttacatc 41460tttttcagtt atttcagcaa gctcatctgt ctgttgttcc atatatattt tattttgtac 41520agctttatta cttaatttct taagtcctac atcaagattg taggacccaa gagtctgctt 41580tatctctgag tcaatatttt catagggaag agcactttta ataccagtct tagaatattt 41640ctccattttc tcattaatac ttttaccttt gcttttcaag tcaacaattg ctaaagggat 41700atcacaaaca tcccttctga tttctagtat tgttatttcc aacacatctt ctgttattat 41760ttcatagaaa tagtcatttc cctcttgaga acataatcct tctgaaatgt taaaccctga 41820gaggcaacat ggaaaggctt gcatgggaac cgacagaagt tcagaaggaa tggcccagag 41880tgcttttggg tccacacagt cttcaatgtt tccaaagtcc acaagcctga cagatacaag 41940ataatcttca caaatattag tgataagtgc cctataataa tgtccatctt ctctgtatct 42000tactatacaa ggatctccaa tataaggaca tgggatacaa tttctcctgt ctgctacctg 42060ttctccagca gtctgtactt ctacttctaa acactgaagt ttctccgtat cagcaaactg 42120acaccaaaag tactcaggtc catctatcac agtggcataa gctcttatca tttttttttc 42180tggattatac cagttaagaa atactgaagt gtcaatgtct gatttgttaa cagactttga 42240actggcactt ttaattactt gggtagaaag ttctacttga gatttttcac tgagagcata 42300cctgctaatc atatcatctg ctatgatccc atgttcatca gcaagaataa cttcccatct 42360gtcttgaaat ttaacaaatt cacatcttat tgcagcctcg ctggtccgtt gggaaaagta 42420atgcatcatt ttcttagaat ttttattgtc aggaacctca

aatccctgca aggagcaatg 42480aatgcacaac cccggcaata ttgcattaac aaggtcaagc ctacctattt tgttagtatg 42540aaccacagaa acattgccat aatctataaa ctgcacagag agaaggtcat tgggttgttg 42600ctccttgatc a 42611

Claims

1. An isolated polynucleotide probe or primer, wherein said probe or primer is a fragment of a polynucleotide which is at least 99% identical to SEQ ID NO:2 and wherein said polynucleotide probe or primer comprises at least 20 contiguous nucleotides of SEQ ID NO:2 or its full complement which contains a transcription factor binding site at an AP1_C site at nucleotides 3731-3741, 4338-4348, 5013-5025, 15556-15566, 15596-15606, 19533-19543, 35142-35152, 38600-38610, 40256-40266, 41192-41202 or 41585-41595 of SEQ ID NO:2; an AP4_Q5 site at nucleotides 32929-32945or 38920-38936 of SEQ ID NO:2; a GATA1.sub.--04 site at nucleotides 25798-25810, 27827-27839, 28501-28513, 31629-31641, 32034-32046, 32169-32181, 33274-33286, 33538-33550or 41958-41970 of SEQ ID NO:2; a GATA1.sub.--06 site at nucleotides 30717-30729, 31464-31476, 35301-35313 or 41365-41377 of SEQ ID NO:2; a GATA2.sub.--02 site at nucleotides 3486-3498, 3609-3621, 9107-9119, 10637-10659, 18349-18361, 20858-20870, 32236-32248, 34609-34621, 35665-35677, 36781-36793 or 42161-42173 of SEQ ID NO:2; a GATA3.sub.--02 site at nucleotides 24794-24806, 25834-25846, 31071-31083, 31973-31985 or 35551-35563 of SEQ ID NO:2; a GATA_C site at nucleotides 343-355, 650-662;3370-3382, 4507-4519, 5160-5172, 7500-7512, 8422-8434, 10595-10607, 17905-17917, 22600-22612, 24775-24797, 27763-27775, 30027-30039, 30541-30553, 33695-33707, 33935-33947 or 41929-41941 of SEQ ID NO:2; a MZF1.sub.--01 site at nucleotides 2907-2915, 4766-4774;10854-10862, 11216-11224, 12675-12683, 15186-15194, 27471-27479, 28548-28556 or 33107-33115 of SEQ ID NO:2; an NFAT_Q6 site at nucleotides 1557-1575, 3717-3735, 3779-3797, 4621-4639, 5588-5606, 7380-7398, 9288-9306, 11523-11541, 12221-12239, 13103-13121, 13236-13254, 14475-14793, 16579-16597, 19812-19830, 21660-21678, 26158-26176, 27106-27124, 27872-27890, 28104-28122, 29107-29125, 32393-32411, 32816-32834, 33285-33303, 34446-35564 or 40661-40679 of SEQ ID NO:2; a NKX25.sub.--01 site at nucleotides 4402-4416, 8109-8123 or 33218-33232; a 58.sub.--01 site at nucleotides 2836-2848, 3390-3402, 3393-3405, 3872-3884, 3875-3887, 4227-4239, 12123-12137, 13569-13583, 15144-151458, 17041-17055, 21032-21046 or 28959-28973 of SEQ ID NO:2; a S8.sub.--01 site at nucleotides 12775-12787, 13388-13400, 14000-14012, 18248-18260, 25893-25905, 25896-25908, 25948-25960, 27231-27243 or 27497-27509 of SEQ ID NO:2; a SOX5.sub.--01 site at nucleotides 42438-42454 of SEQ ID NO:2; a TATA_C site at nucleotides 25236-25242, 25245-25261, or 41494-41510 of SEQ ID NO:2 or a TCF11.sub.--01 site at nucleotides 2093-2099, 10078-10084, 10792-10798, 14200-14206, 15162-15168, 28408-28414, 32852-32858, 34893-34899, 38599-38905 or 41773-41779 of SEQ ID NO:2.

2. A method for isolating the polynucleotide of claim 1 comprising (a) isolating genomic DNA from a sample; (b) providing primers, probes and optionally polymerase and (c) incubating (a) and (b) under conditions promoting the isolation of said nucleic acid molecule.

3. A kit comprising the polynucleotide of claim 1.

4. The polynucleotide according to claim 1, wherein said polynucleotide is an RNA or DNA.

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