CA2384499A1 - Ovarian tumor sequences and methods of use therefor - Google Patents

Abstract

Compositions and methods for the therapy and diagnosis of cancer, such as ovarian cancer, are disclosed. Compositions may comprise one or more ovarian carcinoma proteins, portions thereof, polynucleotides that encode such portions or antibodies or immune system cells specific for such proteins. Su ch compositions may be used, for example, for the prevention and treatment of diseases such as ovarian cancer. Polypeptides and polynucleotides as provide d herein may further be used for the detection and monitoring of ovarian cance r.

Description

OVARIAN TUMOR SEQUENCES AND METHODS OF USE THEREFOR
TECHNICAL FIELD
The present invention relates generally to ovarian cancer therapy. The invention is more specifically related to polypeptides comprising at least a portion of an ovarian carcinoma protein, and to polynucleotides encoding such polypeptides, as well as antibodies and immune system cells that specifically recognize such polypeptides.
Such polypeptides, polynucleotides, antibodies and cells may be used in vaccines and pharmaceutical compositions for treatment of ovarian cancer.
1o BACKGROUND OF THE INVENTION
Ovarian cancer is a significant health problem for women in the United States and throughout the world. , Although advances have been made in detection and therapy of this cancer, no vaccine or other universally successful method for prevention or treatment is currently available. Management of the disease currently relies on a combination of early diagnosis and aggressive treatment, which may include one or more of a variety of treatments such as surgery, radiotherapy, chemotherapy and hormone therapy. The course of treatment for a particular cancer is often selected based on a variety of prognostic parameters, including an analysis of specific tumor markers.
However, the use of established markers often leads to a result that is difficult to 2o interpret, and high mortality continues to be observed in many cancer patients.
Immunotherapies have the potential to substantially improve cancer treatment and survival. Such therapies may involve the generation or enhancement of an immune response to an ovarian carcinoma antigen. However, to date, relatively few ovarian carcinoma antigens are known and the generation of an immune response against such antigens has not been shown to be therapeutically beneficial.
Accordingly, there is a need in the art for improved methods for identifying ovarian tumor antigens and for using such antigens in the therapy of ovarian cancer. The present invention fulfills these needs and further provides other related advantages.

SUMMARY OF THE INVENTION
Briefly stated, this invention provides compositions and methods for the therapy of cancer, such as ovarian cancer. In one aspect, the present invention provides polypeptides comprising an immunogenic portion of an ovarian carcinoma protein, or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with ovarian carcinoma protein-specific antisera is not substantially diminished. Within certain embodiments, the ovarian carcinoma protein comprises a sequence that is encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NOs:l, 2, S, 9, 10, 13, 16, 19, l0 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185 and 193-199, and complements of such polynucleotides.
The present invention further provides polynucleotides that encode a polypeptide as described above or a portion thereof, expression vectors comprising such polynucleotides and host cells transformed or transfected with such expression vectors.
Within other aspects, the present invention provides pharmaceutical compositions and vaccines. Pharmaceutical compositions may comprise a physiologically acceptable carrier or excipient in combination with one or more of: (i) a 2o polypeptide comprising an immunogenic portion of an ovarian carcinoma protein, or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with ovarian carcinoma protein-specific antisera is not substantially diminished, wherein the ovarian carcinoma protein comprises an amino acid sequence encoded by a polynucleotide that comprises a sequence recited in any one of SEQ ID NOs:I-185 and 187-199; (ii) a polynucleotide encoding such a polypeptide; (iii) an antibody that specifically binds to such a polypeptide; (iv) an antigen-presenting cell that expresses such a polypeptide and/or (v) a T cell that specifically reacts with such a polypeptide. Vaccines may comprise a non-specific immune response enhancer in combination with one or more of: (i) a polypeptide comprising ax immunogenic portion of an ovarian carcinoma protein, or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with ovarian carcinoma protein-specific antisera is not substantially diminished, wherein the ovarian carcinoma protein comprises an amino acid sequence encoded by a polynucleotide that comprises a sequence recited in any one of SEQ ID NOs:I-185 and 187-196, (ii) a polynucleotide encoding such a polypeptide; (iii) an anti-idiotypic antibody that is specifically bound by an antibody that specifically binds to such a polypeptide; (iv) an antigen-presenting cell that expresses such a polypeptide and/or (v) a T cell that specifically reacts with such a polypeptide. An exemplary polypeptide comprises an amino acid sequence recited in SEQ ID N0:186.
l0 The present invention further provides, in other aspects, fusion proteins that comprise at least one polypeptide as described above, as well as polynucleotides encoding such fusion proteins.
Within related aspects, pharmaceutical compositions comprising a fusion protein or polynucleotide encoding a fusion protein in combination with a physiologically acceptable carrier are provided.
Vaccines are further provided, within other aspects, comprising a fusion protein or polynucleotide encoding a fusion protein in combination with a non-specific immune response enhancer.
Within further aspects, the present invention provides methods for 2o inhibiting the development of a cancer in a patient, comprising administering to a patient a pharmaceutical composition or vaccine as recited above.
The present invention further provides, within other aspects, methods for stimulating and/or expanding T cells, comprising contacting T cells with (a) a polypeptide comprising an immunogenic portion of an ovarian carcinoma protein, or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with ovarian carcinoma protein-specific antisera is not substantially diminished, wherein the ovarian carcinoma protein comprises an amino acid sequence encoded by a polynucleotide that comprises a sequence recited in any one of SEQ ID NOs:I-185 and 187-199; (b) a polynucleotide encoding such a polypeptide and/or (c) an antigen presenting cell that expresses such a polypeptide under conditions and for a time sufficient to permit the stimulation and/or expansion of T cells. Such polypeptide, polynucleotide and/or antigen presenting cells) may be present within a pharmaceutical composition or vaccine, for use in stimulating and/or expanding T cells in a mammal.
Within other aspects, the present invention provides methods for inhibiting the development of ovarian cancer in a patient, comprising administering to a patient T cells prepared as described above.
Within further aspects, the present invention provides methods for inhibiting the development of ovarian cancer in a patient, comprising the steps of: (a) incubating CD4+ and/or CD8+ T cells isolated from a patient with one or more of: (i) a t o polypeptide comprising an immunogenic portion of an ovarian carcinoma protein, or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with ovarian carcinoma protein-specific antisera is not substantially diminished, wherein the ovarian carcinoma protein comprises an amino acid sequence encoded by a polynucleotide that comprises a is sequence recited in any one of SEQ ID NOs:I-185 and 187-199; (ii) a polynucleotide encoding such a polypeptide; or (iii) an antigen-presenting cell that expresses such a polypeptide; such that T cells proliferate; and (b) administering to the patient an effective amount of the proliferated T cells, and thereby inhibiting the development of ovarian cancer in the patient. The proliferated cells may be cloned prior to 2o administration to the patient.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings.
All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.

The present invention is directed generally to compositions and their use in the therapy and diagnosis of cancer, particularly ovarian cancer. As described further below, illustrative compositions of the present invention include, but are not restricted to, polypeptides, particularly immunogenic polypeptides, polynucleotides encoding 3o such polypeptides, antibodies and other binding agents, antigen presenting cells (APCs) and immune system cells (e.g., T cells).
The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration.
Such techniques are explained fully in the literature. See, e.g., Sambrook, et al.
Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al. Molecular Cloning:
A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D.
Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B.
Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986);
Perbal, A Practical Guide to Molecular Cloning (1984).
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise.
POLYPEPTIDE COMPOSITIONS
2o As used herein, the term "polypeptide" " is used in its conventional meaning, i.e. as a sequence of amino acids. The polypeptides are not limited to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A polypeptide may be an entire protein, or a subsequence thereof. Particular polypeptides of interest in the context of this invention are amino acid subsequences comprising epitopes, i.e. antigenic 3o determinants substantially responsible for the immunogenic properties of a polypeptide and being capable of evoking an immune response.
Particularly illustrative polypeptides of the present invention comprise those encoded by a polynucleotide sequence set forth herein, or a sequence that hybridizes under moderately stringent conditions, or, alternatively, under highly stringent conditions, to a polynucleotide sequence set forth herein.
The polypeptides of the present invention are sometimes herein referred to as ovarian tumor proteins or ovarian tumor polypeptides, as an indication that their identification has been based at least in part upon their increased levels of expression in ovarian tumor samples. Thus, a " ovarian tumor polypeptide" or "ovarian tumor 1 o protein," refers generally to a polypeptide sequence of the present invention, or a polynucleotide sequence encoding such a polypeptide, that is expressed in a substantial proportion of ovarian tumor samples, for example preferably greater than about 20%, more preferably greater than about 30%, and most preferably greater than about 50% or more of ovarian tumor samples tested, at a level that is at least two fold, and preferably at least five fold, greater than the level of expression in normal tissues, as determined using a representative assay provided herein. A ovarian tumor polypeptide sequence of the invention, based upon its increased level of expression in tumor cells, has particular utility both as a diagnostic marker as well as a therapeutic target, as further described below.
In certain preferred embodiments, the polypeptides of the invention are immunogenic, i.e., they react detectably within an immunoassay (such as an ELISA or T-cell stimulation assay) with antisera and/or T-cells from a patient with ovarian cancer.
Screening for immunogenic activity can be performed using techniques well known to the skilled artisan. For example, such screens can be performed using methods such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In one illustrative example, a polypeptide may be immobilized on a solid support and contacted with patient sera to allow binding of antibodies within the sera to the immobilized polypeptide. Unbound sera may then be removed and bound antibodies detected using, for example,'z5I-labeled Protein A.
3o As would be recognized by the skilled artisan, immunogenic portions of the polypeptides disclosed herein are also encompassed by the present invention. An "immunogenic portion," as used herein, is a fragment of an immunogenic polypeptide of the invention that itself is immunologically reactive (i. e., specifically binds) with the B-cells and/or T-cell surface antigen receptors that recognize the polypeptide.
Immunogenic portions may generally be identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides for the ability to react with antigen-specific antibodies, antisera and/or T-cell lines or clones. As used herein, antisera and antibodies are "antigen-specific" if they specifically bind to an antigen (i.e., they react with the protein in an ELISA
or other immunoassay, and do not react detectably with unrelated proteins). Such antisera and antibodies may be prepared as described herein, and using well-known techniques.
In one preferred embodiment, an immunogenic portion of a polypeptide of the present invention is a portion that reacts with antisera and/or T-cells at a level that is not substantially less than the reactivity of the full-length polypeptide (e.g., in an ELISA and/or T-cell reactivity assay). Preferably, the level of immunogenic activity of the immunogenic portion is at least about 50%, preferably at least about 70%
and most preferably greater than about 90% of the immunogenicity for the full-length polypeptide. In some instances, preferred immunogenic portions will be identified that have a level of immunogenic activity greater than that of the corresponding full-length polypeptide, e.g., having greater than about 100% or 150% or more immunogenic activity.
In certain other embodiments, illustrative immunogenic portions may include peptides in which an N-terminal leader sequence and/or transmembrane domain have been deleted. Other illustrative immunogenic portions will contain a small N-and/or C-terminal deletion (e.g., 1-30 amino acids, preferably 5-15 amino acids), relative to the mature protein.
In another embodiment, a polypeptide composition of the invention may also comprise one or more polypeptides that are immunologically reactive with T cells 3o and/or antibodies generated against a polypeptide of the invention, particularly a polypeptide having an amino acid sequence disclosed herein, or to an immunogenic fragment or variant thereof.
In another embodiment of the invention, polypeptides are provided that comprise one or more polypeptides that are capable of eliciting T cells and/or antibodies that are immunologically reactive with one or more polypeptides described herein, or one or more polypeptides encoded by contiguous nucleic acid sequences contained in the polynucleotide sequences disclosed herein, or immunogenic fragments or variants thereof, or to one or more nucleic acid sequences which hybridize to one or more of these sequences under conditions of moderate to high stringency.
to The present invention, in another aspect, provides polypeptide fragments comprising at least about 5, 10, 15, 20, 25, 50, or 100 contiguous amino acids, or more, including all intermediate lengths, of a polypeptide compositions encoded by a polynucleotide sequence set forth herein.
In another aspect, the present invention provides variants of the polypeptide compositions described herein. Polypeptide variants generally encompassed by the present invention will typically exhibit at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity (determined as described below), along its length, to a polypeptide sequences set forth herein.
2o In one preferred embodiment, the polypeptide fragments and variants provide by the present invention are immunologically reactive with an antibody and/or T-cell that reacts with a full-length polypeptide specifically set for the herein.
In another preferred embodiment, the polypeptide fragments and variants provided by the present invention exhibit a level of immunogenic activity of at least about 50%, preferably at least about 70%, and most preferably at least about 90% or more of that exhibited by a full-length polypeptide sequence specifically set forth herein.
A polypeptide "variant," as the term is used herein, is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the invention and evaluating their immunogenic activity as described herein and/or using any of a number of techniques well known in the art.
For example, certain illustrative variants of the polypeptides of the invention include those in which one or more portions, such as an N-terminal leader sequence or transmembrane domain, have been removed. Other illustrative variants include variants in which a small portion (e.g., 1-30 amino acids, preferably 5-15 amino 1o acids) has been removed from the N- andlor C-terminal of the mature protein.
In many instances, a variant will contain conservative substitutions. A
"conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. As described above, modifications may be made in the structure of the polynucleotides and polypeptides of the present invention and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics, e.g., with immunogenic characteristics. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or 2o even an improved, immunogenic variant or portion of a polypeptide of the invention, one skilled in the art will typically change one or more of the codons of the encoding DNA sequence according to Table 1.
For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA
coding sequence, and nevertheless obtain a protein with like properties. It is thus 3o contemplated that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.

Amino Acids Codons Alanine Ala A GCA GCC GCG GCU

Cysteine Cys C UGC UGU

Aspartic acid Asp D GAC GAU

Glutamic acid Glu E GAA GAG

Phenylalanine Phe F UUC UUU

Glycine Gly G GGA GGC GGG GGU

Histidine His H CAC CAU

Isoleucine Ile I AUA AUC AUU

Lysine Lys K AAA AAG

Leucine Leu L UUA UUG CUA CUC CUG CUU

Methionine Met M AUG

Asparagine Asn N AAC AAU

Proline Pro P CCA CCC CCG CCU

Glutamine Gln Q CAA CAG

Arginine Arg R AGA AGG CGA CGC CGG CGU

Serine Ser S AGC AGU UCA UCC UCG UCU

Threonine Thr T ACA ACC ACG ACU

Valine Val V GUA GUC GUG GUU

Tryptophan Trp W UGG

Tyrosine Tyr Y UAC UAU

In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). It is accepted that the relative 1 o hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other to molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values are:
isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);
cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7);
serine (-0.8);
tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2);
glutamate (-3.5);
glutariiine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a l0 protein with similar biological activity, i. e. still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ~2 is preferred, those within ~1 are particularly preferred, and those within ~0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U. S. Patent 4,554,101 (specifically incorporated herein by reference in its entirety), states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.
As detailed in U. S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0);
aspartate (+3.0 ~ 1 ); glutamate (+3.0 ~ 1 ); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ~ 1); alanine (-0.5);
histidine (-0.5);
cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8);
tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ~2 is preferred, those within ~1 are particularly preferred, and those within ~0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well .known to those of skill in the art and include: arginine and lysine; glutamate and aspartate;
serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
In addition, any polynucleotide may be further modified to increase stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl 1 o rather than phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl-methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.
Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine;
2o and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: ( 1 ) ala, pro, gly, glu, asp, gln, asn, ser, thr;
(2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his;
and (5) phe, tyr, trp, his. A variant may also, or alternatively, contain nonconservative changes. In a preferred embodiment, variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.
As noted above, polypeptides may comprise a signal (or leader) 3o sequence at the N-terminal end of the protein, which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support.
For example, a polypeptide may be conjugated to an immunoglobulin Fc region.
When comparing polypeptide sequences, two sequences are said to be "identical" if the sequence of amino acids in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A
"comparison I o window" as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
Optimal alignment of sequences for comparison may be conducted using I S the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M.O. (1978) A
model of evolutionary change in proteins - Matrices for detecting distant relationships.
In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical 2o Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J.
(1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M.
(1989) CABIOS 5:151-153; Myers, E.W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E.D. (1971) Comb. Theor 11:105; Santou; N. Nes, M. (1987) Mol. Biol. Evol.
4:406-25 425; Sneath, P.H.A. and Sokal, R.R. (1973) Numerical Taxonomy - the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J.
and Lipman, D.J. (1983) Proc. Natl. Acad , Sci. USA 80:726-730.
Alternatively, optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add.
APL.
3o Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J.

Mol. Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by inspection.
One preferred example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res.
25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST
2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the invention.
Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction are halted 1 s when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
In one preferred approach, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.

Within other illustrative embodiments, a polypeptide may be a fusion polypeptide that comprises multiple polypeptides as described herein, or that comprises at least one polypeptide as described herein and an unrelated sequence, such as a known tumor protein. A fusion partner may, for example, assist in providing T helper epitopes (an immunological fusion partner), preferably T helper epitopes recognized by humans, or may assist in expressing the protein (an expression enhancer) at higher yields than the native recombinant protein. Certain preferred fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners may be selected so as to increase the solubility of the polypeptide or to enable the polypeptide to be targeted to desired intracellular compartments. Still further fusion partners include affinity tags, which facilitate purification of the polypeptide.
Fusion polypeptides may generally be prepared using standard techniques, including chemical conjugation. Preferably, a fusion polypeptide is expressed as a recombinant polypeptide, allowing the production of increased levels, relative to a non-fused polypeptide, in an expression system. Briefly, DNA
sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector. The 3' end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion polypeptide that retains the biological activity of both component polypeptides.
A peptide linker sequence may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence is incorporated into the fusion polypeptide using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors:
(1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with 3o the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Patent No. 4,935,233 and U.S.
Patent No. 4,751,180. The linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
The ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements. The regulatory elements to responsible for expression of DNA are located only 5' to the DNA sequence encoding the first polypeptides. Similarly, stop codons required to end translation and transcription termination signals are only present 3' to the DNA sequence encoding the second polypeptide.
The fusion polypeptide can comprise a polypeptide as described herein together with an unrelated immunogenic protein, such as an immunogenic protein capable of eliciting a recall response: Examples of such proteins include tetanus, tuberculosis and hepatitis proteins (see, for example, Stoute et al. New Engl.
J. Med., 336:86-91, 1997).
In one preferred embodiment, the immunological fusion partner is 2o derived from a Mycobacterium sp., such as a Mycobacterium tuberculosis-derived Ral2 fragment. Ral2 compositions and methods for their use in enhancing the expression and/or immunogenicity of heterologous polynucleotide/polypeptide sequences is described in U.S. Patent Application 60/158,585, the disclosure of which is incorporated herein by reference in its entirety. Briefly, Ral2 refers to a polynucleotide region that is a subsequence of a Mycobacterium tuberculosis MTB32A nucleic acid.
MTB32A is a serine protease of 32 KD molecular weight encoded by a gene in virulent and avirulent strains of M. tuberculosis. The nucleotide sequence and amino acid sequence of MTB32A have been described (for example, U.S. Patent Application 60/158,585; see also, Skeiky et al., Infection and Immun. (1999) 67:3998-4007, 3o incorporated herein by reference). C-terminal fragments of the MTB32A
coding sequence express at high levels and remain as a soluble polypeptides throughout the purification process. Moreover, Ral2 may enhance the immunogenicity of heterologous immunogenic polypeptides with which it is fused. One preferred Ral2 fusion polypeptide comprises a 14 KD C-terminal fragment corresponding to amino acid residues 192 to 323 of MTB32A. Other preferred Ral2 polynucleotides generally comprise at least about 15 consecutive nucleotides, at least about 30 nucleotides, at least about 60 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, or at least about 300 nucleotides that encode a portion of a Ral2 polypeptide. Ral2 polynucleotides may comprise a native sequence (i. e., an endogenous sequence that to encodes a Ral2 polypeptide or a portion thereof) or may comprise a variant of such a sequence. Ral2 polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions such that the biological activity of the encoded fusion polypeptide is not substantially diminished, relative to a fusion polypeptide comprising a native Ral2 polypeptide. Variants preferably exhibit at least about 70%
identity, more preferably at least about 80% identity and most preferably at least about 90% identity to a polynucleotide sequence that encodes a native Ral2 polypeptide or a portion thereof.
Within other preferred embodiments, an immunological fusion partner is derived from protein D, a surface protein of the gram-negative bacterium Haemophilus 2o influenza B (WO 91/18926). Preferably, a protein D derivative comprises approximately the first third of the protein (e.g., the first N-terminal 100-110 amino acids), and a protein D derivative may be lipidated. Within certain preferred embodiments, the first 109 residues of a Lipoprotein D fusion partner is included on the N-terminus to provide the polypeptide with additional exogenous T-cell epitopes and to increase the expression level in E coli (thus functioning as an expression enhancer).
The lipid tail ensures optimal presentation of the antigen to antigen presenting cells.
Other fusion partners include the non-structural protein from influenzae virus, NS 1 (hemaglutinin). Typically, the N-terminal 81 amino acids are used, although different fragments that include T-helper epitopes may be used.
3o In another embodiment, the immunological fusion partner is the protein known as LYTA, or a portion thereof (preferably a C-terminal portion). LYTA is derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene; Gene 43:265-292, 1986). LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE.
This property has been exploited for the development of E. coli C-LYTA expressing plasmids useful for expression' of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at the amino terminus has been described (see Biotechnology 10:795-798, 1992). Within a preferred embodiment, a repeat portion of LYTA may be incorporated into a fusion polypeptide. A repeat portion is found in the C-terminal region starting at residue 178. A particularly preferred repeat portion incorporates residues 188-305.
Yet another illustrative embodiment involves fusion polypeptides, and the polynucleotides encoding them, wherein the fusion partner comprises a targeting signal capable of directing a polypeptide to the endosomal/lysosomal compartment, as described in U.S. Patent No. 5,633,234. An immunogenic polypeptide of the invention, when fused with this targeting signal, will associate more efficiently with MHC class II
molecules and thereby provide enhanced in vivo stimulation of CD4+ T-cells specific for the polypeptide.
Polypeptides of the invention are prepared using any of a variety of well known synthetic and/or recombinant techniques, the latter of which are further described below. Polypeptides, portions and other variants generally less than about 150 amino acids can be generated by synthetic means, using techniques well known to those of ordinary skill in the art. In one illustrative example, such polypeptides are synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963.
Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems Division (Foster City, CA), and may be operated according to the manufacturer's instructions.

In general, polypeptide compositions (including fusion polypeptides) of the invention are isolated. An "isolated" polypeptide is one that is removed from its original environment. For example, a naturally-occurring protein or polypeptide is isolated if it is separated from some or all of the coexisting materials in the natural system. Preferably, such polypeptides are also purified, e.g., are at least about 90%
pure, more preferably at least about 95% pure and most preferably at least about 99%
pure.
POLYNUCLEOTIDE COMPOSITIONS
to The present invention, in other aspects, provides polynucleotide compositions. The terms "DNA" and "polynucleotide" are used essentially interchangeably herein to refer to a DNA molecule that has been isolated free of total genomic DNA of a particular species. "Isolated," as used herein, means that a polynucleotide is substantially away from other coding sequences, and that the DNA
molecule does not contain large portions of unrelated coding DNA, such as large chromosomal fragments or other functional genes or polypeptide coding regions.
Of course, this refers to the DNA molecule as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
As will be understood by those skilled in the art, the polynucleotide 2o compositions of this invention can include genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, peptides and the like. Such segments may be naturally isolated, or modified synthetically by the hand of man.
As will be also recognized by the skilled artisan, polynucleotides of the invention may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules may include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the 3o present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.
Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a polypeptide/protein of the invention or a portion thereof) or may comprise a sequence that encodes a variant or derivative, preferably and immunogenic variant or derivative, of such a sequence.
Therefore, according to another aspect of the present invention, polynucleotide compositions are provided that comprise some or all of a polynucleotide sequence set forth in any one of SEQ ID NOs: 1-185 and 187-196, complements of a polynucleotide sequence set forth in any one of SEQ ID NOs: 1-185 and 187-196, and degenerate variants of a polynucleotide sequence set forth in any one of SEQ
ID NOs:
1-185 and 187-196. In certain preferred embodiments, the polynucleotide sequences set forth herein encode immunogenic polypeptides, as described above.
In other related embodiments, the present invention provides polynucleotide variants having substantial identity to the sequences disclosed herein in SEQ ID NOs: 1-185 and 187-196, for example those comprising at least 70%
sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
or higher, sequence identity compared to a polynucleotide sequence of this invention using the methods described herein, (e.g., BLAST analysis using standard parameters, as described below). One skilled in this art will recognize that these values can be 2o appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.
Typically, polynucleotide variants will contain one or more substitutions, additions, deletions and/or insertions, preferably such that the immunogenicity of the polypeptide encoded by the variant polynucleotide is not substantially diminished relative to a polypeptide encoded by a polynucleotide sequence specifically set forth herein). The term "variants" should also be understood to encompasses homologous genes of xenogenic origin.
In additional embodiments, the present invention provides 3o polynucleotide fragments comprising various lengths of contiguous stretches of sequence identical to or complementary to one or more of the sequences disclosed herein. For example, polynucleotides are provided by this invention that comprise at least about 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, S00 or 1000 or more contiguous nucleotides of one or more of the sequences disclosed herein as well as all intermediate lengths there between. It will be readily understood that "intermediate lengths", in this context, means any length between the quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50,51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through 200-500; 500-1,000, and the like.
l0 In another embodiment of the invention, polynucleotide compositions are provided that are capable of hybridizing under moderate to high stringency conditions to a polynucleotide sequence provided herein, or a fragment thereof, or a complementary sequence thereof. Hybridization techniques are well known in the art of molecular biology. For purposes of illustration, suitable moderately stringent conditions for testing the hybridization of a polynucleotide of this invention with other polynucleotides include prewashing in a solution of S X SSC, 0.5% SDS, 1.0 mM
EDTA (pH 8.0); hybridizing at 50°C-60°C, 5 X SSC, overnight;
followed by washing twice at 65°C for 20 minutes with each of 2X, O.SX and 0.2X SSC
containing 0.1%
SDS. One skilled in the art will understand that the stringency of hybridization can be readily manipulated, such as by altering the salt content of the hybridization solution and/or the temperature at which the hybridization is performed. For example, in another embodiment, suitable highly stringent hybridization conditions include those described above, with the exception that the temperature of hybridization is increased, e.g., to 60-65°C or 65-70°C.
In certain preferred embodiments, the polynucleotides described above, e.g., polynucleotide variants, fragments and hybridizing sequences, encode polypeptides that are immunologically cross-reactive with a polypeptide sequence specifically set forth herein. In other preferred embodiments, such polynucleotides encode polypeptides that have a level of immunogenic activity of at least about 50%, preferably at least about 70%, and more preferably at least about 90% of that for a polypeptide sequence specifically set forth herein.

The polynucleotides of the present invention, or fragments thereof, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA
protocol.
For example, illustrative polynucleotide segments with total lengths of about 10,000, about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, 1o about 50 base pairs in length, and the like, (including all intermediate lengths) are contemplated to be useful in many implementations of this invention.
When comparing polynucleotide sequences, two sequences are said to be "identical" if the sequence of nucleotides in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A
"comparison window" as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences 2o are optimally aligned.
Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M.O. (1978) A
model of evolutionary change in proteins - Matrices for detecting distant relationships.
In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J.
(1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M.
(1989) 3o CABIOS 5:151-153; Myers, E.W. and Muller W. (1988) CABIOS 4:11-17;
Robinson, E.D. (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol.
4:406-425; Sneath, P.H.A. and Sokal, R.R. (1973) Numerical Taxonomy - the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J.
and Lipman, D.J. (1983) Proc. Natl. Acad , Sci. USA 80:726-730.
Alternatively, optimal alignment of sequences for comparison may be s conducted by the local identity algorithm of Smith and Waterman (1981) Add.
APL.
Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J.
Mol. Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics 1o Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by inspection.
One preferred example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res.
25:3389-3402 15 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST
and BLAST
2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. In one illustrative example, cumulative scores can be calculated using, for 20 nucleotide sequences, the parameters M (reward score for a pair of matching residues;
always >0) and N (penalty score for mismatching residues; always <0).
Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments;
25 or the end of either sequence is reached. The BLAST algorithm parameters W, T and X
determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc.
Natl.
Acad. Sci. USA 89:10915) alignments, (B) of 50, expectation (E) of 10, M=5, N=-4 and 3o a comparison of both strands.

Preferably, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence to (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.
It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Further, alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions 2o and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison).
Therefore, in another embodiment of the invention, a mutagenesis approach, such as site-specific mutagenesis, is employed for the preparation of immunogenic variants and/or derivatives of the polypeptides described herein.
By this approach, specific modifications in a polypeptide sequence can be made through mutagenesis of the underlying polynucleotides that encode them. These techniques provides a straightforward approach to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the polynucleotide.

Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Mutations may be employed in a selected polynucleotide sequence to improve, alter, decrease, modify, or otherwise change the properties of the polynucleotide itself, and/or alter the properties, activity, composition, stability, or primary sequence of the encoded polypeptide.
In certain embodiments of the present invention, the inventors l0 contemplate the mutagenesis of the disclosed polynucleotide sequences to alter one or more properties of the encoded polypeptide, such as the immunogenicity of a polypeptide vaccine. The techniques of site-specific mutagenesis are well-known in the art, and are widely used to create variants of both polypeptides and polynucleotides.
For example, site-specific mutagenesis is often used to alter a specific portion of a DNA
molecule. In such embodiments, a primer comprising typically about 14 to about nucleotides or so in length is employed, with about 5 to about 10 residues on both sides of the junction of the sequence being altered.
As will be appreciated by those of skill in the art, site-specific mutagenesis techniques have often employed a phage vector that exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M 13 phage. These phage are readily commercially-available and their use is generally well-known to those skilled in the art.
Double-stranded plasmids are also routinely employed in site directed mutagenesis that eliminates the step of transferring the gene of interest from a plasmid to a phage.
In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double-stranded vector that includes within its sequence a DNA sequence that encodes the desired peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded 3o vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I

Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.
The preparation of sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis provides a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants. of peptides and the DNA sequences encoding them may be obtained. For example, recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants. Specific details regarding these methods and protocols are found in the teachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994;
and Maniatis et al., 1982, each incorporated herein by reference, for that purpose.
As used herein, the term "oligonucleotide directed mutagenesis procedure" refers to template-dependent processes and vector-mediated propagation which result in an increase in the concentration of a specific nucleic acid molecule relative to its initial concentration, or in an increase in the concentration of a detectable signal, such as amplification. As used herein, the term "oligonucleotide directed 2o mutagenesis procedure" is intended to refer to a process that involves the template-dependent extension of a primer molecule. The term template dependent process refers to nucleic acid synthesis of an RNA or a DNA molecule wherein the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing (see, for example, Watson, 1987).
Typically, vector mediated methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by U. S. Patent No. 4,237,224, specifically incorporated herein by reference in its entirety.
In another approach for the production of polypeptide variants of the present invention, recursive sequence recombination, as described in U.S.
Patent No.

5,837,458, may be employed. In this approach, iterative cycles of recombination and screening or selection are performed to "evolve" individual polynucleotide variants of the invention having, for example, enhanced immunogenic activity.
In other embodiments of the present invention, the polynucleotide sequences provided herein can be advantageously used as probes or primers for nucleic acid hybridization. As such, it is contemplated that nucleic acid segments that comprise a sequence region of at least about 15 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 15 nucleotide long contiguous sequence disclosed herein will find particular utility. Longer contiguous identical or 1o complementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediate lengths) and even up to full length sequences will also be of use in certain embodiments.
The ability of such nucleic acid probes to specifically hybridize to a sequence of interest will enable them to be of use in detecting the presence of complementary sequences in a given sample. However, other uses are also envisioned, such as the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.
Polynucleotide molecules having sequence regions consisting of contiguous nucleotide stretches of 10-14, 15-20, 30, S0, or even of 100-200 nucleotides or so (including intermediate lengths as well), identical or complementary to a polynucleotide sequence disclosed herein, are particularly contemplated as hybridization probes for use in, e.g., Southern and Northern blotting. This would allow a gene product, or fragment thereof, to be analyzed, both in diverse cell types and also in various bacterial cells. The total size of fragment, as well as the size of the complementary stretch(es), will ultimately depend on the intended use or application of the particular nucleic acid segment. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the contiguous complementary region may be varied, such as between about 15 and about 100 nucleotides, but larger contiguous complementarity stretches may be used, according to the length 3o complementary sequences one wishes to detect.

The use of a hybridization probe of about 15-25 nucleotides in length allows the formation of a duplex molecule that is both stable and selective.
Molecules having contiguous complementary sequences over stretches greater than 15 bases in length are generally preferred, though, in order to increase stability and selectivity of the s hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of 1 S to 25 contiguous nucleotides, or even longer where desired.
Hybridization probes may be selected from any portion of any of the l0 sequences disclosed herein. All that is required is to review the sequences set forth herein, or to any continuous portion of the sequences, from about 15-25 nucleotides in length up to and including the full length sequence, that one wishes to utilize as a probe or primer. The choice of probe and primer sequences may be governed by various factors. For example, one may wish to employ primers from towards the termini of the 15 total sequence.
Small polynucleotide segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCRTM
2o technology of U. S. Patent 4,683,202 (incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.
The nucleotide sequences of the invention may be used for their ability to 25 selectively form duplex molecules with complementary stretches of the entire gene or gene fragments of interest. Depending on the application envisioned, one will typically desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, 3o e.g., one will select relatively low salt and/or high temperature conditions, such as provided by a salt concentration of from about 0.02 M to about 0.15 M salt at temperatures of from about 50°C to about 70°C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating related sequences.
Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template, less stringent (reduced stringency) hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ salt conditions such as those of from about 0.15 M to about 0.9 M
l0 salt, at temperatures ranging from about 20°C to about 55°C.
Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature.
Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
According to another embodiment of the present invention, polynucleotide compositions comprising antisense oligonucleotides are provided.
Antisense oligonucleotides have been demonstrated to be effective and targeted inhibitors of protein synthesis, and, consequently, provide a therapeutic approach by which a disease can be treated by inhibiting the synthesis of proteins that contribute to the disease. The efficacy of antisense oligonucleotides for inhibiting protein synthesis is well established. For example, the synthesis of polygalactauronase and the muscarine type 2 acetylcholine receptor are inhibited by antisense oligonucleotides directed to their respective mRNA sequences (U. S. Patent 5,739,119 and U. S. Patent 5,759,829).
Further, examples of antisense inhibition have been demonstrated with the nuclear protein cyclin, the multiple drug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABAA receptor and human EGF (Jaskulski et al., Science. 1988 Jun 10;240(4858):1544-6; Vasanthakumar and Ahmed, Cancer Commun. 1989;1(4):225-32; Peris et al., Brain Res Mol Brain Res. 1998 Jun 15;57(2):310-20; U. S.
Patent 5,801,154; U.S. Patent 5,789,573; U. S. Patent 5,718,709 and U.S. Patent 5,610,288).

Antisense constructs have also been described that inhibit and can be used to treat a variety of abnormal cellular proliferations, e.g. cancer (U. S. Patent 5,747,470; U. S.
Patent 5,591,317 and U. S. Patent 5,783,683).
Therefore, in certain embodiments, the present invention provides oligonucleotide sequences that comprise all, or a portion of, any sequence that is capable of specifically binding to polynucleotide sequence described herein, or a complement thereof. In one embodiment, the antisense oligonucleotides comprise DNA
or derivatives thereof. In another embodiment, the oligonucleotides comprise RNA or derivatives thereof. In a third embodiment, the oligonucleotides are modified DNAs l0 comprising a phosphorothioated modified backbone. In a fourth embodiment, the oligonucleotide sequences comprise peptide nucleic acids or derivatives thereof. In each case, preferred compositions. comprise a sequence region that is complementary, and more preferably substantially-complementary, and even more preferably, completely complementary to one or more portions of polynucleotides disclosed herein.
Selection of antisense compositions specific for a given gene sequence is based upon analysis of the chosen target sequence (i. e. in these illustrative examples the rat and human sequences) and determination of secondary structure, Tm, binding energy, relative stability, and antisense compositions were selected based upon their relative inability to form dimers, hairpins, or other secondary structures that would reduce or 2o prohibit specific binding to the target mRNA in a host cell.
Highly preferred target regions of the mRNA, are those which are at or near the AUG translation initiation codon, and those sequences which are substantially complementary to 5' regions of the mRNA. These secondary structure analyses and target site selection considerations can be performed, for example, using v.4 of the OLIGO primer analysis software and/or the BLASTN 2Ø5 algorithm software (Altschul et al., Nucleic Acids Res. 1997 Sep 1;25(17):3389-402).
The use of an antisense delivery method employing a short peptide vector, termed MPG (27 residues), is also contemplated. The MPG peptide contains a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic 3o domain from the nuclear localization sequence of SV40 T-antigen (Morris et al., Nucleic Acids Res. 1997 Jul 15;25(14):2730-6). It has been demonstrated that several molecules of the MPG peptide coat the antisense oligonucleotides and can be delivered into cultured mammalian cells in less than 1 hour with relatively high efficiency (90%).
Further, the interaction with MPG strongly increases both the stability of the oligonucleotide to nuclease and the ability to cross the plasma membrane.
According to another embodiment of the invention, the polynucleotide compositions described herein are used in the design and preparation of ribozyme molecules for inhibiting expression of the tumor polypeptides and proteins of the present invention in tumor cells. Ribozymes are RNA-protein complexes that cleave to nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cech, Proc Natl Acad Sci U S A. 1987 Dec;84(24):8788-92; Forster and Symons, Cell. 1987 Apr 24;49(2):211-20). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cech et al., Cell. 1981 Dec;27(3 Pt 2):487-96;
Michel and Westhof, J Mol Biol. 1990 Dec 5;216(3):585-610; Reinhold-Hurek and Shub, Nature.
1992 May 14;357(6374):173-6). This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.
2o Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in traps (and thus can cleave other RNA molecules) under physiological conditions. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA.
Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA
through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and 3o cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over many technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the concentration of ribozyme necessary to affect a therapeutic treatment is lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-1o substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme. Similar mismatches in antisense molecules do not prevent their action (Woolf et al., Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7305-9). Thus, the specificity of action of a ribozyme is greater than that of an antisense oligonucleotide binding the same RNA site.
~ 5 The enzymatic nucleic acid molecule may be formed in a hammerhead, hairpin, a hepatitis S virus, group I intron or RNaseP RNA (in association with an RNA
guide sequence) or Neurospora VS RNA motif. Examples of hammerhead motifs are described by Rossi et al. Nucleic Acids Res. 1992 Sep 11;20(17):4559-65.
Examples of hairpin motifs are described by Hampel et al. (Eur. Pat. Appl. Publ. No. EP
0360257), 2o Hampel and Tritz, Biochemistry 1989 Jun 13;28(12):4929-33; Hampel et al., Nucleic Acids Res. 1990 Jan 25;18(2):299-304 and U. S. Patent 5,631,359. An example of the hepatitis b virus motif is described by Perrotta and Been, Biochemistry. 1992 Dec 1;31 (47):11843-52; an example of the RNaseP motif is described by Guerrier-Takada et al., Cell. 1983 Dec;35(3 Pt 2):849-57; Neurospora VS RNA ribozyme motif is 25 described by Collins (Saville and Collins, Cell. 1990 May 18;61(4):685-96;
Saville and Collins, Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8826-30; Collins and Olive, Biochemistry. 1993 Mar 23;32(11):2795-9); and an example of the Group I intron is described in (U. S. Patent 4,987,071). All that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is 30 complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule. Thus the ribozyme constructs need not be limited to specific motifs mentioned herein.
Ribozymes may be designed as described in Int. Pat. Appl. Publ. No.
WO 93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595, each specifically incorporated herein by reference) and synthesized to be tested in vitro and in vivo, as described. Such ribozymes can also be optimized for delivery. While specific examples are provided, those in the art will recognize that equivalent RNA
targets in other species can be utilized when necessary.
Ribozyme activity can be optimized by altering the length of the 1 o ribozyme binding arms, or chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see e.g., Int. Pat. Appl.
Publ. No. WO
92/07065; Int. Pat. Appl. Publ. No. WO 93/15187; Int. Pat. Appl. Publ. No. WO
91/03162; Eur. Pat. Appl. Publ. No. 92110298.4; U. S. Patent 5,334,711; and Int. Pat.
Appl. Publ. No. WO 94/13688, which describe various chemical modifications that can ~ 5 be made to the sugar moieties of enzymatic RNA molecules), modifications which enhance their efficacy in cells, and removal of stem II bases to shorten RNA
synthesis times and reduce chemical requirements.
Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describes the general methods for delivery of enzymatic RNA molecules. Ribozymes may be 20 administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres. For some indications, ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles.
25 Alternatively, the RNA/vehicle combination may be locally delivered by direct inhalation, by direct injection or by use of a catheter, infusion pump or stmt. Other routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. More detailed descriptions 30 of ribozyme delivery and administration are provided in Int. Pat. Appl.
Publ. No. WO

94/02595 and Int. Pat. Appl. Publ. No. WO 93/23569, each specifically incorporated herein by reference.
Another means of accumulating high concentrations of a ribozyme(s) within cells is to incorporate the ribozyme-encoding sequences into a DNA
expression vector. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA
polymerase III (pol III). Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc. ) present nearby.
Prokaryotic RNA polymerase promoters may also be used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells .
Ribozymes expressed from such promoters have been shown to function in mammalian cells.
Such transcription units can be incorporated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA
vectors (such as adenovirus or adeno-associated vectors), or viral RNA vectors (such as retroviral, semliki forest virus, sindbis virus vectors).
In another embodiment of the invention, peptide nucleic acids (PNAs) compositions are provided. PNA is a DNA mimic in which the nucleobases are attached to a pseudopeptide backbone (Good and Nielsen, Antisense Nucleic Acid Drug 2o Dev. 1997 7(4) 431-37). PNA is able to be utilized in a number methods that traditionally have used RNA or DNA. Often PNA sequences perform better in techniques than the corresponding RNA or DNA sequences and have utilities that are not inherent to RNA or DNA. A review of PNA including methods of making, characteristics of, and methods of using, is provided by Corey (Trends Biotechnol 1997 Jun;lS(6):224-9). As such, in certain embodiments, one may prepare PNA
sequences that are complementary to one or more portions of the ACE mRNA sequence, and such PNA compositions may be used to regulate, alter, decrease, or reduce the translation of ACE-specific mRNA, and thereby alter the level of ACE activity in a host cell to which such PNA compositions have been administered.

PNAs have 2-aminoethyl-glycine linkages replacing the normal phosphodiester backbone of DNA (Nielsen et al., Science 1991 Dec 6;254(5037):1497-500; Hanvey et al., Science. 1992 Nov 27;258(5087):1481-5; Hyrup and Nielsen, Bioorg Med Chem. 1996 Jan;4(1):S-23). This chemistry has three important consequences: firstly, in contrast to DNA or phosphorothioate oligonucleotides, PNAs are neutral molecules; secondly, PNAs are achiral, which avoids the need to develop a stereoselective synthesis; and thirdly, PNA synthesis uses standard Boc or Fmoc protocols for solid-phase peptide synthesis, although other methods, including a modified Merrifield method, have been used.
to PNA monomers or ready-made oligomers are commercially available from PerSeptive Biosystems (Framingham, MA). PNA syntheses by either Boc or Fmoc protocols are straightforward using manual or automated protocols (Norton et al., Bioorg Med Chem. 1995 Apr;3(4):437-45). The manual protocol lends itself to the production of chemically modified PNAs or the simultaneous synthesis of families of closely related PNAs.
As with peptide synthesis, the success of a particular PNA synthesis will depend on the properties of the chosen sequence. For example, while in theory PNAs can incorporate any combination of nucleotide bases, the presence of adjacent purines can lead to deletions of one or more residues in the product. In expectation of this 2o difficulty, it is suggested that, in producing PNAs with adjacent purines, one should repeat the coupling of residues likely to be added inefficiently. This should be followed by the purification of PNAs by reverse-phase high-pressure liquid chromatography, providing yields and purity of product similar to those observed during the synthesis of peptides.
Modifications of PNAs for a given application may be accomplished by coupling amino acids during solid-phase synthesis or by attaching compounds that contain a carboxylic acid group to the exposed N-terminal amine.
Alternatively, PNAs can be modified after synthesis by coupling to an introduced lysine or cysteine. The ease with which PNAs can be modified facilitates optimization for better solubility or 3o for specific ftmctional requirements. Once synthesized, the identity of PNAs and their derivatives can be confirmed by mass spectrometry. Several studies have made and utilized modifications of PNAs (for example, Norton et al., Bioorg Med Chem.

Apr;3(4):437-45; Petersen et al., J Pept Sci. 1995 May-Jun;l(3):175-83; Orum et al., Biotechniques. 1995 Sep;l9(3):472-80; Footer et al., Biochemistry. 1996 Aug 20;35(33):10673-9; Griffith et al., Nucleic Acids Res. 1995 Aug 11;23(15):3003-8;
Pardridge et al., Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5592-6; Boffa et al., Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):1901-5; Gambacorti-Passerini et al., Blood. 1996 Aug 15;88(4):1411-7; Armitage et al., Proc Natl Acad Sci U S A.

Nov 11;94(23):12320-S; Seeger et al., Biotechniques. 1997 Sep;23(3):512-7).
U.5.
t o Patent No. 5,700,922 discusses PNA-DNA-PNA chimeric molecules and their uses in diagnostics, modulating protein in organisms, and treatment of conditions susceptible to therapeutics.
Methods of characterizing the antisense binding properties of PNAs are discussed in Rose (Anal Chem. 1993 Dec 15;65(24):3545-9) and Jensen et al.
(Biochemistry. 1997 Apr 22;36(16):5072-7). Rose uses capillary gel electrophoresis to determine binding of PNAs to their complementary oligonucleotide, measuring the relative binding kinetics and stoichiometry. Similar types of measurements were made by Jerisen et al. using BIAcoreTM technology.
Other applications of PNAs that have been described and will be 2o apparent to the skilled artisan include use in DNA strand invasion, antisense inhibition, mutational analysis, enhancers of transcription, nucleic acid purification, isolation of transcriptionally active genes, blocking of transcription factor binding, genome cleavage, biosensors, in situ hybridization, and the like.
POLYNUCLEOTIDE IDENTIFICATION ~ CHARACTERIZATION AND EXPRESSION
Polynucleotides compositions of the present invention may be identified, prepared and/or manipulated using any of a variety of well established techniques (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989, and other like references).
For 3o example, a polynucleotide may be identified, as described in more detail below, by screening a microarray of cDNAs for tumor-associated expression (i.e., expression that is at least two fold greater in a tumor than in normal tissue, as determined using a representative assay provided herein). Such screens may be performed, for example, using a Synteni microarray (Palo Alto, CA) according to the manufacturer's instructions (and essentially as described by Schena et al., Proc. Natl. Acad. Sci. USA
93:10614-10619, 1996 and Heller et al., Proc. Natl. Acad. Sci. USA 94:2150-2155, 1997).
Alternatively, polynucleotides may be amplified from cDNA prepared from cells expressing the proteins described herein, such as tumor cells.
Many template dependent processes are available to amplify a target sequences of interest present in a sample. One of the best known amplification methods is the polymerase chain reaction (PCRT~ which is described in detail in U.S.
Patent Nos. 4,683,195, 4,683,202 and 4,800,159, each of which is incorporated herein by reference in its entirety. Briefly, in PCRTM, two primer sequences are prepared which are complementary to regions on opposite complementary strands of the target sequence. An excess of deoxynucleoside triphosphates is added to a reaction mixture along with a DNA polymerase (e.g., Taq polymerase). If the target sequence is present in a sample, the primers will bind to the target and the polymerase will cause the primers to be extended along the target sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will 2o dissociate from the target to form reaction products, excess primers will bind to the target and to the reaction product and the process is repeated. Preferably reverse transcription and PCRTM amplification procedure may be performed in order to quantify the amount of mRNA amplified. Polymerase chain reaction methodologies are well known in the art.
z5 Any of a number of other template dependent processes, many of which are variations of the PCR TM amplification technique, are readily known and available in the art. Illustratively, some such methods include the ligase chain reaction (referred to as LCR), described, for example, in Eur. Pat. Appl. Publ. No. 320,308 and U.S.
Patent No. 4,883,750; Qbeta Replicase, described in PCT Intl. Pat. Appl. Publ. No.
3o PCT/LJS87/00880; Strand Displacement Amplification (SDA) and Repair Chain Reaction (RCR). Still other amplification methods are described in Great Britain Pat.

Appl. No. 2 202 328, and in PCT Intl. Pat. Appl. Publ. No. PCT/US89/01025.
Other nucleic acid amplification procedures include transcription-based amplification systems (TAS) (PCT Intl. Pat. Appl. Publ. No. WO 88/10315), including nucleic acid sequence based amplification (NASBA) and 3SR. Eur. Pat. Appl. Publ. No. 329,822 describes a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA). PCT Intl. Pat. Appl.
Publ. No. WO 89/06700 describes a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA
("ssDNA") followed by transcription of many RNA copies of the sequence. Other t 0 amplification methods such as "RACE" (Frohman, 1990), and "one-sided PCR"
(Ohara, 1989) are also well-known to those of skill in the art.
An amplified portion of a polynucleotide of the present invention may be used to isolate a full length gene from a suitable library (e.g., a tumor cDNA
library) using well known techniques. Within such techniques, a library (cDNA or genomic) is ~ 5 screened using one or more polynucleotide probes or primers suitable for amplification.
Preferably, a library is size-selected to include larger molecules. Random primed libraries may also be preferred for identifying S' and upstream regions of genes.
Genomic libraries are preferred for obtaining introns and extending 5' sequences.
For hybridization techniques, a partial sequence may be labeled (e.g., by 20 nick-translation or end-labeling with'ZP) using well known techniques. A
bacterial or bacteriophage library is then generally screened by hybridizing filters containing denatured bacterial colonies (or lawns containing phage plaques) with the labeled probe (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989). Hybridizing colonies or plaques are 25 selected and expanded, and the DNA is isolated for further analysis. cDNA
clones may be analyzed to determine the amount of additional sequence by, for example, PCR using a primer from the partial sequence and a primer from the vector. Restriction maps and partial sequences may be generated to identify one or more overlapping clones.
The complete sequence may then be determined using standard techniques, which may 30 involve generating a series of deletion clones. The resulting overlapping sequences can then assembled into a single contiguous sequence. A full length cDNA molecule can be generated by ligating suitable fragments, using well known techniques.
Alternatively, amplification techniques, such as those described above, can be useful for obtaining a full length coding sequence from a partial cDNA
sequence.
One such amplification technique is inverse PCR (see Triglia et al., Nucl.
Acids Res.
16:8186, 1988), which uses restriction enzymes to generate a fragment in the known region of the gene. The fragment is then circularized by intramolecular ligation and used as a template for PCR with divergent primers derived from the known region.
Within an alternative approach, sequences adjacent to a partial sequence may be retrieved by amplification with a primer to a linker sequence and a primer specific to a 1o known region. The amplified sequences are typically subjected to a second round of amplification with the same linker primer and a second primer specific to the known region. A variation on this procedure, which employs two primers that initiate extension in opposite directions from the known sequence, is described in WO
96/38591. Another such technique is known as "rapid amplification of cDNA
ends" or RACE. This technique involves the use of an internal primer and an external primer, which hybridizes to a polyA region or vector sequence, to identify sequences that are 5' and 3' of a known sequence. Additional techniques include capture PCR
(Lagerstrom et al., PCR Methods Applic. 1:l l l-19, 1991) and walking PCR (Parker et al., Nucl. Acids.
Res. 19:3055-60, 1991). Other methods employing amplification may also be employed to obtain a full length cDNA sequence.
In certain instances, it is possible to obtain a full length cDNA sequence by analysis of sequences provided in an expressed sequence tag (EST) database, such as that available from GenBank. Searches for overlapping ESTs may generally be performed using well known programs (e.g., NCBI BLAST searches), and such ESTs may be used to generate a contiguous full length sequence. Full length DNA
sequences may also be obtained by analysis of genomic fragments.
In other embodiments of the invention, polynucleotide sequences or fragments thereof which encode polypeptides of the invention, or fusion proteins or functional equivalents thereof, may be used in recombinant DNA molecules to direct expression of a polypeptide in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences that encode substantially the same or a functionally equivalent amino acid sequence may be produced and these sequences may be used to clone and express a given polypeptide.
As will be understood by those of skill in the art, it may be advantageous in some instances to produce polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half life which is longer than that of a transcript generated from the naturally occurring 1o sequence.
Moreover, the polynucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter polypeptide encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the gene product.
For example, DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. In addition, site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, or introduce mutations, and so forth.
In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences may be ligated to a heterologous sequence to encode a fusion protein. For example, to screen peptide libraries for inhibitors of polypeptide activity, it may be useful to encode a chimeric protein that can be recognized by a commercially available antibody. A fusion protein may also be engineered to contain a cleavage site located between the polypeptide-encoding sequence and the heterologous protein sequence, so that the polypeptide may be cleaved and purified away from the heterologous moiety.
Sequences encoding a desired polypeptide may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers, M. H.
et al.
(1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al. (1980) Nucl. Acids Res.

Symp. Ser. 225-232). Alternatively, the protein itself may be produced using chemical methods to synthesize the amino acid sequence of a polypeptide, or a portion thereof.
For example, peptide synthesis can be performed using various solid-phase techniques (Roberge, J. Y. et al. (1995) Science 269:202-204) and automated synthesis may be achieved, for example, using the ABI 431A Peptide Synthesizer (Perkin Elmer, Palo Alto, CA).
A newly synthesized peptide may be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, T. (1983) Proteins, Structures and Molecular Principles, WH Freeman and Co., New York, N.Y.) to or other comparable techniques available in the art. The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure). Additionally, the amino acid sequence of a polypeptide, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
In order to express a desired polypeptide, the nucleotide sequences encoding the polypeptide, or functional equivalents, may be inserted into appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well 2o known to those skilled in the art may be used to construct expression vectors containing sequences encoding a polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook, J. et al. (1989) Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F.
M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York.
N.Y.
A variety of expression vector/host systems may be utilized to contain and express polynucleotide sequences. These include, but are not limited to, 3o microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors;
insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
The "control elements" or "regulatory sequences" present in an expression vector are those non-translated regions of the vector--enhancers, promoters, 5' and 3' untranslated regions--which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity.
l0 Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used.
For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL, Gaithersburg, MD) and the like may be used. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are generally preferred. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding a polypeptide, vectors based on SV40 or EBV
may be advantageously used with an appropriate selectable marker.
In bacterial systems, any of a number of expression vectors may be 2o selected depending upon the use intended for the expressed polypeptide. For example, when large quantities are needed, for example for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be used. Such vectors include, but are not limited to, the multifunctional E.
coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding the polypeptide of interest may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of .beta.-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke, G.
and S.
M. Schuster (1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX Vectors (Promega, Madison, Wis.) may also be used to express foreign polypeptides as fusion 3o proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST
moiety at will.
In the yeast, Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al. (supra) and Grant et al. (1987) Methods Enzymol. 153 :516-544.
In cases where plant expression vectors are used, the expression of sequences encoding polypeptides may be driven by any of a number of promoters.
For example, viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J. 6:307-311. Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.
~s 3:1671-1680; Brogue, R. et al. (1984) Science 224:838-843; and Winter, J.
et al. (1991) Results Probl. Cell Differ. 17:85-105). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (see, for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw 20 Hill, New York, N.Y.; pp. 191-185 and 187-196).
An insect system may also be used to express a polypeptide of interest.
For example, in one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The sequences encoding the polypeptide may be cloned into a 25 non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of the polypeptide-encoding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses may then be used to infect, for example, S.
frugiperda cells or Trichoplusia larvae in which the polypeptide of interest may be expressed 30 (Engelhard, E. K. et al. (1994) Proc. Natl. Acad. Sci. 91 :3224-3227).

In mammalian host cells, a number of viral-based expression systems are generally available. For example, in cases where an adenovirus is used as an expression vector, sequences encoding a polypeptide of interest may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing the polypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
l0 Specific initiation signals may also be used to achieve more efficient translation of sequences encoding a polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding the polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic.
The efficiency of expression may be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used, such as those described in the literature (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).
In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation. glycosylation, phosphorylation, lipidation, and acylation.
Post-translational processing which cleaves a "prepro" form of the protein may also be used to facilitate correct insertion, folding and/or function. Different host cells such as CHO, COS, HeLa, MDCK, HEK293, and WI38, which have specific cellular 3o machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stable expression is generally preferred. For example, cell lines which stably express a polynucleotide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed 1 o cells may be proliferated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1990) Cell 22:817-23) genes which can be employed in tk^- or aprt^- cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70);
npt, which confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14); and als or pat, which confer resistance to 2o chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra).
Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad.
Sci.
85:8047-51 ). The use of visible markers has gained popularity with such markers as anthocyanins, beta-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, being widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).
Although the presence/absence of marker gene expression suggests that 3o the gene of interest is also present, its presence and expression may need to be confirmed. For example, if the sequence encoding a polypeptide is inserted within a marker gene sequence, recombinant cells containing sequences can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a polypeptide-encoding sequence under the control of a single promoter.
Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
Alternatively, host cells that contain and express a desired polynucleotide sequence may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include, 1o for example, membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein.
A vaxiety of protocols for detecting and measuring the expression of polynucleotide-encoded products, using either polyclonal or monoclonal antibodies specific for the product are known in the art. Examples include enzyme-linked ~ 5 immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on a given polypeptide may be preferred for some applications, but a competitive binding assay may also be employed.
These and other assays are described, among other places, in Hampton, R. et al. (1990;
2o Serological Methods, a Laboratory Manual, APS Press, St Paul. Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med. 158:1211-1216).
A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to 25 polynucleotides include oligolabeling, nick translation, end-labeling or PCR
amplification using a labeled nucleotide. Alternatively, the sequences, or any portions thereof may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA
probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 3o and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits. Suitable reporter molecules or labels, which may be used include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with a polynucleotide sequence of interest may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides of the invention may be designed to contain signal sequences which direct secretion of the 1 o encoded polypeptide through a prokaryotic or eukaryotic cell membrane.
Other recombinant constructions may be used to join sequences encoding a polypeptide of interest to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS
extension/affinity purification system (Immunex Corp., Seattle, Wash.). The inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen.
San Diego, Calif.) between the purification domain and the encoded polypeptide may be used to 2o facilitate purification. One such expression vector provides for expression of a fusion protein containing a polypeptide of interest and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography) as described in Porath, J. et al. (1992, Prot. Exp. Purif. 3:263-281) while the enterokinase cleavage site provides a means for purifying the desired polypeptide from the fusion protein. A discussion of vectors which contain fusion proteins is provided in Kroll, D. J.
et al. (1993; DNA Cell Biol. 12:441-453).
In addition to recombinant production methods, polypeptides of the invention, and fragments thereof, may be produced by direct peptide synthesis using 3o solid-phase techniques (Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Alternatively, various fragments may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.
ANTIBODY COMPOSITIONS, FRAGMENTS THEREOF AND OTHER BINDING AGENTS
According to another aspect, the present invention further provides binding agents, such as antibodies and antigen-binding fragments thereof, that exhibit immunological binding to a tumor polypeptide disclosed herein, or to a portion, variant l0 or derivative thereof. An antibody, or antigen-binding fragment thereof, is said to "specifically bind," "immunogically bind," and/or is "immunologically reactive" to a polypeptide of the invention if it reacts at a detectable level (within, for example, an ELISA assay) with the polypeptide, and does not react detectably with unrelated polypeptides under similar conditions.
Immunological binding, as used in this context, generally refers to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, wherein a smaller Kd represents a greater 2o affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and on geometric parameters that equally influence the rate in both directions.
Thus, both the "on rate constant" (Ko") and the "off rate constant" (I~~.) can be determined by calculation of the concentrations and the actual rates of association and dissociation.
The ratio of Ko~. /Ko~ enables cancellation of all parameters not related to affinity, and is thus equal to the dissociation constant Ka. See, generally, Davies et al.
(1990) Annual Rev. Biochem. 59:439-473.
3o An "antigen-binding site," or "binding portion" of an antibody refers to the part of the immunoglobulin molecule that participates in antigen binding.
The antigen binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains are referred to as "hypervariable regions" which are interposed between more conserved flanking stretches known as "framework regions," or "FRs". Thus the term "FR" refers to amino acid sequences which are naturally found between and adjacent to hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions," or "CDRs."
Binding agents may be further capable of differentiating between patients with and without a cancer, such as ovarian cancer, using the representative assays provided herein. For example, antibodies or other binding agents that bind to a tumor protein will preferably generate a signal indicating the presence of a cancer in at least about 20% of patients with the disease, more preferably at least about 30% of patients. Alternatively, or in addition, the antibody will generate a negative signal 2o indicating the absence of the disease in at least about 90% of individuals without the cancer. To determine whether a binding agent satisfies this requirement, biological samples (e.g., blood, sera, sputum, urine and/or tumor biopsies) from patients with and without a cancer (as determined using standard clinical tests) may be assayed as described herein for the presence of polypeptides that bind to the binding agent.
Preferably, a statistically significant number of samples with and without the disease will be assayed. Each binding agent should satisfy the above criteria;
however, those of ordinary skill in the art will recognize that binding agents may be used in combination to improve sensitivity.
Any agent that satisfies the above requirements may be a binding agent.
3o For example, a binding agent may be a ribosome, with or without a peptide component, an RNA molecule or a polypeptide. In a preferred embodiment, a binding agent is an antibody or an antigen-binding fragment thereof. Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies. In one technique, an immunogen comprising the polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats). In this step, the polypeptides of this invention may serve as the immunogen to without modification. Alternatively, particularly for relatively short polypeptides, a superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically.
Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.
Monoclonal antibodies specific for an antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur.
J.
2o Immunol. 6:511-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. A variety of fusion techniques may be employed. For example, the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells. A preferred selection technique uses HAT
(hypoxanthine, 3o aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and their culture supernatants tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred.
Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. The polypeptides of this invention may be used in the purification process 1 o in, for example, an affinity chromatography step.
A number of therapeutically useful molecules are known in the art which comprise antigen-binding sites that are capable of exhibiting immunological binding properties of an antibody molecule. The proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the "F(ab)"
fragments) t 5 each comprise a covalent heterodimer that includes an intact antigen-binding site. The enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the "F(ab')2 " fragment which comprises both antigen-binding sites. An "Fv"
fragment can be produced by preferential proteolytic cleavage of an IgM, and on rare occasions IgG or IgA immunoglobulin molecule. Fv fragments are, however, more commonly 2o derived using recombinant techniques known in the art. The Fv fragment includes a non-covalent V,,:: VL heterodimer including an antigen-binding site which retains much of the antigen recognition and binding capabilities of the native antibody molecule.
mbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al.
(1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.
25 A single chain Fv ("sFv") polypeptide is a covalently linked VH::VL
heterodimer which is expressed from a gene fusion including VH and V~ encoding genes linked by a peptide-encoding linker. Huston et al. (1988) Proc. Nat.
Acad. Sci.
USA 85(16):5879-5883. A number of methods have been described to discern chemical structures for converting the naturally aggregated--but chemically separated--light and 3o heavy polypeptide chains from an antibody V region into an sFv molecule which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513 and 5,132,405, to Huston et al.;
and U.S. Pat. No. 4,946,778, to Ladner et al.
Each of the above-described molecules includes a heavy chain and a light chain CDR set, respectively interposed between a heavy chain and a light chain FR
set which provide support to the CDRS and define the spatial relationship of the CDRs relative to each other. As used herein, the term "CDR set" refers to the three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as "CDR1,"
"CDR2," and "CDR3" respectively. An antigen-binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. A
polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3) is referred to herein as a "molecular recognition unit." Crystallographic analysis of a number of antigen-antibody complexes has demonstrated that the amino acid residues of CDRs form extensive contact with bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the molecular recognition units are primarily responsible for the specificity of an antigen-binding site.
As used herein, the term "FR set" refers to the four flanking amino acid sequences which frame the CDRs of a CDR set of a heavy or light chain V
region.
2o Some FR residues may contact bound antigen; however, FRs are primarily responsible for folding the V region into the antigen-binding site, particularly the FR
residues directly adjacent to the CDRS. Within FRs, certain amino residues and certain structural features are very highly conserved. In this regard, all V region sequences contain an internal disulfide loop of around 90 amino acid residues. When the V regions fold into a binding-site, the CDRs are displayed as projecting loop motifs which form an antigen-binding surface. It is generally recognized that there are conserved structural regions of FRs which influence the folded shape of the CDR loops into certain "canonical"
structures--regardless, of the precise CDR amino acid sequence. Further, certain FR
residues are known to participate in non-covalent interdomain contacts which stabilize 3o the interaction of the antibody heavy and light chains.

A number of "humanized" antibody molecules comprising an antigen-binding site derived from a non-human immunoglobulin have been described, including chimeric antibodies having rodent V regions and their associated CDRs fused to human constant domains (Winter et al. (1991) Nature 349:293-299; Lobuglio et al.
(1989) Proc. Nat. Acad. Sci. USA 86:4220-4224; Shaw et al. (1987) J Immunol. 138:4534-4538; and Brown et al. (1987) Cancer Res. 47:3577-3583), rodent CDRs grafted into a human supporting FR prior to fusion with an appropriate human antibody constant domain (Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536; and Jones et al. (1986) Nature 321:522-525), and rodent CDRs to supported by recombinantly veneered rodent FRs (European Patent Publication No.
519,596, published Dec. 23, 1992). These "humanized" molecules are designed to minimize unwanted immunological response toward rodent antihuman antibody molecules which limits the duration and effectiveness of therapeutic applications of those moieties in human recipients.
As used herein, the terms "veneered FRs" and "recombinantly veneered FRs" refer to the selective replacement of FR residues from, e.g., a rodent heavy or light chain V region, with human FR residues in order to provide a xenogeneic molecule comprising an antigen-binding site which retains substantially all of the native FR
polypeptide folding structure. Veneering techniques are based on the understanding that 2o the ligand binding characteristics of an antigen-binding site are determined primarily by the structure and relative disposition of the heavy and light chain CDR sets within the antigen-binding surface. Davies et al. (1990) Ann. Rev. Biochem. 59:439-473.
Thus, antigen binding specificity can be preserved in a humanized antibody only wherein the CDR structures, their interaction with each other, and their interaction with the rest of the V region domains are carefully maintained. By using veneering techniques, exterior (e.g., solvent-accessible) FR residues which are readily encountered by the immune system are selectively replaced with human residues to provide a hybrid molecule that comprises either a weakly immunogenic, or substantially non-immunogenic veneered surface.
The process of veneering makes use of the available sequence data for human antibody variable domains compiled by Kabat et al., in Sequences of Proteins of Immunological Interest, 4th ed., (U.S. Dept. of Health and Human Services, U.S.
Government Printing Office, 1987), updates to the Kabat database, and other accessible U.S. and foreign databases (both nucleic acid and protein). Solvent accessibilities of V
region amino acids can be deduced from the known three-dimensional structure for human and marine antibody fragments. There are two general steps in veneering a marine antigen-binding site. Initially, the FRs of the variable domains of an antibody molecule of interest are compared with corresponding FR sequences of human variable domains obtained from the above-identified sources. The most homologous human V
regions are then compared residue by residue to corresponding marine amino acids. The to residues in the marine FR which differ from the human counterpart are replaced by the residues present in the human moiety using recombinant techniques well known in the art. Residue switching is only carried out with moieties which are at least partially exposed (solvent accessible), and care is exercised in the replacement of amino acid residues which may have a significant effect on the tertiary structure of V
region domains, such as proline, glycine and charged amino acids.
In this manner, the resultant "veneered" marine antigen-binding sites are thus designed to retain the marine CDR residues, the residues substantially adjacent to the CDRs, the residues identified as buried or mostly buried (solvent inaccessible), the residues believed to participate in non-covalent (e.g., electrostatic and hydrophobic) 2o contacts between heavy and light chain domains, and the residues from conserved structural regions of the FRs which are believed to influence the "canonical"
tertiary structures of the CDR loops. These design criteria are then used to prepare recombinant nucleotide sequences which combine the CDRs of both the heavy and light chain of a marine antigen-binding site into human-appearing FRs that can be used to transfect mammalian cells for the expression of recombinant human antibodies which exhibit the antigen specificity of the marine antibody molecule.
In another embodiment of the invention, monoclonal antibodies of the present invention may be coupled to one or more therapeutic agents. Suitable agents in this regard include radionuclides, differentiation inducers, drugs, toxins, and derivatives 3o thereof. Preferred radionuclides include 9°Y, ~z3I, 'zsl, 's'I, '86Re, 'ggRe, z"At, and z'zBi.

Preferred drugs include methotrexate, and pyrimidine and purine analogs.
Preferred differentiation inducers include phorbol esters and butyric acid. Preferred toxins include ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein.
A therapeutic agent may be coupled (e.g., covalently bonded) to a suitable monoclonal antibody either directly or indirectly (e.g., via a linker group). A
direct reaction between an agent and an antibody is possible when each possesses a substituent capable of reacting with the other. For example, a nucleophilic group, such as an amino or sulfhydryl group, on one may be capable of reacting with a carbonyl-1 o containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g., a halide) on the other.
Alternatively, it may be desirable to couple a therapeutic agent and an antibody via a linker group. A linker group can function as a spacer to distance an antibody from an agent in order to avoid interference with binding capabilities. A
linker group can also serve to increase the chemical reactivity of a substituent on an agent or an antibody, and thus increase the coupling efficiency. An increase in chemical reactivity may also facilitate the use of agents, or functional groups on agents, which otherwise would not be possible.
It will be evident to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, IL), may be employed as the linker group. Coupling may be effected, for example, through amino groups, carboxyl groups, sulfhydryl groups or oxidized carbohydrate residues. There are numerous references describing such methodology, e.g., U.S. Patent No. 4,671,958, to Rodwell et al.
Where a therapeutic agent is more potent when free from the antibody portion of the immunoconjugates of the present invention, it may be desirable to use a linker group which is cleavable during or upon internalization into a cell. A
number of different cleavable linker groups have been described. The mechanisms for the intracellular release of an agent from these linker groups include cleavage by reduction of a disulfide bond (e.g., U.S. Patent No. 4,489,710, to Spider), by irradiation of a photolabile bond (e.g., U.S. Patent No. 4,625,014, to Senter et al.), by hydrolysis of derivatized amino acid side chains (e.g., U.S. Patent No. 4,638,045, to Kohn et al.), by serum complement-mediated hydrolysis (e.g., U.S. Patent No. 4,671,958, to Rodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Patent No. 4,569,789, to Blattler et al.).
It may be desirable to couple more than one agent to an antibody. In one embodiment, multiple molecules of an agent are coupled to one antibody molecule. In another embodiment, more than one type of agent may be coupled to one antibody.
Regardless of the particular embodiment, immunoconjugates with more than one agent may be prepared in a variety of ways. For example, more than one agent may be 1 o coupled directly to an antibody molecule, or linkers that provide multiple sites for attachment can be used. Alternatively, a carrier can be used.
A carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group. Suitable carriers include proteins such as albumins (e.g., U.S. Patent No. 4,507,234, to Kato et al.), peptides and polysaccharides such as aminodextran (e.g., U.S. Patent No. 4,699,784, to Shih et al.). A
carrier may also bear an agent by noncovalent bonding or by encapsulation, such as within a liposome vesicle (e.g., U.S. Patent Nos. 4,429,008 and 4,873,088). Carriers specific for radionuclide agents include radiohalogenated small molecules and chelating compounds. For example, U.S. Patent No. 4,735,792 discloses representative radiohalogenated small molecules and their synthesis. A radionuclide chelate may be formed from chelating compounds that include those containing nitrogen and sulfur atoms as the donor atoms for binding the metal, or metal oxide, radionuclide.
For example, U.S. Patent No. 4,673,562, to Davison et al. discloses representative chelating compounds and their synthesis.
T CELLS COMPOSITIONS
The present invention, in another aspect, provides T cells specific for a tumor polypeptide disclosed herein, or for a variant or derivative thereof.
Such cells may generally be prepared in vitro or ex vivo, using standard procedures. For example, 3o T cells may be isolated from bone marrow, peripheral blood, or a fraction of bone marrow or peripheral blood of a patient, using a commercially available cell separation system, such as the IsolexTM System, available from Nexell Therapeutics, Inc.
(Irvine, CA; see also U.S. Patent No. 5,240,856; U.S. Patent No. 5,215,926; WO
89/06280; WO
91/16116 and WO 92/07243). Alternatively, T cells may be derived from related or unrelated humans, non-human mammals, cell lines or cultures.
T cells may be stimulated with a polypeptide, polynucleotide encoding a polypeptide and/or an antigen presenting cell (APC) that expresses such a polypeptide.
Such stimulation is performed under conditions and for a time sufficient to permit the generation of T cells that are specific for the polypeptide of interest.
Preferably, a 1 o tumor polypeptide or polynucleotide of the invention is present within a delivery vehicle, such as a microsphere, to facilitate the generation of specific T
cells.
T cells are considered to be specific for a polypeptide of the present invention if the T cells specifically proliferate, secrete cytokines or kill target cells coated with the polypeptide or expressing a gene encoding the polypeptide. T
cell specificity may be evaluated using any of a variety of standard techniques.
For example, within a chromium release assay or proliferation assay, a stimulation index of more than two fold increase in lysis and/or proliferation, compared to negative controls, indicates T cell specificity. Such assays may be performed, for example, as described in Chen et al., Cancer Res. 54:1065-1070, 1994. Alternatively, detection of the 2o proliferation of T cells may be accomplished by a variety of known techniques. For example, T cell proliferation can be detected by measuring an increased rate of DNA
synthesis (e.g., by pulse-labeling cultures of T cells with tritiated thymidine and measuring the amount of tritiated thymidine incorporated into DNA). Contact with a tumor polypeptide (100 ng/ml - 100 ~g/ml, preferably' 200 ng/ml - 25 ~g/ml) for 3 - 7 days will typically result in at least a two fold increase in proliferation of the T cells.
Contact as described above for 2-3 hours should result in activation of the T
cells, as measured using standard cytokine assays in which a two fold increase in the level of cytokine release (e.g., TNF or IFN-y) is indicative of T cell activation (see Coligan et al., Current Protocols in Immunology, vol. l, Wiley Interscience (Greene 1998)). T
3o cells that have been activated in response to a tumor polypeptide, polynucleotide or polypeptide-expressing APC may be CD4+ and/or CD8+. Tumor polypeptide-specific T

cells may be expanded using standard techniques. Within preferred embodiments, the T
cells are derived from a patient, a related donor or an unrelated donor, and are administered to the patient following stimulation and expansion.
For therapeutic purposes, CD4+ or CD8+ T cells that proliferate in response to a tumor polypeptide, polynucleotide or APC can be expanded in number either in vitro or in vivo. Proliferation of such T cells in vitro may be accomplished in a variety of ways. For example, the T cells can be re-exposed to a tumor polypeptide, or a short peptide corresponding to an immunogenic portion of such a polypeptide, with or without the addition of T cell growth factors, such as interleukin-2, and/or stimulator to cells that synthesize a tumor polypeptide. Alternatively, one or more T
cells that proliferate in the presence of the tumor polypeptide can be expanded in number by cloning. Methods for cloning cells are well known in the art, and include limiting dilution.
PHARMACEUTICAL COMPOSITIONS
In additional embodiments, the present invention concerns formulation of one or more of the polynucleotide, polypeptide, T-cell and/or antibody compositions disclosed herein in pharmaceutically-acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy.
2o It will be understood that, if desired, a composition as disclosed herein may be administered in combination with other agents as well, such as, e.g., other proteins or polypeptides or various pharmaceutically-active agents. In fact, there is virtually no limit to other components that may also be included, given that the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues. The compositions may thus be delivered along with various other agents as required in the particular instance. Such compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesized as described herein. Likewise, such compositions may further comprise substituted or derivatized RNA or DNA compositions.

Therefore, in another aspect of the present invention, pharmaceutical compositions are provided comprising one or more of the polynucleotide, polypeptide, antibody, and/or T-cell compositions described herein in combination with a physiologically acceptable carrier. In certain preferred embodiments, the pharmaceutical compositions of the invention comprise immunogenic polynucleotide and/or polypeptide compositions of the invention for use in prophylactic and theraputic vaccine applications. Vaccine preparation is generally described in, for example, M.F.
Powell and M.J. Newman, eds., "Vaccine Design (the subunit and adjuvant approach),"
Plenum Press (NY, 1995). Generally, such compositions will comprise one or more to polynucleotide and/or polypeptide compositions of the present invention in combination with one or more immunostimulants.
It will be apparent that any of the pharmaceutical compositions described herein can contain pharmaceutically acceptable salts of the polynucleotides and polypeptides of the invention. Such salts can be prepared, for example, from ~ 5 pharmaceutically acceptable non-toxic bases, including organic bases (e.g., salts of primary, secondary and tertiary amines and basic amino acids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium and magnesium salts).
In another embodiment, illustrative immunogenic compositions, e.g., vaccine compositions, of the present invention comprise DNA encoding one or more of 2o the polypeptides as described above, such that the polypeptide is generated in situ. As noted above, the polynucleotide may be administered within any of a variety of delivery systems known to those of ordinary skill in the art. Indeed, numerous gene delivery techniques are well known in the art, such as those described by Rolland, Crit. Rev.
Therap. Drug Carrier Systems 15:143-198, 1998, and references cited therein.
25 Appropriate polynucleotide expression systems will, of course, contain the necessary regulatory DNA regulatory sequences for expression in a patient (such as a suitable promoter and terminating signal). Alternatively, bacterial delivery systems may involve the administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of the polypeptide on its cell surface or secretes such an epitope.
3o Therefore, in certain embodiments, polynucleotides encoding immunogenic polypeptides described herein are introduced into suitable mammalian host cells for expression using any of a number of known viral-based systems.
In one illustrative embodiment, retroviruses provide a convenient and effective platform for gene delivery systems. A selected nucleotide sequence encoding a polypeptide of the 'present invention can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject. A number of illustrative retroviral systems have been described (e.g., U.S.
Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D.
(1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852;
Burns l0 et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.
In addition, a number of illustrative adenovirus-based systems have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham (1986) J. Virol. 57:267-274; Bett et al.
(1993) J.
Virol. 67:5911-5921; Mittereder et al. (1994) Human Gene Therapy 5:717-729;
Seth et al. (1994) J: Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58;
Berkner, K. L.
(1988) BioTechniques 6:616-629; and Rich et al. (1993) Human Gene Therapy 4:461-476).
2o Various adeno-associated virus (AAV) vector systems have also been developed for polynucleotide delivery. AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941;
International Publication Nos. WO 92/01070 and WO 93/03769; Lebkowski et al.
(1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129;
Kotin, R. M. ( 1994) Human Gene Therapy 5:793-801; Shelling and Smith ( 1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp. Med. 179:1867-1875.
Additional viral vectors useful for delivering the nucleic acid molecules 3o encoding polypeptides of the present invention by gene transfer include those derived from the pox family of viruses, such as vaccinia virus and avian poxvirus. By way of example, vaccinia virus recombinants expressing the novel molecules can be constructed as follows. The DNA encoding a polypeptide is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia.
Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the polypeptide of interest into the viral genome. The resulting TK (-) recombinant can be selected by culturing the cells in the presence of 5-to bromodeoxyuridine and picking viral plaques resistant thereto.
A vaccinia-based infection/transfection system can be conveniently used to provide for inducible, transient expression or coexpression of one or more polypeptides described herein in host cells of an organism. In this particular system, cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the polynucleotide or polynucleotides of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA which is then translated into 2o polypeptide by the host translational machinery. The method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al. Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.
Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses, can also be used to deliver the coding sequences of interest. Recombinant avipox viruses, expressing immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species. The use of an Avipox vector is particularly desirable in human and other mammalian species since members of the Avipox genus can only productively replicate in susceptible avian species and 3o therefore are not infective in mammalian cells. Methods for producing recombinant Avipoxviruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccinia viruses. See, e.g., WO
91/12882; WO
89/03429; and WO 92/03545.
Any of a number of alphavirus vectors can also be used for delivery of polynucleotide compositions of the present invention, such as those vectors described in U.S. Patent Nos. 5,843,723; 6,015,686; 6,008,035 and 6,015,694. Certain vectors based on Venezuelan Equine Encephalitis (VEE) can also be used, illustrative examples of which can be found in U.S. Patent Nos. 5,505,947 and 5,643,576.
Moreover, molecular conjugate vectors, such as the adenovirus chimeric vectors described in Michael et al. J. Biol. Chem. (1993) 268:6866-6869 and Wagner et to al. Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery under the invention.
Additional illustrative information on these and other known viral-based delivery systems can be found, for example, in Fisher-Hoch et al., Proc. Natl.
Acad. Sci.
USA 86:317-321, 1989; Flexner et al., Ann. N Y. Acad. Sci. 569:86-103, 1989;
Flexner et al., Vaccine 8:17-21, 1990; U.S. Patent Nos. 4,603,112, 4,769,330, and 5,017,487;
WO 89/01973; U.S. Patent No. 4,777,127; GB 2,200,651; EP 0,345,242; WO
91/02805;
Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434, 1991;
Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994; Kass-Eisler et al., Proc. Natl.
Acad. Sci. USA 90:11498-11502, 1993; Guzman et al., Circulation 88:2838-2848, 1993;
2o and Guzman et al., Cir. Res. 73:1202-1207, 1993.
In certain embodiments, a polynucleotide may be integrated into the genome of a target cell. This integration may be in the specific location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the polynucleotide may be stably maintained in the cell as a separate, episomal segment of DNA. Such polynucleotide segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. The mariner in which the expression construct is delivered to a cell and where in the cell the polynucleotide remains is dependent on the type of expression 3o construct employed.

In another embodiment of the invention, a polynucleotide is administered/delivered as "naked" DNA, for example as described in Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993.
The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.
In still another embodiment, a composition of the present invention can be delivered via a particle bombardment approach, many of which have been described.
In one illustrative example, gas-driven particle acceleration can be achieved with devices such as those manufactured by Powderject Pharmaceuticals PLC (Oxford, UK) 1o and Powderject Vaccines Inc. (Madison, WI), some examples of which are described in U.S. Patent Nos. 5,846,796; 6,010,478; 5,865,796; 5,584,807; and EP Patent No.

799. This approach offers a needle-free delivery approach wherein a dry powder formulation of microscopic particles, such as polynucleotide or polypeptide particles, are accelerated to high speed within a helium gas jet generated by a hand held device, propelling the particles into a target tissue of interest.
In a related embodiment, other devices and methods that may be useful for gas-driven needle-less injection of compositions of the present invention include those provided by Bioject, Inc. (Portland, OR), some examples of which are described in U.S. Patent Nos. 4,790,824; 5,064,413; 5,312,335; 5,383,851; 5,399,163;
5,520,639 2o and 5,993,412.
According to another embodiment, the pharmaceutical compositions described herein will comprise one or more immunostimulants in addition to the immunogenic polynucleotide, polypeptide, antibody, T-cell and/or APC
compositions of this invention. An immunostimulant refers to essentially any substance that enhances or potentiates an immune response (antibody and/or cell-mediated) to an exogenous antigen. One preferred type of immunostimulant comprises an adjuvant. Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins.
3o Certain adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N~; AS-2 (SmithKline Beecham, Philadelphia, PA); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate;
salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine;
acylated sugars; cationically or anionically derivatized polysaccharides;
polyphosphazenes;
biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF, interleukin-2, -7, -12, and other like growth factors, may also be used as adj uvants.
Within certain embodiments of the invention, the adjuvant composition l0 is preferably one that induces an immune response predominantly of the Thl type.
High levels of Thl-type cytokines (e.g., IFN-y, TNFa, IL-2 and IL-12) tend to favor the induction of cell mediated immune responses to an administered antigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction of humoral immune responses. Following application of a vaccine as provided herein, a patient will support an immune response that includes Thl-and Th2-type responses. Within a preferred embodiment, in which a response is predominantly Thl-type, the level of Thl-type cytokines will increase to a greater extent than the level of Th2-type cytokines. The levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffman, 2o Ann. Rev. Immunol. 7:145-173, 1989.
Certain preferred adjuvants for eliciting a predominantly Thl-type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A, together with an aluminum salt. MPL~
adjuvants are available from Corixa Corporation (Seattle, WA; see, for example, US
Patent Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Thl response. Such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and U.S. Patent Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by 3o Sato et al., Science 273:352, 1996. Another preferred adjuvant comprises a saponin, such as Quil A, or derivatives thereof, including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, MA); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins . Other preferred formulations include more than one saponin in the adjuvant combinations of the present invention, for example combinations of at least two of the following group comprising QS21, QS7, Quil A, (3-escin, or digitonin.
Alternatively the saponin formulations may be combined with vaccine vehicles composed of chitosan or other polycationic polymers, polylactide and polylactide-co-glycolide particles, poly-N-acetyl glucosamine-based polymer matrix, particles composed of polysaccharides or chemically modified polysaccharides, liposomes and lipid-based particles, particles composed of glycerol monoesters, etc.
The saponins may also be formulated in the presence of cholesterol to form particulate structures such as liposomes or ISCOMs. Furthermore, the saponins may be formulated together with a polyoxyethylene ether or ester, in either a non-particulate solution or suspension, or in a particulate structure such as a paucilamelar liposome or ISCOM.
~ 5 The saponins may also be formulated with excipients such as CarbopolR to increase viscosity, or may be formulated in a dry powder form with a powder excipient such as lactose.
In one preferred embodiment, the adjuvant system includes the combination of a monophosphoryl lipid A and a saponin derivative, such as the 2o combination of QS21 and 3D-MPL~ adjuvant, as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739. Other preferred formulations comprise an oil-in-water emulsion and tocopherol. Another particularly preferred adjuvant formulation employing QS21, 3D-MPL~ adjuvant and tocopherol in an oil-in-water emulsion is described in WO
25 95/17210.
Another enhanced adjuvant system involves the combination of a CpG-containing oligonucleotide and a saponin derivative particularly the combination of CpG and QS21 as disclosed in WO 00/09159. Preferably the formulation additionally comprises an oil in water emulsion and tocopherol.
Additional illustrative adjuvants for use in the pharmaceutical compositions of the invention include Montanide ISA 720 (Seppic, France), SAF
(Chiron, California, United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS
series of adjuvants (e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium), Detox (Enhanzyn~) (Corixa, Hamilton, MT), RC-529 (Corixa, Hamilton, MT) and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as those described in pending U.S. Patent Application Serial Nos. 08/853,826 and 09/074,720, the disclosures of which are incorporated herein by reference in their entireties, and polyoxyethylene ether adjuvants such as those described in WO 99/52549A1.
Other preferred adjuvants include adjuvant molecules of the general formula (I):
~ o HO(CHZCHzO)~-A-R
Wherein, n is 1-50, A is a bond or -C(O)-, R is C,_SO alkyl or Phenyl C,_SO
alkyl.
One embodiment of the present invention consists of a vaccine formulation comprising a polyoxyethylene ether of general formula (I), wherein n is between 1 and 50, preferably 4-24, most preferably 9; the R component is C,_SO
preferably C4-Czo alkyl and most preferably C,2 alkyl, and A is a bond. The concentration of the polyoxyethylene ethers should be in the range 0.1-20%, preferably from 0.1-10%, and most preferably in the range 0.1-1%. Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether, polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether, polyoxyethylerie-4-lauryl ether; polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.
Polyoxyethylene ethers such as polyoxyethylene lauryl ether are described in the Merck index (12'" edition: entry 7717). These adjuvant molecules are described in WO
99/52549.
The polyoxyethylene ether according to the general formula (I) above z5 may, if desired, be combined with another adjuvant. For example, a preferred adjuvant combination is preferably with CpG as described in the pending UK patent application GB 9820956.2.
According to another embodiment of this invention, an immunogenic composition described herein is delivered to a host via antigen presenting cells (APCs), 3o such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs. Such cells may, but need not, be genetically modified to increase the capacity for presenting the antigen, to improve activation and/or maintenance of the T cell response, to have anti-tumor effects per se and/or to be immunologically compatible with the receiver (i. e., matched HLA haplotype).
APCs may generally be isolated from any of a variety of biological fluids and organs, s including tumor and peritumoral tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells.
Certain preferred embodiments of the present invention use dendritic cells or progenitors thereof as antigen-presenting cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392:245-251, 1998) and have been shown to 1o be effective as a physiological adjuvant for eliciting prophylactic or therapeutic antitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529, 1999).
In general, dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro), their ability to take up, process and present antigens with high efficiency and their ability to activate naive T
15 cell responses: Dendritic cells may, of course, be engineered to express specific cell-surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention. As an alternative to dendritic cells, secreted vesicles antigen-loaded dendritic cells (called exosomes) may be used within a vaccine (see Zitvogel et al., Nature Med. 4:594-600, 20 1998).
Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltrating cells, lymph nodes; spleen, skin, umbilical cord blood or any other suitable tissue or fluid. For example, dendritic cells may be differentiated ex vivo by adding a combination of 25 cytokines such as GM-CSF, IL-4, IL-13 and/or TNFa to cultures of monocytes harvested from peripheral blood. Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNFa, CD40 ligand, LPS, flt3 ligand and/or other compounds) that induce differentiation, 3o maturation and proliferation of dendritic cells.
Dendritic cells are conveniently categorized as "immature" and "mature"

cells, which allows a simple way to discriminate between two well characterized phenotypes. However, this nomenclature should not be construed to exclude all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which correlates with the high expression of Fcy receptor and mannose receptor. The mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1BB).
to APCs may generally be transfected with a polynucleotide of the invention (or portion or other variant thereof) such that the encoded polypeptide, or an immunogenic portion thereof, is expressed on the cell surface. Such transfection may take place ex vivo, and a pharmaceutical composition comprising such transfected cells may then be used for therapeutic purposes, as described herein. Alternatively, a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to a patient, resulting in transfection that occurs in vivo. In vivo and ex vivo transfection of dendritic cells, for example, may generally be performed using any methods known in the art, such as those described in WO 97/24447, or the gene gun approach described by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.
2o Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with the tumor polypeptide, DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors). Prior to loading, the polypeptide may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule). Alternatively, a dendritic cell may be pulsed with a non-conjugated immunological partner, separately or in the presence of the polypeptide.
While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will typically vary depending on the mode of administration. Compositions of 'the 3o present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, mucosal, intravenous, intracranial, intraperitoneal, subcutaneous and intramuscular administration.
Carriers for use within such pharmaceutical compositions are biocompatible, and may also be biodegradable. In certain embodiments, the formulation preferably provides a relatively constant level of active component release.
s In other embodiments, however, a more rapid rate of release immediately upon administration may be desired. The formulation of such compositions is well within the level of ordinary skill in the art using known techniques. Illustrative carriers useful in this regard include microparticles of poly(lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran and the like. Other illustrative delayed-release carriers 1o include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as a phospholipid (see e.g., U.S.
Patent No.
5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO 96/06638). The amount of active compound contained within a sustained release formulation depends ~ 5 upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.
In another illustrative embodiment, biodegradable microspheres (e.g., polylactate polyglycolate) are employed as carriers for the compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S.
2o Patent Nos.4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883;
5,853,763;
5,814,344, 5,407,609 and 5,942,252. Modified hepatitis B core protein carrier systems.
such as described in WO/99 40934, and references cited therein, will also be useful for many applications. Another illustrative carrier/delivery system employs a carrier comprising particulate-protein complexes, such as those described in U.S.
Patent No.
25 5,928,647, which are capable of inducing a class I-restricted cytotoxic T
lymphocyte responses in a host.
The pharmaceutical compositions of the invention will often further comprise one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, 3o polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives.
Alternatively, compositions of the present invention may be formulated as a lyophilizate.
The pharmaceutical compositions described herein may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers are typically sealed in such a way to preserve the sterility and stability of the formulation until use. In general, formulations may be stored as suspensions, solutions or emulsions in oily or aqueous vehicles. Alternatively, a pharmaceutical composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use.
The development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, and intramuscular administration and formulation, is well known in the art, some of which are briefly discussed below for general purposes of illustration.
In certain applications, the pharmaceutical compositions disclosed herein may be delivered via oral administration to an animal. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into 2o tablets, or they may be incorporated directly with the food of the diet.
The active compounds may even be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (see, for example, Mathiowitz et al., Nature 1997 Mar 27;386(6623):410-4; Hwang et al., Crit Rev Ther Drug Carrier Syst 1998;15(3):243-84; U. S. Patent 5,641,515; U. S. Patent 5,580,579 and U. S.
Patent 5,792,451 ). Tablets, troches, pills, capsules and the like may also contain any of a variety of additional components, for example, a binder, such as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.
Typically, these formulations will contain at least about 0.1 % of the active compound or more, although the percentage of the active ingredients) may, of 1o course, be varied and may conveniently be between about 1 or 2% and about 60% or 70% or more of the weight or volume of the total formulation. Naturally, the amount of active compounds) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound.
Factors such as solubility, bioavailability, biological half life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
For oral administration the compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash;
dentifrice, buccal tablet, oral spray, or sublingual orally-administered formulation.
Alternatively, the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically-effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
Alternatively the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.
In certain circumstances it will be desirable to deliver the pharmaceutical compositions disclosed herein parenterally, intravenously, intramuscularly, or even intraperitoneally. Such approaches are well known to the skilled artisan, some of which are further described, for example, in U. S. Patent 5,543,158; U. S. Patent 5,641,515 and U. S. Patent 5,399,363. In certain embodiments, solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.
Under ordinary conditions of storage and use, these preparations generally will contain a preservative to prevent the growth of microorganisms.
Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (for example, see U. S. Patent 5,466,468). In all cases the form must be sterile and must be fluid to the extent that to easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants. The prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.
Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
In one embodiment, for parenteral administration in an aqueous solution, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one 3o dosage may be dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. Moreover, for human administration, preparations will of course preferably meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologics standards.
In another embodiment of the invention, the compositions disclosed herein may be formulated in a neutral or salt form. Illustrative pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for l0 example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
The carriers can further comprise any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art.
Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.
Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
In certain embodiments, the pharmaceutical compositions may be delivered by intranasal sprays, inhalation, and/or other aerosol delivery vehicles.
Methods for delivering genes, nucleic acids, and peptide compositions directly to the lungs via nasal aerosol sprays has been described, e.g., in U. S. Patent 5,756,353 and U.
3o S. Patent 5,804,212. Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al., J Controlled Release 1998 Mar 2;52(1-2):81-7) and lysophosphatidyl-glycerol compounds (U. S. Patent 5,725,871) are also well-known in the pharmaceutical arts. Likewise, illustrative transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U. S. Patent 5,780,045.
In certain embodiments, liposomes, nanocapsules, microparticles, lipid particles, vesicles, and the like, are used for the introduction of the compositions of the present invention into suitable host cells/organisms. In particular, the compositions of the present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
Alternatively, compositions of the present invention can be bound, either covalently or non to covalently, to the surface of such carrier vehicles.
The formation and use of liposome and liposome-like preparations as potential drug carriers is generally known to those of skill in the art (see for example, Lasic, Trends Biotechnol 1998 Ju1;16(7):307-21; Takakura, Nippon Rinsho 1998 Mar;56(3):691-5; Chandran et al., Indian J Exp Biol. 1997 Aug;35(8):801-9;
Margalit, Crit Rev Ther Drug Carrier Syst. 1995;12(2-3):233-61; U.S. Patent 5,567,434;
U.S.
Patent 5,552,157; U.S. Patent 5,565,213; U.S. Patent 5,738,868 and U.S. Patent 5,795,587, each specifically incorporated herein by reference in its entirety).
Liposomes have been used successfully with a number of cell types that are normally difficult to transfect by other procedures, including T cell suspensions, 2o primary hepatocyte cultures and PC 12 cells (Renneisen et al., J Biol Chem.
1990 Sep 25;265(27):16337-42; Muller et al., DNA Cell Biol. 1990 Apr;9(3):221-9). In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, various drugs, radiotherapeutic agents, enzymes, viruses, transcription factors, allosteric effectors and the like, into a variety of cultured cell lines and animals. Furthermore, he use of liposomes does not appear to be associated with autoimmune responses or unacceptable toxicity after systemic delivery.
In certain embodiments, liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric 3o bilayer vesicles (also termed multilamellar vesicles (MLVs).
Alternatively, in other embodiments, the invention provides for pharmaceutically-acceptable nanocapsule formulations of the compositions of the present invention. Nanocapsules can generally entrap compounds in a stable and reproducible way (see, for example, Quintanar-Guerrero et al., Drug Dev Ind Pharm.
1998 Dec;24(12):1113-28). To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 p.m) may be designed using polymers able to be degraded in vivo. Such particles can be made as described, for example, by Couvreur et al., Crit Rev Ther Drug Carrier Syst. 1988;5(1):1-20;
zur Muhlen et al., Eur J Pharm Biopharm. 1998 Mar;45(2):149-55; Zambaux et al. J
Controlled Release. 1998 Jan 2;50(1-3):31-40; and U. S. Patent 5,145,684.
CANCER THERAPEUTIC METHODS
In further aspects of the present invention, the pharmaceutical compositions described herein may be used for the treatment of cancer, particularly for the immunotherapy of ovarian cancer. Within such methods, the pharmaceutical compositions described herein are administered to a patient, typically a warm-blooded animal, preferably a human. A patient may or may not be afflicted with cancer.
Accordingly, the above pharmaceutical compositions may be used to prevent the development of a cancer or to treat a patient afflicted with a cancer.
Pharmaceutical compositions and vaccines may be administered either prior to or following surgical 2o removal of primary tumors and/or treatment such as administration of radiotherapy or conventional chemotherapeutic drugs. As discussed above, administration of the pharmaceutical compositions may be by any suitable method, including administration by intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, intradermal, anal, vaginal, topical and oral routes.
Within certain embodiments, immunotherapy may be active immunotherapy, in which treatment relies on the in vivo stimulation of the endogenous host immune system to react against tumors with the administration of immune response-modifying agents (such as polypeptides and polynucleotides as provided herein). .
Within other embodiments, immunotherapy may be passive immunotherapy, in which treatment involves the delivery of agents with established tumor-immune reactivity (such as effector cells or antibodies) that can directly or indirectly mediate antitumor effects and does not necessarily depend on an intact host immune system. Examples of effector cells include T cells as discussed above, T
lymphocytes (such as CD8+ cytotoxic T lymphocytes and CD4+ T-helper tumor-s infiltrating lymphocytes), killer cells (such as Natural Killer cells and lymphokine-activated killer cells), B cells and antigen-presenting cells (such as dendritic cells and macrophages) expressing a polypeptide provided herein. T cell receptors and antibody receptors specific for the polypeptides recited herein may be cloned, expressed and transferred into other vectors or effector cells for adoptive immunotherapy.
The to polypeptides provided herein may also be used to generate antibodies or anti-idiotypic antibodies (as described above and in U.S. Patent No. 4,918,164) for passive immunotherapy.
Effector cells may generally be obtained in sufficient quantities for adoptive immunotherapy by growth in vitro, as described herein. Culture conditions for 15 expanding single antigen-specific effector cells to several billion in number with retention of antigen recognition in vivo are well known in the art. Such in vitro culture conditions typically use intermittent stimulation with antigen, often in the presence of cytokines (such as IL-2) and non-dividing feeder cells. As noted above, immunoreactive polypeptides as provided herein may be used to rapidly expand 2o antigen-specific T cell cultures in order to generate a sufficient number of cells for immunotherapy. In particular, antigen-presenting cells, such as dendritic, macrophage, monocyte, fibroblast and/or B cells, may be pulsed with immunoreactive polypeptides or transfected with one or more polynucleotides using standard techniques well known in the art. For example, antigen-presenting cells can be transfected with a 25 polynucleotide having a promoter appropriate for increasing expression in a recombinant virus or other expression system. Cultured effector cells for use in therapy must be able to grow and distribute widely, and to survive long term in vivo.
Studies have shown that cultured effector cells can be induced to grow in vivo and to survive long term in substantial numbers by repeated stimulation with antigen supplemented 3o with IL-2 (see, for example, Cheever et al., Immunological Reviews 157:177, 1997).
Alternatively, a vector expressing a polypeptide recited herein may be introduced into antigen presenting cells taken from a patient and clonally propagated ex vivo for transplant back into the same patient. Transfected cells may be reintroduced into the patient using any means known in the art, preferably in sterile form by intravenous, intracavitary, intraperitoneal or intratumor administration.
Routes and frequency of administration of the therapeutic compositions described herein, as well as dosage, will vary from individual to individual, and may be readily established using standard techniques. In general, the pharmaceutical compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally.
t o Preferably, between 1 and 10 doses may be administered over a 52 week period.
Preferably, 6 doses are administered, at intervals of 1 month, and booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients. A suitable dose is an amount of a compound that, when administered as described above, is capable of promoting an anti-tumor immune response, and is at least 10-50% above the basal (i.e., untreated) level. Such response can be monitored by measuring the anti-tumor antibodies in a patient or by vaccine-dependent generation of cytolytic effector cells capable of killing the patient's tumor cells in vitro. Such vaccines should also be capable of causing an immune response that leads to an improved clinical outcome (e.g., more frequent remissions, complete or 2o partial or longer disease-free survival) in vaccinated patients as compared to non-vaccinated patients. In general, for pharmaceutical compositions and vaccines comprising one or more polypeptides, the amount of each polypeptide present in a dose ranges from about 25 ~,g to 5 mg per kg of host. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.
In general, an appropriate dosage and treatment regimen provides the active compounds) in an amount sufficient to provide therapeutic and/or prophylactic benefit. Such a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated patients as compared to non-treated patients. Increases in 3o preexisting immune responses to a tumor protein generally correlate with an improved clinical outcome. Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which may be performed using samples obtained from a patient before and after treatment.
CANCER DETECTION AND DIAGNOSTIC COMPOSITIONS, METHODS AND KITS
s In general, a cancer may be detected in a patient based on the presence of one or more ovarian tumor proteins and/or polynucleotides encoding such proteins in a biological sample (for example, blood, sera, sputum urine and/or tumor biopsies) obtained from the patient. In other words, such proteins may be used as markers to indicate the presence or absence of a cancer such as ovarian cancer. In addition, such 1o proteins may be useful for the detection of other cancers. The binding agents provided herein generally permit detection of the level of antigen that binds to the agent in the biological sample. Polynucleotide primers and probes may be used to detect the level of mRNA encoding a tumor protein, which is also indicative of the presence or absence of a cancer. In general, a ovarian tumor sequence should be present at a level that is at 15 least three fold higher in tumor tissue than in normal tissue There are a variety of assay formats known to those of ordinary skill in the art for using a binding agent to detect polypeptide markers in a sample.
See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, the presence or absence of a cancer in a patient may be determined by 20 (a) contacting a biological sample obtained from a patient with a binding agent; (b) detecting in the sample a level of polypeptide that binds to the binding agent; and (c) comparing the level of polypeptide with a predetermined cut-off value.
In a preferred embodiment, the assay involves the use of binding agent immobilized on a solid support to bind to and remove the polypeptide from the 25 remainder of the sample. The bound polypeptide may then be detected using a detection reagent that contains a reporter group and specifically binds to the binding agent/polypeptide complex. Such detection reagents may comprise, for example, a binding agent that specifically binds to the polypeptide or an antibody or other agent that specifically binds to the binding agent, such as an anti-immunoglobulin, protein G, 3o protein A or a lectin. Alternatively, a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized binding agent after incubation of the binding agent with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the binding agent is indicative of the reactivity of the sample with the immobilized binding agent. Suitable polypeptides for use within such assays include full length ovarian tumor proteins and polypeptide portions thereof to which the binding agent binds, as described above.
The solid support may be any material known to those of ordinary skill in the art to which the tumor protein may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane.
1o Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S.
Patent No. 5,359,681. The binding agent may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature. In the context of the present invention, the term "immobilization" refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the agent and functional groups on the support or may be a linkage by way of a cross-linking agent).
Immobilization by adsorption to a well in a microtiter plate or to a membrane is 2o preferred. In such cases, adsorption may be achieved by contacting the binding agent, in a suitable buffer, with the solid support for a suitable amount of time.
The contact time varies with temperature, but is typically between about 1 hour and about 1 day. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of binding agent ranging from about 10 ng to about 10 fig, and preferably about 100 ng to about 1 fig, is sufficient to immobilize an adequate amount of binding agent.
Covalent attachment of binding agent to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the 3o binding agent. For example, the binding agent may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).
In certain embodiments, the assay is a two-antibody sandwich assay.
This assay may be performed by first contacting an antibody that has been immobilized on a solid support, commonly the well of a microtiter plate, with the sample, such that polypeptides within the sample are allowed to bind to the immobilized antibody.
Unbound sample is then removed from the immobilized polypeptide-antibody complexes and a detection reagent (preferably a second antibody capable of binding to a l0 different site on the polypeptide) containing a reporter group is added.
The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific reporter group.
More specifically, once the antibody is immobilized on the support as described above, the remaining protein binding sites on the support are typically ~ 5 blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin or Tween 20TM (Sigma Chemical Co., St. Louis, MO). The immobilized antibody is then incubated with the sample, and polypeptide is allowed to bind to the antibody. The sample may be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation. In general, an appropriate contact 2o time (i.e., incubation time) is a period of time that is sufficient to detect the presence of polypeptide within a sample obtained from an individual with ovarian cancer.
Preferably, the contact time is sufficient to achieve a level of binding that is at least about 95% of that achieved at equilibrium between bound and unbound polypeptide.
Those of ordinary skill in the art will recognize that the time necessary to achieve 25 equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient.
Unbound sample may then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20TM. The second 3o antibody, which contains a reporter group, may then be added to the solid support.
Preferred reporter groups include those groups recited above.

The detection reagent is then incubated with the immobilized antibody-polypeptide complex for an amount of time sufficient to detect the bound polypeptide.
An appropriate amount of time may generally be determined by assaying the level of binding that occurs over a period of time. Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group. The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a t o different reporter group (commonly a radioactive or fluorescent group or an enzyme).
Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.
To determine the presence or absence of a cancer, such as ovarian cancer, the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value. In one preferred embodiment, the cut-off value for the detection of a cancer is the average mean signal obtained when the immobilized antibody is incubated with samples from patients without the cancer. In general, a sample generating a signal that 2o is three standard deviations above the predetermined cut-off value is considered positive for the cancer. In an alternate preferred embodiment, the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiolo~: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p. 106-7. Briefly, in this embodiment, the cut-off value may be determined from a plot of pairs of true positive rates (i.e., sensitivity) and false positive rates (100%-specificity) that correspond to each possible cut-off value for the diagnostic test result.
The cut-off value on the plot that is the closest to the upper left-hand corner (i.e., the value that encloses the largest area) is the most accurate cut-off value, and a sample generating a signal that is higher than the cut-off value determined by this method may be considered 3o positive. Alternatively, the cut-off value may be shifted to the left along the plot, to minimize the false positive rate, or to the right, to minimize the false negative rate. In general, a sample generating a signal that is higher than the cut-off value determined by this method is considered positive for a cancer.
In a related embodiment, the assay is performed in a flow-through or strip test format, wherein the binding agent is immobilized on a membrane, such as nitrocellulose. In the flow-through test, polypeptides within the sample bind to the immobilized binding agent as the sample passes through the membrane. A second, labeled binding agent then binds to the binding agent-polypeptide complex as a solutiori containing the second binding agent flows through the membrane. The detection of bound second binding agent may then be performed as described above. In the strip test to format, one end of the membrane to which binding agent is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing second binding agent and to the area of immobilized binding agent.
Concentration of second binding agent at the area of immobilized antibody indicates the presence of a cancer. Typically, the concentration of second binding agent at that site generates a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result. In general, the amount of binding agent immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of polypeptide that would be sufficient to generate a positive signal in the two-antibody sandwich assay, in the format discussed above.
Preferred binding agents for use in such assays are antibodies and antigen-binding fragments thereof. Preferably, the amount of antibody immobilized on the membrane ranges from about 25 ng to about lpg, and more preferably from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount of biological sample.
Of course, numerous other assay protocols exist that are suitable for use with the tumor proteins or binding agents of the present invention. The above descriptions are intended to be exemplary only. For example, it will be apparent to those of ordinary skill in the art that the above protocols may be readily modified to use tumor polypeptides to detect antibodies that bind to such polypeptides in a biological 3o sample. The detection of such tumor protein specific antibodies may correlate with the presence of a cancer.

A cancer may also, or alternatively, be detected based on the presence of T cells that specifically react with a tumor protein in a biological sample.
Within certain methods, a biological sample comprising CD4+ and/or CD8+ T cells isolated from a patient is incubated with a tumor polypeptide, a polynucleotide encoding such a polypeptide and/or an APC that expresses at least an immunogenic portion of such a polypeptide, and the presence or absence of specific activation of the T cells is detected.
Suitable biological samples include, but are not limited to, isolated T cells.
For example, T cells may be isolated from a patient by routine techniques (such as by Ficoll/Hypaque density gradient centrifugation of peripheral blood lymphocytes). T
1o cells may be incubated in vitro for 2-9 days (typically 4 days) at 37°C with polypeptide (e.g., 5 - 25 p,g/ml). It may be desirable to incubate another aliquot of a T
cell sample in the absence of ovarian tumor polypeptide to serve as a control. For CD4+ T
cells, activation is preferably detected by evaluating proliferation of the T cells.
For CD8+ T
cells, activation is preferably detected by evaluating cytolytic activity. A
level of proliferation that is at least two fold greater and/or a level of cytolytic activity that is at least 20% greater than in disease-free patients indicates the presence of a cancer in the patient.
As noted above, a cancer may also, or alternatively, be detected based on the level of mRNA encoding a ovarian tumor protein in a biological sample. For example, at least two oligonucleotide primers may be employed in a polymerase chain reaction (PCR) based assay to amplify a portion of a tumor cDNA derived from a biological sample, wherein at least one of the oligonucleotide primers is specific for (i. e., hybridizes to) a polynucleotide encoding the tumor protein. The amplified cDNA
is then separated and detected using techniques well known in the art, such as gel electrophoresis. Similarly, oligonucleotide probes that specifically hybridize to a polynucleotide encoding a tumor protein may be used in a hybridization assay to detect the presence of polynucleotide encoding the tumor protein in a biological sample.
To permit hybridization under assay conditions, oligonucleotide primers and probes should comprise an oligonucleotide sequence that has at least about 60%, 3o preferably at least about 75% and more preferably at least about 90%, identity to a portion of a polynucleotide encoding a tumor protein of the invention that is at least 10 nucleotides, and preferably at least 20 nucleotides, in length. Preferably, oligonucleotide primers and/or probes hybridize to a polynucleotide encoding a polypeptide described herein under moderately stringent conditions, as defined above.
Oligonucleotide primers and/or probes which may be usefully employed in the diagnostic methods described herein preferably are at least 10-40 nucleotides in length.
In a preferred embodiment, the oligonucleotide primers comprise at least 10 contiguous nucleotides, more preferably at least 15 contiguous nucleotides, of a DNA
molecule having a sequence as disclosed herein. Techniques for both PCR based assays and hybridization assays are well known in the art (see, for example, Mullis et al., Cold to Spring Harbor Symp. Quant. Biol., 51:263, 1987; Erlich ed., PCR Technology, Stockton Press, NY, 1989).
One preferred assay employs RT-PCR, in which PCR is applied in conjunction with reverse transcription. Typically, RNA is extracted from a biological sample, such as biopsy tissue, and is reverse transcribed to produce cDNA
molecules.
PCR amplification using at least one specific primer generates a cDNA
molecule, which may be separated and visualized using, for example, gel electrophoresis.
Amplification may be performed on biological samples taken from a test patient and from an individual who is not afflicted with a cancer. The amplification reaction may be performed on several dilutions of cDNA spanning two orders of magnitude. A two-fold or greater increase in expression in several dilutions of the test patient sample as compared to the same dilutions of the non-cancerous sample is typically considered positive.
In another embodiment, the compositions described herein may be used as markers for the progression of cancer. In this embodiment, assays as described above for the diagnosis of a cancer may be performed over time, and the change in the level of reactive polypeptide(s) or polynucleotide(s) evaluated. For example, the assays may be performed every 24-72 hours for a period of 6 months to 1 year, and thereafter performed as needed. In general, a cancer is progressing in those patients in whom the level of polypeptide or polynucleotide detected increases over time. In contrast, the 3o cancer is not progressing when the level of reactive polypeptide or polynucleotide either remains constant or decreases with time.

Certain in vivo diagnostic assays may be performed directly on a tumor.
One such assay involves contacting tumor cells with a binding agent. The bound binding agent may then be detected directly or indirectly via a reporter group. Such binding agents may also be used in histological applications. Alternatively, polynucleotide probes may be used within such applications.
As noted above, to improve sensitivity, multiple tumor protein markers may be assayed within a given sample. It will be apparent that binding agents specific for different proteins provided herein may be combined within a single assay.
Further, multiple primers or probes may be used concurrently. The selection of tumor protein t o markers may be based on routine experiments to determine combinations that results in optimal sensitivity. In addition, or alternatively, assays for tumor proteins provided herein may be combined with assays for other known tumor antigens.
The present invention further provides kits for use within any of the above diagnostic methods. Such kits typically comprise two or more components necessary for performing a diagnostic assay. Components may be compounds, reagents, containers and/or equipment. For example, one container within a kit may contain a monoclonal antibody or fragment thereof that specifically binds to a tumor protein. Such antibodies or fragments may be provided attached to a support material, as described above. One or more additional containers may enclose elements, such as 2o reagents or buffers, to be used in the assay. Such kits may also, or alternatively, contain a detection reagent as described above that contains a reporter group suitable for direct or indirect detection of antibody binding.
Alternatively, a kit may be designed to detect the level of mRNA
encoding a tumor protein in a biological sample. Such kits generally comprise at least one oligonucleotide probe or primer, as described above, that hybridizes to a polynucleotide encoding a tumor protein. Such an oligonucleotide may be used, for example, within a PCR or hybridization assay. Additional components that may be present within such kits include a second oligonucleotide and/or a diagnostic reagent or container to facilitate the detection of a polynucleotide encoding a tumor protein.
3o The following Examples are offered by way of illustration and not by way of limitation.

EXAMPLES
Example 1 Identification of Representative Ovarian Carcinoma cDNA Sequences This Example illustrates the identification of ovarian tumor cDNA
molecules.
Primary ovarian tumor and metastatic ovarian tumor cDNA libraries were each constructed in kanamycin resistant pZErOT""-2 vector (Invitrogen) from pools to of three different ovarian tumor RNA samples. For the primary ovarian tumor library, the following RNA samples were used: (1) a moderately differentiated papillary serous carcinoma of a 41 year old, (2) a stage IIIC ovarian tumor and (3) a papillary serous adenocarcinoma for a 50 year old Caucasian. For the metastatic ovarian tumor library, the RNA samples used were omentum tissue from: (1) a metastatic poorly differentiated papillary adenocarcinoma with psammoma bodies in a 73 year old, (2) a metastatic poorly differentiated adenocarcinoma in a 74 year old and (3) a metastatic poorly differentiated papillary adenocarcinoma in a 68 year old.
The number of clones in each library was estimated by plating serial dilutions of unamplified libraries. Insert data were determined from 32 primary ovarian 2o tumor clones and 32 metastatic ovarian tumor clones. The library characterization results are shown in Table I.
Table I
Characterization of cDNA Libraries # ClonesClones Insert Ave. Insert with Size Library in LibraryInsert Range (bp)Size (bp) (%) Primary Ovarian 1,258,00097 175 - 80002356 Tumor Metastatic Ovarian 1,788,000100 150 - 43001755 Tumor Four subtraction libraries were constructed in ampicillin resistant pcDNA3.1 vector (Invitrogen). Two of the libraries were from primary ovarian tumors and two were from metastatic ovarian tumors. In each case, the number of restriction enzyme cuts within inserts was minimized to generate full length subtraction libraries.
The subtractions were each done with slightly different protocols, as described in more detail below.
A. POTS 2 Library: Primary Ovarian Tumor Subtraction Library Tracer: 10 Pg primary ovarian tumor library, digested with Not I
Driver: 35 ~g normal pancreas in pcDNA3.1(+) 20 Pg normal PBMC in pcDNA3.1 (+) ~g normal skin in pcDNA3.1 (+) 10 35 ~g normal bone marrow in pZErOT""-2 Digested with Bam HI/Xho I/Sca I
Two hybridizations were performed, and Not I-cut pcDNA3.1 (+) was the cloning vector for the subtracted library. Sequence results for previously unidentified clones that were randomly picked from the subtracted library are presented in Table II.
Table II
Ovarian Carcinoma Seauences Sequence SEQ ID NO

B. POTS 7 Library: Primary Ovarian Tumor Subtraction Libr Tracer: 10 ~g primary ovarian tumor library, digested with Not I
Driver: 35 ~g normal pancreas in pcDNA3.1 (+) 20 pg normal PBMC in pcDNA3.1 (+) 10 pg normal skin in pcDNA3.1(+) 35 pg normal bone marrow in pZErOT""-2 Digested with Bam HI/Xho I/Sca I
~25 p,g pZErOT""-2, digested with Bam HI and Xho I
Two hybridizations were performed, and Not I-cut pcDNA3.1 (+) was the l0 cloning vector for the subtracted library. Sequence results for previously unidentified clones that were randomly picked from the subtracted library are presented in Table III.
Table III
Ovarian Carcinoma Sequences Sequence SEQ ID NO

C. OS1D Library: Metastatic Ovarian Tumor Subtraction Library Tracer: 10~g metastatic ovarian library in pZErOT""-2, digested 2o with Not I
Driver: 24.S~g normal pancreas in pcDNA3.1 14~g normal PBMC in pcDNA3.1 l4p,g normal skin in pcDNA3.1 24.Spg normal bone marrow in pZErOT""-2 SOp,g pZErOT""-2, digested with Bam HI/Xho I/Sfu I

Three hybridizations were performed, and the last two hybridizations were done with an additional l5pg of biotinylated pZErOT""-2 to remove contaminating pZErOT""-2 vectors. The cloning vector for the subtracted library was pcDNA3.1/Not I
cut. Sequence results for previously unidentified clones that were randomly picked from the subtracted library are presented in Table IV.
Table IV
Ovarian Carcinoma Sequences Sequence SEQ ID NO

23645.1 13 23660.1 16 23666.1 19 23679.1 23 to D. OS1F Library: Metastatic Ovarian Tumor Subtraction Library Tracer: l Opg metastatic ovarian tumor library, digested with Not I
Driver: 12.8~g normal pancreas in pcDNA3.1 7.3pg normal PBMC in pcDNA3.1 7.3p.g normal skin in pcDNA3.1 12.8~g normal bone marrow in pZErOT""-2 25~g pZErOT""-2, digested with Bam HI/Xho I/Sfu I
One hybridization was performed. The cloning vector for the subtracted library was pcDNA3.1/Not I cut. Sequence results for previously unidentified clones that were randomly picked from the subtracted library are presented in Table V.

Table V
Ovarian Carcinoma Sequences Sequence SEQ ID NO

24359 (78% Human mRNA for KIAA011145 gene, complete cds) 24336 (79% with H. sapiens mitochondria)27 genome (consensus sequence)) 24737 (84%Human ADP/ATP translocase114 mRNA) 24363 (87% Homo Sapiens eukaryotic49 translation elongation factor 1 alpha 1 (EEF 1 A 1 ) 24357 (87% S. scrofa mRNA for 43 UDP glucose pyrophosphorylase) 24362 (88% Homo Sapiens Chromosome48 BAC clone CIT987SK-A-233A7) 24704 (88% Homo Sapiens chromosome92 9, clone hRPK.401_G_18) 24367 (89% Homo Sapiens 12p13.3 52 BAC

Sequence SEQ ID NO

RCPI l 1-935C2) 24717 (89% Homo Sapiens proliferation-103 associated gene A (natural killer-enhancing factor A) (PAGA) 24364 (89%Human DNA sequence from50 PAC

27K14 on chromosome Xp11.3-Xp11.4) 24355 (91% Homo sapiens chromosome41 17, clone hCIT.91_J_4) 24341 (91 %Homo Sapiens chromosome32 5, BAC

clone 249h5 (LBNL H 149) 24714 (91 %Human DNA sequence 100 from clone 125N5 on chromosome 6q26-27) The sequences in Table VI, which correspond to known sequences, were also identified in the above libraries.
Table VI
Ovarian Carcinoma Sequences Identity SEQ ID SequenceLibrar NO Y

Genomic sequence from Human 9q34 56 24634 OS1D

Homo Sapiens 12p13.3 PAC RPCII-96H9 66 24653 OS1D
(Roswell Park Cancer Institute Human PACLibrary) Homo Sapiens annexin II (lipocortin 60 24640 OS
II) (ANX2) 1 mRNA D

Homo sapiens eukaryotic translation 55 24627 OS1D
elongation factor 1 alpha 1 (EEF 1 A 1 ) Homo Sapiens ferritin, heavy polypeptide64 24648 OS1D
1 (FTHI) Homo Sapiens FK506-binding protein 22 23677.1 OS
1 A ( 12kD) 1 (FKBP 1 A) mRNA D

Homo Sapiens growth arrest specific 73 24671 OS
transcript 5 gene 1 D

Homo Sapiens keratin 18 (KRT18) mRNA 68 24657 OS1D

Homo sapiens mRNA; cDNA DKFZp564H182 76 24677 OS1D

Homo Sapiens ribosomal protein S7 74 24673 OS1D
(RPS7) Homo Sapiens ribosomal protein, large,14 23647.1 OS1D
PO (RPLPO) mRNA

Homo Sapiens T cell-specific tyrosine67 24655 OS1D
kinase mRNA

Homo Sapiens tubulin, alpha, ubiquitous61 24642 OS
(K-ALPHA- 1 1) D

HSU78095 Homo Sapiens placental bikunin18 23662.1 OS1D
mRNA

Human BAC clone GSOSSK18 from 7p15-p2111 23636.1 OS1D

Identity SEQ ID SequenceLibrar NO Y

Human insulin-like growth factor-binding58 24636 OS1D
protein-3 gene Human mRNA for ribosomal protein 79 24687 OS

D

Human non-histone chromosomal protein62 24645 OS1D

mRNA

Human ribosomal protein L3 mRNA, 3' 59 24638 OS1D
end Human TSC-22 protein mRNA 77 24679 OS1D

HUMGFIBPA Human growth hormone-dependent12 23637.1 OS1D
insulin-like growth factor-binding protein HUMMTA Homo Sapiens mitochondrial 17 23661.1 OS1D
DNA

~HUMMTCG Human mitochondrion 21 23673.1 OS1D

HUMTI227HC Human mRNA for TI-227H 20 23669.1 OS

D

HUMTRPM2A Human TRPM-2 mRNA 15 23657.1 OS1D

Genomic sequence from Human 13 80 24689 OS

F

H.sapiens CpG island DNA genomic Msel104 24719 OS1F
fragment, clone 84a5 H.sapiens RNA for snRNP protein B 110 24730 OS1F

Homo Sapiens (clone L6) E-cadherin 108 24728 OS
(CDH 1 ) gene 1 F

Homo Sapiens atrophin-1 interacting 37 24350 OS1F
protein 4 (AIP4) mRNA

Homo Sapiens CGI-08 protein mRNA 102 24716 OS1F

Homo Sapiens clone 24452 mRNA sequence54 24374 OS1F

Homo Sapiens clone IMAGE 286356 83 24693 OS

F

Homo sapiens cornichon protein mRNA 113 24735 OS1F

Homo Sapiens hypothetical 43.2 Kd 87 24697 OS
protein mRNA 1 F

Homo Sapiens interleukin 1 receptor 29 24338 OS1F
accessory protein (IL 1 RAP) mRNA.

Homo Sapiens K-Cl cotransporter KCC4 31 24340 OS1F
mRNA, complete cds Homo sapiens keratin 8 (KRTB) mRNA 11 S 24739 OS

Homo Sapiens mRNA for DEPP (decidual 36 24349 OS1F
protein induced by progesterone) Homo Sapiens mRNA for KIAA0287 gene 1 O 1 24715 OS

F

Homo sapiens mRNA for KIAA0762 protein118 24742 OS

F

Homo Sapiens mRNA for zinc-finger 24 24333 OS1F
DNA-binding protein, complete cds Homo Sapiens mRNA; cDNA DKFZp434K114 112 24734 OS1F

Homo Sapiens mRNA; cDNA DKFZp564E196225 24334 OS1F
(from clone DKFZp564E1962) Homo Sapiens nuclear chloride ion 34 24345 OS1F
channel protein (NCC27) mRNA

Homo Sapiens ribosomal protein L13 109 24729 OS1F
(RPL13) Homo Sapiens senescence-associated 94 24706 OS
epithelial 1 F

Identity SEQ ID SequenceLibrar NO

y membrane protein (SEMP1) Homo Sapiens tumor protein, translationally-26 24335 OS1F

controlled 1 (TPT 1 ) mRNA.

Homo Sapiens tumor suppressing subtransferable51 24366 OS1F

candidate 1 (TSSC1) Homo Sapiens v-fos FBJ marine osteosarcoma85 24695 OS
viral 1 F

oncogene homolog(FOS) mRNA

Homo Sapiens zinc finger protein slug106 24722 OS1F
(SLUG) gene Human clone 23722 mRNA 105 24721 OS1F

Human clones 23667 and 23775 zinc 119 24744 OS1F
finger protein mRNA

Human collagenase type IV mRNA, 3' 39 24352 OS1F
end.

Human DNA sequence from PAC 29K1 on 116 24740 OS1F

chromosome 6p21.3-22.2.

Human ferritin H chain mRNA 96 24708 OS

F

Human heat shock protein 27 (HSPB 88 24698 OS
1 ) gene exons 1- 1 F

Human mRNA for KIAA0026 gene 30 24339 OS1F

Human mRNA for T-cell cyclophilin 40 24354 OS1F

Genomic sequence from Human 9q34, 140 25092 POTS2 complete sequence [Homo Sapiens]

H.sapiens DNA for muscle nicotinic 3 21910 POTS2 acetylcholine receptor gene promotor, clone ICRFc105F02104 Homo sapiens breast cancer suppressor142 25098 POTS2 candidate 1 (bcsc-1) mRNA, complete cds Homo sapiens CGI-151 protein mRNA, 8 21916 POTS2 complete cds Homo sapiens complement component 4 21913 POTS2 3 (C3) gene, exons 1-30.

Homo Sapiens mRNA for hepatocyte growth159 25758 POTS2 factor activator inhibitor type 2,complete cds Homo Sapiens preferentially expressed153 25745 POTS2 antigen of melanoma (PRAME) mRNA

Homo Sapiens prepro dipeptidyl peptidase152 25117 POTS2 I (DPP-I) gene, complete cds Homo sapiens SKB 1 (S. cerevisiae) 147 25110 POTS2 homolog (SKB 1 ) mRNA.

Homo Sapiens SWI/SNF related, matrix 6 21914 POTS2 associated, actin dependent regulator of chromatin, subfamily a, member 4 (SMARCA4) Human 12S RNA induced by poly(rI), 155 25749 POTS2 poly(rC) and Newcastle disease virus Human ferritin Heavy subunit mRNA, 7 21915 POTS2 complete cds.

Human glyceraldehyde-3-phosphate dehydrogenase141 25093 POTS2 Identity SEQ ID SequenceLibrar NO Y

(GAPDH) mRNA, complete cds.

Human mRNA for fibronectin (FN precursor)157 25755 POTS2 Human translocated t(8;14) c-myc (MYC)154 25746 POTS2 oncogene, exon 3 and complete cds H.sapiens vegf gene, 3'UTR 169 25799 POTS?

Homo Sapiens 305 ribosomal protein 170 25802 POTS7 S7 homolog mRNA, complete cds Homo Sapiens acetyl-Coenzyme A acetyltransferase172 25808 POTS?

(acetoacetyl Coenzyme A thiolase) (ACAT2) mRNA

Homo Sapiens amyloid beta precursor 138 24959 POTS?
protein-binding protein 1, 59kD (APPBP1) mRNA.

Homo Sapiens arylacetamide deacetylase129 24942 POTS7 (esterase) (AADAC) mRNA.

Homo Sapiens clone 23942 alpha enolase165 25787 POTS7 mRNA, partial cds Homo Sapiens echinoderm microtubule-associated130 24943 POTS?
protein-like EMAP2 mRNA, complete cds Homo Sapiens IMP (inosine monophosphate)164 25775 POTS?
dehydrogenase 2 (IMPDH2) mRNA

Homo Sapiens megakaryocyte potentiating126 24938 POTS?
factor (MPF) mRNA.

Homo Sapiens mRNA for KIAA0552 protein,163 25771 POTS7 complete cds Homo Sapiens Norrie disease protein 173 25809 POTS7 (NDP) mRNA

Homo Sapiens podocalyxin-like (PODXL)131 24944 POTS7 mRNA.

Homo Sapiens synaptogyrin 2 (SYNGR2) 135 24952 POTS7 mRNA.

Human aldose reductase mRNA, complete139 24969 POTS7 cds.

Human cyclooxygenase-1 (PTSG1) mRNA, 124 24935 POTS7 partial cds Human H19 RNA gene, complete cds. 122 24933 POTS?

Human mRNA for Apol Human (MERS(Aopl-127 24939 POTS?
Mouse)-like protein), complete cds Human triosephosphate isomerase mRNA,123 24934 POTS7 complete cds.

Still further ovarian carcinoma polynucleotide and/or polypeptide sequences identified from the above libaries are provided below in Table VII.
Sequences 05745 (SEQ ID NOs: 183 & 185), 05845 (SEQ ID NO: 193) and 05855 (SEQ ID NO: 194) represent novel sequences. The remaining sequences exhibited at least some homology with known genomic and/or EST sequences.

Table VII
SEQ ID: Sequence Library 174: 05655 CRABP OS1D

175 : 05665 Ceruloplasmin POTS2 176: 0567S 41191.SEQ(1>487) POTS2 177: 05685 KIAA0762.seq(1>3999)POTS7 178 : 05695 41220.seq(1>1069) POTS7 179: 05705 41215.seq(1>1817) POTS2 180: 05715 41213.seq(1>2382) POTS2 181 : 05725 41208.seq(1>2377) POTS2 182 : 0573S 41177.seq(1>1370) OS1F

183 : 05745 47807.seq(1>2060) n/a 184 : 05685/VSGF DNA seq n/a 185: 0574S 47807.seq(1>3000) n/a 186: 05685/VSGF protein seq n/a 187 : 449H1(57581) OS1D

188: 451 E 12(575 82) OS 1 D

189: 453C7 3'(57583.1)OsteonectinOS1D

190: 453C7 5'(57583.2) OS1D

191: 45661 3'(57584.1)NeurotensinOS1F

192: 45661 5'(57584.2) OS1F

193: 0584S 46565(57585) OS1F

194: 05855 469B12(57586) POTS2 195: 05695 474C3(57587) POTS7 196: 483B1_3'(24934.1)TriosephosphatePOTS?

197: 57885 Human preferentiallyPOTS2 expressed antigen of melanoma 198: 57886 Chromosome 22q12.1 POTS2 clone 199: 57887 Homologous to mousePOTS2 brain cDNA clone MNCb-0671 From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

SEQUENCE LISTING
<110> Corixa Corporation Xu, Jiangchun Stolk, John A.
<120> OVARIAN TUMOR SEQUENCES AND
METHODS OF USE THEREFOR
<130> 210121.484PC
<140> PCT
<141> 2000-09-08 <160> 199 <170> FastSEQ for Windows Version 3.0 <210> 1 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400>

caacctcactagtaaatgaaagaaatattgtaatttgtatttgatctgctgggtctttgg 60 agtcagaactggttttatcagcagtttgatcttctgaggtctggtatgtagtttgctggc 120 ccacagaaccttcacgtgtattcacagcctcaatgccataaggaaactcttttagaagtt 180 ctgacagctggtcatgtaggtataagacaggtgccttatcactgtggatttcatttcttg 240 caggatcttggggagtatagttgctggatgcatctatttcctgagggtaaatatcctcct 300 ggncgacgcggccgctcgagtctagagggcccgtttaaacccgctgatcagcctcgactg 360 tgccttctanttgccanccatntgttgtttgcccct 396 <210> 2 <211> 396 <212> DNA
<213> Homo sapien <400>

cgaccaaaaagtaaactccaagtgaacatcaaatcaaatctaatccttttggccacatga 60 ctggttgttctttatctcatagttacaatgaatcatataaactgtagactgccactacca 120 cgatacttctgtgacacagaaggaatgtcctatttgcctatctatctgaggaatgttaaa 180 tagagaaaaatagattataaaacaacctggaggtcacaggattctgagataatccctctg 240 ttaaaaaacatctgaacagcaaatgtccaatctgtaataaaatagttaaaggtccaagtc 300 aagtccacttctacttggctggcccagcacaagaaatctaacagcactttgtaatcattt 360 tgcttttctaattttcccggaggacatgggccattg 396 <210> 3 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400>

cgcccttttttttttttttttnattggnnnaantcnctttnantnnaaaaacntgnangg 60 naancccanncccnnggnaccannnccaggagttgggtgganactgagtggggtttgtgt 120 gggtgagggggcatctactcctnttgcaacaagccaaaagtagaacagcctaaggaaaag 180 tgacctgccttggagccttagtccctcccttagggccccctcagcctaccctatccaagt 240 ctgaggctatggaagtctccctcctagttcactagcaggttccccatcttttccaggctg 300 cccctagcactccacgtttttctgaaaaaatctanacaggccctttttgggtacctaaaa 360 cccagctgaggttgtgagcttgtaaggtaaagcaag 396 <210> 4 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 4 gaccaatccttgncncactancaaaangaccccnctnaccnccaggaactgaacctnnnt 60 gtnnacctccnnctgcnnagccntatntccaanatcacccaccgtatccactgggaatct 120 gccagcctcctgcgatcagaagagaccaatcgaaaatgagggtttcacantcacagctga 180 aggaaaaggccaaggcaccttgtcggnggngacaatgtaccatgctaaggccaaagatca 240 actcacctgtaataaattcgacctcaaggtcaccataaaaccagcaccggaacagaaaaa 300 gaggcctnaggatgcccaagaaacacttttgatcctttgaaaactgtaccaaggtaccgg 360 ggggagacccaggaaaggnccnttatgtntnnntnt 396 <210> 5 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 5 gacgccggagctgccgcgccagtcgcctagcaggtcctctaccggcttattcctgtgccg 60 gatcttcatcggcacaggggccactgagacgtttctgcctccctctttcttcctccgctc 120 tttctcttccctctngtttagtttgcctgggagcttgaaaggagaaagcacnggggtcgc 180 cccaaaccctttctgcttctgcccatcacaagtgccactaccgccatgggcctcactatc 240 tcctccctcttctcccgactatttggcaagaagcagatgcgcattttgatggttggattg 300 gatgctgctggcaagacaaccattcttgataaactgaaagtanggganataagnaccacc 360 atttctaccattgggtttaatgggggaaacagtana 396 <210> 6 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 6 acgggaggcgccgggaagtcgacggcgccggcggctcctgcaggaggccactgtctgcag 60 ctcccgtgaagatgtccactccagacccacccctgggcggaactcctcggccaggtcctt 120 ccccgggccctgcccttcccctggagccatgctgggccctagcccgggtccctcgccggg 180 ctccgcccacagcatgatggggcccagcccangggccgccctcagcaggacaccccatcc 240 ccacccaggggcctggagggtaccctcaggacaacatgcaccagatgcacaagcccatgg 300 agtccatgcatgagaagggcatgtcggacgacccgcgctacaaccagatgaaaggaatgg 360 ggatgcggtcagggggccatgctgggatggggcccc 396 <210> 7 <211> 396 <212> DNA
<213> Homo sapien <400> 7 acccgagagtcgtcggggtttcctgcttcaacagtgcttggacggaacccggcgctcgtt 60 ccccaccccggccggccgcccatagccagccctccgtcacctcttcaccgcaccctcgga 120 ctgccccaaggcccccgccgccgctccagcgccgcgcagccaccgccgccgccgccgcct 180 ctccttagtcgccgccatgacgaccgcgtccacctcgcaggtgcgccagaactaccacca 240 ggactcagaggccgccatcaaccgccagatcaacctggagctctacgcctcctacgttta 300 cctgtccatgtcttactactttgaccgcgatgatgtggctttgaagaactttgccaaata 360 ctttcttcaccaatctcatgaggagagggaacatgc 396 <210> 8 <211> 396 <212> DNA
<213> Homo sapien <400>

cgacaacaaggttaataccttagttcttaacattttttttctttatgtgtagtgttttca 60 tgctaccttggtaggaaacttatttacaaaccatattaaaaggctaatttaaatataaat 120 aatataaagtgctctgaataaagcagaaatatattacagttcattccacagaaagcatcc 180 aaaccacccaaatgaccaaggcatatatagtatttggaggaatcaggggtttggaaggag 240 tagggaggagaatgaaggaaaatgcaaccagcatgattatagtgtgttcatttagataaa 300 agtagaaggcacaggagaggtagcaaaggccaggcttttctttggttttcttcaaacata 360 ggtgaaaaaaacactgccattcacaagtcaaggaac 396 <210> 9 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 9 tcgacatcgcggcaactttttgcggattgttcttgcttccaggctttgcgctgcaaatcc 60 agtgctaccagtgtgaagaattccagctgaacaacgactgctcctcccccgagttcattg 120 tgaattgcacggtgaacgttcaagacatgtgtcagaaagaagtgatggagcaaagtgccg 180 ggatcatgtaccgcaagtcctgtgcatcatcagcggcctgtctcatcgcctctgccgggt 240 accagtccttctgctccccagggaaactgaactcagtttgcatcagctgctgcaacaccc 300 ctctttgtaacgggccaaggnccaaaaaaaggggaaagttctgncctcggccctcaggcc 360 agggctccgcaccaccatcctgttcctcaaattagc 396 <210> 10 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 10 cctttttttttttttttttttttttttttttttttttttttttttttttttttttttttt 60 ttttttttttttttttttttttttttttttttttttttttttttaaaaaaaaaannnttt 120 tttttttttnaaaaaaangggnnnnnttttttncccnnnngggnggggggggggnnnnnt 180 ttnaaanaaaaaaaccnnaaannnnnggggnnnannnaannncccnccccnaancnntaa 240 aaaannnggnaaaanagggggggnannnnnnnggggggnaaaanttttttttttttnaag 300 ggnnnggnaaaaaantnnnnnnntttttttttnnaanngggnnaaaaaaaaaaaaaaaaa 360 attttttngggntnaggggnngggggaaaancccna 396 <210> 11 <211> 396 <212> DNA
<213> Homo sapien <400>

agaacacaggtgtcgtgaaaactacccctaaaagccaaaatgggaaaggaaaagactcat 60 atcaacattgtcgtcattggacacgtagattcgggcaagtccaccactactggccatctg 120 atctataaatgcggtggcatcgacaaaagaaccattgaaaaatttgagaaggaggctgct 180 gagatgggaaagggctccttcaagtatgcctgggtcttggataaactgaaagctgagcgt 240 gaacgtggtatcaccattgatatctccttgtggaaatttgagaccagcaagtactatgtg 300 actatcattgatgccccaggacacagagactttatcaaaaacatgattacagggacatct 360 caggctgactgtgctgtcctgattgttgctgctggt 396 <210> 12 <211> 396 <212> DNA
<213> Homo sapien <400>

cgaaaacctttaaaccccggtcatccggacatcccaacgcatgctcctggagctcacagc 60 cttctgtggtgtcatttctgaaacaagggcgtggatccctcaaccaagaagaatgtttat 120 gtcttcaagtgacctgtactgcttggggactattggagaaaataaggtggagtcctactt 180 gtttaaaaaatatgtatctaagaatgttctagggcactctgggaacctataaaggcaggt 240 atttcgggccctcctcttcaggaatcttcctgaagacatggcccagtcgaaggcccagga 300 tggcttttgc tgcggccccg tggggtagga gggacagaga gacagggaga gtcagcctcc 360 acattcagag gcatcacaag taatggcaca attctt 396 <210> 13 <211> 396 <212> DNA
<213> Homo sapien <400>

accacaggctggccacaagaagcgctggagtgtgctggcggctgcaggcctacggggcct 60 ggtccggctgctgcacgtgcgtgccggcttctgctgcggggtcatccgagcccacaagaa 120 ggccatcgccaccctgtgcttcagccccgcccacgagacccatctcttcacggcctccta 180 tgacaagcggatcatcctctgggacatcggggtgcccaaccaggactacgaattccaggc 240 cagccagctgctcacactggacaccacctctatccccctgcgcctctgccctgtcgcctc 300 ctgcccggacgcccgcctgctggccggctgcgagggcggctgctgctgctgggacgtgcg 360 gctggaccagccccaaaagaggagggtgtgtgaagt 396 <210> 14 <211> 396 <212> DNA
<213> Homo sapien <400>

acggcgtcctcgtggaagtgacatcgtctttaaaccctgcgtggcaatccctgacgcacc 60 gccgtgatgcccagggaagacagggcgacctggaagtccaactacttccttaagatcatc 120 caactattggatgattatccgaaatgtttcattgtgggagcagacaatgtgggctccaag 180 cagatgcagcagatccgcatgtcccttcgcgggaaggctgtggtgctgatgggcaagaac 240 accatgatgcgcaaggccatccgagggcacctggaaaacaacccagctctggagaaactg 300 ctgcctcatatccgggggaatgtgggctttgtgttcaccaaggaggacctcactgagatc 360 agggacatgttgctggccaataaggtgccagctgct 396 <210> 15 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 15 accgcgcgggcacagggtgccgctgaccgaggcgtgcaaagactccagaattggaggcat 60 gatgaagactctgctgctgtttgtggggctgctgctgacctgggagagtgggcaggtcct 120 gggggaccagacggtctcagacaatgagctccaggaaatgtccaatcagggaagtaagta 180 cgtcaataaggaaattcaaaatgcttgtcaacggggtgaaacagataaagactctcatag 240 aaaaaacaaacgaagagcgcaagacactgctcagcaacctagaagaagccaagaagaaga 300 aagaggatgccctaaatgagaccagggaatcanagacaaagctgaaggagctcccaggag 360 tgtgcaatgagaccatgatggccctctgggaagagt 396 <210> 16 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 16 tttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt 60 tttttttttttttttttttttttttttttttttttttttttttttttttttttngggggg 120 nnnaaanttttttntnanannnnngggnaaaaaaaaaaaaaanaangggggnnntnnggc 180 ccnnnanaaaaaaanngnnaannaanccccccnnnnnnncccncnnntnnggaaananna 240 aaaccccccccngggnngggnnaaaaanncccnggggnantttttatnnnannccccccc 300 ccnggggggggnggaaaaaaaaaantncccccnannaaaannggggnccccccnttttnc 360 aaaangggggnccgggccccccnnantnttnggggg 396 <210> 17 <211> 396 <212> DNA
<213> Homo sapien <400> 17 accacactaaccatataccaatgatggcgcgatgtaacacgagaaagcacataccaaggc 60 caccacacaccacctgtccaaaaaggccttcgatacgggataatcctatttattacctca 120 gaagtttttttcttcgcaggatttttctgagccttttaccactccagcctagcccctacc 180 ccccaactaggagggcactggcccccaacaggcatcaccccgctaaatcccctagaagtc 240 ccactcctaaacacatccgtattactcgcatcaggagtatcaatcacctgagctcaccat 300 agtctaatagaaaacaaccgaaaccaaataattcaagcactgcttattacaattttactg 360 ggtctctattttaccctcctacaagcctcagagtac 396 <210> 18 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400>

tttttttttttttttttttttttttttttttttttttttttttttttttantcnaaaggg 60 gaaggnccctttttattaaanttggncattttactttncttttttnaaaangctaanaaa 120 aaanttttntttntncttaaaaaaaccctnnatntcacnancaaaaaaaacnattcccnc 180 ntncnttttgtgataaaaaaaaaggcaatggaattcaacntancctaanaaaactttncc 240 tgggaggaaaaaaaattnntccgngggaaacacttggggctntccaaantgnanccatnc 300 tangaggaccntctntaagatttccaaangaaaccccttcctnccaaangnantaccccg 360 ntgcctacnncccataaaaaaaacctcanccntaan 396 <210> 19 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

ttttttttttttttttttttttttttttttttttttttttttttttntggtctgggcttt 60 tattttacnaaaaanctaanggnaaanntncnttaaactaantngaanacaaagtnttaa 120 ngaaaaaggnctgggggnntcntttacaaaaanggncngggncanntttgggcttaaaan 180 ttcaaaaagggnncntcaaangggtttgcatttgcatgtttcancnctaaancgnangaa 240 naaacccnggngnccnctgggaaaagttnttnanctnccaaaanatnaantntttgnanc 300 agggnntttttgggnaaaaaaannanttccanaaactttccatcccctggntttgggttc 360 ggccttgngttttcggnatnatntccnttaangggg 396 <210> 20 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 20 ttttttttttttttttttttttttttctnaacaaaccctgttnttgggngggngngggta 60 taatactaagttganatgatntcatttacgggggaaggcnctttgtgaannaggccttat 120 ttctnttgncctttcgtacagggaggaatttgaagtaaananaaaccnacctggattact 180 ccggtctgaactcaaatcacgtaggactttaatcgttgaacaaacaaacctttaatagcg 240 gctgcnccattgggatgtcctgatccaacatcgaggncgtaaaccctattgttgatatgg 300 actctaaaaataggattgcgctgttatccctagggtaacttgttcccgtggtcaaagtta 360 ttggatcaattgagtataagtagttcgctttgactg 396 <210> 21 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 21 acatanatnttatactancattnaccatctcacttgnaggaanactantatatcnctcac 60 acctnatatcctncntactatgcctagaaggaataatactatngctgttnattatancta 120 ctntnataaccctnaacacccactccctcttanccaatattgtgcctattgccatactag 180 tntttgccgcctgcnaagcagnggngggcctanccntactagnctcaatctccaacacnt 240 atggcctanactacgtacataacctaaacctactcnaatgctaaaactaatcnncccaac 300 anttatnttactaccactgacatgactttccaaaaaacacatantttgaatcaacncanc 360 cacccacancctanttattancatcatcccIntact 396 <210> 22 <211> 396 <212> DNA
<213> Homo sapien <220>

<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

ttttttttttttttganaaaagccggcataaagcacttttattgcaataataaaacttga 60 gactcataaatggtgctgggggaagggtgcagcaacgatttctcaccaaatcactacaca 120 ggacagcaaaggggtgagaaggggctgagggaggaaaagccaggaaactgagatcagcag 180 agggagccaagcatcaaaaaacaggagatgctgaagctgcgatgaccagcatcattttct 240 taanagaacattcaaggatttgtcatgatggctgggctttcactgggtgttaagtctaca 300 aacagcaccttcaattgaaactgtcaattaaagttcttaagatttaggaagtggtggagc 360 ttggaaagttatgagattacaaaattcctgaaagtc 396 <210> 23 <211> 396 <212> DNA
<213> Homo sapien <400> 23 acaaaggcggttccaagctaaggaattccatcagtgcttttttcgcagccaccaaattta 60 gcaggcctgtgaggttttcatatcctgaagagatgtattttaaagctttttttttttaat 120 gaaaaaatgtcagacacacacaaaagtagaatagtaccatggagtccccacgtacccagc 180 ctgcagcttcaacagttaccacatttgccaaccggagagactgccaaggcaggaaaaagc 240 cctggaaagcccacggcccctttttcccttgggtcagaggccttagagctggctgccaaa 300 gcagccaaccaaaggggcagctcagctccttcgtggcaccagcagtgttcctgatgcagt 360 tgaagagttgatgtctttgacaacatacggacactg 396 <210> 24 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 24 cgactatcctctcagattcttatctggcactaatttataactattatattatcagagact 60 atgtagcaatatatcagtgcacaggcgcatcccaggcctgtacagatgtatgtctacacg 120 taagtataaatgaatttgcataccaggttttacacttgcatctctaatagagattaaaaa 180 caacaaattggcctcttcctaagtatattaatatcatttatccttacattttatgcctcc 240 ccctaaattaatgactgagttggtggaaagcggctaggttttattcatactgttttttgt 300 tctcaacttcaanagtaatctacctctgaaaaatttntantttaatattnnnnnnnagga 360 atttgngccactttannncttncnntntnntnnccn 396 <210> 25 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G

<400> 25 ttttttttttttttttttttgtcttttaaaaaatataaaagtgttattattttaaaacat 60 caagcattacagactgtaaaatcaattaanaactttctgtatatgaggacaaaaatacat 120 ttaanacatatacaanaagatgctttttcctgagtagaatgcaaacttttatattaagct 180 tctttgaattttcaaaatgtaaaataccaaggctttttcacatcagacaaaaatcaggaa 240 tgttcaccttcacatccaaaaagaaaaaaaaaaaaaanccaattttcaagttgaagttna 300 ncaanaatgatgtaaaatctgaaaaaagtggccaaaattttaanttncaacanannngnn 360 ncagntttnatggatctntnnnnnnncttcnnntnn 396 <210> 26 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

gacgctcccccctccccccgagcgccgctccggctgcaccgcgctcgctccgagtttcag 60 gctcgtgctaagctagcgccgtcgtcgtctcccttcagtcgccatcatgattatctaccg 120 ggacctcatcagccacgatgagatgttctccgacatctacaagatccgggagatcgcgga 180 cgggttgtgcctggaggtggaggggaagatggtcagtaggacagaaggtaacattgatga 240 ctcgctcattggtggaaatgcctccgctgaaggccccgagggcgaaggtacccgaaagca 300 cagtaatcactgnngncnatnttgtcatgaaccatcacctgcnngaaacaannttnacaa 360 aanaancctncnnnnannncctnnnnnattncnnnn 396 <210> 27 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(396) <223> n = A,T,C or G
<400> 27 tttttttttttttttttttttttttttttttttttttttttggctaaantttatgtatac 60 nggttnttcaaangngggggaggggggggggcatccatntanncncnccaggtttatggn 120 gggntnttntactattannanttttcncttcaaancnaaggnttntcaaatcatnaaaat 180 tattaanattncngctgntaaaaaaangaatgaaccnncnnanganagganntttcatgg 240 ggggnatgcatcggggnannccnaanaaccncggggccattcccganaggcccaaaaaat 300 gtttnnnnaaaaagggtaaanttacccccntnaantttatannnnaaannnnannnnagc 360 ccaannnttnnnnnnnnnnnnnnccnnnnannnnnn 396 <210> 28 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc feature <222> (1)...(396) <223> n = A,T,C or G
<400>

cgacctttttttttttttttatagatgaaagagggtttatttattaatatatgatagcct 60 tggctcaaaaaagacaaatgagggctcaaaaaggaattacagtaactttaaaaaatatat 120 taaacatatccaagatcctaaatatattattctccccaaaagctagctgcttccaaactt 180 gatttgatattttgcatgttttccctacgttgcttggtaaatatatttgcttctcctttc 240 tgcaatcgacgtctgacagctgatttttgctgttttgncaacntgacgtttcaccttntg 300 tttcaccanttctggaggaattgttnaacancttacancactgccttgaanaaannnnan 360 gcctcaaaagntcttgnnctatnctnnttcntnnnt 396 <210> 29 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 29 gacttgctcatttagagtttgcaggaggctccatactaggttcagtctgaaagaaatctc 60 ctaatggtgctatagagagggaggtaacagaaagactcttttagggcatttttctgactc 120 atgaaaagagcacagaaaaggatgtttggcaatttgtcttttaagtcttaaccttgctaa 180 tgtgaatactgggaaagtgatttttttctcactcgtttttgttgctccattgtaaagggc 240 ggaggtcagtcttagtggccttgagagttgcttttggcatttaaatattctaagagaatt 300 aactgtatttcctgtcacctattcactantgcangaaatatacttgctccaaataagtca 360 ntatgagaagtcactgtcaatgaaanttgntttgtt 396 <210> 30 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

tttttttttttttttttttgaaatttanaaacaaattttatttaagatctgaaatacaat 60 tcctaaaatatcaacttttccanaaaaccgtggctacacaataatgcattgcctctatca 120 tgttanaacgtgcattanactcaaatacaaaaaccatgaaacaaatcaccatccttcaac 180 aatttgagcaaagatagaatgcctaagaacaacatagatggacttgcagaggatgggctg 240 ttttacttcaagcnccataaaaaaaaaaaagagcncaaatgcattgggttttcaggtnta 300 tacattaagnngaacctttggcactaggaatcagggcgttttgtcacatagcnttaacac 360 atnttaaaaaattntgtantgtcaaagggatangaa 396 <210> 31 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 31 gacgggccagggccatctggaaagggaactcggcttttccagaacgtggtggatcatctg 60 tcgggtgtgtggtgaacacgttcagttcatcagggcctacgctccgggaaggggccccca 120 gctgtggctctgccatgccgggctgtgtttgcagctgtccgagtctccatccgcctttag 180 aaaaccagccacttcttttcataagcactgacagggcccagcccacagccacaggtgcga 240 tcagtgcctcacgcaggcaaatgcactgaaacccaggggcacacncncgcagagtgaaca 300 gtgagttcccccgacagcccacgacagccaggactgccctccccaccccnccccgacccc 360 angancacggcacacanntcancctctnanctngct 396 <210> 32 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 32 cgactggcctcataccttgtctacacagtccctgcacagggttcctaacctgtggttagt 60 aaagaatgtcactttctaacaggtctggaagctccgagtttatcttgggaactcaagagg 120 agaggatcacccagttcacaggtatttgaggatacaaacccattgctgggctcggcttta 180 aaagtcttatctgaaattccttgtgaaacagagtttcatcaaagccaatccaaaaggcct 240 atgtaaaaataaccattcttgctgcactttatgcaaataatcaggccaaatataagacta 300 cagtttatttacaatttgtttttaccaaaaatgaggactanagagaaaaatggtgctcca 360 aagcttatcatacatttgtcattaagtcctagtctc 396 <210> 33 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400>

cctttttttttttttttttttttttttttttttttttttttttttttttttttttttttt 60 tttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt 120 nngnnntntnnnnnannaaaaaaaaaaaaaaannnnnnnaaaaaaaannnnnnnnnnnnt 180 tttnnggggggnttttnanngnannttnnnnttnnnnnaaanccccnnngggnngggggg 240 nntnnnnnnggnaaaaaaannnnnnggggncnnnngggnccncncccnannnnnaaaann 300 nnnggnttttttnnttttnaaaaaaanngnnnnnnaacaaaantttttnnnnaanttttn 360 gggggaaannncccntttntttttttnnannnnnnn 396 <210> 34 <211> 396 <212> DNA

<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 34 acggaccnagctggaggagctgggtgtggggtgcgttgggctggtggggaggcctagttn 60 gggtgcaagtangtctgattgagcttgtgttgtgctgaagggacagccctgggtctaggg 120 ganagagnccctgagtgtgagacccaccttccccngtcccagcccctcccanttccccca 180 gggacggccacttcctgntccccgacncaaccatggctgaagaacaaccgcaggtcgaat 240 tgttcntgaaggctggcagtgatggggccaagattgggaactgcccattctcccacagac 300 tgttnatggtactgtggctcaaggnagtcaccttcaatgttaccaccnntgacaccaaaa 360 ggcggaccnanacagtgcanaagctgtgcccanngg 396 <210> 35 <211> 396 <212> DNA
<213> Homo sapien <400> 35 tcgaccaaaatcaaatctggcactcacaagccctggccgacccccaatgggttttaccac 60 tccccctctagaccctgtcttgcaaaatcctctccctagccagctagtattttctgggct 120 aaagactgtacaaccagttcctccattttatagaagtttactcactccaggggaaatggt 180 gagtcctccaacctccctttcaaccagtcccatcattccaaccagtggtaccatagagca 240 gcaccccccgccaccctctgagccagtagtgccagcagtgatgatggccacccatgagcc 300 cagtgctgacctggcacccaagaaaaagcccaggaagtcaagcatgcctgtgaagattga 360 gaaggaaattattgataccgccgatgagtttgatga 396 <210> 36 <211> 396 <212> DNA
<213> Homo sapien <400> 36 tcgacgggaagagcctgctacggtggactgtgagactcagtgcactgtcctcctcccagc 60 gaccccacgctggaccccctgccggaccctccacccttcggcccccaagcttcccagggg 120 cttcctttggactggactgtccctgctcatccattctcctgccacccccagacctcctca 180 gctccaggttgccacctcctctcgccagagtgatgaggtcccggcttctgctctccgtgg 240 cccatctgcccacaattcgggagaccacggaggagatgctgcttgggggtcctggacagg 300 agcccccaccctctcctagcctggatgactacgtgaggtctatatctcgactggcacagc 360 ccacctctgtgctggacaaggccacggcccagggcc 396 <210> 37 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 37 cgacggtgtcagcaactggccatgccacagcacataaagattacagtgacaagaaaaaca 60 ttgtttgaggattcctttcaacagataatgagcttcagtccccaagatctgcgaagacgt 120 ttgtgggtgatttttccaggagaagaaggtttagattatggaggtgtagcaagagaatgg 180 ttctttcttttgtcacatgaagtgttgaacccaatgtattgcctgtttgaatatgcaggg 240 aaggataactactgcttgcagataaaccccgcttcttacatcaatccagatcacctgaaa 300 tattttcgttttattggcagatttattgccatggctctgttccatgggaaaattcataga 360 cacgggtttttctttnccattctataagcgtatctt 396 <210> 38 <211> 396 <212> DNA
<213> Homo sapien <400> 38 cgaccaaaatgataaatagctttaagaatgtgctaatgataaatgattacatgtcaattt 60 aatgtacttaatgtttaataccttatttgaataattacctgaagaatatattttttagta 120 ctgcatttcattgattctaagttgcactttttacccccatactgttaacatatctgaaat 180 cagaatgtgtcttacaatcagtgatcgtttaacattgtgacaaagtttaatggacagttt 240 tttcccatatgtatatataaaataatgtgttttacaatcagtggcttagattcagtgaaa 300 tacagtaattcattcaattatgatagtatctttacagacattttaaaaataagttatttt 360 tatatgctaatattctatgttcaagtggaatttgga 396 <210> 39 <211> 396 <212> DNA
<213> Homo sapien <400> 39 tcgaccaagaatagatgctgactgtactcctcccaggcgccccttccccctccaatccca 60 ccaaccctcagagccacccctaaagagatactttgatattttcaacgcagccctgctttg 120 ggctgccctggtgctgccacacttcaggctcttctcctttcacaaccttctgtggctcac 180 agaacccttggagccaatggagactgtctcaagagggcactggtggcccgacagcctggc 240 acagggcaagtgggacagggcatggccaggtggccactccagacccctggcttttcactg 300 ctggctgccttagaacctttcttacattagcagtttgctttgtatgcactttgttttttt 360 ctttgggtcttgttttttttttccacttagaaattg 396 <210> 40 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)'. . (396) <223> n = A,T,C or G
<400> 40 ttttttttttttttgttatttagtttttatttcataatcataaacttaactctgcaatcc 60 agctaggcatgggagggaacaaggaaaacatggaacccaaagggaactgcagcgagagca 120 caaagattctaggatactgcgagcaaatggggtggaggggtgctctcctgagctacagaa 180 ggaatgatctggtggttaanataaaacacaagtcaaacttattcgagttgtccacagtca 240 gcaatggtgatcttcttgctggtcttgccattcctggacccaaagcgctccatggcctcc 300 acaatattcatgccttctttcactttgccaaacaccacatgcttgccatccaaccactca 360 gtcttggcagtgcanatgaaaaactgggaaccattt 396 <210> 41 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

tcgacctcttgtgtagtcacttctgattctgacaatcaatcaatcaatggcctagagcac 60 tgactgttaacacaaacgtcactagcaaagtagcaacagctttaagtctaaatacaaagc 120 tgttctgtgtgagaattttttaaaaggctacttgtataataacccttgtcatttttaatg 180 tacaaaacgctattaagtggcttagaatttgaacatttgtggtctttatttactttgctt 240 cgtgtgtgggcaaagcaacatcttccctaaatatatattacccaaagnaaaagcaagaag 300 ccagattaggtttttgacaaaacaaacaggccaaaagggggctgacctggagcagagcat 360 ggtgagaggcaaggcatgagagggcaagtttgttgt 396 <210> 42 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 42 cttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt 60 aaaanccnnannaanananggnaannnannaaaaaanncaaaccncntntanaaaangcc 120 nntntnagggggggggttcaaaaccaaanggnngntnggangnaaannnaaaanttnnnn 180 gggggnanaaanaaaaagggnngaaanntgacccnanaangaccngaaancccgggaaac 240 cnngggntanaaaaaaagntganccctaaanncccccgnaaaangggggaagggnaannc 300 caaatccnntgngggttgggggnggggaaaaaaaaaaccccnaaaaantgnaaaaaaccg 360 ggnttnaaanatttgggttcgggggntttntnttaa 396 <210> 43 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

ttttttttttttttgcttcactgctttatttttgaaatcacaagcaattcaaagtgatca 60 tcattgaggcttctgttaaaagttcttccaaagttgcccagttttaanattaaacaatat 120 tgcactttaagatgaactaacttttgggattctcttcaaagaaggaaagtattgctccat 180 ctgtgcttttcttanactaaaagcatactgcanaaaactctattttaaaaatcaacactg 240 cagggtacagtaacatagtaaagtacctgcctattttanaatcctanagaacatttcatt 300 gtaagaaactagcccattatttaagtgtccacagtatttttcatttcantggtccaagat 360 gccaaggttt ccaaacacaa tcttgttctc taatac 396 <210> 44 <211> 396 <212> DNA
<213> Homo sapien <400> 44 gacctagttttacctcttaaatatctctgttcccttctaagttgtttgctgtgttttctt 60 cagagcaagaaggttatattttttaaaatttacttagtaatgcacattcaaaacacacat 120 caagtcttcaggataaagttcaaaaccgctgtcatggccccatgtgatctctccctcccc 180 tacccctctatcatttagtttcttctgcgcaagccactctggcttcctttcagttttgtg 240 gttcccgtttttagctagttcagtggttttcaatgggcatttcttgcctttttttttcta 300 aacgacaaatagaaatacatcttctttattatcctccaaatccaattcagaggtaatatg 360 ctccacctacacacaattttagaaataaattaaaaa 396 <210> 45 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 45 ttttttttttttttaaannttntaaatttttaatgaaannganttagaacaatgtattat 60 tnacatgtaaataaaaaaagagancataanccccatatnctcnnnaaaggaaggganacn 120 gcnggccntttatnagaanannnnncatataagaccccattaagaagaatctggatctaa 180 anacttncaaacaggagttcacagtangtgaacagcannccctaatcccactgatgtgat 240 gnttcanataaaatcancancgntgatcgggnatcnnancaatntgancggaanannact 300 gctcnatatntttnagganncngatgtggtcattttttacaaagataatggccacaccct 360 tccngnccgaatcgancnganctcccnnttctgtgn 396 <210> 46 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(396) <223> n = A,T,C or G
<400>

tttttttttttttttttttctganacagagtctcattctgttgcctaggctggattgcag 60 tggtgccatctcggctcactgcaacctccgcctcctgggttccanaaattctcctgcctc 120 agcctcccgggtagctgggactanaggcacacgccaccacgccaggctaatttttatatt 180 tttagtananatggcgtttcaccatgttgaccanactgatctcgaactcccgacctcgtg 240 atccacccacctcggcctcccaaagtgctgggattacaggcgtgaaaccaccaggcccgg 300 cctgaaatatctatttnttttcagattatttttaaaattccatttgatgaatcttttaaa 360 gtgagctananaaagtgngtgtgtacatgcacacac 396 <210> 47 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223 > n = A, T, C or G
<400> 47 tttttttttttttttttgctgttgccaactgtttattcagggccctgaacgggtggtgcg 60 tggacatgcaacacactcgggcccacagcagcgtgaccggccgctcccaagccccgggcg 120 cacaaccacagccaggagcagcccctgccaccactgggccaccgtccagggccccacagg 180 accagccgaaggtgccccgggccgaggccagctgggtcaggtgtacccctagcctggggt 240 tgag.tgaggagcggcacccccagtatcctgtgtaccccaagttgcccagnaggccgaggg 300 ggccttgggctccatctgcactggccaccccgtgccaagcatcacagctgcgtgagcagg 360 tttgtgtgtgagcgtgtggcggggcctggttgtccc 396 <210> 48 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A, T, C or G
<400> 48 ctgggcctgtgccgaagggtctgggcagatcttccaaagatgtacaaaatgtagaaattg 60 ccctcaagcaaatgcaaagatgctcaacacccttagtcatcaagaaaatgcaaatggaat 120 ccacagagagatactgcacactgacaaagatggtcgtattactaaaggtgaataaccagc 180 gcggggggcacgtggagtcactggaacatttgtgcaatgctggtgggaatgtcaacccgt 240 gcggccctctggaataagcctggcagctcctccaagagttacccgtgtgacccagcaatt 300 ccactcctagctccacccacaggaattgaaagcaaagacgcaaacagatgcctgtgcacc 360 aaagttcacggcagcatccttcgccatagtggnaan 396 <210> 49 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400>

accccaaaatgggaaaggaaaagactcatatnaacattgncgtnattggacacgtacatt 60 cggncaagtncaccactactggncatntgatntataaatgcggnggcatcgacanaanaa 120 ccatngnaanatttganaaggaggctgctgatatnggaaagggctccntcnantntgcct 180 gggtcttggatnaactgaaanctgancntgaacgtggnntcaccattgatatctncttgt 240 ggaaatntnagaccancanntactatgtnactatcattgatgccccaggacacaganact 300 ttatcnaaancatgattacnnggacatntanagctgactgtgctngcctgattgtngctg 360 ctggtgttggtgaatttgaanctggtatntccaana 396 <210> 50 <211> 396 <212> DNA
<213> Homo sapien <400> 50 cgacttcttgctggtgggtggggcagtttggtttagtgttatactttggtctaagtattt 60 gagttaaactgcttttttgctaatgagtgggctggttgttagcaggtttgtttttcctgc 120 tgttgattgttactagtggcattaacttttagaatttgggctggtgagattaattttttt 180 taatatcccagctagagatatggcctttaactgacctaaagaggtgtgttgtgatttaat 240 tttttcccgttcctttttcttcagtaaacccaacaatagtctaaccttaaaaattgagtt 300 gatgtccttataggtcactacccctaaataaacctgaagcaggtgttttctcttggacat 360 actaaaaaatacctaaaaggaagcttagatgggctg 396 <210> 51 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

ttttttttttttcagcgnggatttattttatttcattttttactctcaaganaaagaana 60 gttactattgcaggaacagacatttttttaaaaagcgaaactcctgacacccttaaaaca 120 gaaaacattgttattcacataataatgnggggctctgtctctgccgacaggggctgggtt 180 cgggcattagctgtgccgtcgacaatagccccattcaccccattcataaatgctgctgct 240 acaggaagggaacagcggctctcccanagagggatccaccctggaacacgagtcacctcc 300 aaagagctgcgactgtttganaatctgccaanaggaaaaccactcaatgggacctggata 360 acccaggcccgggagtcatagcaggatgtggtactt 396 <210> 52 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 52 acctcgctaagtgttcgctacgcggggctaccggatcggtcggaaatggcagaggtggag 60 gagacactgaagcgactgcanagccagaagggagtgcagggaatcatcgtcgtgaacaca 120 gaaggcattcccatcaagagcaccatggacaaccccaccaccacccagtatgccagcctc 180 atgcacagnttcatcctgaaggcacggagcaccgtgcgtgacatcgacccccagaacgat 240 ctcaccttccttcgaattcgctccaagaaaaatgaaattatggttgcaccagataaagac 300 tatttcctgattgtgattcagaatccaaccgaataagccactctcttggctccctgtgtc 360 attccttaatttaatgccccccaagaatgttaatgt 396 <210> 53 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 53 tttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt 60 tttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt 120 tttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt 180 ttttttttttttttttttttttttttttttttttttttttttannttnttttttnttttn 240 cctttnttttaattcanaaaaagaanaagaaaanataanannnancnnannnnnnnnatn 300 ntncttnatantnnttnnnnnanngggnnngcgagnnnnnnnnnnnnnnnnntctnnnnt 360 tnnnnnncttgcnccccttnnnttngnnnnangcaa 396 <210> 54 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 54 ctcttggggctgctgggactcgcgtcggttggcgactcccggacgtaggtagtttgttgg 60 gccgggttctgaggccttgcttctctttacttttccactctaggccacgatgccgcagta 120 ccagacctgggaggagttcagccgcgctgccgagaagctttacctcgctgaccctatgaa 180 ggcacgtgtggttctcaaatataggcattctgatgggaacttgtgtgttaaagtaacaga 240 tgatttagtttgtttggtgtataaaacagaccaagctcaagatgtaaagaagattgagaa 300 attccacagtcaactaatgcgacttatggtagccaaggaagcccgcaatgttaccatgga 360 aactgantgaatggtttgaaatgaagactttgtcgt 396 <210> 55 <211> 396 <212> DNA
<213> Homo sapien <400>

cgacggtttgccgccagaacacaggtgtcgtgaaaactacccctaaaagccaaaatggga 60 aaggaaaagactcatatcaacattgtcgtcattggacacgtagattcgggcaagtccacc 120 actactggccatctgatctataaatgcggtggcatcgacaaaagaaccattgaaaaattt 180 gagaaggaggctgctgagatgggaaagggctccttcaagtatgcctgggtcttggataaa 240 ctgaaagctgagcgtgaacgtggtatcaccattgatatctccttgtggaaatttgagacc 300 agcaagtactatgtgactatcattgatgccccaggacacagagactttatcaaaaacatg 360 attacagggacatctcaggctgactgtgctgtcctg 396 <210> 56 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

ttttttttttttttttctcatttaacttttttaatgggtctcaaaattctgtgacaaatt 60 tttggtcaagttgtttccattaaaaagtactgattttaaaaactaataacttaaaactgc 120 cacacgcaaaaaanaaaaccaaagnggtccacaaaacattctcctttccttctgaaggtt 180 ttacgatgcattgttatcattaaccagtcttttactactaaacttaaatggccaattgaa 240 acaaacagttctganaccgttcttccaccactgattaanagtggggtggcaggtattagg 300 gataatattcatttagccttctgagctttctgggcanacttggngaccttgccagctcca 360 gcagccttnttgtccactgctttgatgacacccacc 396 <210> 57 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 57 cctttttttttttttttttttttttttttttttttttttttttttttttttnaaaanntt 60 ntttttgcaaanccnancaaaaanggnnggaangaaaaannggaaaaattntttttncnt 120 ntttgggaacnnnnagcccttnntttgaaaaaangnggncttaaaanngntgaannaaag 180 gnnanncccngntncttnnntttaaaaanaanggggnngnttttttttaaanaanatttt 240 ttttttccctaanancnncnanntgaaacnngncccnacnnctnncttnaaagggnnnaa 300 atnanangnnaaaaaanccctnancccccccccttanntttncnannananaaagncntt 360 ttgggncntgnaaaaanaancctttttnntgcnttn 396 <210> 58 <211> 396 <212> DNA
<213> Homo sapien <400> 58 cgacctcaaatatgccttattttgcacaaaagactgccaaggacatgaccagcagctggc 60 tacagcctcgatttatatttctgtttgtggtgaactgattttttttaaaccaaagtttag 120 aaagaggtttttgaaatgcctatggtttctttgaatggtaaacttgagcatcttttcact 180 ttccagtagtcagcaaagagcagtttgaattttcttgtcgcttcctatcaaaatattcag 240 agactcgagcacagcacccagacttcatgcgcccgtggaatgctcaccacatgttggtcg 300 aagcggccgaccactgactttgtgacttaggcggctgtgttgcctatgtagagaacacgc 360 ttcacccccactccccgtacagtgcgcacaggcttt 396 <210> 59 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

cttttttttttttttttttt~tcagnggaaaataacttttattganaccccaccaactgca 60 aaatctgttcctggcattaagctccttcttcctttgcaattcggtctttcttcagnggtc 120 ccatgaatgctttcttctcctccatggtctggaagcggccatggccaaacttggaggngg 180 tgtcaatgaacttaaggncaatcttctccanagcccgccgcttcntctgcaccancaagg 240 acttgcggagggngagcacccgcttnttggttcccaccacncagcctttcagcatgacaa 300 agtcattggtcacttcaccatagnggacaaagccacccaaagggttgatgctccttggca 360 aataggncatagtcacnggaggcattgtncttgatc 396 <210> 60 <211> 396 <212> DNA
<213> Homo sapien <400>

acctcagctctcggcgcacggcccagcttccttcaaaatgtctactgttcacgaaatcct 60 gtgcaagctcagcttggagggtgatcactctacacccccaagtgcatatgggtctgtcaa 120 agcctatactaactttgatgctgagcgggatgctttgaacattgaaacagccatcaagac 180 caaaggtgtggatgaggtcaccattgtcaacattttgaccaaccgcagcaatgcacagag 240 acaggatattgccttcgcctaccagagaaggaccaaaaaggaacttgcatcagcactgaa 300 gtcagccttatctggccacctggagacggtgattttgggcctattgaagacacctgctca 360 gtatgacgcttctgagctaaaagcttccatgaaggg 396 <210> 61 <211> 396 <212> DNA
<213> Homo sapien <400> 61 tagcttgtcggggacggtaaccgggacccggtgtctgctcctgtcgccttcgcctcctaa 60 tccctagccactatgcgtgagtgcatctccatccacgttggccaggctggtgtccagatt 120 ggcaatgcctgctgggagctctactgcctggaacacggcatccagcccgatggccagatg 180 ccaagtgacaagaccattgggggaggagatgactccttcaacaccttcttcagtgagacg 240 ggcgctggcaagcacgtgccccgggctgtgtttgtagacttggaacccacagtcattgat 300 gaagttcgcactggcacctaccgccagctcttccaccctgagcagctcatcacaggcaag 360 gaagatgctgccaataactatgcccgagggcactac 396 <210> 62 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 62 tcgacgtttcctaaagaaaaccactctttgatcatggctctctctgccagaattgtgtgc 60 actctgtaacatctttgtggtagtcctgttttcctaataactttgttactgtgctgtgaa 120 agattacagatttgaacatgtagtgtacgtgctgttgagttgtgaactggtgggccgtat 180 gtaacagctgaccaacgtgaagatactggtacttgatagcctcttaaggaaaatttgctt 240 ccaaattttaagctggaaagncactggantaactttaaaaaagaattacaatacatggct 300 ttttagaatt tcnttacgta tgttaagatt tgngtacaaa ttgaantgtc tgtnctganc 360 ctcaaccaat aaaatctcag tttatgaaan aaannn 396 <210> 63 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 63 t.tntttttttnttttntnttttntcnttgnttgnacngaacccggcgctnnttccccacn 60 nnnnacggccgcccntattcannnntncntcanntannnaccgcaccctcggactgcnnn 120 tngggccccgccgncnanncnccnncncccanttcnccgccgccgccgccgccttttttt 180 attggcnnccatnanaaccggggncacctcncangngcgccnaaantnggggcangactc 240 anagggggccatcaaccnccaagnncaanctgganctctacaaacggcctacgntttntg 300 nccatgngggtagggntttacccgcnatgatgannatgnnaanaactttnncaanccctt 360 tattaaccaatgnggtgnggagacggaacntggtta 396 <210> 64 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

tcgacgtcggggtttcctgcttcaacagtgcttggacggaacccggcgctcgttccccac 60 cccggccggccgcccatagccagccctccgtcacctcttcaccgcaccctcggactgccc 120 caaggcccccgccgccgctccagcgccgcgcagccaccgccgccgccgccgcctntnctt 180 agtcgccgccatgacgaccgcgtccacctcgcaggtgcgccagaactaccaccaggactc 240 agaggccgccatcaaccgccagatcaacctggagctctacgcctcctacgtttacctgtc 300 catgtcttactactttgaccgcgatgatgtggctttgaanaactttgccaaatactttct 360 tcccaatctcatgaggagaaggaacatgctganaaa 396 <210> 65 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 65 tttttttttt tttttttttt tttttnacca ataatgcttt tattttccac atcaanatta 60 atttatatgt tagttttagt acaagtacta aaatgtatac ttnttgccct aatagctaag 120 gnatacataa gcttcaccat acatnttgca nccncctgtc tgtcctatgt cattgttata 180 aatgtanana ttttaggaaa ctnttttatt caacctggga catntatact gtaggagtta 240 gcactgacct gatgtnttat ttaaaagtaa tgnatattac ctttacatat attccttata 300 tattnaaacg tatttccatg ttatccagct taaaatcaca tggnggttaa aagcatgagt 360 tctgagtcaa atctggactg aaatcctgat gctccc 396 <210> 66 <211> 396 <212> DNA
<213> Homo sapien <400> 66 tcgacttttttttttccaggacattgtcataattttttattatgtatcaaattgtcttca 60 atataagttacaacttgattaaagttgatagacatttgtatctatttaaagacaaaaaaa 120 ttcttttatgtacaatatcttgtctagagtctagcaaatatagtacctttcattgcagga 180 tttctgcttaatataacaagcaaaaacaaacaactgaaaaaatataaaccaaagcaaacc 240 aaaccccccgctcaactacaaatgtcaatattgaatgaagcattaaaagacaaacataaa 300 gtaacttcagcttttatctagcaatgcagaatgaatactaaaattagtggcaaaaaaaca 360 aacaacaaacaacaaacaaaacaaaacaaacaaaca 396 <210> 67 <211> 396 <212> DNA
<213> Homo sapien <400> 67 acgcttttgtccttcattttaactgttatgtcatactgttatgttgacatatttctttat 60 aagagaatagaggcaaaagtatagaactgaggatcatttgtatttttgagttggaaatta 120 tgaaacttcaccatattatgatcatacatattttgaagaacagactgaccaaagctcacc 180 tgttttttgtgttaggtgctttggctgaacttgattccagcccccttttccctttggtgt 240 tgtgtatgtctcttcatttcctctcaaatcttcaactcttgccccatgtctccttggcag 300 caggatgctggcatctgtgtagtcctcatactgtttactgataacccacaaattcatttt 360 catggcagacctaagctcagaccctgccttgtcctg 396 <210> 68 <211> 396 <212> DNA
<213> Homo sapien <400>

acctgagtcctgtcctttctctctccccggacagcatgagcttcaccactcgctccacct 60 tctccaccaactaccggtccctgggctctgtccaggcgcccagctacggcgcccggccgg 120 tcagcagcgcggccagcgtctatgcaggcgctgggggctctggttcccggatctccgtgt 180 cccgctccaccagcttcaggggcggcatggggtccgggggcctggccaccgggatagccg 240 ggggtctggcaggaatgggaggcatccagaacgagaaggagaccatgcaaagcctgaacg 300 accgcctggcctcttacctggacagagtgaggagcctggagaccgagaaccggaggctgg 360 agagcaaaatccgggagcacttggagaagaagggac 396 <210> 69 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

ntcncngnngntgtggtnntttttttaatttttatnttttctttttttttctngctagcn 60 cttnctttttttggaattncggtncctttttntntcnattttttngacaaaaanaacctn 1'20 ttntttnanaccanagnnnggnncacncntnnaatntnccccttttncgntngggagctn 180 cncnttnnncgccnacntcantcgagacngtncttttnnntnnancannntnngtncgtt 240 gncngcnttnntncannantnttccctatnnacntgnnntcncncatnnttggacnancn 300 cctagccttnccatnntttnnttntttntnnatnancctngaaaacntcngnntnttcnc 360 nncnttnccncncncnccttcntatgtncnatgncn 396 c210> 70 c211> 396 <212> DNA
<213> Homo sapien c220>
c221> misc_feature c222> (1). .(396) c223> n = A,T,C or G
c400> 70 ttttttttttttttnttttttttttttttttttttttnttttttttttttttttttntnc 60 aannnntnaacttttaannggccnccngcnccccaanggggaccctgcttttgnnggcta 120 aatgccnnaaaactttggggnantnggtatnaaaccccnctttgcccnncannttncngg 180 ggggggggggtttttgnnggggaacangnanaacnttttnncnanggnatcaccaaaaan 240 aaagcccnnccctttttccnanngggggggggnggggggaaantcancccccanattgac 300 cttnatttcaaaanggggcttataatcctgggcntgganncttccctntacccgggggtt 360 gnccacnttttattanaggggnangnggatccccnt 396 c210> 71 <211> 396 <212> DNA
c213> Homo sapien <220>
<221> misc_feature c222> (1) . . (396) c223> n = A,T,C or G
<400> 71 gcatctagagggccngtttantctagaggnccngnntaaacnnnnncatcnacctncnnt 60 gcncctgctngttgccncccntctgtgncttgcnnnncccnngagcgtnccttnaccnnn 120 gaangtgcctnnnnnactgannnnnncnnataanatgngganantncgtcgncattntnt 180 natnnggggtgatgctattctggggggtggggnggngnnatnnnatactnnggggacgtn 240 nnatnangagnnatntcnngnttntctnntgntttntggggggcnatnngnnntctntnn 300 ggactcntcgcncannnatcaatancttnattcngtgtanngtccgnccntagnncngcn 360 ngtactnnanngttgnnntcattactnttcgtnngg 396 <210> 72 <211> 396 <212> DNA
<213> Homo sapien <220>

<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

tntttttttttttctaaaacatnactntttattnnnnangntttntgaacctctnngcnt 60 natggtgagagtttgtctgattaataanaatnggannnttnannanangcntgnncgcaa 120 ngatggcnncnctgtatatcccaccatcccattacactntgaaccttttntttgattaat 180 aaaaggaaggnatgcggggaanggggaaagagaatgcttgaacattnccatgngnccttn 240 gacaaactttccaatggaggcnggaacnaannaccaccanncaactcccctttttgtaat 300 ttnnnaacttncaacnnctanctntttattttggcntccctggnngaaacagnctgtatn 360 annnnnaagnccntgagaacatccctggntnncnna 396 <210> 73 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 73 ntcaacntngactnctgtgaggnatggtgctgggngcntatgcngtgngnttttggatac 60 naccttatggacantngcnntcccnnggaangatnataatncttactgnagnnactnnaa 120 nnttccntntcnaaaangttnaaaancattggatgtgccacaatgatgacagtttatttg 180 ctactcttgagtgctataatgatgaagatcttanccaccattatcttaactgangcaccc 240 aanatggtganttggggaacatatanagtacacctaagttcacatgaagttgtttnttcc 300 caggnnctaaagagcaagcctaactcaagccattgncacacaggtgagacacctctattt 360 tgtacttctcacttttaagggattagaaaatagcca 396 <210> 74 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 74 cctttttttttttttttactgngaatatatactttttatttagtcatttttgtttacaat 60 tgaaactctgggaattcaaaattaacatccttgcccgtgagcttcttatagacaccanaa 120 aaagtttcaaccttgtgttccacattgttctgctgtgctttgtccaaatgaacctttatg 180 agccggctgccatctagtttgacgcggattctcttgcccacaatttcgcttgggaagacc 240 aagtcctcaaggatggcatcgtgcacagctgtcagagtacggctcctgggacgcttttgc 300 ttattttttgtacggctttttcgagttggcttaggcagaattctcctctgagcgataaag 360 acgacatgcttcccactgaactttttctccaattcg 396 <210> 75 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 75 tttttttttttttntttttttttttttttttttttttnaantntaangggganggcccct 60 tttttttaaactngnccnttttnctttccttttttnaaaaggaaaaaaaaanntttnttt 120 ttcnttnaaaaaccctttttcccacnaacaaaaaaaaccnttccccntnccttttnnnna 180 aaaaaaaggggctnggnntttccccttanncaaaaaaccntntccnngggnaaaaaantt 240 ntcnccgggggggaaacnnntgggggtgtnnccnaaatttgggggccntcggaagggggg 300 nnccncncctaaagangtntttcaaaanaaaaacccccntcctnttntaaaaanaaaana 360 aaanaangnnngnnttttttntcnttnnccccccaa 396 <210> 76 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 76 acattcttcagaaatacagtgatgaaaattcattttgaaactcaaatattttcattttgg 60 atattctcctgtttttattaaaccagngattacncctggccntccctntaaatgttctag 120 gaaggcatgtctgttgtnntttnnnnaaaannaaattntttttttttngnnaaaccccaa 180 atcccantttatcaggaagttagncnaatgaaatggaaattggntaatggacaaaagcta 240 gcttgtaaaaaggaccacccnnccacnngnctttacccccttggttngttgggggaaaaa 300 ccatnnttaaccntntggnnaaaattgggnncntaaagtttncntggnnaacagtncntn 360 cngtattnaattgncnttatnggaaaatcngggatt 396 <210> 77 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400>

tttttttttttttttttttttttttttttttatcaacatttatatgctttattgaaagtt 60 ganaanggcaacagttaaatncngggacnccttacaattgtgtaaanaacatgcncanaa 120 acatatgcatataactactatacaggngatntgcaaaaacccctactgggaaatccattt 180 cattagttanaactgagcatttttcaaagtattcaaccagctcaattgaaanacttcagt 240 gaacaaggatttacttcagcgtattcagcagctanatttcaaattacncaaagngagtaa 300 ctgngccaaattcttaaaatttntttaggggnggtttttggcatgtaccagtttttatgt 360 aaatctatntataaaagtccacacctcctcanacag 396 <210> 78 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 78 agctggcnaaaggngnatgngctgcnangcgattangnnnggtaacgtcannggntnncc 60 agtgcangacnttgtaaaacgacggccacatgaattgtaatacgactcactatngggcgn 120 attgggccgtgnaggatngtgntcacactcgaatgtatnctggcngatncananngcttt 180 atngctnttgacggngnntnanccanctngggctttagggggtatcccctcgcccctgct 240 tcnttgatttgcacgggcnnctccganttccttcataataccngacgcttcnatccccta 300 gctcngacctntcantntnttcnntgggttntnnccgntcacngcttncccgnangntat 360 aatctnggctcctttngggatccattantctttact 396 <210> 79 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 79 caccaaccaaaacctggcgccgttggcatcgtagagtgaacacaacccaaaaacgatacg 60 ccatctgttctgccctggctgcctcagccctaccagcactggtcatgtctaaaggncatc 120 gtattgaggaagttcctgaacttcctttggtangttgaagataaagctgaaggctacaag 180 aagaccaangaagntgttttgctccttaanaaacttanacgcctggaatgatatcaaaaa 240 ngctatgcctctcagcgaatgagactgganangcaaaatgagaaaccntcnccgcatcca 300 gcgnaggggccgtgcatctctatnntgangatnntggnancnttcaaggccttcagaacc 360 tccctngaaatnctctnctttaangaaccaaactgn 396 <210> 80 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

tgtacataggcatcttattcactgcaccctgtcacacccagcaccccccgccccgcacat 60 tatttgaaagactgggaatttaatggttagggacagtaaatctacttctttttccaggga 120 cgactgtcccctctaaagttaaagtcaatacaagaaaactgtctatttttagcctaaagt 180 aaaggctgtgaagaaaattcattttacattgggtagacagtaaaaaacaagtaaaataac 240 ttgacatgagcacctttagatccttcccttcatggggctttgggcccagaatgacctttg 300 aggcctgtaaanggattgnaatttcctataagctgtatagtggagggattggngggtcat 360 ttgagtaagccctccaagatacnttcaatacctggg 396 <210> 81 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

gcagctgaagttcagcaggtgctgaatcgattctcctcggcccctctcattccacttcca 60 acccctcccattattccagtactacctcagcaatttgtgccccctacaaatgttagagac 120 tgtatacgccttcgaggtcttccctatgcagccacaattgaggacatcctgcatttcctg 180 ggggagttcgccacagatattcgtactcatggggttcacatggttttgaatcaccagggn 240 ccgccatcaggagatgcctttatccagatgaagtctgcggacagancatttatggctgca 300 cagaagtggcataaaaaaaacatgaaggacagatatgttgaagttttcagtgtcagctga 360 nganagaacattgnngtannngggggnactttaaat 396 <210> 82 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 82 gactcagaaatgtcagtctcatgaagttcaaaagatcgagaatgtttgctatcttggtgg 60 agcagccgcagccaagcaagtaacttgtaaaatgaggaatgccatcacccctcgagtgtc 120 catcccacataacttggggttagagcacaagcgttcccaggaactactcaccttaccatc 180 ttggccgtttcatttgcttccaccagttctggaaagaganggcctagaagttcaaaaaaa 240 aagtaggaaangtgcttttggagaaaatcacctgctcctcagaactgggcttacaanctg 300 ngaagtacnctatgtgccacctaatcctcatatatgacctcaagagacnccaataagcat 360 atttccaccacggaatgaccagtgctttgggtaana 396 <210> 83 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 83 tttgatttaaganatttattatttttttaaaaaaagcaacttccagggttgtcattgtac 60 aggttttgcccagtctcctatagcatggtatagtgataactgattttttataacaatgac 120 tcagaggcattgaagatccataactatcttctgaattatcacagaaagaagaaagttaga 180 agagtttaatgttaagtgtattaaaaatcatattctaattcttttaatttggttatctga 240 gtatgataatataggagagctcagataacaaggaaaaggcattggggtaagaacactcct 300 tcccacaggatggcattaacagactttttctgcatatgctttatatagttgccaactaat 360 tcacctttta cncagcttna ttttttttta ctnggg 396 <210> 84 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 84 tttttacagcaatttttttttattgatgtttaacctgtatacaaccatacccattttaag 60 ngtacagacaaatgaattttgacaaattcattcactcatctaatcatcactataaccatg 120 atacagatttttatcactccaaaagtccatcctgtgctcttttcaagtccatcctcctca 180 tctgataccccaagccaccattgttttgctttctggaactacagttttgggnttttagaa 240 tttcatatatggtngaatcataccatttgnnatttggggctgacgnctttcctccaataa 300 tggatttgagaattatctacattttgcatggatcctgggttatttataccaacnangggt 360 tattatgnaaaatnggaccacaatttggnggcanta 396 <210> 85 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(396) <223> n = A, T, C or G
<400>

cagtgaccgtgctcctacccagctctgctccacagcgcccacctgtctccgcccctcggc 60 ccctcgcccggctttgcctaaccgccacgatgatgttctcgggcttcaacgcagactacg 120 aggcgtcatcctcccgctgcagcagcgcgtccccggccggggatagcctctcttactacc 180 actcacccgcagactccttctccagcatgggctcgcctgcaacgcgcaggacttctgcac 240 ggacctggccgctccagtgccaacttcattccacggcactgcatctcgaccanccggact 300 tgcannggttggggaanccgcccttgtttctccgtggcccatctaanaccaaacccntca 360 ccttttcggagnccccncccctccgntgggnttact 396 <210> 86 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 86 ttttnnactg aatgtttaat acatttgnag gaacagaaga aatgcagtan ggattaanat 60 tttataatta gacattaatg taacagatgn ttcatttttc aaagaagntn cccccttntc 120 cctatctttt tttaatcttc cttanagcaa taantagtaa ttactatatt tgtggacaag 180 ctgctccact gtgntggaca gtaattatta aatctttatg tttcacatca ttattacctt 240 ccanaattct accttcattt ccctgcacag gttcactgga ctggntcaca ancaaattgn 300 actccactca antanaagag cccaaagaaa ttagagtaac gncnantcct atgaattana 360 gacccaaaga tttnaggngn tgattagaaa cataan 396 <210> 87 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 87 atggaggcgctggggaagctgaagcagttcgatgcctaccccaagactttggaggacttc 60 cgggtcaagacctgcgggggcgccaccgtgaccattgtcagtggccttctcatgctgcta 120 ctgttcctgtccgagctgcagtattacctcaccacggaggtgcatcctgagctctacgtg 180 gacaagtcgcggggagataaactgaagatcaacatcgatgtactttttccncacatgcct 240 tgtgcctatctgagtattgatgccatggatgtggccngagaacancagctggatgnggaa 300 cacaacctgtttaagccaccactagataaagatgcatcccngtgagctcanagctgagcg 360 gcatgagcttgngaaantcnaggtgaccgggtttga 396 <210> 88 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400>

tccagagcagagtcagccagcatgaccgagcgccgcgtccccttctcgctcctgcggggc 60 cccagctgggaccccttccgcgactggtacccgcatagccgctcttcgaccaggccttcg 120 ggctgccccggctgccggaggagtggtcgcagtggttaggcggcagcagctggccaggct 180 acgtgcgccccctgccccccgccgcatcgagagccccgcagtggccgcgcccgctacagc 240 cgcgcngctcagccggcaactcacancggggctcggagatccgggacactgcggaccgct 300 ngcgcgtgccctggatgtcaccactttngcccggacaactgacggtnanacaaggatggg 360 gggtgganannccngtaanccaagaangggnaggac 396 <210> 89 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 89 gagagaacag taaacatcca gccttagcat ctctcangag tactgcagat cttcattagc 60 tatattcaca tggagnaatg ctattcaacc tatttctctt atcaaaacta attttgtatt 120 ctttgaccaatgttcctaaattcactctgcttctctatctcaatctttttcccctttctc 180 atctttcctccttttttcagtttctaactttcactggttctttggaatgntttttctttc 240 atctcttttcttttacattttggggtgtcccctctcttttcttaccctctttctncatcc 300 ttcttnttcttttgaattggctgccctttatcntctcatctgctgncatcttcatttctc 360 ctccctcctntttccnntcattctactctctcccnt 396 <210> 90 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 90 gggcgccggcgcgcccccccacccccgccccacgtctcgtcgcgcgcgcgtccgctgggg 60 gcggggagcggtcgggccggcngcggtcggccggcggcagggtggtgcgntttcnttttn 120 nattnnccncnttcttcttnn~tnnncnnnctnntanncnntnncnttcncnnnntttnc 180 tntntcttnaccnnnttttntaatcntcttctncntnnnntctcttnnatntnttnctta 240 nttcctnnnntttnttctntcntttctcncctnnntctcnnnctcnncnctcnncatttt 300 nntnttttntnccttctnntcttnnttctnntnntnntttnnnnttctnttnntcatntt 360 ncctntnttactntcancttntatnnncctcntttt 396 <210> 91 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 91 ntntcctnnatttttnnntcnncttttttttnnaatttttctttnttttntttataaaaa 60 tcnncacntaaaacngcggaanaggggatttnttnttngggngtancncnnggccncaaa 120 naaccccaaaaatancccaaaatgcacaggnccngggnaaangaccnacntgggtntttt 180 ntttntnaacaaggggggttttaaagggnatnggnatcaaagggnataaantttaaacct 240 ttganaaattttttaanaggcttgccccccactttggnccccnccccncngnngggatcc 300 aattttttttcnttggggctcccngncccnnannttccgggttnntggncnntcctnntt 360 tttttttttttgccttcacccntnccattncntttt 396 <210> 92 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 92 ctntttnnntntttttttccccatcatccanaaatgggttttattctcagccgagggaca 60 gcaggactggtaaaaactgtcaggccacacggttgcctgcacagcacccccatgcttggt 120 agggggtgggagggatggcgggggctggntgnccacaggccgggcatgacaaggaggctc 180 actggaggtggcacactttggagtgggatgtcgggggacancttctttggtanttgggcc 240 acaagattcccaaggatancacnnnnactgattnccannctanagncaagcggntggcca 300 tntgtangnnnttntntatntgactatttatagatttttatanaacagggnaagggcata 360 ccncaaaagggnccaantttttaccnccgggcnccc 396 <210> 93 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 93 gctgccacagatctgttcctttgtccgtttttgggatccacaggccctatgtatttgaag 60 ggaaatgtgtatggctcagatcctttttgaaacatatcatacaggttgcagtcctgaccc 120 aagaacagttttaatggaccactatgagcccagttacataaagaaaaaggagtgctaccc 180 atgttctcatccttcagaagaatcctgcgaacggagcttcagtaatatatcgtggcttca 240 catgtgaggaagctacttaacactagttactctcacaatgaaggacctgnaatgaaaaat 300 ctgnttctaaccnagtcctntttanattttagngcanatccagaccancgncggtgctcg 360 agtaattctttcatgggacctttggaaaactttcag 396 <210> 94 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 94 tgccttaaccagtctctcaagtgatgagacagtgaagtaaaattgagtgcactaaacgaa 60 taagattctgaggaagtcttatcttctgcagtgagtatggcccaatgctttctgnggcta 120 aacagatgtaatgggaagaaataaaagcctacgtgttggtaaatccaacagcaagggaga 180 tttttgaatcataataactcatanngtgctatctgtcagtgatgccctcagagctcttgc 240 tgntagctggcagctgacgcttctangatagttagnttggaaatggtcttcataataact 300 acacaaggaaagtcanccnccgggcttatgaggaattggacttaataaatttagngngct 360 tccnacctaaaatatatcttttggaagtaaaattta 396 <210> 95 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G

<400> 95 cctcccacccncttanttcatgagattcganaatgncacttntgtgctntttnctnnttn 60 tattctnacnatttctttcttggngcggnannaatcccntttttnngggcgnctctcccn 120 ncttntnntttcntggngctntcccttttcnnnnnaaacttntacnnngtttanaantnt 180 ttctgnangggggnntccnaaanantttttccncctncctnattccnctctnaannctcn 240 cnaattgtttcccccccccnntagnntattttttctaaaaaattaactccnacgganaaa 300 attttccctaaaatttcncctccanatttngaaaaaacncgcccgganctnntntncgaa 360 tntnaatttttnaaaaaaanttattttcatcnggnn 396 <210> 96 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 96 cctgggtaccaaatttctttatttgaaggaatggtacaaatcaaagaacttaagtggatg 60 ttttggacaacttatagaaaaggtaaaggaaaccccaacatgcatgcactgccttggcga 120 ccagggaagtcaccccacggctatggggaaattagcccgangcttaactttcattatcac 180 tgcttccaagggngtgcttg~gcaaaaaaatattccgccaaccaaatcgggcgctccatct 240 tgcccagttggtnccgggnccccaattcttggatgctttcncctcttnttccggaatgng 300 ctcatgaantcccccaannggggcattttgccagnggccntttngccattcnagnnggcc 360 tgatccattttttccaatgtaatgccncttcattgn 396 <210> 97 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1)...(396) <223> n = A,T,C or G
<400> 97 ctcaccctcctcntnnttntcanaatattgngaacttnntnctgntcgaatcactggcat 60 taaaggancactagctaatggcactaaatttacnnactanggaaacttttttataatant 120 gcaaaaacatntnaaaaagantgnagttcgcccatttctgcttnggaaganctcttcact 180 tntaancccnnatgnngncctttgggtcaaaanctccgcgattattacngngttncccnc 240 tatttgnccttcctttntccccaangccncanatttcnnaactttnccntnaaatgcctt 300 tatttnatnncntttcnacnncttaannttccctttnaanaangatccctncttcaaatn 360 ntttcccngttcctngcattncccnnnnatttctct 396 <210> 98 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc feature <222> (1)...(396) <223> n = A,T,C or G
<400> 98 acagggacaatgaagcctttgaagtgccagtctatgaagaggccgtggtgggactagaat 60 cccagtgccgcccccaagagttggaccaaccaccccctacagcactgttgtgataccccc 120 agcacctgangaggaacaacctaccatccagaggggccaggaaaagccaaactggaacag 180 aggcgaatggctcagaggggtncatggccaagaaggaagccctggaagaacttcaatcac 240 cttcggtttcgggaccaccggcttgtgtccctgttctgactgcanaacttggcgcngtnc 300 cccattanaacctntgactcnncccttgctataagnctgttttggcccctgatgatgata 360 gggtttttatgangacacttgggcacccccttaatg 396 <210> 99 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 99 nttntttttccgncnaaagggcaagngtttncatctttcctgnccncncaananngggtn 60 tntgtgcntttnttttttcccaaaacccgggtnggggacaccttttgagganccactnnt 120 cntccggggcnnnnttttagaaggngnctaanaagcntcttgnngggggaaaaacatctt 180 tttgcncccnacatacccccaaggggggggggtgtctgggagganactaangacttttnt 240 tttttnnccncaaanaactganggcccccattgctccccccccantctttaaaaaacccc 300 ttcaatttccttgncnggnaaaaanggttggnaaaaaangagngngcntcnnttncnttt 360 natggaaggnaaaaggtttttggttgnaaaaccccg 396 <210> 100 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400>

ctaacacggtgaaaccctgtctctactaaaaatacaaaaaaattagccaggcgtggtggc 60 gggcacctgtagtcccagctgctcaggaagctgaggcaggagaatggcgtgaacccagaa 120 ggcggagcttgcagtgagctgagatcgtgtcagtgcactccagcctgggcgacagagcga 180 gactcccgctcaaaaaaaaaaaaaaaaagagaaaagaaaaagctgcagngagctgggaat 240 gggccctatcccctccttggggatcaatgagaccccttttcaaaanaaaaaaaaaaataa 300 tgngattttggnaacatatggcactggtgcttcnnggaattctgtttntnggcatgnccc 360 cctntgactgnggaaaaatccagcaggaggcccana 396 <210> 101 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 101 agttataactcaacagttcatttatatgctgttcatttaacagttcatttaaacagttca 60 ttataactgtttaaaaatatatatgcttatagncaaaanntgttgtggcgnagttgttgc 120 cgcttatagctgagcattatttcttaaattcttgaatgttcttttggngggntnctaaaa 180 ccgtatatgatccattttnatgggaaacngaattcntnncattatcncaccttggaaata 240 cnnaacgtgggggaaaaaaatcattcccnccntccaaaactatacttcttttatctngan 300 nttcttgntcctgcncnggtttngaatatanctgggcaaanggntttnccaaatccntnt 360 acnntnctttgggaantancggcaantcntcncttt 396 <210> 102 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

actatacataagaacangctcacatgggaggctggaggtgggtacccagctgctgtggaa 60 cgggtatggacaggtcataaacctagagtcagngtcctgttggcctagcccatttcagca 120 ccctgccacttggagnggacccctctactcttcttagcgcctaccctcatacctatctcc 180 ctnctcccatctcctacggactggcgccaaatggctttcctgccaattttgggatcttct 240 ctggctctccagcctgcttactcctctatttttaaagggccaaacaaatcccttctcttt 300 ctcaaacacagtaatgnggcactgaccctaccacacctcatgaagggggcttgttgcttt 360 tatttgggcccgatctggggggggcaaaatattttg 396 <210> 103 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 103 ttgtgttgggactgctgataggaagatgtcttcaggaaatgctaaaattgggcaccctgc 60 cccaacttcaaagccacagctggtatgccanatggtcaggttaaagatatcaacctgctg 120 actacaaaggaaaatatggtggggtcttcttttaccctcttgacttccctttgngngccc 180 cccgagancattgctttccgngatagggcaaaanaaattaaaaaacttaactggccagtg 240 aatggggcttctgnggatctccttctggcattacatnggcaatccctaaaaaacaagang 300 actgggacccataacattcttttgnatcaaccgaagcccccattgttangatatngggct 360 taaangctgatnaagcatctcgtccgggcnttttat 396 <210> 104 <211> 396 <212> DNA

<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 104 aagggagggcgcgccaagaccttcccactcgngcacactgggggcgccgacangacgcaa 60 cccagtccaacttggatacccttggntttagttctcggacacttcttttatctctccgtc 120 gcaacttgtcaagttctcaanactgtctctctgngntatcttttttcttcgctgctcttc 180 nncccccgacgtatttntcaaaangtctgcaattgttgnatacntnganctncaccactg 240 ttacnaggtcatnaatttcncntcaactctntnccncttgttccctgatatntcggccgg 300 ngncnccaattctgtattttnctcntcaacgntctcacttttncctcctccnggccactt 360 tctccccttccttattccggcnttgtttgccnccat 396 <210> 105 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 105 tcaatagccagccagtgttcatttttatccttgagcttttagtaaaaacttcctggnttt 60 atttttagtcattgggtcatacagcactaaagtctgctatttatggaaactaactttttt 120 gtttttaatccaggccaacatgtatgtaaattaaatttttagataattgattatctcttt 180 gtactacttgagatttgattatgagatgtgcatattgctttgggaagagctcgaggaagg 240 aaataattctctcctttggtttgaacctcaactagataaaccctaggaattgttaactgc 300 acaagnattttcattccacaaaacctgaggcagctcttttgccagagcgttcctgnaccc 360 ccccaccccacttgccttgggtctttanaangagcc 396 <210> 106 <211> 396 <212> DNA
<213> Homo sapien <400>

gctgtgtagcacactgagtgacgcaatcaatgtttactcgaacagaatgcatttcttcac 60 tccgaagccaaatgacaaataaagtccaaaggcattttctcctgtgctgaccaaccaaat 120 aatatgtatagacacacacacatatgcacacacacacacacacacccacagagagagagc 180 tgcaagagcatggaattcatgtgtttaaagataatcctttccatgtgaagtttaaaatta 240 ctatatatttgctgatggctagattgagagaataaaagacagtaacctttctcttcaaag 300 ataaaatgaaaagcaattgctcttttcttcctaaaaaatgcaaaagatttacattgctgc 360 caaatcatttcaactgaaaagaacagtattgctttg 396 <210> 107 <211> 396 <212> DNA
<213> Homo sapien <220>

<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

ttcacagaacanggtggtttattatttcaatagcaaagagctgaaaaatgtcgggtccca 60 taaaggagcagaacctgacccagagcctgcagtacatttccaccccacaggggtgcaggc 120 tgggccaggcagggccaaaggcagcagaaatgggagtaagagactgtgcccactgagaag 180 ctctgctgggtgtgggcaggtgggcatganatgatgatgatgtagtgtaaggaccaggta 240 ggcaaaacctgtcaggnttgntgaatgtcanagtggatccaaaaggctgagggggtcgtc 300 anaaggccggnggncccncccttgcccgtatgggccttcaaaaagtatgcttgctcatcc 360 gttgtttnccccanggagctgccangganaaggctn 396 <210> 108 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 108 gcctgcttttgatgatgtctacagaaaatgctggctgagctgaacacatttgcccaattc 60 caggtgtgcacagaaaaccgagaatattcaaaattccaaatttttttcttaggagcaaga 120 agaaaatgtggccctaaagggggttagttgaggggtagggggtagtgaggatcttgattt 180 ggatctctttttatttaaatgtgaatttcaacttttgacaatcaaagaaaagacttttgt 240 tgaaatagctttactgcttctcacgtgttttggagaaaannatcanccctgcaatcactt 300 tttgnaactgncnttgattttcngcnnccaagctatatcnaatatcgtctgngtanaaaa 360 tgncctggncttttgaangaatacatgngtgntgct 396 <210> 109 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 109 ggccgtaggcagccatggcgcccagcccggaatggcatggtcttgaagccccacttccac 60 aaggactggcagcggcgcgtggccacgtggttcaaccagccggcccggaagatccgcaga 120 cgtaaggcccggcaagccaaggcgcgccgcatcgctccgcgccccgcgtcgggtcccatc 180 cggcccatcgtgcgctgcccacggttcggtaccacacgaagggcgcgccggcgcggnttc 240 agcctggaggagctcagggtggccggatttacaagaagnggccngacatcngtattcttg 300 ggatncnngaagnggaacaagtcacngagtccttgcagccacntcagcggntgatgacac 360 cgttcnaactcatctnttcccaagaaacctcngnnc 396 <210> 110 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223 > n = A, T, C or G
<400>

nntgggctcctnncantnataataaaccngactcatacnccacaaggagatgaacaggan 60 tatgtncatnctgacgcggaaacagngcanggagctgaggaggngccaagatgagaccta 120 nnggccnnggtgggcgcattcccggnggagggggccactaaggantacgannntcnagcg 180 gctcttgnnggcngncctcctcacncctgnntattcgattgtcncnnatgncntcctatn 240 atnntcannattctntnntnatctcntntacnncntcncnttcatgnttacngntccctc 300 tcnttctnaccnttntctgnanctcctttctnnnnctttcatctntnttcngctttcttt 360 ctnnaatcntnntttaacntnntctnctttntnatt 396 <210> 111 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 111 taangancatnctggnttntgcctnnccgnctnattgantgttaaaggcaattntgtggn 60 tgtcccagngaatgncggctnattttctttccacattgngcncattcactcctcccactc 120 ttggcatgtngngacataagcanggtacataatngnaaaaatctgnatttctgatgccan 180 angggtanancntnttgnatntcattccattgatatacagccactnttttatttttgatc 240 ancggccttcggntcactgcncanggtacttgacctcagtgtcactattatgggntttgg 300 tttcnctcttttncnggccnttntntttcncacnttncancttncttnntnnaaaannna 360 nncactctctcttgctctctngatacnnngtctnaa 396 <210> 112 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 112 tcaacgtcaccaattactgccatttagcccacgagctgcgtctcagctgcatggagagga 60 aaaaggtccagattcgaagcatggatccctccgccttggcaagcgaccgatttaacctca 120 tactggcagataccaacagtgaccggctcttcacagtgaacgatgttaaagntggaggct 180 ccaagnatggtatcatcaacctgcaaagtctgaagacccctacgctcaaggtgttcatgc 240 acgaaaacctctacttcaccaaccggaaggtgaattcggggggctgggcctcgctgaatc 300 acttggattccacattctgctatgcctcatgggactcgcagaacttcaggctggccaccc 360 tgctcccaccatcactgntngncaatantcacccag 396 <210> 113 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 113 nnnnttnnnnnggagccttaatttcagagttttattgtattgcactaaaggaacagcagg 60 atggntatacaattttctctcattcagttttgaaaatctgtagtacctgcaaattcttaa 120 gaatacctttaccaccagattagaacagtaagcataataaccaatttcttaataagtaat 180 gtcttacaaataaaaacacatttaaaatagctttaaatgcattcttcacaagtaattcag 240 catatattttatatcatggttacttatgcttangaattnnagcaggatntttattctttt 300 gatggaaatatgggaaaactntattcatgcatatacanggataatattcagcgaagggaa 360 aatcccgtttttattttggnaatgattcatatataa 396 <210> 114 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 114 aaatgggacaacgtgattcttttgttttaaataaatactnagaacacggacttggctcct 60 acaagcatttggactctaaggnttagaactggagagtcttacccatgggccccncncagg 120 gacgccacggttccctcccaccccgngatcaagacacggaatcngntggcgatngttgga 180 tcgcnatgtgccccttatctatagccttcccnggncatntacangcaggatgcggntggg 240 anaactacaactgnaatntctcnaacggtnatggtccccaccgatnaagattctacctng 300 tcttttcntcccctggagtgtgagtgnnngaggaagaagcccttnccttacatcaccttt 360 tgnacttctgaacaagancaanacnatggccccccc 396 <210> 115 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

ccgcctggttcggcccgcctgcctccactcctgcctctaccatgtccatcagggtgaccc 60 agaagtcctacaaggtgtccacctctggcccccgggccttcagcagccgctcctacacga 120 gtgggcccggttcccgcatcagctcctcgagcttctcccgagtgggcagcagcaactttc 180 gcggtggcctggcggcggctatggtggggccagcggcatgggaggcatcacccgcagtta 240 cggcaaccagagcctgctgagccccttgcctggaggnggaccccaacatcaagccgngcg 300 cacccaggaaaaggagcagancaagaccctcaacaacaagnttgcttcttcatagacaag 360 ggaccggtccttgaacagcanaacaagatgntggag 396 <210> 116 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

atctcagtttactagctaagtgactttgggcaagggatttaacctctcgtccctcagttt 60 cctcctatgtaaaatgacaaggataatagtaccaacccaatgtagattaaatgagtttac 120 gaagtgttagaatagtgcttggcacattagtgctttacaactgctattttgattgttgtt 180 gtgggctctctcaaatgcattgtctctagatgccagtgacccaggtcaaaatttaccttt 240 aaccaagctgcatgtttcccagactgntgcacagtcctctaccctgaganaaagcttcca 300 cccaaggatacttttactttctgctggaaaactgatgagcaanggcaacangggacactt 360 atcgccaactggaaangagaaattcttccttttgct 396 <210> 117 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 117 aaacattttttaataaaattcctatagaaagctcagtcatagggcaaatactcagttctc 60 tttcccatatcaccgaggattgagagctcccaatattctttggagaataagcagtagttt 120 tgctggatgttgccaggactcagagagatcacccatttacacattcaaaccagtagttcc 180 tattgcacatattaacattacttgcccctagcaccctaaatatatggnacctcaacaaat 240 aacttaaagatttccgtggggcgcganaccatttcaatttgaactaatatccttgaaaaa 300 aatcacattattacaagntttaataaatacnggaagaagagctggcatttttctaanatc 360 tgaattcngacttggntttattccataaatacggtt 396 <210> 118 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

accnncacctgntnnnttttaacnattacaacttctttatatggcagtttttactgggng 60 cctaacactctctttactgnctcaagnggaagtccaaacaaatttcatttttgtagtaaa 120 aaatctttatttccaaaatgatttgttagccaaaagaactataaaccacctaacaagact 180 ttggaagaaagagacttgatgcttcttataaattccccattgcanacaaaaaataacaat 240 ccaacaagagcatggtacccattcttaccattaacctggntttaannctccaaancnnga 300 tttaaaaatgaccccactgggcccaatccaacatganacctaggggggnttgccttgatt 360 angaatcccc cttanggact ttatctnggc tganaa 396 <210> 119 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A, T, C or G
<400> 119 atggccagctcactttaaataccacctcaagactcatcgaaatgaccgctccttcatctg 60 tcctgcagaaggttgtgggaaaagcttctatgtgctgcagaggctgaaggtgcacatgag 120 gacccacaatggagagaagccctttatgtgccatgagtctggctgtggtaagcagtttac 180 tacagctggaaacctgaagaaccaccggcgcatccacacaggagagaaacctttcctttg 240 tgaagcccaangatgtggccgtcctttgctgagtattctancttcgaaaacatctggngg 300 ntactcangagagaaagcctcattantgccantctgngggaaaaccttctntcagagngg 360 angcaggaatgtgcatattaaaaagctnccttgnac 396 <210> 120 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 120 catgggtcagtcggtcctgagagttcgaagagggcacattcccaaagacattcccagtca 60 tgaaatgtagaagactggaaaattaagacattatgtaaaggtagatatggcttttagagt 120 tacattatgcttggcatgaataaggtgccaggaaaacagtttaaaattatacatcagcat 180 acagactgctgttagaaggtatgggatcatattaagataatctgcagctctactacgcat 240 ttattgttaattgagttacanangncattcannactgagtttataganccatattgctct 300 atctctgngnagaacatttgattccattgngaagaatgcagtttaaaatatctgaatgcc 360 atctagatgtattgtaccnaaaggggaaaaataaca 396 <210> 121 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 121 tttttttttt ttttttttaa aatcaagtta tgtttaataa acattaataa atgtttactt 60 aaaagggtta ataaacnttt actacatggc aaattatttt agctagaatg cttttggctt 120 caagncatan aaaccagatt cnaatgccct taaanaattt tnaaanatcc attgangggg 180 ataactgtaa tccccaaggg gaanagggtt gggtatgaca ggtacanggg gccagcccag 240 tnntnncana nncagactct taccntcttt ctgctgtgnc accctcaggc attggctcca 300 ttctcngggn tgcncatggg aagatggctt tggacntaac nacacccttt tgtncacgta 360 aaggccngat gcagggtcaa anagnttccn ccatnt 396 <210> 122 <211> 396 <212> DNA
<213> Homo sapien <400>

gtcgacatggctgccctctgggctcccagaacccacaacatgaaagaaatggtgctaccc 60 agctcaagcctgggcctttgaatccggacacaaaaccctctagcttggaaatgaatatgc 120 tgcactttacaaccactgcactacctgactcaggaatcggctctggaaggtgaagctaga 180 ggaaccagacctcatcagcccaacatcaaagacaccatcggaacagcagcgcccgcagca 240 cccaccccgcaccggcgactccatcttcatggccaccccctgcggtggacggttgaccac 300 cagccaccacatcatcccagagctgagctcctccagcgggatgacgccgtccccaccacc 360 tccctcttcttctttttcatccttctgtctctttgt 396 <210> 123 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 123 gccctttttttttttttttttttcctagtgccaggtttattccctcacatgggtggttca 60 catacacagcacanaggcacgggcaccatggganagggcagcactcctgccttctgaggg 120 gatcttggcctcacggtgtaanaaggganaggatggtttctcttctgccctcactagggc 180 ctagggaacccagnagcaaatcccaccacgccttccatntctcagccaagganaagccac 240 cttggtgacgtttagttccaaccattatagtaagtgganaagggattggcctggtcccaa 300 ccattacagggtgaanatataaacagtaaaggaanatacagtttggatgaggccacagga 360.

aggagcanatgacaccatcaaaagcatatgcaggga 396 <210> 124 <211> 396 <212> DNA
<213> Homo sapien <400> 124 gaccattgccccagacctggaagatataacattcagttcccaccatctgattaaaacaac 60 ttcctcccttacagagcatacaacagagggggcacccggggaggagagcacatactgtgt 120 tccaatttcacgcttttaattctcatttgttctcacaccaacagtgtgaagtgcgtggta 180 taatctccatttcaaaaccaaggaagcagcctcagagtggtcgagtgacacacctcacgc 240 aggctgagtccagagcttgtgctcctcttgattcctggtttgactcagttccaggcctga 300 tcttgcctgtctggctcagggtcaaagacagaatggtggagtgtagcctccacctgatat 360 tcaggctactcattcagtcccaaatatgtattttcc 396 <210> 125 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400>

ccctttttttttttttttttttttttttttttttttactttgnaacaaaaatttattagg 60 attaagtcaaattaaaaaacttcatgcnccnccncttgtcatatttacctgaaatgacaa 120 agttatacttagcttgagngnaaaacttgngccccaaaaattntgtttggaaagcaaaaa 180 aataattgatgcncatagcagngggcctgatnccnccacagngaatgttgtttaaggnct 240 aacaaacaggggncancaaagcatacattacttttaagctttgggnccaaggaaaangtc 300 attccctacctccttcaaaagcaaactcatnatagcctgggcncctaggnctggagcctn 360 ttttttcgagtctaanatgaacatntggatttcaan 396 <210> 126 <211> 396 <212> DNA
<213> Homo sapien <400> 126 cgcgtcgactcgcaagtggaatgtgacgtccctggagaccctgaaggctttgcttgaagt 60 caacaaagggcacgaaatgagtcctcaggtggccaccctgatcgaccgctttgtgaaggg 120 aaggggccagctagacaaagacaccctagacaccctgaccgccttctaccctgggtacct 180 gtgctccctcagccccgaggagctgagctccgtgccccccagcagcatctgggcggtcag 240 gccccacgacctggacacgctggggctacggctacagggcggcatccccaacggctacct 300 ggtcctagacctcagcatgcaagaggccctctcggggacgccctgcctcctaggacctgg 360 acctgttctcaccgtcctggcactgctcctagcctc 396 <210> 127 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 127 ttttttttttttggnggtaaaatgcaaatgttttaaaatatgtttattttgtatgtttta 60 caatgaatacttcagcaaagaaaataattataatttcaaaatgcaatccctggatttgat 120 aaatatcctttataatcgattacactaatcaatatctagaaatatacatagacaaagtta 180 gctaatgaataaaataagtaaaatgactacataaactcaatttcagggatgagggatcat 240 gcatgatcagttaagtcactctgccactttttaaaataatacgattcacatttgcttcaa 300 tcacataaacattcattgcaggagttacacggctaatcattgaaaattatgatctttgtt 360 agcttaaaagaaaattcagtttaatacaaagacatt 396 <210> 128 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc feature <222> (1)...(396) <223> n = A,T,C or G
<400> 128 gccctttttttttttttttaaaggcaaataaaataagtttattgggatgtaaccccatca 60 taaattgaggagcatccatacaggcaagctataaaatctggaaaatttaaatcaaattaa 120 attctgcttttaaaaaggtgccttaagttaaccaagcattttgataacacattcaaattt 180 aatatataaaaatagatgta.tcctggaagatataatgaanaacatgccatgtgtataaat 240 tcanaatacgctttttacacaaagaactacaaaaagttacaaagacagccttcaggaacc 300 acacttaggaaaagtgagccgagcagccttcacgcaaagcctccttcaaanaagtctcac 360 aaagactccagaaccagccgagtntgtgaaaaagga 396 <210> 129 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

gcccttttttttttttttttttttactcagacaggcaatatttgctcacatttattctct 60 tgcatcgtaaatagtagccaactcacaaaaataaagtatacaanaatgtaatatttttta 120 aaataagattaacagtgtaagaaggaaaatctcaaaaaaagcanatagacaatgtanaaa 180 attgaaatgaaatcccacagtaanaaaaaaaaaacanaaaagtgcctatttaanaattat 240 gctacatgtggaacttaactagaccattttaanaaagaccaatttctaatgcaaattttc 300 tgaggttttcanattttatttttaaaatatgttatagctacatgttgtcnacncggccgc 360 tcgagtctanagggcccgtttaaacccgctgatcag 396, <210> 130 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 130 cgcccttttttttttttttttanngnacgtgnctttatttctggatgatataaaanaaaa 60 aacttaaaaaacaccccaaaccaaacaccaatggatccccaaagcgatgtgactccctct 120 tcccacccggataaatagagacttctgtatgtcagtctaccctcccgcccccataacccc 180 ctctgctatanacatactctgggtatatattactctactcggcaatagacatctcccgaa 240 aatagaattcctgccctgacacctgactcttccctggccgcatcanaccacccgccactg 300 tagcacactggtgtccttgccccctgtggtcagggccatgctgtcatcccacaanaaggc 360 cacatttgtcacatggctgctgtgtccaccgtactt 396 <210> 131 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 131 gcccttttttttttttttttttttttttttttcagtttacacaaaaacnctttaattgac 60 agtatacnnttttccaaaatatnttttngtaanaaaatgcaataattattaactatagtt 120 tttacaaacaagtttntcantaaattccagtgtncttnaaaccccnnncnannaaaacat 180 atatgancccccagttcctgggcaaactgttgaacattcactgcanacaaaaagaccanc 240 nccaaanagtcatctgngncctccatgctgngtttgcaccaaacctgagggancagctag 300 ngaccgtgacaaaagctntgctacagttttactntngccctntntgcctcccccatnatg 360 tttccttggtccctcantcctgtnggagtaagttcc 396 <210> 132 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 132 cgcgtcgaccgcggccgtagcagccgggctggtcctgctgcgagccggcggcccggagtg 60 gggcggcgntatgtaccttccacattgagtattcagaaagaagtgatctgaactctgacc 120 attctttatggatacattaagtcaaatataagagtctgactacttgacacactggctcgg 180 tgagttctgctttttctttttaatataaatttattatgttggtaaatttagcttttggct 240 tttcactttgctctcatgatataagaaaatgtaggttttctctttcagtttgaattttcc 300 tattcagtaaaacaacatgctagaaaacaaacttttggaaaggcattgtaactatttttt 360 caaatagaaccataataacaagtcttgtcttaccct 396 <210> 133 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 133 ntattacccctcctggnnanntggnnatannctgcaaggngatnnncccgnngaacttca 60 ctgatnnnccaatnaaaactgctttaaanctgactgcacatatgaattntaatacttact 120 tngcgggaggggtggggcagggacagcaagggggaggattgggaanacaatagacaggca 180 tgctggggatgcngcgggctctatggcttctgangcgnaaagaaccagctggggctctag 240 ggggtatccccacgcgccctgtagcngcncattaaacgcggcgggtgtggnggttacttc 300 gcaaagngaccgatncacttgccagcgccctagctgcccgctcctttngctttcttccct 360 tcctttctcgccacnttnnccggctntccccgncaa 396 <210> 134 <211> 396 <212> DNA

<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 134 ttttttttttttctgctttttatatgtttaaaaatctctcattctattgctgctttattt 60 aaagaaagattactttcttccctacaagatctttattaattgtaaagggaaaatgaataa 120 ctttacaatgganacacctggcanacaccatcttaaccaaagcttgaagttaacataacc 180 agtaatagaactgatcaatatcttgtgcctcctgatatggngtactaanaaaaacacaac 240 atcatgccatgatagtcttgccaaaagtgcataacctaaatctaatcataaggaaacatt 300 anacaaactcaaattgaaggacattctacaaagtgccctgtattaaggaattattcanag 360 taaaggagacttaaaagacatggcaacaatgcagta 396 <210> 135 <211> 396 <212> DNA
<213> Homo sapien <400> 135 gcgtcgacgctggcagagccacaccccaagtgcctgtgcccagagggcttcagtcagctg 60 ctcactcctccagggcacttttaggaaagggtttttagctagtgtttttcctcgctttta 120 atgacctcagccccgcctgcagtggctagaagccagcaggtgcccatgtgctactgacaa 180 gtgcctcagcttccccccggcccgggtcaggccgtgggagccgctattatctgcgttctc 240 tgccaaagactcgtgggggccatcacacctgccctgtgcagcggagccggaccaggctct 300 tgtgtcctcactcaggtttgcttcccctgtgcccactgctgtatgatctgggggccacca 360 ccctgtgccggtggcctctgggctgcctcccgtggt 396 <210> 136 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400>

ttatgcttccggctcgtntgttgtgtggaattgtgagcggataacaatttcacacaggaa 60 acagctatgaccatgattacgccaagctatttaggtgacactatagaatactcaagctat 120 gcatcaagcttggtaccgagctcggatccactagtaacggccgccagtgtgctggaattc 180 gcggncgntcnantctagagggcccgtttaaacccgctgatcagcctcgactgtgccttc 240 tagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgc 300 cactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtg 360 tcattctattctggggggtggggtggggcaggacan 396 <210> 137 <211> 396 <212> DNA
<213> Homo sapien <220>

<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 137 ttttttttttttctgctttgtacttgagtttatttcacaaaaccacggagaaagatactg 60 aaatggagctctttccagcctccaagcaaggaggccccagcagccagtctccagcccctt 120 gagccctttttgttaggcccacacccaaaagagganaaccagtgtgtgcgcgaaggtaca 180 tggcaaggcacttttgaaaacatcccagtttaccgnggtgaaattgaacttactctgaaa 240 cagatgaaaagggacatgcaaaattgctgagcacatggaggtgtttgttagtaggtgaaa 300 atcatgtcctgggtataacccagcttctccaggttagggtgagccgccgtctggatcagt 360 ggtggcgggccacacaccaggatgagcgtggacttc 396 <210> 138 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400>

ccctttttttttttttttacaaatgagaaaaatgtttattaagaaaacaatttagcagct 60 ctcctttanaattttacagactaaagcacaacccgaaggcaattacagtttcaatcatta 120 acacactacttaaggngcttgcttactctacaactggaaagttgctgaagtttgtgacat 180 gccactgtaaatgtaagtattattaaaaattacaaattgtttggtgattattttgatgac 240 ctcttgagcagcagctccccccaanaatgcancaatggtatgtggctcaccagctccata 300 tcggcaaaattcgtggacataatcatctttcaccattacagataaaccatattcctgaag 360 gaagccagtgagacaagacttcaactttcctatatc 396 <210> 139 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 139 ccgcccttttttttttttttttcacaaaagcactttttatttgaggcaaanagaagtctt 60 gctgaaaggattccagttccaagcagtcaaaactcaaccgttagnggcactattttgacc 120 tggtanattttgcttctctttggtcanaaaagggtattcaggttgtactttccccagcag 180 ggtaaaaagaagggcaaagcaaactggaananacttctactctactgacagggctnttga 240 natccaacatcaagctanacacnccctcgctggccactctacaggttgctgtcccactgc 300 tgagtgacacaggcc.atactacatttgcaaggaaaaaaatgaggcaanaaacacaggtat 360 aggtcacttggggacgagcaggcaaccacagcttca 396 <210> 140 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400>

tttttttttttttttttttttttttttctcatttaacttttttaatgggnctcaaaattn 60 tgngacaaatttttggtcaagttgtttccattaaaaagtnctgattttaaaaactaataa 120 cttaaaactgccncncccaaaaaaaaaaaccaaaggggtccacaaaacattntcctttcc 180 ttntgaaggntttacnatgcattgttatcattaaccagtnttttactactaaacttaaan 240 ggccaattgaaacaaacagttntganaccgttnttccnccactgattaaaagnggggggg 300 caggtattagggataatattcatttanccttntgagctttntgggcanacttggngacct 360 tgccagctccagcagccttnttgtccactgntttga 396 <210> 141 <211> 396 <212> DNA
<213> Homo sapien <400>

acgccgagccacatcgctcagacaccatggggaaggtgaaggtcggagtcaacggatttg 60 gtcgtattgggcgcctggtcaccagggctgcttttaactctggtaaagtggatattgttg 120 ccatcaatgaccccttcattgacctcaactacatggtttacatgttccaatatgattcca 180 cccatggcaaattccatggcaccgtcaaggctgagaacgggaagcttgtcatcaatggaa 240 atcccatcaccatcttccaggagcgagatccctccaaaatcaagtggggcgatgctggcg 300 ctgagtacgtcgtggagtccactggcgtcttcaccaccatggagaaggctggggctcatt 360 tgcaggggggagccaaaagggtcatcatctctgccc 396 <210> 142 <211> 396 <212> DNA
<213> Homo sapien <400> 142 acgcaggagaggaagcccagcctgttctaccagagaacttgcccaggtcagaggtctgcg 60 tagaagcccttttctgagcatcctctcctctcctcacacctgccactgtcctctgcgttg 120 ctgtcgaattaaatcttgcatcaccatggtgcacttctgtggcctactcaccctccaccg 180 ggagccagtgccgctgaagagtatctctgtgagcgtgaacatttacgagtttgtggctgg 240 tgtgtctgcaactttgaactacgagaatgaggagaaagttcctttggaggccttctttgt 300 gttccccatggatgaagactctgctgtttacagctttgaggccttggtggatgggaagaa 360 aattgtagcagaattacaagacaagatgaaggcccg 396 <210> 143 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 143 tttttttttt tttccatana aaataggatt tattttcaca tttaaggnga acacaaatcc 60 atgttccanaaatgttttatgcataacacatcatgagtagattgaatttctttaacacac 120 anaaaaatcaaagcctaccaggaaatgcttccctccggagcacaggagcttacaggccac 180 ttntgttagcaacacaggaattcacattgtctaggcacagctcaagngaggtttgttccc 240 aggttcaactgctcctacccccatgggccctcctcaaaaacgacagcagcaaaccaacag 300 gcttcacagtaaccaggaggaaagatctcagngggggaaccttcacaaaagccctgagtt 360 gtgtttcaaaagccaagctctggggtctgnggcctg 396 <210> 144 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 144 tttttttttttttcgctctttggtctgacaagaaaagagttttaggtgtgtgaagtaggg 60 tgggaaaaaaggtcagtttcaaattcagtaacatatggtaacactaagttaggctgctgc 120 attcttttctttgggtacttaagccagctggcacttccactttgtaaccaattatattat 180 gatcaacaactaatcagttagttcctcagcttcaactgaanagttcctgattacctgatg 240 aaggacatacttgctctggcttcaattagcatgctgtcaagcatccctctccatgcttaa 300 catggcaacacaaaacccaagagtccttctntttttttcattagccatgaataaacactc 360 acaaaggggaagagtagacactgcttttagtaaacg 396 <210> 145 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 145 tttttttttttttttttcaatggatccgttagctttactactaanatcttgctganatca 60 nanaagggcttctgggcaggctgagcactgggggtgtgcaacatggtaactctgaataan 120 anaaaccctgagttttactgggcaaanaaanaacaagnggtaggtatgatttctgaacct 180 ggaaatagcgaaaatgaaggaaattccaaaagcgcgtatttccaaataatgacaggccag 240 caagaggacaccaaacctntanaaagaggtattntttcttccagctactgatggctttgg 300 catcccacaggcacattcctttggccttcaggatcttanatgcanatgtgganagtcaag 360 aggtaggctgactctgagtcttcagctaaattcttt 396 <210> 146 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G

<400>

ttttttttttttttcattagcaaggaaggatttattttttcttttgaggggagggcggaa 60 cagccgggatttttggaacactacctttgtctttcactttgttgtttgtgtgttaacacn 120 aataaatcanaagcgactttaaatctcccttcgcaggactgtcttcacgtatcagngcan 180 acaanaaaacagtggctttacaaaaaanatgttcaagtaggctgcactttgcctctgngg 240 gtgaggcacactgngggananacaaggtcccctgnaaccagaggngggaaggacanagct 300 ggctgactccctgctctcccgcattctctcctccatgtgttttgaanagggaagcaacat 360 gttgaggtctgatcatttctacccagggaacctgtt 396 <210> 147 <211> 396 <212> DNA
<213> Homo sapien <400> 147 acggggaagccaagtgaccgtagtctcatcagacatgagggaatgggtggctccagagaa 60 agcagacatcattgtcagtgagcttctgggctcatttgctgacaatgaattgtcgcctga 120 gtgcctggatggagcccagcacttcctaaaagatgatggtgtgagcatccccggggagta 180 cacttcctttctggctcccatctcttcctccaagctgtacaatgaggtccgagcctgtag 240 ggagaaggaccgtgaccctgaggcccagtttgagatgccttatgtggtacggctgcacaa 300 cttccaccagctctctgcaccccagccctgtttcaccttcagccatcccaacagagatcc 360 tatgattgacaacaaccgctattgcaccttggaatt 396 <210> 148 <211> 396 <212> DNA
<213> Homo sapien <400> 148 acgtcccatgattgttccagaccatgactcttcctggttgtgggtttgttacagagcagg 60 agaagcagaggttatgacagttatgcagactttccccctcctttttctcttttctcttcc 120 ccttgcttttccactgtttcttcctgctgccacctgggccttgaattcctgggctgtgaa 180 gacatgtagcagctgcagggtttaccacacgtgggagggcagcccagtactgtccctctg 240 ccttccccactttgagaatatggcagcccctttcattcctggcttggggtaggggagacc 300 attgaagtagaagcctcaaagcagacttttccctttactgtgtgtactccaggacgaaga 360 aggaagatcatgcttgatacttagattggttttccc 396 <210> 149 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

tttttttttttttaaagagtcacattttattcaatgcctatttgtacatgttactagcaa 60 taaactcttttatctttaattttgagaagttttacaaatacagcaaagcagaatgactaa 120 tagagccggtaaccaggacacagatttggaaaaataggtctaattggttgttacactgtg 180 tttatgtcatacatttcgcttatttttatcaaanaaaaatcagaatttataaaatgttaa 240 ttaaaaggaaaacattctgagtaaatttagtcccgtgtttcttcctccaaatctntttgt 300 tctacactaacaggtcaggataagtatggatggggaggctggaaaaagggcatccttccc 360 catgcggtccccagagccaccctctccaagcaggac 396 <210> 150 <211> 396 <212> DNA
<213> Homo sapien <400> 150 acgcctctcttcagttggcacccaaacatctggattggcaaatcagtggcaagaagttcc 60 agcatctggacttttcagaattgatcttaagtctactgtcatttccagatgcattatttt 120 acaactgtatccttggaaatatatttctagggagaatattattgaagaaaatgttaatag 180 cctgagtcaaatttcagcagacttaccagcatttgtatcagtggtagcaaatgaagccaa 240 actgtatcttgaaaaacctgttgttcctttaaatatgatgttgccacaagctgcattgga 300 gactcattgcagtaatatttccaatgtgccacctacaagagagatacttcaagtctttct 360 tactgatgtacacatgaaggaagtaattcagcagtt 396 <210> 151 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

acaaaatgcccagcctacagagtctgagaaggaaatttataatcaggtgaatgtagtatt 60 aaaagatgcagaaggcatcttggaggacttgcagtcatacagaggagctggccacgaaat 120 acgagaggcaatccagcatccagcanatgagaagttgcaagagaaggcatggggtgcagt 180 tgttccactagtaggcaaattaaagaaattttacgaattttctcagaggttagaagcagc 240 attaagaggtcttctgggagccttaacaagtaccccatattctcccacccagcatctana 300 gcgagagcaggctcttgctaaacagtttgcanaaattcttcatttcacactccggtttga 360 tgaactcaagatgacaaatcctgccatacagaatga 396 <210> 152 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 152 acgcagcgctcggcttcctggtaattcttcacctcttttctcagctccctgcagcatggg 60 tgctgggccctccttgctgctcgccgccctcctgctgcttctctccggcgacggcgccgt 120 gcgctgcgacacacctgccaactgcacctatcttgacctgctgggcacctgggtcttcca 180 ggtgggctccagcggttcccagcgcgatgtcaactgctcggttatgggaccacaagaaaa 240 aaaagtagnggtgtaccttcagaagctggatacagcatatgatgaccttggcaattctgg 300 ccatttcaccatcatttacaaccaaggctttgagattgtgttgaatgactacaagtggtt 360 tgccttttttaagtataaagaagagggcagcaaggt 396 <210> 153 <211> 396 <212> DNA
<213> Homo sapien <400> 153 ccagagacaacttcgcggtgtggtgaactctctgaggaaaaacacgtgcgtggcaacaag 60 tgactgagacctagaaatccaagcgttggaggtcctgaggccagcctaagtcgcttcaaa 120 atggaacgaaggcgtttgcggggttccattcagagccgatacatcagcatgagtgtgtgg 180 acaagcccacggagacttgtggagctggcagggcagagcctgctgaaggatgaggccctg 240 gccattgccgccctggagttgctgcccagggagctcttcccgccactcttcatggcagcc 300 tttgacgggagacacagccagaccctgaaggcaatggtgcaggcctggcccttcacctgc 360 ctccctctgggagtgctgatgaagggacaacatctt 396 <210> 154 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 154 acagcaaacctcctcacagcccactggtcctcaagaggggcnacntcttcacacatcanc 60 acaactacgcattgcctccctncactcggaaggactatcctgctgccaagagggtcaagt 120 tggacagtgtcagagtcctgagacagatcagcaacaaccgaaaatgcaccagccccaggt 180 cctcggacaccgaggagaatgtcaagaggcgaacacacaacgtcttggagcgccagagga 240 ggaacgagctaaaacggagcttttttgccctgcgtgaccagatcccggagttggaaaaca 300 atgaaaaggcccccaaggtagttatccttaaaaaagccacagcatacatcctgtccgtcc 360 aagcagaggagcaaaagctcatttctgaagaggact 396 <210> 155 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400>

tttttttttttgaananacaggtctttaatgtacggagtctcacaaggcacaaacaccct 60 caccaggaccaaataaataactccacggttgcaggaaggcgcggtctggggaggatgcgg 120 catctgagctctcccagggctggtgggcgagccgggggtctgcagtctgtgaggggcctc 180 ctgggtgtgtccgggcctctanagcgggtccagtctccaggatggggatcgctcactcac 240 tctccgagtcggagtagtccgccacgagggaggagccganactgcaggggtgccgcgtgt 300 cgggggtgtcagctgcctcctgggaggagcctgctggcnacaggggcttgtcctgacggc 360 tcccttcctgccccctcgggctgctgcacttggggg 396 <210> 156 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 156 gaaggggggcngggcaggggcggaatgtananattantgccatgattgaagatttaagaa 60 acgtgagattcaggattttcaccacatccccatttagttagcttgctcgtttggctggtg 120 caaatgccagatggattatgaacaatgacagtaaattaatgcaacataatcaggtaatga 180 tgccaagcgtatctggtgttccaggtattgtacctttaccggaacaaatcagtaaatcca 240 caatccctggcacctgttaggcagctattaacctagtaaatgctcccccatcccatctca 300 atcagcaangacaatcaaaaacatttgctttnagtggcaggaacactggtacatttttac 360 ttgctccaagggctgtgccaacgctccctctctctg 396 <210> 157 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

tttttttttttttttggggaatgtaaatcttttattaaaacagttgtctttccacagtag 60 taaagctttggcacatacagtataaaaaataatcacccaccataattataccaaattcct 120 nttatcaactgcatactaagtgttttcaatacaattttttccgtataaaaatactgggaa 180 aaattgataaataacaggtaananaaagatatttctaggcaattactaggatcatttgga 240 aaaagtgagtactgnggatatttaaaatatcacagtaacaagatcatgcttgttcctaca 300 gtattgcgggccanacacttaagtgaaagcanaagtgtttgggtgactttcctacttaaa 360 attttggncatatcatttcaaaacatttgcatcttg 396 <210> 158 <211> 396 <212> DNA
<213> Homo sapien <400> 158 tttccgaagacgggcagcttcagagaagaggattattcgggagattgctggtgtggccca 60 tagactctttggcatagactctttcgcaggcagccactctgagtgtggccagttctataa 120 ccatccccaaactagctggagcctgatggataggaacgggtagtctgtcctcttccccat 180 aaaaatgttccaaaaagttatctccagagagagtcccttatgaagacagttgccaagctg 240 tattctcattctttaaaccaatacccaggtcagggctagttcacactagcactgttaggg 300 acatggtgtggctagaaatgaattgagtgtgacttctccctacaaccccaggcccaggga 360 taggaggaggcagaggggtgcctggagtttctgcac 396 <210> 159 <211> 396 <212> DNA
<213> Homo sapien <400> 159 tccgcgcgtt gggaggtgta gcgcggctct gaacgcgctg agggccgttg agtgtcgcag 60 gcggcgaggg cgcgagtgag gagcagaccc aggcatcgcg cgccgagaag gccgggcgtc 120 cccacactgaaggtccggaaaggcgacttccgggggctttggcacctggcggaccctccc 180 ggagcgtcggcacctgaacgcgaggcgctccattgcgcgtgcgcgttgaggggcttcccg 240 cacctgatcgcgagaccccaacggctggtggcgtcgcctgcgcgtctcggctgagctggc 300 catggcgcagctgtgcgggctgaggcggagccgggcgtttctcgccctgctgggatcgct 360 gctcctctctggggtcctggcggccgaccgagaacg 396 <210> 160 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 160 ggaaaccttctcaactaagagaacatcatttctggcaaactatttttgttagctcacaat 60 atatgtcgtacactctacaatgtaaatagcactganccacancttacagaaggtaaaaag 120 angnataanaacttcctttacaaaananttcctgttgttcttaatactccccattgctta 180 tganaattntctatangtctctcangantgttcgcacccatttcttttntaacttctact 240 aaaaanccatttacattgnanagtgtacnacntatatttgngagctaacaaaaaatngtt 300 ttccnganatgatgttcttttagtttnaganggttcnnncaanttnctactccngcccgc 360 cactgnncnccacatttnnnnaattacaccncacng 396 <210> 161 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400>

tttttgtttgattatttttattataatgaaattaaacttatgactattacagtatgctca 60 gcttaaaacatttatgagtactgcaaggactaacagaaacaggaaaaatcctactaaaaa 120 tatttgttgatgggaaatcattgtgaaagcaaacctccaaatattcatttgtaagccata 180 agaggataagcacaaccatatgggaggagataaccagtctctcccttcatatatattctt 240 ttttatttcttggtataccttcccaaaacananacattcaacagtagttagaatggccat 300 ctcccaacattttaaaaaaactgcnccccccaatgggtgaacaaagtaaagagtagtaac 360 ctanagttcagctgagtaagccactgtggagcctta 396 <210> 162 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 162 ttttttttttttttttttttttttttttttttnggggnccaaatttttttntttgaagga 60 angggacaaannaaaaaacttaaggggntgttttggnncnacttanaaaaaagggaaagg 120 aaaccccaacatgcatgccctnccttggggaccanggaanncnccccncnggtntgggga 180 aantaacccnaggnttaactttnattatcactgncncccagggggggcttnnaaaaaaaa 240 nnttcccccaanccaaantngggnncncccattttncncaanttggncnccnggncnccc 300 nattttttgangggtttcnccngcncattnagggaangggnntcaannaaaccncncaaa 360 ngggggnnatttttntcangggccnatttgngcnnt 396 <210> 163 <211> 396 <212> DNA
<213> Homo sapien <400>

cactgtccggctctaacacagctattaagtgctacctgcctctcaggcactctcctcgcc 60 cagtttctgaggtcagacgagtgtctgcgatgtcttcccgcactctattcccccagcctc 120 tttctgctttcatgctcagcacatcatcttcctaggcagtctcttccccaaagtctcacc 180 ttttcttccaatagaaaattccgcttgacctttggtgcactgcccacttcccagctccac 240 tggcccaagtctgagccggaggcccttgttttgggggcggggggagagttggatgtgatt 300 gcccttgaagaacaaggctgacctgagaggttcctggcgccctgaggtggctcagcacct 360 gcccagggtaggcctggcatgaggggttaggtcagc 396 <210> 164 <211> 396 <212> DNA
<213> Homo sapien <400> 164 gacacgcggcggtgtcctgtgttggccatggccgactacctgattagtgggggcacgtcc 60 tacgtgccagacgacggactcacagcacagcagctcttcaactgcggagacggcctcacc 120 tacaatgactttctcattctccctgggtacatcgacttcactgcagaccaggtggacctg 180 acttctgctctgaccaagaaaatcactcttaagaccccactggtttcctctcccatggac 240 acagtcacagaggctgggatggccatagcaatggcgcttacaggcggtattggcttcatc 300 caccacaactgtacacctgaattccaggccaatgaagttcggaaagtgaagaaatatgaa 360 cagggattcatcacagaccctgtggtcctcagcccc 396 <210> 165 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400>

ttttttttttttttttttttttttttcangggncactgaggctttttattttgancncaa 60 aaccnccggggatctancctgnggccnccccggaaatnacncnaggctcacatnactnta 120 aacncttgggggaaagggaggcaaaaaaaacaatgacttgggccaattncncnactgcaa 180 agntananctgccaacagggctccagggagcttggnttntgtaaaanttntaaggaagcg 240 gnncnaactccncggggggggggcnctaactancagggacccctgcaagngttggncggg 300 ggcctcaacctgcctgagctnacncaaggggnggggtntntntanccaacaggggaccna 360 agggcttgcctncccacagnttacttggccaagggg 396 <210> 166 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 166 ttttttcaaattcagagcatttttattaaaagaacaaaatattaaggcacaaaatacatc 60 aatttttcaaatgaaaacccttcaaacggttatgtcctacattcaacgaaacttcttcca 120 aattacggaataatttaactttttaaaatanaaaaatacaagttcttaaatgcctaaaat 180 ttctccccaaataaatgttttcttagttttaatgaagtctcttcatgcagtactgagctc 240 caatattataatgtncacttccttaaaaatctagttttgccacttatatacattcaatat 300 gtttaaccagtatattaaccagtatattaaccaatatgttaaacttcttttaagtataag 360 gcttggtattttgtattgcttattgcatgctttgat 396 <210> 167 <211> 396 <212> DNA
<213> Homo sapien <400> 167 tggcggcagcggcggtggcggtggctgagcagaggacccggcgggcggcctcgcgggtca 60 ggacacaatgtttgcacgaggactgaagaggaaatgtgttggccacgaggaagacgtgga 120 gggagccctggccggcttgaagacagtgtcctcatacagcctgcagcggcagtcgctcct 180 ggacatgtctctggtgaagttgcagctttgccacatgcttgtggagcccaatctgtgccg 240 ctcagtcctcattgccaacacggtccggcagatccaagaggagatgacgcaggatgggac 300 gtggcgcacagtggcaccccaggctgcagagcgggcgccgctcgaccgcttggtctccac 360 ggagatcctgtgccgtgcagcgtgggggcaagaggg 396 <210> 168 <211> 396 <212> DNA
<213> Homo sapien <400>

taggatggtaagagtattataaggattggtacaaggcatgatgagtccttttgcttttag 60 gcttttgacttctggttttagactttctttagcttctgttgttagacaacattgtgcaag 120 cttggtttttataagtttgcatggattaaactgaacttaatgaaattgtccctcccccca 180 aattctcagcacaatttttaggcccacaaggagtcaagcacctcaaggagatcttcagtt 240 tgaacttggtgtagacacagggatactgatgaatcaatattcaaattagctgttacctac 300 ttaagaaagagaggagaccttggggatttcgaggaagggttcataagggagattttagct 360 gagaaataccatttgcacagtcaatcacttctgacc 396 <210> 169 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1). .(396) <223> n = A,T,C or G
<400> 169 tttttttttttttcanaattaaattctttaatacaaaatgcttttttttttttaaaanat60 atctgtatttctttgncgttgttnaaaaataaatatgtnctacggaatatntcnaaaaac120 tgcnctaaaaacaaanacgngatgttaatatcttttccccncaattnttacggataaaca180 gtanccccnataaataaatgatancnaatnttaaaattaaaaaagganananatttagta240 tgnaaaattctctattttttcttggtttggttttncntataaaaaacanaatagcaatgt300 ntnttttatcanaatcccntntntncctaaacntttttttttttntttncccccnaatnc360 aagnngccaaanatntntntagnatgnanatgtntn 396 <210> 170 <211> 396 <212> DNA
<213> Homo sapien <400> 170 tgagaagtac catgccgcttctgcagaggaacaggcaaccatcgaacgcaacccctacac 60 catcttccat caagcactgaaaaactgtgagcctatgattgggctggtacccatcctcaa 120 gggaggccgt ttctaccaggtccctgtacccctacccgaccggcgtcgccgcttcctagc 180 catgaagtgg atgatcactgagtgccgggataaaaagcaccagcggacactgatgccgga 240 gaagctgtca cacaagctgctggaggctttccataaccagggccccgtgatcaagaggaa 300 gcatgacttg cacaagatggcagaggccaaccgtgccctggcccactaccgctggtggta 360 gagtctccag gaggagcccagggccctctgcgcaag 396 <210> 171 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 171 ggtcctcgtcgtggtgagcgcagccactcaggctggtcctgggggtggggctgtagggga60 aagtgctaaagccgctgagtgaagtaagaactctgctagagaggaaaatgggcttgcttt120 catcatcatcctnctcagctggtggggtcaagtgggaagttctgtcactgggatctggtt180 cagtgtctcaagaccttgccccaccacggaaagcctttttcacntaccccaaaggacttg240 gagagatgttagaagatggntctnaaanattcctctgcnaatntgtttttagctatcaag300 tggcttccccccttaancaggnaaaacatgatcagcangttgctcggatggaaaaactan360 cttggtttgnnaaaaaanctggaggcttgacaatgg 396 <210> 172 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 172 agccttgggccaccctcttggagcatctggctgtcgaattcttgtgaccctgttacacac 60 actggagagaatgggcagaagtcgtggtgttgcagccctgtgcattgggggtgggatggg 120 aatagcaatgtgtgttcagagagaatgaattgcttaaactttgaacaacctcaatttctt 180 tttaaactaataaagtactaggttgcaatatgtgaaaaaaaaaaaaaaagggcggccgnt 240 cnantntanagggcccnttnaaacccgttgatcaacctcgactgtgccttctagttgcca 300 gccatctgttgttngcccctcccccgtgnctttcttgaccttgaaaggggccccncccct 360 gtctttcctaanaaaaangaagaantnnccttccnt 396 <210> 173 <211> 396 <212> DNA
<213> Homo sapien <220>
<221> misc_feature <222> (1) . . (396) <223> n = A,T,C or G
<400> 173 aagcatgtggatatgtttagctacgtttactcacagccagcgaactgacattaaaataac 60 taacaaacagattcttttatgtgatgctggaactcttgacagctataattattattcaga 120 aatgactttttgaaagtaaaagcagcataaagaatttgtcacaggaaggctgtctcagat 180 aaattatggtaaaattttgcaggggacannctttttaagacttgcacaattnccggatcc 240 tgcnctgactttggaaaaggcatatatgtnctagnggcatgganaatgccccatactcat 300 gcatgcaaattaaacaaccaagtttgaatctttttgggggngngctatnctttaacccng 360 tacnggcnttattatntaangnccctgnnncntgtg 396 <210> 174 <211> 924 <212> DNA
<213> Homo sapiens <400> 174 cctgacgacc cggcgacggc gacgtctctt ttgactaaaa gacagtgtcc agtgctccag 60 cctaggagtc tacggggacc gcctcccgcg ccgccaccat gcccaacttc tctggcaact 120 ggaaaatcat ccgatcggaa aacttcgagg aattgctcaa agtgctgggg gtgaatgtga 180 tgctgaggaa gattgctgtg gctgcagcgt ccaagccagc agtggagatc aaacaggagg 240 gagacacttt ctacatcaaa acctccacca ccgtgcgcac cacagagatt aacttcaagg 300 ttggggagga gtttgaggag cagactgtgg atgggaggcc ctgtaagagc ctggtgaaat 360 gggagagtga gaataaaatg gtctgtgagc agaagctcct gaagggagag ggccccaaga 420 cctcgtggac cagagaactg accaacgatg gggaactgat cctgaccatg acggcggatg 480 acgttgtgtg caccagggtc tacgtccgag agtgagtggc cacaggtaga accgcggccg 540 aagcccacca ctggccatgc tcaccgccct gcttcactgc cccctccgtc ccaccccctc 600 cttctaggat agcgctcccc ttaccccagt cacttctggg ggtcactggg atgcctcttg 660 cagggtcttg ctttctttga cctcttctct cctcccctac accaacaaag aggaatggct 720 gcaagagccc agatcaccca ttccgggttc actccccgcc tccccaagtc agcagtccta 780 gccccaaacc agcccagagc agggtctctc taaaggggac ttgagggcct gagcaggaaa 840 gactggccct ctagcttcta ccctttgtcc ctgtagccta tacagtttag aatatttatt 900 tgttaatttt attaaaatgc.ttta 924 <210> 175 <211> 3321 <212> DNA
<213> Homo sapiens <400> 175 atgaagattt tgatacttgg tatttttctg tttttatgta gtaccccagc ctgggcgaaa 60 gaaaagcatt attacattgg aattattgaa acgacttggg attatgcctc tgaccatggg 120 gaaaagaaac ttatttctgt tgacacggaa cattccaata tctatcttca aaatggccca 180 gatagaattg ggagactata taagaaggcc ctttatcttc agtacacaga tgaaaccttt 240 aggacaacta tagaaaaacc ggtctggctt gggtttttag gccctattat caaagctgaa 300 actggagata aagtttatgt acacttaaaa aaccttgcct ctaggcccta cacctttcat 360 tcacatggaa taacttacta taaggaacat gagggggcca tctaccctga taacaccaca 420 gattttcaaa gagcagatga caaagtatat ccaggagagc agtatacata catgttgctt 480 gccactgaag aacaaagtcc tggggaagga gatggcaatt gtgtgactag gatttaccat 540 tcccacattg atgctccaaa agatattgcc tcaggactca tcggaccttt aataatctgt 600 aaaaaagatt ctctagataa agaaaaagaa aaacatattg accgagaatt tgtggtgatg 660 ttttctgtgg tggatgaaaa tttcagctgg tacctagaag acaacattaa aacctactgc 720 tcagaaccag agaaagttga caaagacaac gaagacttcc aggagagtaa cagaatgtat 780 tctgtgaatg gatacacttt tggaagtctc ccaggactct ccatgtgtgc tgaagacaga 840 gtaaaatggt acctttttgg tatgggtaat gaagttgatg tgcacgcagc tttctttcac 900 gggcaagcac tgactaacaa gaactaccgt attgacacaa tcaacctctt tcctgctacc 960 ctgtttgatg cttatatggt ggcccagaac cctggagaat ggatgctcag ctgtcagaat 1020 ctaaaccatc tgaaagccgg tttgcaagcc tttttccagg tccaggagtg taacaagtct 1080 tcatcaaagg ataatatccg tgggaagcat gttagacact actacattgc cgctgaggaa 1140 atcatctgga actatgctcc ctctggtata gacatcttca ctaaagaaaa cttaacagca 1200 cctggaagtg actcagcggt gttttttgaa caaggtacca caagaattgg aggctcttat 1260 aaaaagctgg tttatcgtga gtacacagat gcctccttca caaatcgaaa ggagagaggc 1320 cctgaagaag agcatcttgg catcctgggt cctgtcattt gggcagaggt gggagacacc 1380 atcagagtaa ccttccataa caaaggagca tatcccctca gtattgagcc gattggggtg 1440 agattcaata agaacaacga gggcacatac tattccccaa attacaaccc ccagagcaga 1500 agtgtgcctc cttcagcctc ccatgtggca cccacagaaa cattcaccta tgaatggact 1560 gtccccaaag aagtaggacc cactaatgca gatcctgtgt gtctagctaa gatgtattat 1620 tctgctgtgg atcccactaa agatatattc actgggctta ttgggccaat gaaaatatgc 1680 aagaaaggaa gtttacatgc aaatgggaga cagaaagatg tagacaagga attctatttg 1740 tttcctacag tatttgatga gaatgagagt ttactcctgg aagataatat tagaatgttt 1800 acaactgcac ctgatcaggt ggataaggaa gatgaagact ttcaggaatc taataaaatg 1860 cactccatga atggattcat gtatgggaat cagccgggtc tcactatgtg caaaggagat 1920 tcggtcgtgt ggtacttatt cagcgccgga aatgaggccg atgtacatgg aatatacttt 1980 tcaggaaaca catatctgtg gagaggagaa cggagagaca cagcaaacct cttccctcaa 2040 acaagtctta cgctccacat gtggcctgac acagagggga cttttaatgt tgaatgcctt 2100 acaactgatc attacacagg cggcatgaag caaaaatata ctgtgaacca atgcaggcgg 2160 cagtctgagg attccacctt ctacctggga gagaggacat actatatcgc agcagtggag 2220 gtggaatggg attattcccc acaaagggag tgggaaaagg agctgcatca tttacaagag 2280 cagaatgttt caaatgcatt tttagataag ggagagtttt acataggctc aaagtacaag 2340 aaagttgtgt atcggcagta tactgatagc acattccgtg ttccagtgga gagaaaagct 2400 gaagaagaac atctgggaat tctaggtcca caacttcatg cagatgttgg agacaaagtc 2460 aaaattatct ttaaaaacat ggccacaagg ccctactcaa tacatgccca tggggtacaa 2520 acagagagtt ctacagttac tccaacatta ccaggtgaaa ctctcactta cgtatggaaa 2580 atcccagaaa gatctggagc tggaacagag gattctgctt gtattccatg ggcttattat 2640 tcaactgtgg atcaagttaa ggacctctac agtggattaa ttggccccct gattgtttgt 2700 cgaagacctt acttgaaagt attcaatccc agaaggaagc tggaatttgc ccttctgttt 2760 ctagtttttg atgagaatga atcttggtac ttagatgaca acatcaaaac atactctgat 2820 caccccgaga aagtaaacaa agatgatgag gaattcatag aaagcaataa aatgcatgct 2880 attaatggaa gaatgtttgg aaacctacaa ggcctcacaa tgcacgtggg agatgaagtc 2940 aactggtatc tgatgggaat gggcaatgaa atagacttac acactgtaca ttttcacggc 3000 catagcttcc aatacaagca caggggagtt tatagttctg atgtctttga cattttccct 3060 ggaacatacc aaaccctaga aatgtttcca agaacacctg gaatttggtt actccactgc 3120 catgtgaccg accacattca tgctggaatg gaaaccactt acaccgttct acaaaatgaa 3180 gacaccaaat ctggctgaat gaaataaatt ggtgataagt ggaaaaaaga gaaaaaccaa 3240 tgattcataa caatgtatgt gaaagtgtaa aatagaatgt tactttggaa tgactataaa 3300 cattaaaaga gactggagca t 3321 <210> 176 <211> 487 <212> DNA
<213> Homo sapiens <400> 176 gaaatacttt ctgtcttatt aaaattaata aattattggt ctttacaaga cttggataca 60 ttacagcaga catggaaata taattttaaa aaatttctct ccaacctcct tcaaattcag 120 tcaccactgt tatattacct tctccaggaa ccctccagtg gggaaggctg cgatattaga 180 tttccttgta tgcaaagttt ttgttgaaag ctgtgctcag aggaggtgag aggagaggaa 240 ggagaaaact gcatcataac tttacagaat tgaatctaga gtcttccccg aaaagcccag 300 aaacttctct gcagtatctg gcttgtccat ctggtctaag gtggctgctt cttccccagc 360 catgagtcag tttgtgccca tgaataatac acgacctgtt atttccatga ctgctttact 420 gtatttttaa ggtcaatata ctgtacattt gataataaaa taatattctc ccaaaaaaaa 480 aaaaaaa 487 <210> 177 <211> 3999 <212> DNA
<213> Homo sapiens <400> 177 caagattcca catttgatgg ggtgactgac aaacccatct tagactgctg tgcctgcgga 60 actgccaagt acagactcac attttatggg aattggtccg agaagacaca cccaaaggat 120 taccctcgtc gggccaacca ctggtctgcg atcatcggag gatcccactc caagaattat 180 gtactgtggg aatatggagg atatgccagc gaaggcgtca aacaagttgc agaattgggc 240 tcacccgtga aaatggagga agaaattcga caacagagtg atgaggtcct caccgtcatc 300 aaagccaaag cccaatggcc agcctggcag cctctcaacg tgagagcagc accttcagct 360 gaattttccg tggacagaac gcgccattta atgtccttcc tgaccatgat gggccctagt 420 cccgactgga acgtaggctt atctgcagaa gatctgtgca ccaaggaatg tggctgggtc 480 cagaaggtgg tgcaagacct gattccctgg gacgctggca ccgacagcgg ggtgacctat 540 gagtcaccca acaaacccac cattccccag gagaaaatcc ggcccctgac cagcctggac 600 catcctcaga gtcctttcta tgacccagag ggtgggtcca tcactcaagt agccagagtt 660 gtcatcgaga gaatcgcacg gaagggtgaa caatgcaata ttgtacctga caatgtcgat 720 gatattgtag ctgacctggc tccagaagag aaagatgaag atgacacccc tgaaacctgc 780 atctactcca actggtcccc atggtccgcc tgcagctcct ccacctgtga caaaggcaag 840 aggatgcgac agcgcatgct gaaagcacag ctggacctca gcgtcccctg ccctgacacc 900 caggacttcc agccctgcat gggccctggc tgcagtgacg aagacggctc cacctgcacc 960 atgtccgagt ggatcacctg gtcgccctgc agcatctcct gcggcatggg catgaggtcc 1020 cgggagaggt atgtgaagca gttcccggag gacggctccg tgtgcacgct gcccactgag 1080 gaaacggaga agtgcacggt caacgaggag tgctctccca gcagctgcct gatgaccgag 1140 tggggcgagt gggacgagtg cagcgccacc tgcggcatgg gcatgaagaa gcggcaccgc 1200 atgatcaaga tgaaccccgc agatggctcc atgtgcaaag ccgagacatc acaggcagag 1260 aagtgcatga tgccagagtg ccacaccatc ccatgcttgc tgtccccatg gtccgagtgg 1320 agtgactgca gcgtgacctg cgggaagggc atgcgaaccc gacagcggat gctcaagtct 1380 ctggcagaac ttggagactg caatgaggat ctggagcagg tggagaagtg catgctccct 1440 gaatgcccca ttgactgtga gctcaccgag tggtcccagt ggtcggaatg taacaagtca 1500 tgtgggaaag gccacgtgat tcgaacccgg atgatccaaa tggagcctca gtttggaggt 1560 gcaccctgcc cagagactgt gcagcgaaaa aagtgccgca tccgaaaatg ccttcgaaat 1620 ccatccatcc aaaagctacg ctggagggag gcccgagaga gccggcggag tgagcagctg 1680 aaggaagagt ctgaagggga gcagttccca ggttgtagga tgcgcccatg gacggcctgg 1740 tcagaatgca ccaaactgtg cggaggtgga attcaggaac gttacatgac tgtaaagaag 1800 agattcaaaa gctcccagtt taccagctgc aaagacaaga aggagatcag agcatgcaat 1860 gttcatcctt gttagcaagg gtacgagttc cccagggctg cactctagat tccagagtca 1920 ccaatggctg gattatttgc ttgtttaaga caatttaaat tgtgtacgct agttttcatt 1980 tttgcagtgt ggttcgccca gtagtcttgt ggatgccaga gacatccttt ctgaatactt 2040 cttgatgggt acaggctgag tggggcgccc tcacctccag ccagcctctt cctgcagagg 2100 agtagtgtca gccaccttgt actaagctga aacatgtccc tctggagctt ccacctggcc 2160 agggaggacg gagactttga cctactccac atggagaggc aaccatgtct ggaagtgact 2220 atgcctgagt cccagggtgc ggcaggtagg aaacattcac agatgaagac agcagattcc 2280 ccacattctc atctttggcc tgttcaatga aaccattgtt tgcccatctc ttcttagtgg 2340 aactttaggt ctcttttcaa gtctcctcag tcatcaatag ttcctgggga aaaacagagc 2400 tggtagactt gaagaggagc attgatgttg ggtggctttt gttctttcac tgagaaattc 2460 ggaatacatt tgtctcaccc ctgatattgg ttcctgatgc ccccccaaca aaaataaata 2520 aataaattat ggctgcttta tttaaatata aggtagctag tttttacacc tgagataaat 2580 aataagctta gagtgtattt ttcccttgct tttgggggtt cagaggagta tgtacaattc 2640 ttctgggaag ccagccttct gaactttttg gtactaaatc cttattggaa ccaagacaaa 2700 ggaagcaaaa ttggtctctt tagagaccaa tttgcctaaa ttttaaaatc ttcctacaca 2760 catctagacg ttcaagtttg caaatcagtt tttagcaaga aaacattttt gctatacaaa 2820 cattttgcta agtctgccca aagccccccc aatgcattcc ttcaacaaaa tacaatctct 2880 gtactttaaa gttattttag tcatgaaatt ttatatgcag agagaaaaag ttaccgagac 2940 agaaaacaaa tctaagggaa aggaatatta tgggattaag ctgagcaagc aattctggtg 3000 gaaagtcaaa cctgtcagtg ctccacacca gggctgtggt cctcccagac atgcatagga 3060 atggccacag gtttacactg ccttcccagc aattataagc acaccagatt cagggagact 3120 gaccaccaag ggatagtgta aaaggacatt ttctcagttg ggtccatcag cagtttttct 3180 tcctgcattt attgttgaaa actattgttt catttcttct tttataggcc ttattactgc 3240 ttaatccaaa tgtgtaccat tggtgagaca catacaatgc tctgaataca ctacgaattt 3300 gtattaaaca catcagaata tttccaaata caacatagta tagtcctgaa tatgtacttt 3360 taacacaaga gagactattc aataaaaact cactgggtct ttcatgtctt taagctaagt 3420 aagtgttcag aaggttcttt tttatattgt cctccacctc catcattttc aataaaagat 3480 agggcttttg ctcccttgtt cttggaggga ccattattac atctctgaac tacctttgta 3540 tccaacatgt tttaaatcct taaatgaatt gctttctccc aaaaaaagca caatataaag 3600 aaacacaaga tttaattatt tttctacttg gggggaaaaa agtcctcatg tagaagcacc 3660 cacttttgca atgttgttct aagctatcta tctaactctc agcccatgat aaagttcctt 3720 aagctggtga ttcctaatca aggacaagcc accctagtgt ctcatgtttg tatttggtcc 3780 cagttgggta cattttaaaa tcctgatttt ggagacttaa aaccaggtta atggctaaga 3840 atgggtaaca tgactcttgt tggattgtta ttttttgttt gcaatgggga atttataaga 3900 agcatcaagt ctctttctta ccaaagtctt gttaggtggt ttatagttct tttggctaac 3960 aaatcatttt ggaaataaag attttttact acaaaaatg 3999 <210> 178 <211> 1069 <212> DNA
<213> Homo Sapiens <400> 178 aaaaaagatg aataaatgaa taagagagat gaataaacaa atttacatta catgtgatag 60 ttatcatggt atggccttca tgacaagatg gatgagaata tcactgatag gatattagcc 120 ttctttcata tctttatatt gaaatatggg ctttacttca atttgaaggt ctttcatgaa 180 caataaaaga gagtagaagg actgtctgag aaggcaggag acatataaaa cagatgactg 240 aaagactgac tagctcctgg aaagggaaac atttggaaca tccagagtaa gggcaaatgg 300 gcttctacca gcacaacaaa gagcctccag gtggcaacat ggaagcaggt tatcagagaa 360 aataaatgtg caaattcctt atttacaatg actcacttaa ccccacaaac atgtttcact 420 gctgccttcc ccagttgtcg cttatgtact gttgttacct ttcagttaca tgcctttgat 480 cctaaaattc tctacttttg gtgccttatc agttctttgc aatctgcctg tggttatcag 540 cacttaaagc acaattttga aggggaaaaa aatgataatc accttagtcc caaagaaata 600 atttgtcaaa ctgccttatt agtattaaaa acagacacac tgaatgaagt agcatgatac 660 gcatatatcc tactcagtat cattggcctt ttatcaaatg gggaaactat acttttgtat 720 tacatagttt tagaaatcga aagttagaga ctctttataa gtaatgtcaa ggaacagtaa 780 tttaaaaaca aagttctaac aaatatattg tttgcttaat cacaatgccc tcaacttgta 840 tttgaataac taaataggac atgtcttcct tggagctgtg ggcattagtt cagaagcact 900 acctgcatct taattttcaa aacttaagtt ttattagcaa atcctcttct ctgtaagact 960 tagctatgaa gtggtatatt ttttccaaat atttttctga aaacatttgt tgttgtaact 1020 gcacaataaa agtccagttg caattaaaaa aaaaaaaaaa aaaaaaaaa 1069 <210> 179 <211> 1817 <212> DNA
<213> Homo Sapiens <400> 179 tgctattctg ccaaaagaca atttctagag tagttttgaa tgggttgatt tcccccactc 60 ccacaaactc tgaagccagt gtctagctta ctaaaaaaag agttgtatat aatatttaag 120 atgctgagta tttcatagga aagctgaatg ctgctgtaaa gtgctcttta agtctttttt 180 ttttttaatc cccttctaat gaatgaaact aggggaattt caggggacag agatgggatt 240 tgttgtatga taaactgtat gtagttttta gtctttctgt tttgagaagc agtggttggg 300 gcatttttaa gatggctggc tactcttgtt ttccctcatg ataataaatt tgtcataact 360 cagtaacatg aacttgcccc tagaggtagt tgttaataat tttgaaatat taaggtcttg 420 ccaagcttct gatgattcac acctgtacta ctgattatta agcaggacag actgagcttt 480 ctgttgcaaa taccttggag gagaaagtaa tttctaaata tacagagagg taacttgact 540 atatatgttg catcctgtgc ctcccttcat attaatattt gataaagatt ttaatttatg 600 taaaacttct aaagcagaat caaagctcct cttggggaaa tggcaagtct ttaggatagg 660 caagaccctg tatgaatagt accaaagcat taccgcatgg tagagaacac actcgattaa 720 aaatgttaag ctatctgaaa aataaaatgt gcaagtcttc aggatggcac aaaacaaagg 780 ttaatgcttc ttggggcaca tttcttagag ggcttgctga gtgtgtaaat ataatcgact 840 tttgtttgtg ttacatgact tctgtgactt cattgaaaat ctgcacaatt cagtttcagc 900 tctggattac ttcagttgac ctttgtgaag gtttttatct gtgtagaatg ggtgtttgac 960 ttgttttagc ctattaaatt tttattttct ttcactctgt attaaaagta aaacttacta 1020 aaagaaaaga ggtttgtgtt cacattaaat ggttttggtt tggcttcttt tagtcaggct 1080 ttctgaacat tgagatatcc tgaacttaga gctcttcaat cctaagattt tcatgaaaag 1140 cctctcactt gaacccaaac cagagtactc ttactgcctc ttttctaaat gttcaggaaa 1200 agcattgcca gttcagtctt ttcaaaatga gggagaaaca tttgcctgcc ttgtaataac 1260 aagactcagt gcttattttt taaactgcat tttaaaaatt ggatagtata ataacaataa 1320 ggagtaagcc accttttata ggcaccctgt agttttatag ttcttaatct aaacatttta 1380 tatttccttc ttttggaaaa aacctacatg ctacaagcca ccatatgcac agactataca 1440 gtgagttgag ttggctctcc cacagtcttt gaggtgaatt acaaaagtcc agccattatc 1500 atcctcctga gttatttgaa atgatttttt ttgtacattt tggctgcagt attggtggta 1560 gaatatacta taatatggat catctctact tctgtattta tttatttatt actagacctc 1620 aaccacagtc ttctttttcc ccttccacct ctctttgcct gtaggatgta ctgtatgtag 1680 tcatgcactt tgtattaata tattagaaat ctacagatct gttttgtact ttttatactg 1740 ttggatactt ataatcaaaa cttttactag ggtattgaat aaatctagtc ttactagaaa 1800 aaaaaaaaaa aaaaaaa 1817 <210> 180 <211> 2382 <212> DNA
<213> Homo Sapiens <400> 180 acttttattg gaagcagcag ccacatccct gcatgatttg cattgcaata caaccataac 60 cgggcagcca ctcctgagtg ataaccagta taacataaac gtagcagcct caatttttgc 120 ctttatgacg acagcttgtt atggttgcag tttgggtctg gctttacgaa gatggcgacc 180 gtaacactcc ttagaaactg gcagtcgtat gttagtttca cttgtctact ttatatgtct 240 gatcaatttg gataccattt tgtccagatg caaaaacatt ccaaaagtaa tgtgtttagt 300 agagagagac tctaagctca agttctggtt tatttcatgg atggaatgtt aattttatta 360 tgatattaaa gaaatggcct tttattttac atctctcccc tttttccctt tcccccttta 420 ttttcctcct tttctttctg aaagtttcct tttatgtcca taaaatacaa atatattgtt 480 cataaaaaat tagtatccct tttgtttggt tgctgagtca cctgaacctt aattttaatt 540 ggtaattaca gcccctaaaa aaaacacatt tcaaataggc ttcccactaa actctatatt 600 ttagtgtaaa ccaggaattg gcacactttt tttagaatgg gccagatggt aaatatttat 660 gcttcacggt ccatacagtc tctgtcacaa ctattcagtt ctgctagtat agcgtgaaag 720 cagctataca caatacagaa atgaatgagt gtggttatgt tctaataaaa cttatttata 780 aaaacaaggg gaggctgggt ttagcctgtg ggccatagtt tgtcaaccac tggtgtaaaa 840 ccttagttat atatgatctg cattttcttg aactgatcat tgaaaactta taaacctaac 900 agaaaagcca cataatattt agtgtcatta tgcaataatc acattgcctt tgtgttaata 960 gtcaaatact tacctttgga gaatacttac ctttggagga atgtataaaa tttctcaggc 1020 agagtcctgg atataggaaa aagtaattta tgaagtaaac ttcagttgct taatcaaact 1080 aatgatagtc taacaactga gcaagatcct. catctgagag tgcttaaaat gggatcccca 1140 gagaccatta accaatactg gaactggtat ctagctactg atgtcttact ttgagtttat 1200 ttatgcttca gaatacagtt gtttgccctg tgcatgaata tacccatatt tgtgtgtgga 1260 tatgtgaagc ttttccaaat agagctctca gaagaattaa gtttttactt ctaattattt 1320 tgcattactt tgagttaaat ttgaatagag tattaaatat aaagttgtag attcttatgt 1380 gtttttgtat tagcccagac atctgtaatg tttttgcact ggtgacagac aaaatctgtt 1440 ttaaaatcat atccagcaca aaaactattt ctggctgaat agcacagaaa agtattttaa 1500 cctacctgta gagatcctcg tcatggaaag gtgccaaact gttttgaatg gaaggacaag 1560 taagagtgag gccacagttc ccaccacacg agggcttttg tattgttcta ctttttcagc 1620 cctttacttt ctggctgaag catccccttg gagtgccatg tataagttgg gctattagag 1680 ttcatggaac atagaacaac catgaatgag tggcatgatc cgtgcttaat gatcaagtgt 1740 tacttatcta ataatcctct agaaagaacc ctgttagatc ttggtttgtg ataaaaatat 1800 aaagacagaa gacatgagga aaaacaaaag gtttgaggaa atcaggcata tgactttata 1860 cttaacatca gatcttttct ataatatcct actactttgg ttttcctagc tccataccac 1920 acacctaaac ctgtattatg aattacatat tacaaagtca taaatgtgcc atatggatat 1980 acagtacatt ctagttggaa tcgtttactc tgctagaatt taggtgtgag attttttgtt 2040 tcccaggtat agcaggctta tgtttggtgg cattaaattg gtttctttaa aatgctttgg 2100 tggcactttt gtaaacagat tgcttctaga ttgttacaaa ccaagcctaa gacacatctg 2160 tgaatactta gatttgtagc ttaatcacat tctagacttg tgagttgaat gacaaagcag 2220 ttgaacaaaa attatggcat ttaagaattt aacatgtctt agctgtaaaa atgagaaagt 2280 gttggttggt tttaaaatct ggtaactcca tgatgaaaag aaatttattt tatacgtgtt 2340 atgtctctaa taaagtattc atttgataaa aaaaaaaaaa as 2382 <210> 181 <211> 2377 <212> DNA
<213> Homo Sapiens <400> 181 atctttatgc aagacaagag tcagccatca gacactgaaa tatattatga tagattatga 60 agaattttct ctgtagaatt atattcttcc tggaacctgg tagagtagat tagactcaaa 120 ggctttttct tccttttctt actcctgttt tttccactca ctcttcccaa gagatttcct 180 aaagcttcaa gcttaataag cctaatagtg aaaaataact gaatttaatg gtataatgaa 240 gttcttcatt tccagacatc tttaattgat cttaaagctc atttgagtct ttgcccctga 300 acaaagacag acccattaaa atctaagaat tctaaatttt cacaactgtt tgagcttctt 360 ttcattttga aggatttgga atatatatgt tttcataaaa gtatcaagtg aaatatagtt 420 acatgggagc tcaatcatgt gcagattgca ttctgttatg ttgactcaat atttaattta 480 caactatcct tatttatatt gacctcaaga actccatttt atgcaatgca gaccactgag 540 atatagctaa cattctttca aataattttc cttttctttt ataattcctc tatagcaaat 600 ttttatgtat aactgattat acatatccat atttatattt cattgattcc aagacatcac 660 tttttcaatt taacatctct gaaattgtga catttcttgc aactgttggc acttcagatg 720 cagtgtttaa aattatgctt gaataaatat tacactaatc caactttacc taaatgttta 780 tgcatctagg caaattttgt tttcttataa agatttgaga gcccatttat gacaaaatat 840 gaaggcgaaa tttaaggaca actgagtcac gcacaactca acatggagcc taactgatta 900 tcagctcaga tcccgcatat cttgagttta caaaagctct ttcaggtccc catttatact 960 ttacgtgagt gcgaatgatt tcagcaaacc ctaacttaac taacaagaat gggtaggtat 1020 gtctacgttt cattaacaaa tttttattat ttttattcta ttatatgaga tccttttata 1080 ttatcatctc acttttaaac aaaattaact ggaaaaatat tacatggaac tgtcatagtt 1140 aggttttgca gcatcttaca tgtcttgtat caatggcagg agaaaaatat gataaaaaca 1200 atcagtgctg tgaaaaacaa ctttcttcta gagtcctctt actttttatt cttctttatc 1260 atttgtgggt ttttccccct tggctctcac tttaacttca agcttatgta acgactgtta 1320 taaaactgca tatttaaatt atttgaatta tatgaaataa ttgttcagct atctgggcag 1380 ctgttaatgt aaacctgaga gtaataacac tactctttta tctacctgga atacttttct 1440 gcataaaatt tatctttgta agctaactct attaatcagg tttcttctag cctctgcaac 1500 ctacttcagt tagaattgtc taatactgct ctattaatca ggtttctacc ctctacaacc 1560 tacttcagtt aaaattgtct aatacagcaa tatttaaaaa aaaaacactg caattgtcaa 1620 ggatggaaaa tgtgtgattt gtgtaaacaa tttttaccaa ctttacattt tcctacagat 1680 aaatgtgaaa ttttgataag aagtctacgc aatgacaagt acggtacata aattttatta 1740 agaatattga gtataaagta ctttaattct aaattataag aaaatataca tttgcacata 1800 ttaatataga aattcatttt gtgtatattt aacatagctt ttaaactatt ttacattagc 1860 tacttcatta tggtttcttg aacttctgaa aaaaattaga aatgtattaa acttatcagt 1920 aacataaaaa cttattttgt ttcacctaac gaatactgcg tttgtaaaaa taaatttaat 1980 atagaatata tttttaaatt aaatatttga atataaaata gctctaagaa agaagcaaat 2040 tatcactgaa catatttctt attatttctg gctttgaatt atacgtaact taaattgtct 2100 taaatgatac agaatattgg agaatatgat actttcacat aatatactat gaacctgttc 2160 atataactct gattgactac taacttctgt tttatgtatt tattaaagag ctgacactgt 2220 agtttgtggt gagatgttta tttttctaac agagcttata acagttagga caaggcattt 2280 aattaatgca tcattctgtt tagtagtagg tgttaatcaa tatgaaattc tctgttttaa 2340 aataaaaatg taaaaatcta aaaaaaaaaa aaaaaaa 2377 <210> 182 <211> 1370 <212> DNA
<213> Homo Sapiens <400> 182 tgtgagcatg gtattttgtc tcggaagaaa aaaatatggg tcaggcgcaa agtaagccca 60 ccccactggg aactatgtta aaaaaaaatt tcaagattta agggagatta cggtgttact 120 atgacaccag aaaaacttag aactttgtgt gaaatagact ggctaacatt agaggtgggt 180 tggctatcag aagaaagcct ggagaggtcc cttgtttcaa aggtatggca caaggtaacc 240 tgtaagccaa agcacccgga ccagtttcta tacatagaca gttacagctg gtttagaccc 300 cttccccctc tccccacagt agttaagaga acagcagcat aagcagctgg cagaggcaag 360 gaaagaccag cagagagaaa aaaaggccat ctataccaat tttaagttaa tttagactga 420 acaagggctt attaatagca aaggataatt gaaatcacaa acttataagg gtttcaacaa 480 aagtgaagtt tgctaaaagt taacagtgta acatgtatta tggtaacttc taatcttgtg 540 gccttagaca gtctagtcaa aacacataaa gaaagtttgc tttaaaaaaa caatggttat 600 cttcaaaaat aaaggggaga ggcagaattt atataaaaag agttatatga taaattcttg 660 tcctgaaata aattaactgg ttgtttaaag aaaagaatgt ttgtaataag tcaaaaagtt 720 aaaacatgtt taaaaaattg tctgcaaaag tcataaaaga aaaaatttta ttaaaaaaat 780 tttaagcaaa aaatgttgta taatttaaaa gtaataaggc ctcctgtgta ctattaagac 840 agatgcaaat tcctggttga aatggatcaa atattccatc tgcacattaa acaaaagcaa 900 ttgttatgct tgtgcacatg gcaggccaga ggccctgatt gtcccccttc cactaaggtg 960 gtcctctagt cgaccaggcg tggactgcat ggtagctctt ttccaggatt ctacagcctg 1020 gagtaataag tcatgccaag ctctctctgc tatatcccaa agtctctgcg ggtcagcccc 1080 caagggccat gcagcttctg tctcccaaca ctaagttcac ttcgtgtctc tcacggcaga 1140 gaggaaactt agtattcctt ggagacctga agggatgcag tgagcttaag aattttcaag 1200 agcttatcaa tcagtcagcc cttgttcatc cccgagtgga tgtgtggtgg tattgtggtg 1260 gacctttact gggcactctg ccaaataact agtgtggcac ttgtgcttta gtccatttgg 1320 ctatcccttt caccctggca tttcatcaac caaaaaaaaa aaaaaaaaaa 1370 <210> 183 <211> 2060 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <222> (1). .(2060) <223> n=A,T,C or G
<400> 183 gtttcagggg aggagacaag gtttcttgtt tgccgtatat gctcctgcag agaagaggaa 60 gtgaccgtgg aggccatctg gccctgtgtt ttgatatggc aaaattaatg aatgcaatca 120 gaagaccttt gagcaagaaa gtaccctgga acaacccaat ttggactgca agtattagtt 180 gggtcttcca ggtgcctctc acagcagcag tcatggcagc agtgactcta gccatgtcca 240 tgaccaactg ctgcataaca aatagccccg agactcagca gcttacaaca gggtccccag 300 cccacagact ggcactggtc catggcttgt taggaacctg actgcgcagc agaaggtgag 360 tgagcattac tgcctgagct ctgcctcctg tcagatcatc aggggcatta gattctcata 420 ggagcgtgaa ccctattgca aaccgcgcat gcgaaggatg tacgttgcgt gctccttatg 480 agaatctaac taatgcctga tgatttgagg tggggcagtt tcatccccaa accatctctc 540 tcccttcatg tccatggaaa aattgtcttc tacaaaacca gtccgtggtg ccaaaaaggt 600 tggagactgc tggtttacaa ccgcaatgaa cattcatcat cccacacagt gtcagagggt 660 cgggaacacg ggtgccctgc ctgtgtgctt ccggttccag atttctcagt gggttgtgat 720 caaggtatca gcggaggccg tattcatctg caagcttgac caggaataga agagccactt 780 catgggtggc tcactcagat gccagcaggt cagtgctggt ggctggcagg cagcctcagc 840 tcctcacctc atggatctct cctgagcaca gttttcctgt ccttacaacc tggtagctgg 900 cttctccaga gcaggtgact caggagagga caaggtgaga gcccagcacc ttatggtcta 960 gtctcagaag tcacacgcca tcatttctgc aatgtcattt tggggttcca ggtcagctgt 1020 atcactgtgg gaggtgagta tatagatgtc ctagaccatt caggctgcta tgacagaaca 1080 ccatgaactg agtggctcat gaacaacaga aatttcccac agttctgtag gctgggaaat 1140 ccaagatcaa ggtggcagca ggttcagcgt ctgctaagct cctgcttttc atggattgca 1200 tcttctcact gtgtcctcac gtgatggaca gagcaaatga gctctcaggc actagtccca 1260 gccatgagga ctctgctttc atgactcatc actccgcaaa ggcccacctc catcagaaga 1320 cagctgctaa ctgcagctgc catcctccaa gacgggagac acagaattgg gggacatata 1380 cattgagatc tgaaaggcct ggacagcaac aggtggggat cgtgggggca tcttggaggg 1440 tggctgccgc agtaacattt ctgacccatg ctttctgctt gcactcatct cctgcctttg 1500 atcttcatta tctcargcag tccccacaac gactgtatct aggagttcat tttaccctca 1560 ttttacagat gaaacgtctc agagggtaat gtgcttgccc agtgtctcac aaatgcaaag 1620 tcactgaggt aggatttcaa cctaggtcca atcatctctg cagcattagg ggttcaccat 1680 tgccatagac ttaactgtgt cccccaaaat ttgtatgttg aagccctacc agcctccccc 1740 ccccaatgtg ctgatgtttg gagaaagggc ctttgggagg taattaggtt tagatgagat 1800 catgagggtg ggactctcat aatggcatta atgccatcag gtgaagagat accagagacc 1860 ttgtgtcctc tctctctgca atgtgaggac acagtgagaa ggcagctgtc tgcaagctgg 1920 gaagagagta ctgaccagga acttaatcag agggcatctt gatcttggac ttcccagcct 1980 ccagaactct gaaaagttaa tgnctattat ttaagccacg cagtctatgg aattttgtta 2040 gagccaaccc caagcttact 2060 <210> 184 <211> 3079 <212> DNA
<213> Homo sapiens <400> 184 ggcacaaagt tgggggccgc gaagatgagg ctgtccccgg cgcccctgaa gctgagccgg 60 actccggcac tgctggccct ggcgctgccc ctggccgcgg cgctggcctt ctccgacgag 120 accctggaca aagtgcccaa gtcagagggc tactgtagcc gtatcctgcg cgcccagggc 180 acgcggcgcg agggctacac cgagttcagc ctccgcgtgg agggcgaccc cgacttctac 240 aagccgggaa ccagctaccg cgtaacactt tcagctgctc ctccctccta cttcagagga 300 ttcacattaa ttgccctcag agagaacaga gagggtgata aggaagaaga ccatgctggg 360 accttccaga tcatagacga agaagaaact cagtttatga gcaattgccc tgttgcagtc 420 actgaaagca ctccacggag gaggacccgg atccaggtgt tttggatagc accaccagcg 480 ggaacaggct gcgtgattct gaaggccagc atcgtacaaa aacgcattat ttattttcaa 540 gatgagggct ctctgaccaa gaaactttgt gaacaagatt ccacatttga tggggtgact 600 gacaaaccca tcttagactg ctgtgcctgc ggaactgcca agtacagact cacattttat 660 gggaattggt ccgagaagac acacccaaag gattaccctc gtcgggccaa ccactggtct 720 gcgatcatcg gaggatccca ctccaagaat tatgtactgt gggaatatgg aggatatgcc 780 agcgaaggcg tcaaacaagt tgcagaattg ggctcacccg tgaaaatgga ggaagaaatt 840 cgacaacaga gtgatgaggt cctcaccgtc atcaaagcca aagcccaatg gccagcctgg 900 cagcctctca acgtgagagc agcaccttca gctgaatttt ccgtggacag aacgcgccat 960 ttaatgtcct tcctgaccat gatgggccct agtcccgact ggaacgtagg cttatctgca 1020 gaagatctgt gcaccaagga atgtggctgg gtccagaagg tggtgcaaga cctgattccc 1080 tgggacgctg gcaccgacag cggggtgacc tatgagtcac ccaacaaacc caccattccc 1140 caggagaaaa tccggcccct gaccagcctg gaccatcctc agagtccttt ctatgaccca 1200 gagggtgggt ccatcactca agtagccaga gttgtcatcg agagaatcgc acggaagggt 1260 gaacaatgca atattgtacc tgacaatgtc gatgatattg tagctgacct ggctccagaa 1320 gagaaagatg aagatgacac ccctgaaacc tgcatctact ccaactggtc cccatggtcc 1380 gcctgcagct cctccacctg tgacaaaggc aagaggatgc gacagcgcat gctgaaagca 1440 cagctggacc tcagcgtccc ctgccctgac acccaggact tccagccctg catgggccct 1500 ggctgcagtg acgaagacgg ctccacctgc accatgtccg agtggatcac ctggtcgccc 1560 tgcagcatct cctgcggcat gggcatgagg tcccgggaga ggtatgtgaa gcagttcccg 1620 gaggacggct ccgtgtgcac gctgcccact gaggaaatgg agaagtgcac ggtcaacgag 1680 gagtgctctc ccagcagctg cctgatgacc gagtggggcg agtgggacga gtgcagcgcc 1740 acctgcggca tgggcatgaa gaagcggcac cgcatgatca agatgaaccc cgcagatggc 1800 tccatgtgca aagccgagac atcacaggca gagaagtgca tgatgccaga gtgccacacc 1860 atcccatgct tgctgtcccc atggtccgag tggagtgact gcagcgtgac ctgcgggaag 1920 ggcatgcgaa cccgacagcg gatgctcaag tctctggcag aacttggaga ctgcaatgag 1980 gatctggagc aggtggagaa gtgcatgctc cctgaatgcc ccattgactg tgagctcacc 2040 gagtggtccc agtggtcgga atgtaacaag tcatgtggga aaggccacgt gattcgaacc 2100 cggatgatcc aaatggagcc tcagtttgga ggtgcaccct gcccagagac tgtgcagcga 2160 aaaaagtgcc gcatccgaaa atgccttcga aatccatcca tccaaaagcc acgctggagg 2220 gaggcccgag agagccggcg gagtgagcag ctgaaggaag agtctgaagg ggagcagttc 2280 ccaggttgta ggatgcgccc atggacggcc tggtcagaat gcaccaaact gtgcggaggt 2340 ggaattcagg aacgttacat gactgtaaag aagagattca aaagctccca gtttaccagc 2400 tgcaaagaca agaaggagat cagagcatgc aatgttcatc cttgttagca agggtacgag 2460 ttccccaggg ctgcactcta gattccagag tcaccaatgg ctggattatt tgcttgttta 2520 agacaattta aattgtgtac gctagttttc atttttgcag tgtggttcgc ccagtagtct 2580 tgtggatgcc agagacatcc tttctgaata cttcttgatg ggtacaggct gagtggggcg 2640 ccctcacctc cagccagcct cttcctgcag aggagtagtg tcagccacct tgtactaagc 2700 tgaaacatgt ccctctggag cttccacctg gccagggagg acggagactt tgacctactc 2760 cacatggaga ggcaaccatg tctggaagtg actatgcctg agtcccaggg tgcggcaggt 2820 aggaaacatt cacagatgaa gacagcagat tccccacatt ctcatctttg gcctgttcaa 2880 tgaaaccatt gtttgcccat ctcttcttag tggaacttta ggtctctttt caagtctcct 2940 cagtcatcaa tagttcctgg ggaaaaacag agctggtaga cttgaagagg agcattgatg 3000 ttgggtggct tttgttcttt cactgagaaa ttcggaatac atttgtctca cccctgatat 3060 tggttcctga tgccccagc 3079 <210> 185 <211> 3000 <212> DNA
<213> Homo Sapiens <400> 185 gtttcagggg aggagacaag gtttcttgtt tgccgtatat gctcctgcag agaagaggaa 60 gtgaccgtgg aggccatctg gccctgtgtt ttgatatggc aaaattaatg aatgcaatca 120 gaagaccttt gagcaagaaa gtaccctgga acaacccaat ttggactgca agtattagtt 180 gggtcttcca ggtgcctctc acagcagcag tcatggcagc agtgactcta gccatgtcca 240 tgaccaactg ctgcataaca aatagccccg agactcagca gcttacaaca gggtccccag 300 cccacagact ggcactggtc catggcttgt taggaacctg actgcgcagc agaaggtgag 360 tgagcattac tgcctgagct ctgcctcctg tcagatcatc aggggcatta gattctcata 420 ggagcgtgaa ccctattgca aaccgcgcat gcgaaggatg tacgttgcgt gctccttatg 480 agaatctaac taatgcctga tgatttgagg tggggcagtt tcatccccaa accatctctc 540 tcccttcatg tccatggaaa aattgtcttc tacaaaacca gtccgtggtg ccaaaaaggt 600 tggagactgc tggtttacaa ccgcaatgaa cattcatcat cccacacagt gtcagagggt 660 cgggaacacg ggtgccctgc ctgtgtgctt ccggttccag atttctcagt gggttgtgat 720 caaggtatca gcggaggccg tattcatctg caagcttgac caggaataga agagccactt 780 catgggtggc tcactcagat gccagcaggt cagtgctggt ggctggcagg cagcctcagc 840 tcctcacctc atggatctct cctgagcaca gttttcctgt ccttacaacc tggtagctgg 900 cttctccaga gcaggtgact caggagagga caaggtgaga gccacagcac cttatggtct 960 agtctcagaa gtcacacgcc atcatttctg caatgtcatt ttggggttcc aggtcagctg 1020 tatcactgtg ggaggtgagt atatagatgt cctagaccat tcaggctgct atgacagaac 1080 accatgaact gagtggctca tgaacaacag aaatttccca cagttctgta ggctgggaaa 1140 tccaagatca aggtggcagc aggttcagcg tctgctaagc tcctgctttt catggattgc 1200 atcttctcac tgtgtcctca cgtgatggac agagcaaatg agctctcagg cactagtccc 1260 agccatgagg actctgcttt catgactcat cactccgcaa aggcccacct ccatcagaag 1320 acagctgcta actgcagctg ccatcctcca agacgggaga cacagaattg ggggacatat 1380 acattgagat ctgaaaggcc tggacagcaa caggtgggga tcgtgggggc atcttggagg 1440 gtggctgccg cagtaacatt tctgacccat gctttctgct tgcactcatc tcctgccttt 1500 gatcttcatt atctcaggca gtccccacaa cgactgtatc taggagttca ttttaccctc 1560 attttacaga tgaaacgtct cagagggtaa tgtgcttgcc cagtgtctca caaatgcaaa 1620 gtcactgagg taggatttca acctaggtcc aatcatctct gcagcattag gggttcacca 1680 ttgccataga cttaactgtg tcccccaaaa tttgtatgtt gaagccctac cagcctcccc 1740 cccccaatgt gctgatgttt ggagaaaggg cctttgggag gtaattaggt ttagatgaga 1800 tcatgagggt gggactctca taatggcatt aatgccatca ggtgaagaga taccagagac 1860 cttgtgtcct ctctctctgc aatgtgagga cacagtgaga aggcagctgt ctgcaagctg 1920 ggaagagagt actgaccagg aacttaatca gagggcatct tgatcttgga cttcccagcc 1980 tccagaactc tgaaaagtta atgtctatta tttaagccac gcagtctatg gaattttgtt 2040 agagccaacc caagcttact aagataatca gtatgctgca ctttctataa atgtaatttt 2100 tacatttata aaaacaaaac aagagatttg ctgctctata acaactgtac ctacattgta 2160 gatggaataa caaatctaca tacagattta gtaatctcta tgtagatata gaacatagtg 2220 tatctaatag agacatagtg tctgtggtct gatgttaatt ttaggaatta gccgtcactg 2280 attgggcctt gtccaggtat tcttctccct tgtcctggct ctgtaaccta gttatccttg 2340 tctttgctaa cccataacca actattgtat caggactatt atgccactac agatgatgca 2400 gtttgggttt actgtttctc accatttaga caatacttca tcaaatatat ttctgtatga 2460 ctttagtgat atcagttttt gattcattcc tgcatagatc tgggcaaatt gtagacctta 2520 ggaggtgtat tcaccatcca gttctctgga actgcttatg acatttttct ctgagctttc 2580 ttgtcccaaa aggagccttc ctaaaatagt ctttaagtgc ctttaaaaag agaaagagaa 2640 attaagagaa aaaaaacccc aaactcattc ctttactctg atgtgacagt cctcccagga 2700 cactgcagtg gcctgagttt tgctgttaat ttcattcact tatgtttggg ctatgtaaat 2760 tctgcctaga gctggaatgt cattatgtaa agaaatattt tttgtttata ttctttaata 2820 gtaccagtaa tgtatatctt attcagcttc gagaatataa ttgggttgtt tataaaaacc 2880 acacatcatc aaactcacat tgtaacgatt atttcacttt tcaaaaaaaa tggcattaga 2940 aaaacttgaa tgatgttagt tatcttaaag aagtgtgtac tatgtttaaa aaaaaaaaaa 3000 <210> 186 <211> 807 <212> PRT
<213> Homo Sapiens <400> 186 Met Arg Leu Ser Pro Ala Pro Leu Lys Leu Ser Arg Thr Pro Ala Leu Leu Ala Leu Ala Leu Pro Leu Ala Ala Ala Leu Ala Phe Ser Asp Glu Thr Leu Asp Lys Val Pro Lys Ser Glu Gly Tyr Cys Ser Arg Ile Leu Arg Ala Gln Gly Thr Arg Arg Glu Gly Tyr Thr Glu Phe Ser Leu Arg Val Glu Gly Asp Pro Asp Phe Tyr Lys Pro Gly Thr Ser Tyr Arg Val Thr Leu Ser Ala Ala Pro Pro Ser Tyr Phe Arg Gly Phe Thr Leu Ile Ala Leu Arg Glu Asn Arg Glu Gly Asp Lys Glu Glu Asp His Ala Gly Thr Phe Gln Ile Ile Asp Glu Glu Glu Thr Gln Phe Met Ser Asn Cys Pro Val Ala Val Thr Glu Ser Thr Pro Arg Arg Arg Thr Arg Ile Gln Val Phe Trp Ile Ala Pro Pro Ala Gly Thr Gly Cys Val Ile Leu Lys Ala Ser Ile Val Gln Lys Arg Ile Ile Tyr Phe Gln Asp Glu Gly Ser Leu Thr Lys Lys Leu Cys Glu Gln Asp Ser Thr Phe Asp Gly Val Thr Asp Lys Pro Ile Leu Asp Cys Cys Ala Cys Gly Thr Ala Lys Tyr Arg Leu Thr Phe Tyr Gly Asn Trp Ser Glu Lys Thr His Pro Lys Asp Tyr Pro Arg Arg Ala Asn His Trp Ser Ala Ile Ile Gly Gly Ser His Ser Lys Asn Tyr Val Leu Trp Glu Tyr Gly Gly Tyr Ala Ser Glu Gly Val Lys Gln Val Ala Glu Leu Gly Ser Pro Val Lys Met Glu Glu Glu Ile Arg Gln Gln Ser Asp Glu Val Leu Thr Val Ile Lys Ala Lys Ala Gln Trp Pro Ala Trp Gln Pro Leu Asn Val Arg Ala Ala Pro Ser Ala Glu Phe Ser Val Asp Arg Thr Arg His Leu Met Ser Phe Leu Thr Met Met Gly Pro Ser Pro Asp Trp Asn Val Gly Leu Ser Ala Glu Asp Leu Cys Thr Lys Glu Cys Gly Trp Val Gln Lys Val Val Gln Asp Leu Ile Pro Trp Asp Ala Gly Thr Asp Ser Gly Val Thr Tyr Glu Ser Pro Asn Lys Pro Thr Ile Pro Gln Glu Lys Ile Arg Pro Leu Thr Ser Leu Asp His Pro Gln Ser Pro Phe Tyr Asp Pro Glu Gly Gly Ser Ile Thr Gln Val Ala Arg Val Val Ile Glu Arg Ile Ala Arg Lys Gly Glu Gln Cys Asn Ile Val Pro Asp Asn Val Asp Asp Ile Val Ala Asp Leu Ala Pro Glu Glu Lys Asp Glu Asp Asp Thr Pro Glu Thr Cys Ile Tyr Ser Asn Trp Ser Pro Trp Ser Ala Cys Ser Ser Ser Thr Cys Asp Lys Gly Lys Arg Met Arg Gln Arg Met Leu Lys Ala Gln Leu Asp Leu Ser Val Pro Cys Pro Asp Thr Gln Asp Phe Gln Pro Cys Met Gly Pro Gly Cys Ser Asp Glu Asp Gly Ser Thr Cys Thr Met Ser Glu Trp Ile Thr Trp Ser Pro Cys Ser Ile Ser Cys Gly Met Gly Met Arg Ser Arg Glu Arg Tyr Val Lys Gln Phe Pro Glu Asp Gly Ser Val Cys Thr Leu Pro Thr Glu Glu Met Glu Lys Cys Thr Val Asn Glu Glu Cys Ser Pro Ser Ser Cys Leu Met Thr Glu Trp Gly Glu Trp Asp Glu Cys Ser Ala Thr Cys Gly Met Gly Met Lys Lys Arg His Arg Met Ile Lys Met Asn Pro Ala Asp Gly Ser Met Cys Lys Ala Glu Thr Ser Gln Ala Glu Lys Cys Met Met Pro Glu Cys His Thr Ile Pro Cys Leu Leu Ser Pro Trp Ser Glu Trp Ser Asp Cys Ser Val Thr Cys Gly Lys Gly Met Arg Thr Arg Gln Arg Met Leu Lys Ser Leu Ala Glu Leu Gly Asp Cys Asn Glu Asp Leu Glu Gln Val Glu Lys Cys Met Leu Pro Glu Cys Pro Ile Asp Cys Glu Leu Thr Glu Trp Ser Gln Trp Ser Glu Cys Asn Lys Ser Cys Gly Lys Gly His Val Ile Arg Thr Arg Met Ile Gln Met Glu Pro Gln Phe Gly Gly Ala Pro Cys Pro Glu Thr Val Gln Arg Lys Lys Cys Arg Ile Arg Lys Cys Leu Arg Asn Pro Ser Ile Gln Lys Pro Arg Trp Arg Glu Ala Arg Glu Ser Arg Arg Ser Glu Gln Leu Lys Glu Glu Ser Glu Gly Glu Gln Phe Pro Gly Cys Arg Met Arg Pro Trp Thr Ala Trp Ser Glu Cys Thr Lys Leu Cys Gly Gly Gly Ile Gln Glu Arg Tyr Met Thr Val Lys Lys Arg Phe Lys Ser Ser Gln Phe Thr Ser Cys Lys Asp Lys Lys Glu Ile Arg Ala Cys Asn Val His Pro Cys <210> 187 <211> 892 <212> DNA
<213> Homo sapiens <400> 187 tttattgatg tttcaacagg cacttattca aataagttat atatttgaaa acagccatgg 60 taagcatcct tggcttctca cccattcctc atgtggcatg ctttctagac tttaaaatga 120 ggtaccctga atagcactaa gtgctctgta agctcaagga atctgtgcag tgctacaaag 180 cccacaggca gagaaagaac tcctcaagtg cttgtggtca gagactaggt tccatatgag 240 gcacacctat gatgaaggtc ttcacctcca gaaggtgaca ctgttcagag atcctcattt 300 cctggagagt gggagaaaat ccctcctttg ggaaatccct tttcccagca gcagagccca 360 cctcattgct tagtgatcat ttggaaggca ctgagagcct tcaggggctg acagcagaga 420 aatgaaaatg agtacagttc agatggtgga agaagcatgg cagtgacatc ttccatgctc 480 tttttctcag tgtctgcaac tccaaagatc aaggccataa cccaggagac catcaacgga 540 agattagttc tttgtcaagt gaatgaaatc caaaagcacg catgagacca atgaaagttt 600 ccgcctgttg taaaatctat tttcccccaa ggaaagtcct tgcacagaca ccagtgagtg 660 agttctaaaa gatacccttg gaattatcag actcagaaac ttttattttt tttttctgta 720 acagtctcac cagacttctc ataatgctct taatatattg cacttttcta atcaaagtgc 780 gagtttatga gggtaaagct ctactttcct actgcagcct tcagattctc atcattttgc 840 atctattttg tagccaataa aactccgcac tagcaaaaaa aaaaaaaaaa as 892 <210> 188 <211> 1448 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <222> (1). .(1448) <223> n = A,T,C or G
<400> 188 tgtgactcac atttctttta ctgtgacaca ataatgtgat cctaaaactg gcttatcctt 60 gagtgtttac aactcaaaca actttttgaa tgcagtagtt tttttttttt aaaaacaaac 120 ttttatgtca aatttttttt cttagaagta gtcttcatta ttataaattt gtacaccaaa 180 aggccatggg gaactttgtg caagtacctc atcgctgagc aaatggagct tgctatgttt 240 taatttcaga aaatttcctc atatacgtag tgtgtagaat caagtctttt aataattcat 300 tttttcttca taatatttac tcaaagttaa gcttaaaaat aagttttatc ttaaaatcat 360 atttgaagac agtaagacag taaactattt taggaagtca acccccattg cactctgtgg 420 cagttattct ggtaaaaata ggcaaaagtg acctgaatct acaatggtgt cccaaagtaa 480 ccaagtaaga gagattgtaa atgataaacc gagctttaaa ggataaagtg ttaataaaga 540 aaggaagctg ggcacatgtc aaaaagggag atcgaaatgt taggtaatca tttagaaagg 600 acagaaaata tttaaagtgg ctcataggta atgaatattt ctgacttaga tgtaaatcca 660 tctggaatct ttacatcctt tgccagctga aacaagaaag tgaagggaca atgatatttc 720 atggtcagtt tattttgtaa gagacagaag aaattatatc tatacattac cttgtagcag 780 cagtacctgg aagccccagc ccgtcacaga agtgtggagg ggggctcctg actagacaat 840 ttccctagcc cttgtgattt gaagcatgaa agttctggca ggttatgagc agcactaggg 900 ataaagtatg gttttatttt ggtgtaattt aggtttttca acaaagccct tgtctaaaat 960 aaaaggcatt attggaaata tttgaaaact agaaaatgat ggataaaagg gctgataaga 1020 aaatttctga ctgtcagtag aagtgagata agatcctcag aggaaacagt aagaagggat 1080 aatcattaag atagtaaaac aggcaaagca gaatcacatg tgcncacaca catacacatg 1140 taaacattgg aatgcataag ttttaatatt ttagcgctat cagtttctaa atgcattaat 1200 tactaactgc cctctcccaa gattcattta gttcaaacag tatccgtaaa ctaggaataa 1260 tgccacatgc attcaatggg atcttttaag tactcttcag tttgttccaa gaaatgtgcc 1320 tactgaaatc aaattaattt gtattcaatg tgtacttcaa gactgctaat tgtttcatct 1380 gaaagcctac aatgaatcat tgttcamcct tgaaaaataa aattttgtaa atcaaaaaaa 1440 aaaaaaaa 1448 <210> 189 <211> 460 <212> DNA
<213> Homo sapiens <400> 189 ttttgggagc acggactgtc agttctctgg gaagtggtca gcgcatcctg cagggcttct 60 cctcctctgt cttttggaga accagggctc ttctcagggg ctctagggac tgccaggctg 120 tttcagccag gaaggccaaa atcaagagtg agatgtagaa agttgtaaaa tagaaaaagt 180 ggagttggtg aatcggttgt tctttcctca catttggatg attgtcataa ggtttttagc 240 atgttcctcc ttttcttcac cctccccttt tttcttctat taatcaagag aaacttcaaa 300 gttaatggga tggtcggatc tcacaggctg agaactcgtt cacctccaag catttcatga 360 aaaagctgct tcttattaat catacaaact ctcaccatga tgtgaagagt ttcacaaatc 420 cttcaaaata aaaagtaatg acttaaaaaa aaaaaaaaaa 460 <210> 190 <211> 481 <212> DNA
<213> Homo sapiens <400> 190 aggtggtgga agaaactgtg gcacgaggtg actgaggtat ctgtgggagc taatcctgtc 60 caggtggaag taggagaatt tgatgatggt gcagaggaaa ccgaagagga ggtggtggcg 120 gaaaatccct gccagaacca ccactgcaaa cacggcaagg tgtgcgagct ggatgagaac 180 aacaccccca tgtgcgtgtg ccaggacccc accagctgcc cagcccccat tggcgagttt 240 gagaaggtgt gcagcaatga caacaagacc ttcgactctt cctgccactt ctttgccaca 300 aagtgcaccc tggagggcac caagaagggc cacaagctcc acctggacta catcgggcct 360 tgcaaataca tccccccttg cctggactct gagctgaccg aattccccct gcgcatgcgg 420 gactggctca agaacgtcct ggtcaccctg tatgagaggg atgaggacaa caaccttctg 480 a 481 <210> 191 <211> 489 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <222> (1) . . (489) <223> n = A,T,C or G
<400> 191 atataaatta gactaagtgt tttcaaataa atctaaatct tcagcatgat gtgttgtgta 60 taattggagt agatattaat taagtcccct gtataatgtt ttgtaatttt gcaaaacata 120 tcttgagttg tttaaacagt caaaatgttt gatattttat accagcttat gagctcaaag 180 tactacagca aagcctagcc tgcatatcat tcacccaaaa caaagtaata gcgcctcttt 240 tattattttg actgaatgtt ttatggaatt gaaagaaaca tacgttcttt tcaagacttc 300 ctcatgaatc tntcaattat aggaaaagtt attgtgataa aataggaaca gctgaaagat 360 tgattaatga actattgtta attcttccta ttttaatgaa tgacattgaa ctgaattttt 420 tgtctgttaa atgaacttga tagctaataa aaagncaact agccatcaaa aaaaaaaaaa 480 aaaaaaaaa 489 <210> 192 <211> 516 <212> DNA
<213> Homo Sapiens <400> 192 acttcaaagc cagctgaagg aaagaggaag tgctagagag agcccccttc agtgtgcttc 60 tgacttttac ggacttggct tgttagaagg ctgaaagatg atggcaggaa tgaaaatcca 120 gcttgtatgc atgctactcc tggctttcag ctcctggagt ctgtgctcag attcagaaga 180 ggaaatgaaa gcattagaag cagatttctt gaccaatatg catacatcaa agattagtaa 240 agcacatgtt ccctcttgga agatgactct gctaaatgtt tgcagtcttg taaataattt 300 gaacagccca gctgaggaaa caggagaagt tcatgaagag gagcttgttg caagaaggaa 360 cttcttactg ctttagatgg ctttagcttg gaagcaatgt tgacaatata ccagctccac 420 aaaatctgtc acagcagggc ttttcaacac tgggagttaa tccaggaaga tattcttgat 480 actggaaatg acaaaaatgg aaaggaagaa gtcata 516 <210> 193 <211> 1409 <212> DNA
<213> Homo Sapiens <400> 193 tgattctttt ccaaaacttt tagccatagg gtcttttata gacagggata gtaaaatgaa 60 aattgagaaa tataagatga aaaggaatgg taaaaatatc ttttaggggg cttttaattg 120 gtgatctgaa atcttgggag aagctgttct tttcaggcct gaggtgctct tgactgtcgc 180 ctgcgcactg tgtaccccga gcaacattct aagggtgtgc tttcgccttg gctaactcct 240 ttgacctcat tcttcatata gtagtctagg aaaaagttgc aggtaattta aactgtctag 300 tggtacatag taactgaatt tctattccta tgagaaatga gaattattta tttgccatca 360 acacatttta tactttgcat ctccaaattt attgcggcga gacttgtcca ttgtgaaagt 420 tagagaacat tatgtttgta tcatttcttt cataaaacct caagagcatt tttaagccct 480 tttcatcaga cccagtgaaa actaaggata gatgtttttt aactggaggt ctcctgataa 540 ggagaacaca atccaccatt gtcatttaag taataagaca ggaaattgac cttgacgctt 600 tcttgttaaa tagatttaac aggaacatct gcacatcttt tttccttgtg cactatttgt 660 ttaattgcag tggattaata cagcaagagt gccacattat aactaggcaa ttatccattc 720 ttcaagactt agttattgtc acactaattg atcgtttaag gcataagatg gtctagcatt 780 aggaacatgt gaagctaatc tgctcaaaaa gatcaacaaa ttaatattgt tgctgatatt 840 tgcataattg gctgcaatta tttaatgttt aattgggttg atcaaatgag attcagcaat 900 tcacaagtgc attaatataa acagaactgg ggcacttaaa atgataatga ttaacttata 960 ttgcatgttc tcttcctttc acttttttca gtgtctacat ttcagaccga gtttgtcagc 1020 ttttttgaaa acacatcagt agaaaccaag attttaaaat gaagtgtcaa gacgaaggca 1080 aaacctgagc agttcctaaa aagatttgct gttagaaatt ttctttgtgg cagtcattta 1140 ttaaggattc aactcgtgat acaccaaaag aagagttgac ttcagagatg tgttccatgc 1200 tctctagcac aggaatgaat aaatttataa cacctgcttt agcctttgtt ttcaaaagca 1260 caaaggaaaa gtgaaaggga aagagaaaca agtgactgag aagtcttgtt aaggaatcag 1320 gttttttcta cctggtaaac attctctatt cttttctcaa aagattgttg taagaaaaaa 1380 tgtaagmcaa aaaaaaaaaa aaaaaaaaa 1409 <210> 194 <211> 441 <212> DNA
<213> Homo Sapiens <400> 194 cagatttcgg tagccatctc cctccaaata tgtctctttc tgctttctta gtgcccatta 60 tttccccttc tcctttcttc tgtcactgcc atctccttct tggtcttccc attgttcttt 120 aactggccgt aatgtggaat tgatatttac attttgatac ggtttttttc ttggcctgtg 180 tacgggattg cctcatttcc tgctctgaat tttaaaatta gatattaaag ctgtcatatg 240 gtttcctcac aaaagtcaac aaagtccaaa caaaaatagt ttgccgtttt actttcatcc 300 attgaaaaag gaaattgtgc ctcttgcagc ctaggcaaag gacatttagt actatcgatt 360 ctttccaccc tcacgatgac ttgcggttct ctctgtagaa aagggatggc ctaagaaata 420 caactaaaaa aaaaaaaaaa a 441 <210> 195 <211> 707 <212> DNA
<213> Homo Sapiens <400> 195 cagaaaaata tttggaaaaa atataccact tcatagctaa gtcttacaga gaagaggatt 60 tgctaataaa acttaagttt tgaaaattaa gatgcaggta gagcttctga actaatgccc 120 acagctccaa ggaagacatg tcctatttag ttattcaaat acaagttgag ggcattgtga 180 ttaagcaaac aatatatttg ttagaacttt gtttttaaat tactgttcct tgacattact 240 tataaagagt ctctaacttt cgatttctaa aactatgtaa tacaaaagta tagtttcccc 300 atttgataaa aggccaatga tactgagtag gatatatgcg tatcatgcta cttcattcag 360 tgtgtctgtt tttaatacta ataaggcagt ttgacagaaa ttatttcttt gggactaagg 420 tgattatcat ttttttcccc ttcaaaattg tgctttaagt gctgataacc acaggcagat 480 tgcaaagaac tgataaggca acaaaagtag agaattttag gatcaaaggc atgtaactga 540 aaggtaacaa cagtacataa gcgacaactg gggaaggcag cagtgaaaca tgtttgtggg 600 gttaagtgag tcattgtaaa taaggaattt gcacatttat tttctgtcga cgcggccgcc 660 actgtgctgg atatctgcag aattccacca cactggacta gtggatc 707 <210> 196 <211> 552 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <222> (1). .(552) <223> n = A,T,C or G
<400> 196 tggccagcca gcctgatgtg gatggcttcc ttggggtggt gcttccctca agcccgaatt 60 ngtggacatc atcaatgcca aacaatgagc cccatccatt ttccctaccc ttcctgccaa 120 gccagggant aagcagccca gaagcccagt aactgccctt tccctgcata tgcttttgat 180 ggtgtcatnt gctccttcct gtggcctcat ccaaactgta tnttccttta ctgtttatat 240 nttcaccctg taatggttgg gaccaggcca atcccttntc cacttactat aatggttgga 300 actaaacgtc accaaggtgg cttntccttg gctgaganat ggaaggcgtg gtgggatttg 360 ctnctgggtt ccctaggccc tagtgagggc agaagagaaa ccatcctntc ccttnttaca 420 ccgtgaggcc aagatcccct cagaaggcag gagtgctgcc ctntcccatg gtgcccgtgc 480 ctntgtgctg tgtatgtgaa ccacccatgt gagggaataa acctggcact aggaaaaaaa 540 aaaaaaaaaa as 552 <210> 197 <211> 449 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <222> (1). .(449) <223> n = A,T,C or G
<400> 197 ctccagagac aacttcgcgg tgtggtgaac tctctgagga aaaacacgtg cgtggnanca 60 agtgactgag acctanaaat ccaagcgttg gaggtcctga ggccagccta agtcgcttca 120 aaatggaacg aaggcgtttg cggggttcca ttcagagccg atacatcagc atgagtgtgt 180 ggacaagccc acggagactt gtggagctgg cagggcagag cctgctgaag gatgaggccc 240 tggccattgc ccgccctgga gttgctgccc agggagctct tcccgccact cttcatggca 300 gcctttgacg ggagacacag ccagaccctg aaggcaatgg tgcaggcctg gcccttcacc 360 tgcctccctc tgggagtgct gatgaaggga caacatcttc acctggagac cttcaaagct 420 gtgcttgatg gacttgatgt gctccttgc 449 <210> 198 <211> 606 <212> DNA
<213> Homo Sapiens <400> 198 tgagtttgcc cccttacccc catcccagtg aatatttgca attcctaaag acgtgttttg 60 attgtcacac ctgggtgggg aacatgctac tggcatctaa tgcatagagg gcagtaatgc 120 tgctaaacat ctttcaacgc acaggacaga gccccacaaa agagaattat ctagccccaa 180 atgtccataa cactgctgtt gagaaaacct accgcaggat cttactgggc ttcataggta 240 agcttgcctt tgttctggct tctgtagata tataaaataa agacactgcc cagtccctcc 300 ctcaacgtcc cgagccaggg ctcaaggcaa ttccaataac agtagaatga acactaaata 360 ttgatttcaa aatctcagca actagaagaa tgaccaacca tcctggttgg cctgggactg 420 tcctagtttt agcattgaaa gtttcaggtt ccaggaaagc cctcaggcct gggctgctgg 480 tcaccctagc agctgaggga ctcttcaata cagaattagt ctttgtgcac tggagatgaa 540 tatactttaa tttgtaacat gtgaaaacat ctataaacat ctactgaagc ctgttcttgt 600 ctgcac 606 <210> 199 <211> 369 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <222> (1). .(369) <223> n = A,T,C or G
<400> 199 ggcaactttt tgcggattgt tcttgcttnc aggctttgcg ctgcaaatcc agtgctacca 60 gtgtgaagaa ttccagctga acaacgactg ctcctccccc gagttcattg tgaattgcac 120 ggtgaacgtt caagacatgt gtcagaaaga agtgatggag caaagtgccg ggatcatgta 180 ccgcaagtcc tgtgcatcat cagcggcctg tctcatcgcc tctgccgggt accagtcctt 240 ctgctcccca gggaaactga actcagtttg catcagctgc tgcaacaccc ctctttgtaa 300 cgggccaagg cccaagaaaa ggggaagttc tgcctcggcc ctcangccat ggctccgcac 360 caccatcct 369

Claims (65)

1. An isolated polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein, or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera is not substantially diminished, wherein the ovarian carcinoma protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
(a) polynucleotides recited in any one of SEQ ID NOs:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193, 194; and (b) complements of the foregoing polynucleotides.

2. A polypeptide according to claim 1, wherein the polypeptide comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
(a) polynucleotides recited in any one of SEQ ID NOs:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193, 194; and (b) complements of such polynucleotides.

3. An isolated polynucleotide encoding at least 5 amino acid residues of a polypeptide according to claim polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein, or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera is not substantially diminished, wherein the ovarian carcinoma protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
(a) polynucleotides recited in any one of SEQ ID NOs:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 57, 63, 65, 69-72, 75, 78, 81, 82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 143-146, 148-151, 156, 158, 160-162, 166-168 or 171, 174-183, 185, 193, 194; and (b) complements of the foregoing polynucleotides

4. A polynucleotide according to claim 3, wherein the polynucleotide encodes an immunogenic portion of the polypeptide.

5. A polynucleotide according to claim 3, wherein the polynucleotide comprises a sequence recited in any one of SEQ ID NOs:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 57, 63, 65, 69-72, 75, 78, 81, 82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 143-146, 148-151, 156, 158, 160-162, 166-168, 171 or 174-183, 185, 193, 194 or a complement of any of the foregoing sequences.

6. An isolated polynucleotide complementary to a polynucleotide according to claim 3.

7. An expression vector comprising a polynucleotide according to claim 3 or claim 6.

8. A host cell transformed or transfected with an expression vector according to claim 7.

9. A pharmaceutical composition comprising a polypeptide according to claim 1, in combination with a physiologically acceptable carrier.

10. A pharmaceutical composition according to claim 9, wherein the polypeptide comprises an amino acid sequence encoded by a polynucleotide that comprises a sequence recited in any one of SEQ ID NOs:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193 and 194.

11. A vaccine comprising a polypeptide according to claim 1, in combination with a non-specific immune response enhancer.

12. A vaccine according to claim 11, wherein the polypeptide comprises an amino acid sequence encoded by a polynucleotide that comprises a sequence recited in any one of SEQ ID NOs:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193 and 194.

13. A pharmaceutical composition comprising:
(a) a polynucleotide encoding an ovarian carcinoma polypeptide, wherein the polypeptide comprises at least an immunogenic portion of an ovarian carcinoma protein or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera is not substantially diminished, wherein the ovarian carcinoma protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
(i) polynucleotides recited in any one of SEQ ID NOs:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193, 194; and (ii) complements of the foregoing polynucleotides; and (b) a physiologically acceptable carrier.

14. A pharmaceutical composition according to claim 13, wherein the polynucleotide comprises a sequence recited in any one of SEQ ID NOs:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193, 194 or a complement of any of the foregoing sequences.

15. A vaccine comprising:
(a) a polynucleotide encoding an ovarian carcinoma polypeptide, wherein the polypeptide comprises at least an immunogenic portion of an ovarian carcinoma protein or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera is not substantially diminished, wherein the ovarian carcinoma protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
(i) polynucleotides recited in any one of SEQ ID NOs:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193, 194; and (ii) complements of the foregoing polynucleotides; and

16. A vaccine according to claim 15, wherein the polynucleotide comprises a sequence recited in any one of SEQ ID NOs:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193, 194.

17. A pharmaceutical composition comprising:
(a) an antibody that specifically binds to an ovarian carcinoma protein, wherein the ovarian carcinoma protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
(i) polynucleotides recited in any one of SEQ ID NOs:1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193, 194; and (ii) complements of such polynucleotides; and (b) a physiologically acceptable carrier.

18. A method for inhibiting the development of ovarian cancer in a patient, comprising administering to a patient an effective amount of an agent selected from the group consisting of:
(a) an ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera is not substantially diminished, wherein the ovarian carcinoma protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
(i) polynucleotides recited in any one of SEQ ID NOs:1-185 and 187-199; and (ii) complements of such polynucleotides;
(b) a polynucleotide encoding a polypeptide as recited in (a); and (c) an antibody that specifically binds to an ovarian carcinoma protein that comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
(i) polynucleotides recited in any one of SEQ ID NOs:1-185 and 187-199; and (ii) complements of such polynucleotides;
and thereby inhibiting the development of ovarian cancer in the patient.

19. A method according to claim 18, wherein the agent is present within a pharmaceutical composition according to any one of claims 9, 13 or 17.

20. A method according to claim 18, wherein the agent is present within a vaccine according to any one of claims 11, 15 or 18.

21. A fusion protein comprising at least one polypeptide according to claim 1.

22. A polynucleotide encoding a fusion protein according to claim 21.

23. A pharmaceutical composition comprising a fusion protein according to claim 21 in combination with a physiologically acceptable carrier.

24. A vaccine comprising a fusion protein according to claim 21 in combination with a non-specific immune response enhancer.

25. A pharmaceutical composition comprising a polynucleotide according to claim 22 in combination with a physiologically acceptable carrier.

26. A vaccine comprising a polynucleotide according to claim 22 in combination with a non-specific immune response enhancer.

27. A method for inhibiting the development of ovarian cancer in a patient, comprising administering to a patient an effective amount of a pharmaceutical composition according to claim 23 or claim 25.

28. A method for inhibiting the development of ovarian cancer in a patient, comprising administering to a patient an effective amount of a vaccine according to claim 23 or claim 26.

29. A pharmaceutical composition, comprising:
(a) an antigen presenting cell that expresses an ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera is not substantially diminished, wherein the ovarian carcinoma protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
(i) polynucleotides recited in any one of SEQ ID NOs:1-185 and 187-199; and (ii) complements of such polynucleotides; and (b) a pharmaceutically acceptable carrier or excipient.

30. A vaccine, comprising:
(a) an antigen presenting cell that expresses an ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera is not substantially diminished, wherein the ovarian carcinoma protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
(i) polynucleotides recited in any one of SEQ ID NOs:1-185 and 187-199; and (ii) complements of such polynucleotides; and (b) a non-specific immune response enhancer.

31. A vaccine comprising:
(a) an anti-idiotypic antibody or antigen-binding fragment thereof that is specifically bound by an antibody that specifically binds to an ovarian carcinoma protein that comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
(i) polynucleotides recited in any one of SEQ ID NOs:1-185 and 187-199; and (ii) complements of such polynucleotides; and (b) non-specific immune response enhancer.

32. A vaccine according to claim 30 or claim 31, wherein the immune response enhancer is an adjuvant.

33. A pharmaceutical composition, comprising:
(a) a T cell that specifically reacts with an ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera is not substantially diminished, wherein the ovarian carcinoma protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:

(i) polynucleotides recited in any one of SEQ ID NOs:1-185 and 187-199; and (ii) complements of such polynucleotides; and (b) a physiologically acceptable carrier.

34. A vaccine, comprising:
(a) a T cell that specifically reacts with an ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera is not substantially diminished, wherein the ovarian carcinoma protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
(i) polynucleotides recited in any one of SEQ ID NOs:1-185 and 187-199 and (ii) complements of such polynucleotides; and (b) a non-specific immune response enhancer.

35. A method for inhibiting the development of ovarian cancer in a patient, comprising administering to the patient an effective amount of a pharmaceutical composition according to claim 29 or claim 33.

36. A method for inhibiting the development of ovarian cancer in a patient, comprising administering to the patient an effective amount of a vaccine according to any one of claims 30, 31 or 34.

37. A method for stimulating and/or expanding T cells, comprising contacting T cells with:

(a) an ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera is not substantially diminished, wherein the ovarian carcinoma protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
(i) polynucleotides recited in any one of SEQ ID NOs:1-185 and 187-199; and (ii) complements of such polynucleotides;
(b) a polynucleotide encoding such a polypeptide; and/or (c) an antigen presenting cell that expresses such a polypeptide under conditions and for a time sufficient to permit the stimulation and/or expansion of T cells.

38. A method according to claim 37, wherein the T cells are cloned prior to expansion.

39. A method for stimulating and/or expanding T cells in a mammal, comprising administering to a mammal a pharmaceutical composition comprising:
(a) one or more of:
(i) an ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera is not substantially diminished, wherein the ovarian carcinoma protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
polynucleotides recited in any one of SEQ ID NOs:1-185 and 187-199; and complements of such polynucleotides;
(ii) a polynucleotide encoding an ovarian carcinoma polypeptide;
or (iii) an antigen-presenting cell that expresses an ovarian carcinoma polypeptide; and (b) a physiologically acceptable carrier or excipient;
and thereby stimulating and/or expanding T cells in a mammal.

40. A method for stimulating and/or expanding T cells in a mammal, comprising administering to a mammal a vaccine comprising:
(a) one or more of:
(i) an ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera is not substantially diminished, wherein the ovarian carcinoma protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
polynucleotides recited in any one of SEQ ID NOs:1-185 and 187-199; and complements of such polynucleotides;
(ii) a polynucleotide encoding an ovarian carcinoma polypeptide;
or (iii) an antigen-presenting cell that expresses an ovarian carcinoma polypeptide; and (b) a non-specific immune response enhancer;
and thereby stimulating and/or expanding T cells in a mammal.

41. A method for inhibiting the development of ovarian cancer in a patient, comprising administering to a patient T cells prepared according to the method of claim 39 or claim 40.

42. A method for inhibiting the development of ovarian cancer in a patient, comprising the steps of:

(a) incubating CD4+ T cells isolated from a patient with one or more of:
(i) an ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera is not substantially diminished, wherein the ovarian carcinoma protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
polynucleotides recited in any one of SEQ ID NOs:1-185 and 187-199; and complements of such polynucleotides;
(ii) a polynucleotide encoding an ovarian carcinoma polypeptide;
or (iii) an antigen-presenting cell that expresses an ovarian carcinoma polypeptide;
such that T cells proliferate; and (b) administering to the patient an effective amount of the proliferated T cells, and therefrom inhibiting the development of ovarian cancer in the patient.

43. A method for inhibiting the development of ovarian cancer in a patient, comprising the steps of:
(a) incubating CD4+ T cells isolated from a patient with one or more of:
(i) an ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera is not substantially diminished, wherein the ovarian carcinoma protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
polynucleotides recited in any one of SEQ ID NOs:1-185 and 187-199; and complements of such polynucleotides;

(ii) a polynucleotide encoding an ovarian carcinoma polypeptide;
or (iii) an antigen-presenting cell that expresses an ovarian carcinoma polypeptide;
such that T cells proliferate;
(b) cloning one or more proliferated cells; and (c) administering to the patient an effective amount of the cloned T cells.

44. A method for inhibiting the development of ovarian cancer in a patient, comprising the steps of:
(a) incubating CD8+T cells isolated from a patient with one or more of:
(i) an ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera is not substantially diminished, wherein the ovarian carcinoma protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
polynucleotides recited in any one of SEQ ID NOs:1-185 and 187-199; and complements of such polynucleotides;
(ii) a polynucleotide encoding an ovarian carcinoma polypeptide;
or (iii) an antigen-presenting cell that expresses an ovarian carcinoma polypeptide;
such that T cells proliferate; and (b) administering to the patient an effective amount of the proliferated T cells, and therefrom inhibiting the development of ovarian cancer in the patient.

45. A method for inhibiting the development of ovarian cancer in a patient, comprising the steps of:

(a) incubating CD8+T cells isolated from a patient with one or more of:
(i) an ovarian carcinoma polypeptide comprising at least an immunogenic portion of an ovarian carcinoma protein or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera is not substantially diminished, wherein the ovarian carcinoma protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
polynucleotides recited in any one of SEQ ID NOs:1-185 and 187-199; and complements of such polynucleotides;
(ii) a polynucleotide encoding an ovarian carcinoma polypeptide;
or (iii) an antigen-presenting cell that expresses an ovarian carcinoma polypeptide;
such that the T cells proliferate;
(b) cloning one or more proliferated cells; and (c) administering to the patient an effective amount of the cloned T cells.

46. A method for determining the presence or absence of a cancer in a patient, comprising the steps of:
(a) contacting a biological sample obtained from a patient with a binding agent that binds to an ovarian carcinoma protein, wherein the ovarian carcinoma protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
(i) polynucleotides recited in any one of SEQ ID NOs:1-185 and 187-199; and (ii) complements of the foregoing polynucleotides;

(b) detecting in the sample an amount of polypeptide that binds to the binding agent; and (c) comparing the amount of polypeptide to a predetermined cut-off value, and therefrom determining the presence or absence of a cancer in the patient.

47. A method according to claim 46, wherein the binding agent is an antibody.

48. A method according to claim 47, wherein the antibody is a monoclonal antibody.

49. A method according to claim 46, wherein the cancer is ovarian cancer.

50. A method for monitoring the progression of a cancer in a patient, comprising the steps of:
(a) contacting a biological sample obtained from a patient at a first point in time with a binding agent that binds to an ovarian carcinoma protein, wherein the ovarian carcinoma protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
(i) polynucleotides recited in any one of SEQ ID NOs:1-185 and 187-199; and (ii) complements of the foregoing polynucleotides;
(b) detecting in the sample an amount of polypeptide that binds to the binding agent;
(c) repeating steps (a) and (b) using a biological sample obtained from the patient at a subsequent point in time; and (d) comparing the amount of polypeptide detected in step (c) to the amount detected in step (b) and therefrom monitoring the progression of the cancer in the patient.

51. A method according to claim 50, wherein the binding agent is an antibody.

52. A method according to claim 51, wherein the antibody is a monoclonal antibody.

53. A method according to claim 50, wherein the cancer is ovarian cancer.

54. A method for determining the presence or absence of a cancer in a patient, comprising the steps of:

(a) contacting a biological sample obtained from a patient with an oligonucleotide that hybridizes to a polynucleotide that encodes an ovarian carcinoma protein, wherein the ovarian carcinoma protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
(i) polynucleotides recited in any one of SEQ ID NOs:1-185 and 187-199; and (ii) complements of the foregoing polynucleotides;
(b) detecting in the sample an amount of a polynucleotide that hybridizes to the oligonucleotide; and (c) comparing the amount of polynucleotide that hybridizes to the oligonucleotide to a predetermined cut-off value, and therefrom determining the presence or absence of a cancer in the patient.

55. A method according to claim 54, wherein the amount of polynucleotide that hybridizes to the oligonucleotide is determined using a polymerase chain reaction.

56. A method according to claim 54, wherein the amount of polynucleotide that hybridizes to the oligonucleotide is determined using a hybridization assay.

57. A method for monitoring the progression of a cancer in a patient, comprising the steps of:
(a) contacting a biological sample obtained from a patient with an oligonucleotide that hybridizes to a polynucleotide that encodes an ovarian carcinoma protein, wherein the ovarian carcinoma protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
(i) polynucleotides recited in any one of SEQ ID NOs:1-185 and 187-199; and (ii) complements of the foregoing polynucleotides;
(b) detecting in the sample an amount of a polynucleotide that hybridizes to the oligonucleotide;
(c) repeating steps (a) and (b) using a biological sample obtained from the patient at a subsequent point in time; and (d) comparing the amount of polynucleotide detected in step (c) to the amount detected in step (b) and therefrom monitoring the progression of the cancer in the patient.

58. A method according to claim 57, wherein the amount of polynucleotide that hybridizes to the oligonucleotide is determined using a polymerase chain reaction.

59. A method according to claim 57, wherein the amount of polynucleotide that hybridizes to the oligonucleotide is determined using a hybridization assay.

60. A diagnostic kit, comprising:
(a) one or more antibodies or antigen-binding fragments thereof that specifically bind to an ovarian carcinoma protein that comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
(i) polynucleotides recited in any one of SEQ ID NOs:1-185 and 187-199; and (ii) complements of the foregoing polynucleotides; and (b) a detection reagent comprising a reporter group.

61. A kit according to claim 60, wherein the antibodies are immobilized on a solid support.

62. A kit according to claim 61, wherein the solid support comprises nitrocellulose, latex or a plastic material.

63. A kit according to claim 60, wherein the detection reagent comprises an anti-immunoglobulin, protein G, protein A or lectin.

64. A kit according to claim 60, wherein the reporter group is selected from the group consisting of radioisotopes, fluorescent groups, luminescent groups, enzymes, biotin and dye particles.

65. A diagnostic kit, comprising:
(a) an oligonucleotide comprising 10 to 40 nucleotides that hybridize under moderately stringent conditions to a polynucleotide that encodes an ovarian carcinoma protein, wherein the ovarian carcinoma protein comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of:
(i) polynucleotides recited in any one of SEQ ID NOs:1-185 and 187-199; and (ii) complements of the foregoing polynucleotides; and (b) a diagnostic reagent for use in a polymerase chain reaction or hybridization assay.