US20040063134A1 - Novel isoforms of human pregnancy-associated protein-E - Google Patents

Novel isoforms of human pregnancy-associated protein-E Download PDF

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US20040063134A1
US20040063134A1 US10/675,685 US67568503A US2004063134A1 US 20040063134 A1 US20040063134 A1 US 20040063134A1 US 67568503 A US67568503 A US 67568503A US 2004063134 A1 US2004063134 A1 US 2004063134A1
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Yizhong Gu
Mark Shannon
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GE Healthcare Ltd
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Yizhong Gu
Shannon Mark E.
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Abstract

The invention provides isolated nucleic acids that encode three novel isoforms of human pregnancy associated plasma protein E, hPAPP-E, and fragments thereof, vectors for propagating and expressing PAPP-E nucleic acids, host cells comprising the nucleic acids and vectors of the present invention, proteins, protein fragments, and protein fusions of the novel PAPP-E isoforms, and antibodies thereto. The invention further provides transgenic cells and non-human organisms comprising human PAPP-E isoform nucleic acids, and transgenic cells and non-human organisms with targeted disruption of the endogenous orthologue of the human PAPP-E gene. The invention further provides pharmaceutical formulations of the nucleic acids, proteins, and antibodies of the present invention, and diagnostic, investigational, and therapeutic methods based on the PAPP-E nucleic acids, proteins, and antibodies of the present invention.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • The present application claims priority to U.S. provisional patent application No. 60/207,456, filed May 26, 2000, and No. 60/236,359, filed Sep. 27, 2000, the disclosures of which are incorporated herein by reference in their entireties.[0001]
  • REFERENCE TO SEQUENCE LISTING SUBMITTED ON COMPACT DISC
  • The present application includes a Sequence Listing filed on one (1) CD-R disc, provided in duplicate, containing a single file named pto_MDhMORF-8.txt, having 349 kilobytes, last modified on Apr. 3, 2001 and recorded Apr. 5, 2001. The Sequence Listing contained in said file on said disc is incorporated herein by reference in its entirety. [0002]
  • FIELD OF THE INVENTION
  • The present invention relates to novel isoforms of a human protein, and particularly relates to novel isoforms of human pregnancy-associated plasma protein-E. [0003]
  • BACKGROUND OF THE INVENTION
  • Pregnancy-Associated Plasma Protein-A (PAPP-A) was first identified as a component of a circulating protein complex uniquely present in the serum of pregnant women. Principally of placental, that is, fetal, origin, and detectable in maternal serum as early as 4 weeks into gestation, PAPP-A has proven useful as a readily sampled marker for prenatal monitoring of fetal health and diagnosis of a number of human fetal abnormalities. [0004]
  • Maternal serum PAPP-A levels normally increase throughout gestation. Failure of PAPP-A levels to increase at the normal rate—that is, PAPP-A levels lower than the average for the respective gestational age—has been associated with a variety of fetal disorders. [0005]
  • For example, PAPP-A levels have been shown to be significantly lower than normal at 10-14 weeks gestation in pregnancies that subsequently result in miscarriage, pregnancy-induced hypertension, growth restriction, and pre-existing or gestational diabetes mellitus. Ong et al., [0006] Brit. J. Obstet. Gynaec. 107: 1265-70 (2000).
  • As another example, a statistical measure of maternal serum PAPP-A levels (the median multiple of the median (MoM)) is significantly decreased (less than the 5th centile of normal in 78% of cases) during the first trimester in cases of fetal trisomy 18. Tul et al., [0007] Prenat. Diagn. 19: 1035-42 (1999). When measurement of PAPP-A is combined with measurement of maternal serum free βhCG, fetal nuchal translucency, and maternal age, 89% of cases of trisomy 18 can be detected with a 1% false-positive rate.
  • In the second trimester of pregnancy, maternal serum levels of PAPP-A are reduced more markedly than either alpha fetoprotein (αFP) or free beta-hCG in cases of trisomy 18; one study reports levels of PAPP-A lower than the 5% centile of normal in 93% of the cases. Spencer et al., [0008] Prenat. Diagn. 19: 1127-34 (1999).
  • Median maternal serum levels of PAPP-A are also significantly reduced at 8 to 14 weeks in trisomy 21 gestations. Screening using maternal age, serum-free β-hCG, and PAPP-A at 10 weeks of pregnancy has been demonstrated to provide better prediction of fetal trisomy 21 than one standard test (levels of alpha-fetoprotein and hCG, in conjuction with maternal age), and equal predictive value to the test of αFP, unconjugated estriol, hCG, and maternal age at 15-22 weeks. Wald et al., [0009] Br. J. Obstet. Gynaecol. 103(5):407-412 (1996).
  • Although first discovered based upon its primary expression in placental tissue, PAPP-A has also been detected in ovaries, with expression restricted to healthy antral follicles in granulosa cells and healthy corpora lutea (CL) in a subset of large luteal cells, a pattern of expression consistent with a role for PAPP-A at the very outset of pregnancy, through control of survival, growth, and/or differentiation of the dominant ovarian follicle. Hourvitz et al., [0010] J. Clin. Endocrinol. Metab. 85:4916-4920 (2000).
  • PAPP-A is a member of the metzincin superfamily of metalloproteinases. [0011]
  • The metzincin gene superfamily was first identified based upon topological and sequence relationships among the astacins, adamalysins, serralysins, and matrix metalloproteinases. These zinc endopeptidases share topological similarity with respect to a five-stranded beta-sheet and three alpha-helices arranged in typical sequential order. The common consensus motif, HEXXHXXGXXH, found in PAPP-A at residues 482-492, contains three histidine residues which are involved in binding of the catalytically essential zinc ion. Stocker et. al., [0012] Protein Sci. 4:823-40 (1995). Metzincins also possess a conserved methionine residue in spaced relationship to the zinc-binding motif; in PAPP-A, the conserved methionine is believed to be the methionine at residue 556.
  • PAPP-A has been demonstrated specifically to cleave insulin-like growth factor binding protein 4 (IGFBP-4), Lawrence et al., [0013] Proc. Natl. Acad. Sci. USA 96:3149-3153 (1999), and to be the dominating, if not sole, IGFBP-4 protease present in the circulation, Overgaard et al., J. Biol. Chem. 275:41128-31133 (2000). IGFBP-4 is one of six known inhibitors of IGF action in vitro; like other IGFBPs, cleavage of IGFBP-4 has been shown to abolish its ability to inhibit IGF activity. The cleavage specificity of PAPP-A for IGFBP-4 implicates PAPP-A in normal and pathological physiology of insulin-like growth factor (IGF). Overgaard et al., J. Biol. Chem. 275: 31128-33 (2000).
  • PAPP-A exists in pregnancy serum as a covalent, heterotetrameric 2:2 complex with the proform of eosinophil major basic protein (proMBP); pro-MBP appears to inhibit PAPP-A's protease activity. Overgaard et al., [0014] J. Biol. Chem. 275:31128-33 (2000). Conversely, IGF appears to be a necessary cofactor or agonist for PAPP-A protease activity, leading to a feedback network controlling IGF availability.
  • Reports in the literature of pregnancy-related proteins related in sequence to PAPP-A, termed PAPP-B, PAPP-C, and PAPP-D, have proven spurious. Farr et al., [0015] Biochim. Biophys. Acta 1493:356-362 (2000) recently identified a cDNA that encodes a protein related in primary sequence and protein domain structure to PAPP-A and that is expressed primarily in placenta, which they term PAPP-E. FARR et al. report that the cDNA encodes a complete open reading frame.
  • Recent reports suggest that at least one-third, and likely a higher percentage, of human genes are alternatively spliced. Hanke et al., [0016] Trends Genet. 15(1):389-390 (1999); Mironov et al., Genome Res. 9:1288-93 (1999); Brett et al., FEBS Lett. 474(1):83-6 (2000). Alternative splicing has been proposed to account for at least part of the difference between the number of genes recently called from the completed human genome draft sequence—30,000 to 40,000 (Genome International Sequencing Consortium, Nature 409:860-921 (Feb. 15, 2001)—and earlier predictions of human gene number that routinely ranged as high as 120,000, Liang et al., Nature Genet. 25(2):239-240 (2000). With the Drosophila homolog of one human gene reported to have 38,000 potential alternatively spliced variants, Schmucker et al., Cell 101:671 (2000), it now appears that alternative splicing may permit the relatively small number of human coding regions to encode millions, perhaps tens of millions, of structurally distinct proteins and protein isoforms.
  • With increasing age, women experience decrease in ovarian reserve and, upon conception, an increased incidence of aneuploid gestations. Given a likely role of PAPP-A in controlling ovarian follicular maturation, and its proven clinical utility as a predictor of fetal abnormality during gestation, PAPP-A has potential therapeutic as well as diagnostic roles in clinical infertility practice. [0017]
  • With the recent identification of a protein that is related to the clinically useful prenatal diagnostic marker, human PAPP-A, and the recognition that alternatively spliced isoforms of proteins are as critical to metabolic and physiologic function as proteins that are separately encoded, there is a need to identify and to characterize additional isoforms of the PAPP-E protein. [0018]
  • SUMMARY OF THE INVENTION
  • The present invention solves these and other needs in the art by providing isolated nucleic acids that encode three novel isoforms of hPAPP-E, and fragments thereof. [0019]
  • In other aspects, the invention provides vectors for propagating and expressing the nucleic acids of the present invention, host cells comprising the nucleic acids and vectors of the present invention, proteins, protein fragments, and protein fusions of the novel PAPP-E isoforms, and antibodies thereto. [0020]
  • The invention further provides pharmaceutical formulations of the nucleic acids, proteins, and antibodies of the present invention. [0021]
  • In other aspects, the invention provides transgenic cells and non-human organisms comprising human PAPP-E isoform nucleic acids, and transgenic cells and non-human organisms with targeted disruption of the endogenous orthologue of the human PAPP-E gene. [0022]
  • The invention additionally provides diagnostic, investigational, and therapeutic methods based on the PAPP-E nucleic acids, proteins, and antibodies of the present invention.[0023]
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • The above and other objects and advantages of the present invention will be apparent upon consideration of the following detailed description taken in conjunction with the accompanying drawings, in which like characters refer to like parts throughout, and in which: [0024]
  • FIG. 1 schematizes the protein domain structure of the three novel isoforms of PAPP-E and the earlier-described PAPP-A; [0025]
  • FIG. 2 is a map showing the genomic structure and alternative exon usage of three novel isoforms of human PAPP-E that are encoded at chromosome 1q24.1, termed PAPP-Ea, PAPP-Eb, and PAPP-Ec; [0026]
  • FIG. 3 presents the nucleotide and predicted amino acid sequences of PAPP-Ea; [0027]
  • FIG. 4 presents the nucleotide and predicted amino acid sequences of PAPP-Eb; and [0028]
  • FIG. 5 presents the nucleotide and predicted amino acid sequences of PAPP-Ec. [0029]
  • DETAILED DESCRIPTION OF THE INVENTION
  • Mining the sequence of the human genome for novel human genes, the present inventors have identified three novel isoforms of the recently cloned human pregnancy-associated protein E (PAPP-E), a protein expressed predominantly in placenta and related to the clinically useful prenatal diagnostic marker, human PAPP-A. [0030]
  • As schematized in FIG. 1, the newly isolated isoforms—PAPP-Ea, PAPP-Eb, and PAPP-Ec—share certain protein domains and an overall structural organization with PAPP-A; in conjunction with a pattern of expression strikingly similar to that of PAPP-A, with high level expression in placenta, the shared structural features strongly imply that the three PAPP-E isoforms play a role similar to that of PAPP-A in regulating the activity of a plasma borne growth factor(s), likely IGF, which in turn is important for maintenance of pregnancy and/or normal fetal development, thus making the PAPP-E isoforms clinically useful diagnostic markers and potential therapeutic agents. [0031]
  • Like PAPP-A, all three novel isoforms have the zinc-binding domain (“zinc”) characteristic of metzincin superfamily metalloproteases, defined by the degenerate motif “[0032] HEXXHXXGXXH”, where invariant residues are shown underlined and variable residues are shown as “X”. In PAPP-Ea, the longest isoform, the zinc binding domain occurs at residues 733-743 with sequence HEVGHVLGLYH.
  • In common with PAPP-A, all three novel isoforms of PAPP-E have an at least four-fold repetition near the C-terminus of the short consensus repeat (“SCR”; alternatively denominated “sushi” domain) (relaxation of certain bioinformatic parameters causes bioinformatic algorithms to suggest a potential five-fold repetition). [0033]
  • In common with PAPP-A, all three novel isoforms of PAPP-E also have at least one “NL” (notch-lin, also termed lin notch repeat, or “LNR”) domain, so-called due to its presence in Notch and Lin-12 proteins, both of which proteins regulate early tissue differentiation. As shown in FIG. 1, PAPP-Ea possesses three NL domains in the same general spaced relationship to the zinc domain as is found in PAPP-A. PAPP-Eb, in contrast, lacks the C-terminal NL domain, whereas PAPPE-c, the shortest of the novel isoforms, lacks the two NL domains on the N-terminal side of the zinc-binding domain. [0034]
  • The four-fold repetition of SCR (“sushi”) domains is characteristic of complement proteins and selecting. Five-fold repetition of SCR domains with further presence of at least one NL domain has been previously identified in complement decay-accelerating factor and P-selectin. [0035]
  • In contrast to PAPP-A, two of the novel isoforms of PAPP-E—PAPP-Ea and PAPP-Eb—have a laminin G domain. Laminin G domains are found in a number of extracellular and receptor proteins, and are implicated in interactions with cellular receptors (integrins, alpha-dystroglycan), sulfated carbohydrates and other extracellular ligands. [0036]
  • In contrast to PAPP-A, all three novel isoforms of PAPP-E contain nuclear localization signals (“NLS”); with concurrent presence of a leader sequence (not shown), these signals suggest that all three PAPP-E isoforms can be secreted and also localize to the cell nucleus. [0037]
  • FIG. 2 shows the genomic organization of the three PAPP-E isoforms. [0038]
  • At the top is shown the four bacterial artificial chromosomes (BACs), with GenBank accession numbers, that span the PAPP-E locus. The genome-derived single-exon probe first used to demonstrate expression from this locus, as further described in commonly owned and copending provisional patent application No. 60/207,456, filed May 26, 2000, the disclosure of which is incorporated herein by reference in its entirety, is shown below the BACs and labeled “500”. The 500 bp probe includes sequence drawn solely from exon two. [0039]
  • As shown in FIG. 2, PAPP-Ea, encoding a protein of 1791 amino acids, is the longest PAPP-E isoform, comprising exons 1-20, 22 and 23. Insertion of the 85 bp exon 21 in PAPP-Eb leads to a downstream frame shift and earlier termination, thus encoding a protein of 1770 amino acids. PAPP-Ec lacks exons 2, 3 and 21, encoding a protein of 1385 amino acids. Predicted molecular weights, prior to any post-translational modification, are 199 kD, 196 kD and 152 kD, respectively. [0040]
  • As further discussed in the examples herein, expression of PAPP-E was assessed using hybridization to genome-derived single exon microarrays and northern blot. Microarray analysis of the first two exons showed high level expression in placenta, and little expression in other tissues. This was confirmed by northern blot of 12 tissues (blood leukocyte, lung, placenta, small intestine, liver, kidney, spleen, thymus, colon, skeletal muscle, heart and brain). [0041]
  • As more fully described below, the present invention provides isolated nucleic acids that encode each of the novel isoforms of hPAPP-E, and fragments thereof. The invention further provides vectors for propagation and expression of the nucleic acids of the present invention, host cells comprising the nucleic acids and vectors of the present invention, proteins, protein fragments, and protein fusions of the present invention, and antibodies specific for all or any one of the isoforms. The invention provides pharmaceutical formulations of the nucleic acids, proteins, and antibodies of the present invention. The invention further provides transgenic cells and non-human organisms comprising human PAPP-E isoform nucleic acids, and transgenic cells and non-human organisms with targeted disruption of the endogenous orthologue of the human PAPP-E gene. The invention additionally provides diagnostic, investigational, and therapeutic methods based on the PAPP-E nucleic acids, proteins, and antibodies of the present invention. [0042]
  • Definitions [0043]
  • As used herein, “nucleic acid” includes polynucleotides having natural nucleotides in native 5′-3′ phosphodiester linkage—e.g., DNA or RNA—as well as polynucleotides that have nonnatural nucleotide analogues, normative internucleoside bonds, or both, so long as the nonnatural polynucleotide is capable of sequence-discriminating basepairing under experimentally desired conditions. Unless otherwise specified, the term “nucleic acid” includes any topological conformation; the term thus explicitly comprehends single-stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular, and padlocked conformations. [0044]
  • As used herein, an “isolated nucleic acid” is a nucleic acid molecule that exists in a physical form that is nonidentical to any nucleic acid molecule of identical sequence as found in nature; “isolated” does not require, although it does not prohibit, that the nucleic acid so described has itself been physically removed from its native environment. [0045]
  • For example, a nucleic acid can be said to be “isolated” when it includes nucleotides and/or internucleoside bonds not found in nature. When instead composed of natural nucleosides in phosphodiester linkage, a nucleic acid can be said to be “isolated” when it exists at a purity not found in nature, where purity can be adjudged with respect to the presence of nucleic acids of other sequence, with respect to the presence of proteins, with respect to the presence of lipids, or with respect the presence of any other component of a biological cell, or when the nucleic acid lacks sequence that flanks an otherwise identical sequence in an organism's genome, or when the nucleic acid possesses sequence not identically present in nature. [0046]
  • As so defined, “isolated nucleic acid” includes nucleic acids integrated into a host cell chromosome at a heterologous site, recombinant fusions of a native fragment to a heterologous sequence, recombinant vectors present as episomes or as integrated into a host cell chromosome. [0047]
  • As used herein, an isolated nucleic acid “encodes” a reference polypeptide when at least a portion of the nucleic acid, or its complement, can be directly translated to provide the amino acid sequence of the reference polypeptide, or when the isolated nucleic acid can be used, alone or as part of an expression vector, to express the reference polypeptide in vitro, in a prokaryotic host cell, or in a eukaryotic host cell. [0048]
  • As used herein, the term “exon” refers to a nucleic acid sequence found in genomic DNA that is bioinformatically predicted and/or experimentally confirmed to contribute contiguous sequence to a mature mRNA transcript. [0049]
  • As used herein, the phrase “open reading frame” and the equivalent acronym “ORF” refer to that portion of a transcript-derived nucleic acid that can be translated in its entirety into a sequence of contiguous amino acids. As so defined, an ORF has length, measured in nucleotides, exactly divisible by 3. As so defined, an ORF need not encode the entirety of a natural protein. [0050]
  • As used herein, the phrase “ORF-encoded peptide” refers to the predicted or actual translation of an ORF. [0051]
  • As used herein, the phrase “degenerate variant” of a reference nucleic acid sequence intends all nucleic acid sequences that can be directly translated, using the standard genetic code, to provide an amino acid sequence identical to that translated from the reference nucleic acid sequence. [0052]
  • As used herein, the term “microarray” and equivalent phrase “nucleic acid microarray” refer to a substrate-bound collection of plural nucleic acids, hybridization to each of the plurality of bound nucleic acids being separately detectable. The substrate can be solid or porous, planar or non-planar, unitary or distributed. [0053]
  • As so defined, the term “microarray” and phrase “nucleic acid microarray” include all the devices so called in Schena (ed.), [0054] DNA Microarrays: A Practical Approach (Practical Approach Series), Oxford University Press (1999) (ISBN: 0199637768); Nature Genet. 21(1)(suppl):1-60 (1999); and Schena (ed.), Microarray Biochip: Tools and Technology, Eaton Publishing Company/BioTechniques Books Division (2000) (ISBN: 1881299376), the disclosures of which are incorporated herein by reference in their entireties.
  • As so defined, the term “microarray” and phrase “nucleic acid microarray” also include substrate-bound collections of plural nucleic acids in which the plurality of nucleic acids are distributably disposed on a plurality of beads, rather than on a unitary planar substrate, as is described, inter alia, in Brenner et al., [0055] Proc. Natl. Acad. Sci. USA 97(4):166501670 (2000), the disclosure of which is incorporated herein by reference in its entirety; in such case, the term “microarray” and phrase “nucleic acid microarray” refer to the plurality of beads in aggregate.
  • As used herein with respect to solution phase hybridization, the term “probe”, or equivalently, “nucleic acid probe” or “hybridization probe”, refers to an isolated nucleic acid of known sequence that is, or is intended to be, detectably labeled. As used herein with respect to a nucleic acid microarray, the term “probe” (or equivalently “nucleic acid probe” or “hybridization probe”) refers to the isolated nucleic acid that is, or is intended to be, bound to the substrate. In either such context, the term “target” refers to nucleic acid intended to be bound to probe by sequence complementarity. [0056]
  • As used herein, the expression “probe comprising SEQ ID NO:X”, and variants thereof, intends a nucleic acid probe, at least a portion of which probe has either (i) the sequence directly as given in the referenced SEQ ID NO:X, or (ii) a sequence complementary to the sequence as given in the referenced SEQ ID NO:X, the choice as between sequence directly as given and complement thereof dictated by the requirement that the probe be complementary to the desired target. [0057]
  • As used herein, the phrases “expression of a probe” and “expression of an isolated nucleic acid” and their linguistic equivalents intend that the probe or, respectively, the isolated nucleic acid, can hybridize detectably under high stringency conditions to a sample of nucleic acids that derive from mRNA from a given source. For example, and by way of illustration only, expression of a probe in “liver” means that the probe can hybridize detectably under high stringency conditions to a sample of nucleic acids that derive from mRNA obtained from liver. [0058]
  • As used herein, the terms “protein”, “polypeptide”, and “peptide” are used interchangeably to refer to a naturally-occurring or synthetic polymer of amino acid monomers (residues), irrespective of length, where amino acid monomer here includes naturally-occurring amino acids, naturally-occurring amino acid structural variants, and synthetic non-naturally occurring analogs that are capable of participating in peptide bonds. The terms “protein”, “polypeptide”, and “peptide” explicitly permits of post-translational and post-synthetic modifications, such as glycosylation. [0059]
  • The term “oligopeptide” herein denotes a protein, polypeptide, or peptide having 25 or fewer monomeric subunits. [0060]
  • The phrases “isolated protein”, “isolated polypeptide”, “isolated peptide” and “isolated oligopeptide” refer to a protein (equally, to a polypeptide, peptide, or oligopeptide) that is nonidentical to any protein molecule of identical amino acid sequence as found in nature; “isolated” does not require, although it does not prohibit, that the protein so described has itself been physically removed from its native environment. [0061]
  • For example, a protein can be said to be “isolated” when it includes amino acid analogues or derivatives not found in nature, or includes linkages other than standard peptide bonds. [0062]
  • When instead composed entirely of natural amino acids linked by peptide bonds, a protein can be said to be “isolated” when it exists at a purity not found in nature—where purity can be adjudged with respect to the presence of proteins of other sequence, with respect to the presence of non-protein compounds, such as nucleic acids, lipids, or other components of a biological cell, or when it exists in a composition not found in nature, such as in a host cell that does not naturally express that protein. [0063]
  • A “purified protein” (equally, a purified polypeptide, peptide, or oligopeptide) is an isolated protein, as above described, present at a concentration of at least 95%, as measured on a mass basis with respect to total protein in a composition. A “substantially purified protein” (equally, a substantially purified polypeptide, peptide, or oligopeptide) is an isolated protein, as above described, present at a concentration of at least 70%, as measured on a mass basis with respect to total protein in a composition. [0064]
  • As used herein, the phrase “protein isoforms” refers to a plurality of proteins having nonidentical primary amino acid sequence but that share amino acid sequence encoded by at least one common exon. [0065]
  • As used herein, the phrase “alternative splicing” and its linguistic equivalents includes all types of RNA processing that lead to expression of plural protein isoforms from a single gene; accordingly, the phrase “splice variant(s)” and its linguistic equivalents embraces mRNAs transcribed from a given gene that, however processed, collectively encode plural protein isoforms. For example, and by way of illustration only, splice variants can include exon insertions, exon extensions, exon truncations, exon deletions, alternatives in the 5′ untranslated region (“5′ UT”) and alternatives in the 3′ untranslated region (“3′ UT”). Such 3′ alternatives include, for example, differences in the site of RNA transcript cleavage and site of poly(A) addition. See, e.g., Gautheret et al., [0066] Genome Res. 8:524-530 (1998).
  • As used herein, “orthologues” are separate occurrences of the same gene in multiple species. The separate occurrences have similar, albeit nonidentical, amino acid sequences, the degree of sequence similarity depending, in part, upon the evolutionary distance of the species from a common ancestor having the same gene. [0067]
  • As used herein, the term “paralogues” indicates separate occurrences of a gene in one species. The separate occurrences have similar, albeit nonidentical, amino acid sequences, the degree of sequence similarity depending, in part, upon the evolutionary distance from the gene duplication event giving rise to the separate occurrences. [0068]
  • As used herein, the term “homologues” is generic to “orthologues” and “paralogues”. [0069]
  • As used herein, the term “antibody” refers to a polypeptide, at least a portion of which is encoded by at least one immunoglobulin gene, or fragment thereof, and that can bind specifically to a desired target molecule. The term includes naturally-occurring forms, as well as fragments and derivatives. [0070]
  • Fragments within the scope of the term include those produced by digestion with various proteases, those produced by chemical cleavage and/or chemical dissociation, and those produced recombinantly, so long as the fragment remains capable of specific binding to a target molecule. Among such fragments are Fab, Fab′, Fv, F(ab)′[0071] 2, and single chain Fv (scFv) fragments.
  • Derivatives within the scope of the term include antibodies (or fragments thereof) that have been modified in sequence, but remain capable of specific binding to a target molecule, including: interspecies chimeric and humanized antibodies; antibody fusions; heteromeric antibody complexes and antibody fusions, such as diabodies (bispecific antibodies), single-chain diabodies, and intrabodies (see, e.g., Marasco (ed.), [0072] Intracellular Antibodies: Research and Disease Applications, Springer-Verlag New York, Inc. (1998) (ISBN: 3540641513), the disclosure of which is incorporated herein by reference in its entirety).
  • As used herein, antibodies can be produced by any known technique, including harvest from cell culture of native B lymphocytes, harvest from culture of hybridomas, recombinant expression systems, and phage display. [0073]
  • As used herein, “antigen” refers to a ligand that can be bound by an antibody; an antigen need not itself be immunogenic. The portions of the antigen that make contact with the antibody are denominated “epitopes”. [0074]
  • “Specific binding” refers to the ability of two molecular species concurrently present in a heterogeneous (inhomogeneous) sample to bind to one another in preference to binding to other molecular species in the sample. Typically, a specific binding interaction will discriminate over adventitious binding interactions in the reaction by at least two-fold, more typically by at least 10-fold, often at least 100-fold; when used to detect analyte, specific binding is sufficiently discriminatory when determinative of the presence of the analyte in a heterogeneous (inhomogeneous) sample. Typically, the affinity or avidity of a specific binding reaction is least about 10[0075] −7 M, with specific binding reactions of greater specificity typically having affinity or avidity of at least 10−8 M to at least about 10−9 M.
  • As used herein, “molecular binding partners”—and equivalently, “specific binding partners”—refer to pairs of molecules, typically pairs of biomolecules, that exhibit specific binding. Nonlimiting examples are receptor and ligand, antibody and antigen, and biotin to any of avidin, streptavidin, neutrAvidin and captAvidin. [0076]
  • Nucleic Acid Molecules [0077]
  • In a first aspect, the invention provides isolated nucleic acids that encode three novel isoforms of the PAPP-E protein, variants having at least 90% sequence identity thereto, degenerate variants thereof, variants that encode PAPP-E proteins having conservative or moderately conservative substitutions, cross-hybridizing nucleic acids, and fragments thereof. [0078]
  • FIGS. 3, 4, and [0079] 5 present the nucleotide sequences of PAPP-Ea, PAPP-Eb, and PAPP-Ec cDNA clones, with predicted amino acid translations; the nucleotide sequences are further presented, respectively, in SEQ ID NOs:1 (full length nucleotide sequence of PAPP-Ea cDNA), 3 (full length amino acid coding sequence of PAPP-Ea), 8 (nucleotide sequence encoding the entirety of PAPP-Eb), 10 (full length amino acid coding sequence of PAPP-Eb), 15 (nucleotide sequence encoding the entirety of PAPP-Ec), and 16 (full length amino acid coding sequence of PAPP-Ec).
  • Unless otherwise indicated, each nucleotide sequence is set forth herein as a sequence of deoxyribonucleotides. It is intended, however, that the given sequence be interpreted as would be appropriate to the polynucleotide composition: for example, if the isolated nucleic acid is composed of RNA, the given sequence intends ribonucleotides, with uridine substituted for thymidine. [0080]
  • Unless otherwise indicated, nucleotide sequences of the isolated nucleic acids of the present invention were determined by sequencing a DNA molecule that had resulted, directly or indirectly, from at least one enzymatic polymerization reaction (e.g., reverse transcription and/or polymerase chain reaction) using an automated sequencer (such as the MegaBACE™ 1000, Molecular Dynamics, Sunnyvale, Calif., USA), or by reliance upon such sequence or upon genomic sequence prior-accessioned into a public database. Unless otherwise indicated, all amino acid sequences of the polypeptides of the present invention were predicted by translation from the nucleic acid sequences so determined. [0081]
  • As a consequence, any nucleic acid sequence presented herein may contain errors introduced by erroneous incorporation of nucleotides during polymerization, by erroroneous base calling by the automated sequencer (although such sequencing errors have been minimized for the nucleic acids directly determined herein, unless otherwise indicated, by the sequencing of each of the complementary strands of a duplex DNA), or by similar errors accessioned into the public database. [0082]
  • Accordingly, each of PAPP-Ea, PAPP-Eb, and PAPP-Ec cDNA clones described herein has been deposited in a public repository (American Type Culture Collection, Manassas, Va., USA) under accession numbers ______ (PAPP-Ea), ______ (PAPP-Eb), ______ (PAPP-Ec). Any errors in sequence reported herein can be determined and corrected by sequencing nucleic acids propagated from the deposited clones using standard techniques. [0083]
  • Single nucleotide polymorphisms (SNPs) occur frequently in eukaryotic genomes—more than 1.4 million SNPs have already identified in the human genome, International Human Genome Sequencing Consortium, [0084] Nature 409:860-921 (2001)—and the sequence determined from one individual of a species may differ from other allelic forms present within the population. Additionally, small deletions and insertions, rather than single nucleotide polymorphisms, are not uncommon in the general population, and often do not alter the function of the protein.
  • Accordingly, it is an aspect of the present invention to provide nucleic acids not only identical in sequence to those described with particularity herein, but also to provide isolated nucleic acids at least about 90% identical in sequence to those described with particularity herein, typically at least about 91%, 92%, 93%, 94%, or 95% identical in sequence to those decribed with particularity herein, usefully at least about 96%, 97%, 98%, or 99% identical in sequence to those described with particularity herein, and, most conservatively, at least about 99.5%, 99.6%, 99.7%, 99.8% and 99.9% identical in sequence to those described with particularity herein. These sequence variants can be naturally occurring or can result from human intervention, as by random or directed mutagenesis. [0085]
  • For purposes herein, percent identity of two nucleic acid sequences is determined using the procedure of Tatiana et al., “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, [0086] FEMS Microbiol Lett. 174:247-250 (1999), which procedure is effectuated by the computer program BLAST 2 SEQUENCES, available online at
  • http://www.ncbi.nlm.nih.gov/blast/b12seq/b12.html. [0087]
  • To assess percent identity of nucleic acids, the BLASTN module of BLAST 2 SEQUENCES is used with default values of (i) reward for a match: 1; (ii) penalty for a mismatch: −2; (iii) open gap [0088] 5 and extension gap 2 penalties; (iv) gap X_dropoff 50 expect 10 word size 11 filter, and both sequences are entered in their entireties.
  • As is well known, the genetic code is degenerate, with each amino acid except methionine translated from a plurality of codons, thus permitting a plurality of nucleic acids of disparate sequence to encode the identical protein. As is also well known, codon choice for optimal expression varies from species to species. The isolated nucleic acids of the present invention being useful for expression of PAPP-E proteins and protein fragments, it is, therefore, another aspect of the present invention to provide isolated isolated nucleic acids that encode PAPP-E isoforms, and portions thereof, not only identical in sequence to those described with particularity herein, but degenerate variants thereof as well. [0089]
  • As is also well known, amino acid substitutions occur frequently among natural allelic variants, with conservative substitutions often occasioning only de minimis change in protein function. [0090]
  • Accordingly, it is an aspect of the present invention to provide nucleic acids not only identical in sequence to those described with particularity herein, but also to provide isolated nucleic acids that encode PAPP-E isoforms, and portions thereof, having conservative amino acid substitutions, and also to provide isolated nucleic acids that encode PAPP-E isoforms, and portions thereof, having moderately conservative amino acid substitutions. [0091]
  • Although there are a variety of metrics for calling conservative amino acid substitutions, based primarily on either observed changes among evolutionarily related proteins or on predicted chemical similarity, for purposes herein a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix reproduced herein below (see Gonnet et al., [0092] Science 256(5062):1443-5 (1992)):
    A R N D C Q E G H I L K H F P S T W Y V
    A 2 −1 0 0 0 0 0 0 −1 −1 −1 0 −1 −2 0 1 1 −4 −2 0
    R −1 5 0 0 −2 2 0 −1 1 −2 −2 3 −2 −3 −1 0 0 −2 −2 −2
    N 0 0 4 2 −2 1 1 0 1 −3 −3 1 −2 −3 −1 1 0 −4 −1 −2
    D 0 0 2 5 −3 1 3 0 0 −4 −4 0 −3 −4 −1 0 0 −5 −3 −3
    C 0 −2 −2 −3 12 −2 −3 −2 −1 −1 −2 −3 −1 −1 −3 0 0 −1 0 0
    Q 0 2 1 1 −2 3 −2 −1 1 −2 −2 2 −1 −3 0 0 0 −3 −2 −2
    E 0 0 1 3 −3 2 4 −1 0 −3 −3 1 −2 −4 0 0 0 −4 −3 −2
    G 0 −1 0 0 −2 −1 −1 7 −1 −4 −4 −1 −4 −5 −2 0 −1 −4 −4 −3
    H −1 1 1 0 −1 1 0 −1 6 −2 −2 1 −1 0 −1 0 0 −1 2 −2
    I −1 −2 −3 −4 −1 −2 −3 −4 −2 4 3 −2 2 1 −3 −2 −1 −2 −1 3
    L −1 −2 −3 −4 −2 −2 −3 −4 −2 3 4 −2 3 2 −2 −2 −1 −1 0 2
    K 0 3 1 0 −3 2 1 −1 1 −2 −2 3 −1 −3 −1 0 0 −4 −2 −2
    M −1 −2 −2 −3 −1 −1 −2 −4 −1 2 3 −1 4 2 −2 −1 −1 −1 0 2
    F −2 −3 −3 −4 −1 −3 −4 −5 0 1 2 −3 2 7 −4 −3 −2 4 5 0
    P 0 −1 −1 −1 −3 0 0 −2 −1 −3 −2 −1 −2 −4 8 0 0 −5 −3 −2
    S 1 0 1 0 0 0 0 0 0 −2 −2 0 −1 −3 0 2 2 −3 −2 −1
    T 1 0 0 0 0 0 0 −1 0 −1 −1 0 −1 −2 0 2 2 −4 −2 0
    W −4 −2 −4 −5 −1 −3 −4 −4 −1 −2 −1 −4 −1 4 −5 −3 −4 14 4 −3
    Y −2 −2 −1 −3 0 −2 −3 −4 2 −1 0 −2 0 5 −3 −2 −2 4 8 −1
    V 0 −2 −2 −3 0 −2 −2 −3 −2 3 2 −2 2 0 −2 −1 0 −3 −1 3
  • For purposes herein, a “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix reproduced herein above. [0093]
  • As is also well known in the art, relatedness of nucleic acids can also be characterized using a functional test, the ability of the two nucleic acids to base-pair to one another at defined hybridization stringencies. [0094]
  • It is, therefore, another aspect of the invention to provide isolated nucleic acids not only identical in sequence to those described with particularity herein, but also to provide isolated nucleic acids (“cross-hybridizing nucleic acids”) that hybridize under high stringency conditions (as defined herein below) to all or to a portion of various of the isolated PAPP-E nucleic acids of the present invention (“reference nucleic acids”), as well as cross-hybridizing nucleic acids that hybridize under moderate stringency conditions to all or to a portion of various of the isolated PAPP-E nucleic acids of the present invention. [0095]
  • Such cross-hybridizing nucleic acids are useful, inter alia, as probes for, and to drive expression of, proteins related to the proteins of the present invention as alternative isoforms, homologues, paralogues, and orthologues. Particularly preferred orthologues are those from other primate species, such as chimpanzee, rhesus macaque, baboon, and gorilla, from rodents, such as rats, mice, guinea pigs, and from livestock, such as cow, pig, sheep, horse, goat. [0096]
  • For purposes herein, high stringency conditions are defined as aqueous hybridization (i.e., free of formamide) in 6×SSC (where 20×SSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65° C. for at least 8 hours, followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C. For purposes herein, moderate stringency conditions are defined as aqueous hybridization (i.e., free of formamide) in 6×SSC, 1% SDS at 65° C. for at least 8 hours, followed by one or more washes in 2×SSC, 0.1% SDS at room temperature. [0097]
  • The hybridizing portion of the reference nucleic acid is typically at least 15 nucleotides in length, often at least 17 nucleotides in length. Often, however, the hybridizing portion of the reference nucleic acid is at least 20 nucleotides in length, 25 nucleotides in length, and even 30 nucleotides, 35 nucleotides, 40 nucleotides, and 50 nucleotides in length. Of course, cross-hybridizing nucleic acids that hybridize to a larger portion of the reference nucleic acid—for example, to a portion of at least 50 nt, at least 100 nt, at least 150 nt, 200 nt, 250 nt, 300 nt, 350 nt, 400 nt, 450 nt, or 500 nt or more—or even to the entire length of the reference nucleic acid, are also useful. [0098]
  • The hybridizing portion of the cross-hybridizing nucleic acid is at least 75% identical in sequence to at least a portion of the reference nucleic acid. Typically, the hybridizing portion of the cross-hybridizing nucleic acid is at least 80%, often at least 85%, 86%, 87%, 88%, 89% or even at least 90% identical in sequence to at least a portion of the reference nucleic acid. Often, the hybridizing portion of the cross-hybridizing nucleic acid will be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in sequence to at least a portion of the reference nucleic acid sequence. At times, the hybridizing portion of the cross-hybridizing nucleic acid will be at least 99.5% identical in sequence to at least a portion of the reference nucleic acid. [0099]
  • The invention also provides fragments of various of the isolated nucleic acids of the present invention. [0100]
  • By “fragments” of a reference nucleic acid is here intended isolated nucleic acids, however obtained, that have a nucleotide sequence identical to a portion of the reference nucleic acid sequence, which portion is at least 17 nucleotides and less than the entirety of the reference nucleic acid. As so defined, “fragments” need not be obtained by physical fragmentation of the reference nucleic acid, although such provenance is not thereby precluded. [0101]
  • In theory, an oligonucleotide of 17 nucleotides is of sufficient length as to occur at random less frequently than once in the three gigabase human genome, and thus to provide a nucleic acid probe that can uniquely identify the reference sequence in a nucleic acid mixture of genomic complexity. As is well known, further specificity can be obtained by probing nucleic acid samples of subgenomic complexity, and/or by using plural fragments as short as 17 nucleotides in length collectively to prime amplification of nucleic acids, as, e.g., by polymerase chain reaction (PCR). [0102]
  • As further described herein below, nucleic acid fragments that encode at least 6 contiguous amino acids (i.e., fragments of 18 nucleotides or more) are useful in directing the expression or the synthesis of peptides that have utility in mapping the epitopes of the protein encoded by the reference nucleic acid. See, e.g., Geysen et al., “Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid,” [0103] Proc. Natl. Acad. Sci. USA 81:3998-4002 (1984); and U.S. Pat. Nos. 4,708,871 and 5,595,915, the disclosures of which are incorporated herein by reference in their entireties.
  • As further described herein below, fragments that encode at least 8 contiguous amino acids (i.e., fragments of 24 nucleotides or more) are useful in directing the expression or the synthesis of peptides that have utility as immunogens. See, e.g., Lerner, “Tapping the immunological repertoire to produce antibodies of predetermined specificity,” Nature 299:592-596 (1982); Shinnick et al., “Synthetic peptide immunogens as vaccines,” [0104] Annu. Rev. Microbiol. 37:425-46 (1983); Sutcliffe et al., “Antibodies that react with predetermined sites on proteins,” Science 219:660-6 (1983), the disclosures of which are incorporated herein by reference in their entireties.
  • The nucleic acid fragment of the present invention is thus at least 17 nucleotides in length, typically at least 18 nucleotides in length, and often at least 24 nucleotides in length. Often, the nucleic acid of the present invention is at least 25 nucleotides in length, and even 30 nucleotides, 35 nucleotides, 40 nucleotides, or 45 nucleotides in length. Of course, larger fragments having at least 50 nt, at least 100 nt, at least 150 nt, 200 nt, 250 nt, 300 nt, 350 nt, 400 nt, 450 nt, or 500 nt or more are also useful, and at times preferred. [0105]
  • Having been based upon the mining of genomic sequence, rather than upon surveillance of expressed message, the present invention further provides isolated genome-derived nucleic acids that include portions of the PAPP-E gene. [0106]
  • The invention particularly provides genome-derived single exon probes. [0107]
  • As further described in commonly owned and copending U.S. patent application Ser. No. 09/774,203, filed Jan. 29, 2001 and 09/632,366, filed Aug. 3, 2000, and provisional U.S. patent application No. 60/236,359, filed May 26, 2000 and 60/236,359, filed Sep. 27, 2000, the disclosures of which are incorporated herein by reference in their entireties, single exon probes comprise a portion of no more than one exon of the reference gene; the exonic portion is of sufficient length to hybridize under high stringency conditions to transcript-derived nucleic acids—such as mRNA or cDNA—that contain the exon or a portion thereof. [0108]
  • Genome-derived single exon probes typically further comprise, contiguous to a first end of the exon portion, a first intronic and/or intergenic sequence that is identically contiguous to the exon in the genome. Often, the genome-derived single exon probe further comprises, contiguous to a second end of the exonic portion, a second intronic and/or intergenic sequence that is identically contiguous to the exon in the genome. [0109]
  • The minimum length of genome-derived single exon probes is defined by the requirement that the exonic portion be of sufficient length to hybridize under high stringency conditions to transcript-derived nucleic acids. Accordingly, the exon portion is at least 17 nucleotides, typically at least 18 nucleotides, 20 nucleotides, 24 nucleotides, 25 nucleotides or even 30, 35, 40, 45, or 50 nucleotides in length, and can usefully include the entirety of the exon, up to 100 nt, 150 nt, 200 nt, 250 nt, 300 nt, 350 nt, 400 nt or even 500 nt or more in length. [0110]
  • The maximum length of genome-derived single exon probes is defined by the requirement that the probes contain portions of no more than one exon. Given variable spacing of exons through eukaryotic genomes, the maximum length is typically no more than 25 kb, often no more than 20 kb, 15 kb, 10 kb or 7.5 kb, or even no more than 5 kb, 4 kb, 3 kb, or even no more than about 2.5 kb in length. [0111]
  • Genome-derived single exon probes can usefully include at least a first terminal priming sequence not found in contiguity with the rest of the probe sequence in the genome, and often will contain a second terminal priming sequence not found in contiguity with the rest of the probe sequence in the genome. [0112]
  • The present invention also provides isolated genome-derived nucleic acids that include nucleic acid sequence elements that control transcription of the PAPP-E gene and its various isoforms. [0113]
  • The isolated nucleic acids of the present invention can be composed of natural nucleotides in native 5′-3′ phosphodiester internucleoside linkage—e.g., DNA or RNA—or can contain any or all of nonnatural nucleotide analogues, normative internucleoside bonds, or post-synthesis modifications, either throughout the length of the nucleic acid or localized to one or more portions thereof. As is well known in the art, when the isolated nucleic acid is used as a hybridization probe, the range of such nonnatural analogues, normative internucleoside bonds, or post-synthesis modifications will be limited to those that permit sequence-discriminating basepairing of the resulting nucleic acid. When used to direct expression or RNA or protein in vitro or in vivo, the range of such nonnatural analogues, normative internucleoside bonds, or post-synthesis modifications will be limited to those that permit the nucleic acid to function properly as a polymerization substrate. When the isolated nucleic acid is used as a therapeutic agent, the range of such changes will be limited to those that do not confer toxicity upon the isolated nucleic acid. [0114]
  • For example, when desired to be used as probes, the isolated nucleic acids of the present invention can usefully include nucleotide analogues that incorporate labels that are directly detectable, such as radiolabels or fluorophores, or nucleotide analogues that incorporate labels that can be visualized in a subsequent reaction, such as biotin or various haptens. [0115]
  • Common radiolabeled analogues include those labeled with [0116] 33P, 32P, and 35S, such as α-32P-dATP, α-32P-dCTP, α-32P-dGTP, α-32P-dTTP, α-32P-3′dATP, α-32P-ATP, α-32P-CTP, α-32P-GTP, α-32P-UTP, α-35S-dATP, γ-35S-GTP, γ-33P-dATP, and the like.
  • Commercially available fluorescent nucleotide analogues readily incorporated into the nucleic acids of the present invention include Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy3-dUTP (Amersham Pharmacia Biotech, Piscataway, N.J., USA), fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, Texas Red®-5-dUTP, Cascade Blue®-7-dUTP, BODIPY® FL-14-dUTP, BODIPY® TMR-14-dUTP, BODIPY® TR-14-dUTP, Rhodamine Green™-5-dUTP, Oregon Green® 488-5-dUTP, Texas Red®-12-dUTP, BODIPY® 630/650-14-dUTP, BODIPY® 650/665-14-dUTP, Alexa Fluor® 488-5-dUTP, Alexa Fluor® 532-5-dUTP, Alexa Fluor® 568-5-dUTP, Alexa Fluor® 594-5-dUTP, Alexa Fluor® 546-14-dUTP, fluorescein-12-UTP, tetramethylrhodamine-6-UTP, Texas Red®-5-UTP, Cascade Blue®-7-UTP, BODIPY® FL-14-UTP, BODIPY® TMR-14-UTP, BODIPY® TR-14-UTP, Rhodamine Green™-5-UTP, Alexa Fluor® 488-5-UTP, Alexa Fluor® 546-14-UTP (Molecular Probes, Inc. Eugene, Oreg., USA). [0117]
  • Protocols are available for custom synthesis of nucleotides having other fluorophores. Henegariu et al., “Custom Fluorecent-Nucleotide Synthesis as an Alternative Method for Nucleic Acid Labeling,” [0118] Nature Biotechnol. 18:345-348 (2000), the disclosure of which is incorporated herein by reference in its entirety.
  • Haptens that are commonly conjugated to nucleotides for subsequent labeling include biotin (biotin-11-dUTP, Molecular Probes, Inc., Eugene, Oreg., USA; biotin-21-UTP, biotin-21-dUTP, Clontech Laboratories, Inc., Palo Alto, Calif., USA), digoxigenin (DIG-11-dUTP, alkali labile, DIG-11-UTP, Roche Diagnostics Corp., Indianapolis, Ind., USA), and dinitrophenyl (dinitrophenyl-11-dUTP, Molecular Probes, Inc., Eugene, Oreg., USA). [0119]
  • As another example, when desired to be used for antisense inhibition of translation, the isolated nucleic acids of the present invention can usefully include altered, often nuclease-resistant, internucleoside bonds. See Hartmann et al. (eds.), [0120] Manual of Antisense Methodology (Perspectives in Antisense Science), Kluwer Law International (1999) (ISBN:079238539X); Stein et al. (eds.), Applied Antisense Oligonucleotide Technology, Wiley-Liss (cover (1998) (ISBN: 0471172790); Chadwick et al. (eds.), Oligonucleotides as Therapeutic Agents—Symposium No. 209, John Wiley & Son Ltd (1997) (ISBN: 0471972797), or for targeted gene correction, Gamper et al., Nucl. Acids Res. 28(21):4332-9 (2000), the disclosures of which are incorporated herein by reference in their entireties.
  • Modified oligonucleotide backbones often preferred when the nucleic acid is to be used for antisense purposes are, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, the disclosures of which are incorporated herein by reference in their entireties. [0121]
  • Preferred modified oligonucleotide backbones for antisense use that do not include a phosphorus atom have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH[0122] 2 component parts. Representative U.S. patents that teach the preparation of the above backbones include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, the disclosures of which are incorporated herein by reference in their entireties.
  • In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage are replaced with novel groups, such as peptide nucleic acids (PNA). [0123]
  • In PNA compounds, the phosphodiester backbone of the nucleic acid is replaced with an amide-containing backbone, in particular by repeating N-(2-aminoethyl) glycine units linked by amide bonds. Nucleobases are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone, typically by methylene carbonyl linkages. [0124]
  • The uncharged nature of the PNA backbone provides PNA/DNA and PNA/RNA duplexes with a higher thermal stability than is found in DNA/DNA and DNA/RNA duplexes, resulting from the lack of charge repulsion between the PNA and DNA or RNA strand. In general, the Tm of a PNA/DNA or PNA/RNA duplex is 1° C. higher per base pair than the Tm of the corresponding DNA/DNA or DNA/RNA duplex (in 100 mM NaCl). [0125]
  • The neutral backbone also allows PNA to form stable DNA duplexes largely independent of salt concentration. At low ionic strength, PNA can be hybridized to a target sequence at temperatures that make DNA hybridization problematic or impossible. And unlike DNA/DNA duplex formation, PNA hybridization is possible in the absence of magnesium. Adjusting the ionic strength, therefore, is useful if competing DNA or RNA is present in the sample, or if the nucleic acid being probed contains a high level of secondary structure. [0126]
  • PNA also demonstrates greater specificity in binding to complementary DNA. A PNA/DNA mismatch is more destabilizing than DNA/DNA mismatch. A single mismatch in mixed a PNA/DNA 15-mer lowers the Tm by 8-20° C. (15° C. on average). In the corresponding DNA/DNA duplexes, a single mismatch lowers the Tm by 4-16° C. (11° C. on average). Because PNA probes can be significantly shorter than DNA probes, their specificity is greater. [0127]
  • Additionally, nucleases and proteases do not recognize the PNA polyamide backbone with nucleobase sidechains. As a result, PNA oligomers are resistant to degradation by enzymes, and the lifetime of these compounds is extended both in vivo and in vitro. In addition, PNA is stable over a wide pH range. [0128]
  • Because its backbone is formed from amide bonds, PNA can be synthesized using a modified peptide synthesis protocol. PNA oligomers can be synthesized by both Fmoc and tBoc methods. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference; automated PNA synthesis is readily achievable on commercial synthesizers (see, e.g., “PNA User's Guide,” Rev. 2, February 1998, Perseptive Biosystems Part No. 60138, Applied Biosystems, Inc., Foster City, Calif.). [0129]
  • PNA chemistry and applications are reviewed, inter alia, in Ray et al., [0130] FASEB J. 14(9):1041-60 (2000); Nielsen et al., Pharmacol Toxicol. 86(1):3-7 (2000); Larsen et al., Biochim Biophys Acta. 1489(1):159-66 (1999); Nielsen, Curr. Opin. Struct. Biol. 9(3):353-7 (1999), and Nielsen, Curr. Opin. Biotechnol. 10(1):71-5 (1999), the disclosures of which are incorporated herein by reference in their entireties.
  • Differences from nucleic acid compositions found in nature—e.g., normative bases, altered internucleoside linkages, post-synthesis modification—can be present throughout the length of the nucleic acid or can, instead, usefully be localized to discrete portions thereof. As an example of the latter, chimeric nucleic acids can be synthesized that have discrete DNA and RNA domains and demonstrated utility for targeted gene repair, as further described in U.S. Pat. Nos. 5,760,012 and 5,731,181, the disclosures of which are incorporated herein by reference in their entireties. As another example, chimeric nucleic acids comprising both DNA and PNA have been demonstrated to have utility in modified PCR reactions. See Misra et al., [0131] Biochem. 37: 1917-1925 (1998); see also Finn et al., Nucl. Acids Res. 24: 3357-3363 (1996), incorporated herein by reference.
  • Unless otherwise specified, nucleic acids of the present invention can include any topological conformation appropriate to the desired use; the term thus explicitly comprehends, among others, single-stranded, double-stranded, triplexed, quadruplexed, partially double-stranded, partially-triplexed, partially-quadruplexed, branched, hairpinned, circular, and padlocked conformations. Padlock conformations and their utility are further described in Baner et al., [0132] Curr. Opin. Biotechnol. 12:11-15 (2001); Escude et al., Proc. Natl. Acad. Sci. USA 14;96(19):10603-7 (1999); Nilsson et al., Science 265(5181):2085-8 (1994), the disclosures of which are incorporated herein by reference in their entireties. Triplex and quadruplex conformations, and their utility, are reviewed in Praseuth et al., Biochim. Biophys. Acta. 1489(1):181-206 (1999); Fox, Curr. Med. Chem. 7(1):17-37 (2000); Kochetkova et al., Methods Mol. Biol. 130:189-201 (2000); Chan et al., J. Mol. Med. 75(4):267-82 (1997), the disclosures of which are incorporated herein by reference in their entireties.
  • The nucleic acids of the present invention can be detectably labeled. Commonly-used labels include radionuclides, such as [0133] 32P, 33P, 35S, 3H (and for nmr detection, 13C and 15N), haptens that can be detected by specific antibody or high affinity binding partner (such as avidin), and fluorophores.
  • As noted above, detectable labels can be incorporated by inclusion of labeled nucleotide analogues in the nucleic acid. Such analogues can be incorporated by enzymatic polymerization, such as by nick translation, random priming, polymerase chain reaction (PCR), terminal transferase tailing, and end-filling of overhangs, for DNA molecules, and in vitro transcription driven, e.g., from phage promoters, such as T7, T3, and SP6, for RNA molecules. Commercial kits are readily available for each such labeling approach. [0134]
  • Analogues can also be incorporated during automated solid phase chemical synthesis. [0135]
  • As is well known, labels can also be incorporated after nucleic acid synthesis, with the 5′ phosphate and 3′ hydroxyl providing convenient sites for post-synthetic covalent attachment of detectable labels. [0136]
  • Various other post-synthetic approaches permit internal labeling of nucleic acids. [0137]
  • For example, fluorophores can be attached using a cisplatin reagent that reacts with the N7 of guanine residues (and, to a lesser extent, adenine bases) in DNA, RNA, and PNA to provide a stable coordination complex between the nucleic acid and fluorophore label (Universal Linkage System) (available from Molecular Probes, Inc., Eugene, Oreg., USA and Amersham Pharmacia Biotech, Piscataway, N.J., USA); see Alers et al., [0138] Genes, Chromosomes & Cancer, Vol. 25, pp. 301-305 (1999); Jelsma et al., J. NIH Res. 5:82 (1994); Van Belkum et al., BioTechniques 16:148-153 (1994), incorporated herein by reference. As another example, nucleic acids can be labeled using a disulfide-containing linker (FastTag™ Reagent, Vector Laboratories, Inc., Burlingame, Calif., USA) that is photo- or thermally coupled to the target nucleic acid using aryl azide chemistry; after reduction, a free thiol is available for coupling to a hapten, fluorophore, sugar, affinity ligand, or other marker.
  • Multiple independent or interacting labels can be incorporated into the nucleic acids of the present invention. For example, both a fluorophore and a moiety that in proximity thereto acts to quench fluorescence can be included to report specific hybridization through release of fluorescence quenching, Tyagi et al., [0139] Nature Biotechnol. 14: 303-308 (1996); Tyagi et al., Nature Biotechnol. 16, 49-53 (1998); Sokol et al., Proc. Natl. Acad. Sci. USA 95: 11538-11543 (1998); Kostrikis et al., Science 279:1228-1229 (1998); Marras et al., Genet. Anal. 14: 151-156 (1999); U.S. Pat. Nos. 5,846,726, 5,925,517, 5,925,517, or to report exonucleotidic excision, U.S. Pat. No. 5,538,848; Holland et al., Proc. Natl. Acad. Sci. USA 88:7276-7280 (1991); Heid et al., Genome Res. 6(10):986-94 (1996); Kuimelis et al., Nucleic Acids Symp Ser. (37):255-6 (1997); U.S. Pat. No. 5,723,591, the disclosures of which are incorporated herein by reference in their entireties.
  • So labeled, the isolated nucleic acids of the present invention can be used as probes, as further described below. [0140]
  • Nucleic acids of the present invention can also usefully be bound to a substrate. The substrate can porous or solid, planar or non-planar, unitary or distributed; the bond can be covalent or noncovalent. Bound to a substrate, nucleic acids of the present invention can be used as probes in their unlabeled state. [0141]
  • For example, the nucleic acids of the present invention can usefully be bound to a porous substrate, commonly a membrane, typically comprising nitrocellulose, nylon, or positively-charged derivatized nylon; so attached, the nucleic acids of the present invention can be used to detect PAPP-E nucleic acids present within a labeled nucleic acid sample, either a sample of genomic nucleic acids or a sample of transcript-derived nucleic acids, e.g. by reverse dot blot. [0142]
  • The nucleic acids of the present invention can also usefully be bound to a solid substrate, such as glass, although other solid materials, such as amorphous silicon, crystalline silicon, or plastics, can also be used. Such plastics include polymethylacrylic, polyethylene, polypropylene, polyacrylate, polymethylmethacrylate, polyvinylchloride, polytetrafluoroethylene, polystyrene, polycarbonate, polyacetal, polysulfone, celluloseacetate, cellulosenitrate, nitrocellulose, or mixtures thereof. [0143]
  • Typically, the solid substrate will be rectangular, although other shapes, particularly disks and even spheres, present certain advantages. Particularly advantageous alternatives to glass slides as support substrates for array of nucleic acids are optical discs, as described in Demers, “Spatially Addressable Combinatorial Chemical Arrays in CD-ROM Format,” international patent publication WO 98/12559, incorporated herein by reference in its entirety. [0144]
  • The nucleic acids of the present invention can be attached covalently to a surface of the support substrate or applied to a derivatized surface in a chaotropic agent that facilitates denaturation and adherence by presumed noncovalent interactions, or some combination thereof. [0145]
  • The nucleic acids of the present invention can be bound to a substrate to which a plurality of other nucleic acids are concurrently bound, hybridization to each of the plurality of bound nucleic acids being separately detectable. At low density, e.g. on a porous membrane, these substrate-bound collections are typically denominated macroarrays; at higher density, typically on a solid support, such as glass, these substrate bound collections of plural nucleic acids are colloquially termed microarrays. As used herein, the term microarray includes arrays of all densities. It is, therefore, another aspect of the invention to provide microarrays that include the nucleic acids of the present invention. [0146]
  • The isolated nucleic acids of the present invention can be used as hybridization probes to detect, characterize, and quantify PAPP-E nucleic acids in, and isolate PAPP-E nucleic acids from, both genomic and transcript-derived nucleic acid samples. When free in solution, such probes are typically, but not invariably, detectably labeled; bound to a substrate, as in a microarray, such probes are typically, but not invariably unlabeled. [0147]
  • For example, the isolated nucleic acids of the present invention can be used as probes to detect and characterize gross alterations in the PAPP-E genomic locus, such as deletions, insertions, translocations, and duplications of the PAPP-E genomic locus through fluorescence in situ hybridization (FISH) to chromosome spreads. See, e.g., Andreeff et al. (eds.), [0148] Introduction to Fluorescence In Situ Hybridization: Principles and Clinical Applications, John Wiley & Sons (1999) (ISBN: 0471013455), the disclosure of which is incorporated herein by reference in its entirety. The isolated nucleic acids of the present invention can be used as probes to assess smaller genomic alterations using, e.g., Southern blot detection of restriction fragment length polymorphisms. The isolated nucleic acids of the present invention can be used as probes to isolate genomic clones that include the nucleic acids of the present invention, which thereafter can be restriction mapped and sequenced to identify deletions, insertions, translocations, and substitutions (single nucleotide polymorphisms, SNPs) at the sequence level.
  • The isolated nucleic acids of the present invention can be also be used as probes to detect, characterize, and quantify PAPP-E nucleic acids in, and isolate PAPP-E nucleic acids from, transcript-derived nucleic acid samples. [0149]
  • For example, the isolated nucleic acids of the present invention can be used as hybridization probes to detect, characterize by length, and quantify PAPP-E mRNA by northern blot of total or poly-A[0150] +-selected RNA samples. For example, the isolated nucleic acids of the present invention can be used as hybridization probes to detect, characterize by location, and quantify PAPP-E message by in situ hybridization to tissue sections (see, e.g., Schwarchzacher et al., In Situ Hybridization, Springer-Verlag New York (2000) (ISBN: 0387915966), the disclosure of which is incorporated herein by reference in its entirety). For example, the isolated nucleic acids of the present invention can be used as hybridization probes to measure the representation of PAPP-E clones in a cDNA library. For example, the isolated nucleic acids of the present invention can be used as hybridization probes to isolate PAPP-E nucleic acids from cDNA libraries, permitting sequence level characterization of PAPP-E messages, including identification of deletions, insertions, truncations—including deletions, insertions, and truncations of exons in alternatively spliced forms—and single nucleotide polymorphisms.
  • All of the aforementioned probe techniques are well within the skill in the art, and are described at greater length in standard texts such as Sambrook et al., [0151] Molecular Cloning: A Laboratory Manual (3rd ed.), Cold Spring Harbor Laboratory Press (2001) (ISBN: 0879695773); Ausubel et al. (eds.), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology (4th ed.), John Wiley & Sons, 1999 (ISBN: 047132938X); and Walker et al. (eds.), The Nucleic Acids Protocols Handbook, Humana Press (2000) (ISBN: 0896034593), the disclosures of which are incorporated herein by reference in their entirety.
  • As described in the Examples herein below, the nucleic acids of the present invention can also be used to detect and quantify PAPP-E nucleic acids in transcript-derived samples—that is, to measure expression of the PAPP-E gene—when included in a microarray. Measurement of placental PAPP-E expression has particular utility in prenatal diagnosis, as further described in the Examples herein below. [0152]
  • As would be readily apparent to one of skill in the art, each PAPP-E nucleic acid probe—whether labeled, substrate-bound, or both—is thus currently available for use as a tool for measuring the level of PAPP-E expression in each of the tissues in which expression has already been confirmed, notably placenta. The utility is specific to the probe: under high stringency conditions, the probe reports the level of expression of message specifically containing that portion of the PAPP-E gene included within the probe. [0153]
  • Measuring tools are well known in many arts, not just in molecular biology, and are known to possess credible, specific, and substantial utility. For example, U.S. Pat. No. 6,016,191 describes and claims a tool for measuring characteristics of fluid flow in a hydrocarbon well; U.S. Pat. No. 6,042,549 describes and claims a device for measuring exercise intensity; U.S. Pat. No. 5,889,351 describes and claims a device for measuring viscosity and for measuring characteristics of a fluid; U.S. Pat. No. 5,570,694 describes and claims a device for measuring blood pressure; U.S. Pat. No. 5,930,143 describes and claims a device for measuring the dimensions of machine tools; U.S. Pat. No. 5,279,044 describes and claims a measuring device for determining an absolute position of a movable element; U.S. Pat. No. 5,186,042 describes and claims a device for measuring action force of a wheel; and U.S. Pat. No. 4,246,774 describes and claims a device for measuring the draft of smoking articles such as cigarettes. [0154]
  • As for tissues not yet demonstrated to express PAPP-E, the PAPP-E nucleic acid probes of the present invention are currently available as tools for surveying such tissues to detect the presence of PAPP-E nucleic acids. [0155]
  • Survey tools—i.e., tools for determining the presence and/or location of a desired object by search of an area—are well known in many arts, not just in molecular biology, and are known to possess credible, specific, and substantial utility. For example, U.S. Pat. No. 6,046,800 describes and claims a device for surveying an area for objects that move; U.S. Pat. No. 6,025,201 describes and claims an apparatus for locating and discriminating platelets from non-platelet particles or cells on a cell-by-cell basis in a whole blood sample; U.S. Pat. No. 5,990,689 describes and claims a device for detecting and locating anomalies in the electromagnetic protection of a system; U.S. Pat. No. 5,984,175 describes and claims a device for detecting and identifying wearable user identification units; U.S. Pat. No. 3,980,986 (“Oil well survey tool”), describes and claims a tool for finding the position of a drill bit working at the bottom of a borehole. [0156]
  • As noted above, the nucleic acid probes of the present invention are useful in constructing microarrays; the microarrays, in turn, are products of manufacture that are useful for measuring and for surveying gene expression. [0157]
  • When included on a microarray, each PAPP-E nucleic acid probe makes the microarray specifically useful for detecting that portion of the PAPP-E gene included within the probe, thus imparting upon the microarray device the ability to detect a signal where, absent such probe, it would have reported no signal. This utility makes each individual probe on such microarray akin to an antenna, circuit, firmware or software element included in an electronic apparatus, where the antenna, circuit, firmware or software element imparts upon the apparatus the ability newly and additionally to detect signal in a portion of the radio-frequency spectrum where previously it could not; such devices are known to have specific, substantial, and credible utility. [0158]
  • Changes in expression need not be observed for the measurement of expression to have utility. [0159]
  • For example, where gene expression analysis is used to assess toxicity of chemical agents on cells, the failure of the agent to change a gene's expression level is evidence that the drug likely does not affect the pathway of which the gene's expressed protein is a part. Analogously, where gene expression analysis is used to assess side effects of pharmacologic agents—whether in lead compound discovery or in subsequent screening of lead compound derivatives—the inability of the agent to alter a gene's expression level is evidence that the drug does not affect the pathway of which the gene's expressed protein is a part. [0160]
  • WO 99/58720, incorporated herein by reference in its entirety, provides methods for quantifying the relatedness of a first and second gene expression profile and for ordering the relatedness of a plurality of gene expression profiles, without regard to the identity or function of the genes whose expression is used in the calculation. [0161]
  • Gene expression analysis, including gene expression analysis by microarray hybridization, is, of course, principally a laboratory-based art. Devices and apparatus used principally in laboratories to facilitate laboratory research are well-established to possess specific, substantial, and credible utility. For example, U.S. Pat. No. 6,001,233 describes and claims a gel electrophoresis apparatus having a cam-activated clamp; for example, U.S. Pat. No. 6,051,831 describes and claims a high mass detector for use in time-of-flight mass spectrometers; for example, U.S. Pat. No. 5,824,269 describes and claims a flow cytometer—few gel electrophoresis apparatuses, TOF-MS devices, or flow cytometers are sold for consumer use. [0162]
  • Indeed, and in particular, nucleic acid microarrays, as devices intended for laboratory use in measuring gene expression, are well-established to have specific, substantial and credible utility. Thus, the microarrays of the present invention have at least the specific, substantial and credible utilities of the microarrays claimed as devices and articles of manufacture in the following U.S. patents, the disclosures of each of which is incorporated herein by reference: U.S. Pat. No. 5,445,934 (“Array of oligonucleotides on a solid substrate”); U.S. Pat. No. 5,744,305 (“Arrays of materials attached to a substrate”); and U.S. Pat. No. 6,004,752 (“Solid support with attached molecules”). [0163]
  • Genome-derived single exon probes and genome-derived single exon probe microarrays have the additional utility, inter alia, of permitting high-throughput detection of splice variants of the nucleic acids of the present invention, as further described in copending and commonly owned U.S. patent application Ser. No. 09/632,366, filed Aug. 3, 2000, the disclosure of which is incorporated herein by reference in its entirety. [0164]
  • The isolated nucleic acids of the present invention can also be used to prime synthesis of nucleic acid, for purpose of either analysis or isolation, using mRNA, cDNA, or genomic DNA as template. [0165]
  • For use as primers, at least 17 contiguous nucleotides of the isolated nucleic acids of the present invention will be used. Often, at least 18, 19, or 20 contiguous nucleotides of the nucleic acids of the present invention will be used, and on occasion at least 20, 22, 24, or 25 contiguous nucleotides of the nucleic acids of the present invention will be used, and even 30 nucleotides or more of the nucleic acids of the present invention can be used to prime specific synthesis. [0166]
  • The nucleic acid primers of the present invention can be used, for example, to prime first strand cDNA synthesis on an mRNA template. [0167]
  • Such primer extension can be done directly to analyze the message. Alternatively, synthesis on an mRNA template can be done to produce first strand cDNA. The first strand cDNA can thereafter be used, inter alia, directly as a single-stranded probe, as above-described, as a template for sequencing—permitting identification of alterations, including deletions, insertions, and substitutions, both normal allelic variants and mutations associated with abnormal phenotypes- or as a template, either for second strand cDNA synthesis (e.g., as an antecedent to insertion into a cloning or expression vector), or for amplification. [0168]
  • The nucleic acid primers of the present invention can also be used, for example, to prime single base extension (SBE) for SNP detection (see, e.g., U.S. Pat. No. 6,004,744, the disclosure of which is incorporated herein by reference in its entirety). [0169]
  • As another example, the nucleic acid primers of the present invention can be used to prime amplification of PAPP-E nucleic acids, using transcript-derived or genomic DNA as template. [0170]
  • Primer-directed amplification methods are now well-established in the art. Methods for performing the polymerase chain reaction (PCR) are compiled, inter alia, in McPherson, [0171] PCR (Basics: From Background to Bench), Springer Verlag (2000) (ISBN: 0387916008); Innis et al. (eds.), PCR Applications: Protocols for Functional Genomics, Academic Press (1999) (ISBN: 0123721857); Gelfand et al. (eds.), PCR Strategies, Academic Press (1998) (ISBN: 0123721822); Newton et al., PCR, Springer-Verlag New York (1997) (ISBN: 0387915060); Burke (ed.), PCR: Essential Techniques, John Wiley & Son Ltd (1996) (ISBN: 047195697X); White (ed.), PCR Cloning Protocols: From Molecular Cloning to Genetic Engineering, Vol. 67, Humana Press (1996) (ISBN: 0896033430); McPherson et al. (eds.), PCR 2: A Practical Approach, Oxford University Press, Inc. (1995) (ISBN: 0199634254), the disclosures of which are incorporated herein by reference in their entireties. Methods for performing RT-PCR are collected, e.g., in Siebert et al. (eds.), Gene Cloning and Analysis by RT-PCR, Eaton Publishing Company/Bio Techniques Books Division, 1998 (ISBN: 1881299147); Siebert (ed.), PCR Technique:RT-PCR, Eaton Publishing Company/BioTechniques Books (1995) (ISBN:1881299139), the disclosure of which is incorporated herein by reference in its entirety.
  • Isothermal amplification approaches, such as rolling circle amplification, are also now well-described. See, e.g., Schweitzer et al., [0172] Curr. Opin. Biotechnol. 12(1):21-7 (2001); U.S. Pat. Nos. 5,854,033 and 5,714,320 and international patent publications WO 97/19193 and WO 00/15779, the disclosures of which are incorporated herein by reference in their entireties. Rolling circle amplification can be combined with other techniques to facilitate SNP detection. See, e.g., Lizardi et al., Nature Genet. 19(3):225-32 (1998).
  • As further described below, nucleic acids of the present invention, inserted into vectors that flank the nucleic acid insert with a phage promoter, such as T7, T3, or SP6 promoter, can be used to drive in vitro expression of RNA complementary to either strand of the nucleic acid of the present invention. The RNA can be used, inter alia, as a single-stranded probe, to effect subtraction, or for in vitro translation. [0173]
  • As will be further discussed herein below, nucleic acids of the present invention that encode PAPP-E protein or portions thereof can be used, inter alia, to express the PAPP-E proteins or protein fragments, either alone, or as part of fusion proteins. [0174]
  • Expression can be from genomic nucleic acids of the present invention, or from transcript-derived nucleic acids of the present invention. [0175]
  • Where protein expression is effected from genomic DNA, expression will typically be effected in eukaryotic, typically mammalian, cells capable of splicing introns from the initial RNA transcript. Expression can be driven from episomal vectors, such as EBV-based vectors, or can be effected from genomic DNA integrated into a host cell chromosome. As will be more fully described below, where expression is from transcript-derived (or otherwise intron-less) nucleic acids of the present invention, expression can be effected in wide variety of prokaryotic or eukaryotic cells. [0176]
  • Expressed in vitro, the protein, protein fragment, or protein fusion can thereafter be isolated, to be used, inter alia, as a standard in immunoassays specific for the proteins, or protein isoforms, of the present invention; to be used as a therapeutic agent, e.g., to be administered as passive replacement therapy in individuals deficient in the proteins of the present invention, or to be administered as a vaccine; to be used for in vitro production of specific antibody, the antibody thereafter to be used, e.g., as an analytical reagent for detection and quantitation of the proteins of the present invention or to be used as an immunotherapeutic agent. [0177]
  • The isolated nucleic acids of the present invention can also be used to drive in vivo expression of the proteins of the present invention. In vivo expression can be driven from a vector—typically a viral vector, often a vector based upon a replication incompetent retrovirus, an adenovirus, or an adeno-associated virus (AAV)—for purpose of gene therapy. In vivo expression can also be driven from signals endogenous to the nucleic acid or from a vector, often a plasmid vector, for purpose of “naked” nucleic acid vaccination, as further described in U.S. Pat. Nos. 5,589,466; 5,679,647; 5,804,566; 5,830,877; 5,843,913; 5,880,104; 5,958,891; 5,985,847; 6,017,897; 6,110,898; 6,204,250, the disclosures of which are incorporated herein by reference in their entireties. [0178]
  • The nucleic acids of the present invention can also be used for antisense inhibition of translation. See Phillips (ed.), [0179] Antisense Technology, Part B, Methods in Enzymology Vol. 314, Academic Press, Inc. (1999) (ISBN: 012182215X); Phillips (ed.), Antisense Technology, Part A, Methods in Enzymology Vol. 313, Academic Press, Inc. (1999) (ISBN: 0121822141); Hartmann et al. (eds.), Manual of Antisense Methodology (Perspectives in Antisense Science), Kluwer Law International (1999) (ISBN:079238539X); Stein et al. (eds.), Applied Antisense Oligonucleotide Technology, Wiley-Liss (cover (1998) (ISBN: 0471172790); Agrawal et al. (eds.), Antisense Research and Application, Springer-Verlag New York, Inc. (1998) (ISBN: 3540638334); Lichtenstein et al. (eds.), Antisense Technology: A Practical Approach, Vol. 185, Oxford University Press, INC. (1998) (ISBN: 0199635838); Gibson (ed.), Antisense and Ribozyme Methodology: Laboratory Companion, Chapman & Hall (1997) (ISBN: 3826100794); Chadwick et al. (eds.), Oligonucleotides as Therapeutic Agents—Symposium No. 209, John Wiley & Son Ltd (1997) (ISBN: 0471972797), the disclosures of which are incorporated herein by reference in their entireties.
  • Nucleic acids of the present invention that encode full-length human PAPP-E protein isoforms, particularly cDNAs encoding full-length isoforms, have additional, well-recognized, utility as products of manufacture suitable for sale. [0180]
  • For example, cDNAs encoding full length human proteins have immediate, real world utility as commercial products suitable for sale. Invitrogen Corp. (Carlsbad, Calif., USA), through its Research Genetics subsidiary, sells full length human cDNAs cloned into one of a selection of expression vectors as GeneStorm® expression-ready clones; utility is specific for the gene, since each gene is capable of being ordered separately and has a distinct catalogue number, and utility is substantial, each clone selling for $650.00 US. [0181]
  • Nucleic acids of the present invention that include genomic regions encoding the human PAPP-E protein isoforms, or portions thereof, have yet further utilities. [0182]
  • For example, genomic nucleic acids of the present invention can be used as amplification substrates, e.g. for preparation of genome-derived single exon probes of the present invention, described above, and further described in copending and commonly-owned U.S. patent application Ser. No. 09/774,203, filed Jan. 29, 2001, and Ser. No. 09/632,366, filed Aug. 3, 2000 and commonly-owned and copending U.S. provisional patent application No. 60/207,456, filed May 26, 2000, No. 60/234,687, filed Sep. 21, 2000, No. 60/236,359, filed Sep. 27, 2000, the disclosures of which are incorporated herein by reference in their entireties. [0183]
  • As another example, genomic nucleic acids of the present invention can be integrated non-homologously into the genome of somatic cells, e.g. CHO cells, COS cells, or 293 cells, with or without amplification of the insertional locus, in order, e.g., to create stable cell lines capable of producing the proteins of the present invention. [0184]
  • As another example, more fully described herein below, genomic nucleic acids of the present invention can be integrated nonhomologously into embryonic stem (ES) cells to create transgenic non-human animals capable of producing the proteins of the present invention. [0185]
  • Genomic nucleic acids of the present invention can also be used to target homologous recombination to the human PAPP-E locus. See, e.g., U.S. Pat. Nos. 6,187,305; 6,204,061; 5,631,153; 5,627,059; 5,487,992; 5,464,764; 5,614,396; 5,527,695 and 6,063,630; and Kmiec et al. (eds.), [0186] Gene Targeting Protocols, Vol. 133, Humana Press (2000) (ISBN: 0896033600); Joyner (ed.), Gene Targeting: A Practical Approach, Oxford University Press, Inc. (2000) (ISBN: 0199637938); Sedivy et al., Gene Targeting, Oxford University Press (1998) (ISBN: 071677013X); Tymms et al. (eds.), Gene Knockout Protocols, Humana Press (2000) (ISBN: 0896035727); Mak et al. (eds.), The Gene Knockout FactsBook, Vol. 2, Academic Press, Inc. (1998) (ISBN: 0124660444); Torres et al., Laboratory Protocols for Conditional Gene Targeting, Oxford University Press (1997) (ISBN: 019963677X); Vega (ed.), Gene Targeting, CRC Press, LLC (1994) (ISBN: 084938950X), the disclosures of which are incorporated herein by reference in their entireties.
  • Where the genomic region includes transcription regulatory elements, homologous recombination can be used to alter the expression of PAPP-E, both for purpose of in vitro production of PAPP-E protein from human cells, and for purpose of gene therapy. See, e.g., U.S. Pat. Nos. 5,981,214, 6,048,524; 5,272,071. [0187]
  • Fragments of the nucleic acids of the present invention smaller than those typically used for homologous recombination can also be used for targeted gene correction or alteration, possibly by cellular mechanisms different from those engaged during homologous recombination. [0188]
  • For example, partially duplexed RNA/DNA chimeras have been shown to have utility in targeted gene correction, U.S. Pat. Nos. 5,945,339, 5,888,983, 5,871,984, 5,795,972, 5,780,296, 5,760,012, 5,756,325, 5,731,181, the disclosures of which are incorporated herein by reference in their entireties. So too have small oligonucleotides fused to triplexing domains have been shown to have utility in targeted gene correction, Culver et al., “Correction of chromosomal point mutations in human cells with bifunctional oligonucleotides,” [0189] Nature Biotechnol. 17(10):989-93 (1999), as have oligonucleotides having modified terminal bases or modified terminal internucleoside bonds, Gamper et al., Nucl. Acids Res. 28(21):4332-9 (2000), the disclosures of which are incorporated herein by reference.
  • Nucleic acids of the present invention can be obtained by using the labeled probes of the present invention to probe nucleic acid samples, such as genomic libraries, cDNA libraries, and mRNA samples, by standard techniques. Nucleic acids of the present invention can also be obtained by amplification, using the nucleic acid primers of the present invention, as further demonstrated in Example 1, herein below. Nucleic acids of the present invention of fewer than about 100 nt can also be synthesized chemically, typically by solid phase synthesis using commercially available automated synthesizers. [0190]
  • “Full Length” PAPP-E Isoform Nucleic Acids [0191]
  • In a first series of nucleic acid embodiments, the invention provides isolated nucleic acids that encode the entirety of a PAPP-E protein isoform. As discussed above, the “full-length” nucleic acids of the present invention can be used, inter alia, to express full length PAPP-Ea, PAPP-Eb, and PAPP-Ec isoforms. The full-length nucleic acids can also be used as nucleic acid probes; used as probes, the isolated nucleic acids of these embodiments will hybridize to all known isoforms of PAPP-E without discriminating thereamong. [0192]
  • In a first such embodiment, the invention provides an isolated nucleic acid comprising (i) the nucleotide sequence of the nucleic acid of ATCC deposit ______, (ii) the nucleotide sequence of SEQ ID NO:1, or (iii) the complement of (i) or (ii). The ATCC deposit has, and SEQ ID NO:1 presents, the entire cDNA of PAPP-Ea, including the 5′ untranslated (UT) region and 31 UT. [0193]
  • In a second embodiment, the invention provides an isolated nucleic acid comprising (i) the nucleotide sequence of SEQ ID NO:2, (ii) a degenerate variant of the nucleotide sequence of SEQ ID NO:2, or (iii) the complement (i) or (ii). SEQ ID NO:2 presents the open reading frame from SEQ ID NO:1. [0194]
  • In a third embodiment, the invention provides an isolated nucleic acid comprising (i) a nucleotide sequence that encodes a polypeptide with the amino acid sequence of SEQ ID NO:3 or (ii) the complement of a nucleotide sequence that encodes a polypeptide with the amino acid sequence of SEQ ID NO:3. SEQ ID NO:3 provides the 1791 amino acid sequence of PAPP-Ea. [0195]
  • In a fourth embodiment, the invention provides an isolated nucleic acid having a nucleotide sequence that (i) encodes a polypeptide having the sequence of SEQ ID NO:3, (ii) encodes a polypeptide having the sequence of SEQ ID NO:3 with conservative amino acid substitutions, or (iii) that is the complement of (i) or (ii), where SEQ ID NO:3 provides the 1791 amino acid sequence of PAPP-Ea. [0196]
  • In another embodiment, the invention provides an isolated nucleic acid comprising (i) the nucleotide sequence of the nucleic acid of ATCC deposit ______, (ii) the nucleotide sequence of SEQ ID NO:8 or (iii) the complement of (i) or (ii), where the referenced ATCC deposit has, and SEQ ID NO:8 provides, the nucleotide sequence of the entire PAPP-Eb ORF and portions of the 3′ UT. [0197]
  • In another embodiment, the invention provides an isolated nucleic acid comprising (i) the nucleotide sequence of SEQ ID NO:9, (ii) a degenerate variant of the nucleotide sequence of SEQ ID NO:9, or (iii) the complement (i) or (ii), where SEQ ID NO:9 presents the nucleotide seuqence of the open reading frame coding region of PAPP-Eb cDNA. [0198]
  • In a further embodiment, the invention provides an isolated nucleic acid comprising (i) a nucleotide sequence that encodes a polypeptide with the amino acid sequence of SEQ ID NO:10 or (ii) the complement of a nucleotide sequence that encodes a polypeptide with the amino acid sequence of SEQ ID NO:10, where SEQ ID NO:10 provides the full length amino acid coding sequence of PAPP-Eb. The invention further provides an isolated nucleic acid comprising a nucleotide sequence that (i) encodes a polypeptide having the sequence of SEQ ID NO:10, (ii) encodes a polypeptide having the sequence of SEQ ID NO:10 with conservative amino acid substitutions, or (iii) that is the complement of (i) or (ii). [0199]
  • The invention also provides isolated nucleic acids that encode the entirety of the PAPP-Ec isoform. [0200]
  • In a first such embodiment, the invention provides an isolated nucleic acid comprising (i) the nucleotide sequence of the nucleic acid of ATCC deposit ______, (ii) the nucleotide sequence of SEQ ID NO:15, (iii) a degenerate variant of SEQ ID NO:15, or (iv) the complement of (i), (ii) or (iii), where the referenced deposit has, and SEQ ID NO:15 provides, the nucleotide sequence of the PAPP-Ec open reading frame. [0201]
  • In another embodiment, the invention provides an isolated nucleic acid comprising (i) a nucleotide sequence that encodes a polypeptide with the amino acid sequence of SEQ ID NO:16 or (ii) the complement of a nucleotide sequence that encodes a polypeptide with the amino acid sequence of SEQ ID NO:16. SEQ ID NO:16 provides the entire amino acid sequence of PAPP-Ec. The invention further provides an isolated nucleic acid comprising a nucleotide sequence that (i) encodes a polypeptide having the sequence of SEQ ID NO:16, (ii) encodes a polypeptide having the sequence of SEQ ID NO:16 with conservative amino acid substitutions, or (iii) that is the complement of (i) or (ii). [0202]
  • Selected Partial Nucleic Acids [0203]
  • In a second series of nucleic acid embodiments, the invention provides isolated nucleic acids that encode select portions of one or more PAPP-E protein isoforms. As will be further discussed herein below, these “partial” nucleic acids can be used, inter alia, to express specific portions of the PAPP-E protein isoforms—both those portions that are shared by two or more isoforms, and those other portions that are unique to one or another of the isoforms—in vitro and in vivo. These “partial” nucleic acids can also be used, inter alia, as nucleic probes; used as probes, various of these embodiments are able to discriminate among the individual PAPP-E isoforms. [0204]
  • In a first such embodiment, the invention provides isolated nucleic acids comprising (i) the nucleotide sequence of SEQ ID NO:4 or (ii) the complement of the nucleotide sequence of SEQ ID NO:4, wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, more typically no more than about 50 kb in length. SEQ ID NO:4 is the nucleotide sequence, drawn from both 5′ UT and initial coding region, of the PAPP-Ea cDNA clone that is absent from the clone encoding the PAPP-Ef isoform. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0205]
  • In another embodiment, the invention provides an isolated nucleic acid comprising (i) the nucleotide sequence of SEQ ID NO:5 or (ii) the complement of the nucleoide sequence of SEQ ID NO:5, wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, more typically no more than about 50 kb in length. SEQ ID NO:5 presents the 5′ untranslated region of the PAPP-Ea cDNA, which is not found in the PAPP-Ef cDNA. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0206]
  • In another embodiment, the invention provides an isolated nucleic acid comprising (i) the nucleotide sequence of SEQ ID NO:6, (ii) a degenerate variant of the nucleotide sequence of SEQ ID NO:6, or (iii) the complement (i) or (ii), wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, more typically no more than about 50 kb in length. SEQ ID NO:6 presents the nucleotide sequence of the 5′ portion of the coding region of the PAPP-Ea cDNA not found in the PAPP-Ef cDNA. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0207]
  • In yet another embodiment, the invention provides isolated nucleic acids comprising (i) a nucleotide sequence that encodes a polypeptide with the amino acid sequence of SEQ ID NO:7 or (ii) the complement of a nucleotide sequence that encodes a polypeptide with the amino acid sequence of SEQ ID NO:7, wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, more typically no more than about 50 kb in length. SEQ ID NO:7 is the amino acid sequence of the N-terminal coding region of the PAPP-Ea isoform absent from the PAPP-Ef cDNA. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0208]
  • In yet a further embodiment, the invention provides an isolated nucleic acid comprising a nucleotide sequence (i) that encodes a polypeptide having the sequence of SEQ.ID NO:7, (ii) that encodes a polypeptide having the sequence of SEQ ID NO:7 with conservative amino acid substitutions, or (iii) the complement of (i) or (ii), wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, more typically no more than about 50 kb in length. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0209]
  • In a further embodiment, the invention provides an isolated nucleic acid comprising (i) the nucleotide sequence of SEQ ID NO:11, (ii) a degenerate variant of SEQ ID NO:11, or (iii) the complement of (i) or (ii), wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, more typically no more than about 50 kb length. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. SEQ ID NO:11 provides the portion of the PAPP-E cDNA sequence drawn from exon 21, which appears uniquely in the PAPP-Eb isoform; probes that include SEQ ID NO:11 and no other portions of the PAPP-E gene will be useful in discriminating expression of the PAPP-Eb isoform. [0210]
  • In another embodiment, the invention provides an isolated nucleic acid comprising (i) a nucleotide sequence that encodes SEQ ID NO:12 or (ii) the complement of a nucleotide sequence that encodes SEQ ID NO:12, wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, frequently no more than about 50 kb in length. SEQ ID NO:12 provides the amino acid sequence encoded by exon 21 that is uniquely present in the PAPP-Eb isoform (aa 1735-1762). Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0211]
  • In another embodiment, the invention provides an isolated nucleic acid comprising (i) a nucleotide sequence that encodes SEQ ID NO:12, (ii) a nucleotide sequence that encodes SEQ ID NO:12 with conservative substititions, or (iii) the complement of (i) or (ii), wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, and often no more than about 50 kb in length. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0212]
  • In a further embodiment, the invention provides an isolated nucleic acid comprising (i) a nucleotide sequence that encodes a polypeptide with the amino acid sequence of SEQ ID NO:14 or (ii) the complement of a nucleotide sequence that encodes a polypeptide with the amino acid sequence of SEQ ID NO:14, wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, often no more than about 50 kb in length. SEQ ID NO:14 is the amino acid sequence unique to the PAPP-Eb isoform, both that encoded by exon 21 and that caused by subsequent frameshit (aa 1735-1770). Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0213]
  • In yet another embodiment, the invention provides isolated nucleic acids comprising a nucleotide sequence (i) that encodes a polypeptide having the sequence of SEQ ID NO:14, (ii) that encodes a polypeptide having the sequence of SEQ ID NO:14 with conservative amino acid substitutions, or (iii) the complement of (i) or (ii), wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, often no more than about 50 kb in length. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0214]
  • In another embodiment, the invention provides an isolated nucleic acid comprising (i) the nucleotide sequence of SEQ ID NO:17, (ii) a degenerate variant of SEQ ID NO:17, or (iii) the complement of (i) or (ii). SEQ ID NO:17 provides the nucleotide sequence surrounding the junction of exons 1 and 4, a junction unique to PAPP-Ec among the PAPP-E isoforms. [0215]
  • In another embodiment, the invention provides isolated nucleic acids comprising (i) a nucleotide sequence that encodes SEQ ID NO:18, (ii) a nucleotide sequence that encodes SEQ ID NO:18 with conservative amino acid substititions, or (iii) the complement of (i) or (ii). SEQ ID NO:18 presents the 20 amino acid sequence centered at the junction between exons 1 and 4, a sequence unique to PAPP-Ec among the PAPP-E isoforms. [0216]
  • Cross-Hybridizing Nucleic Acids [0217]
  • In another series of nucleic acid embodiments, the invention provides isolated nucleic acids that hybridize to various of the PAPP-E nucleic acids of the present invention. These cross-hybridizing nucleic acids can be used, inter alia, as probes for, and to drive expression of, proteins that are related to the PAPP-E isoforms of the present invention as further isoforms, homologues, paralogues, or orthologues. [0218]
  • In a first such embodiment, the invention provides an isolated nucleic acid comprising a sequence that hybridizes under high stringency conditions to a probe the nucleotide sequence of which consists of SEQ ID NO:4 or the complement of SEQ ID NO:4, wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, and often no more than about 50 kb in length. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0219]
  • In a further embodiment, the invention provides an isolated nucleic acid comprising a sequence that hybridizes under moderate stringency conditions to a probe the nucleotide sequence of which consists of SEQ ID NO:4 or the complement of SEQ ID NO:4, wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, and often no more than about 50 kb in length. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0220]
  • In another embodiment, the invention provides an isolated nucleic acid comprising a sequence that hybridizes under high stringency conditions to a hybridization probe that consists of a nucleotide sequence that encodes SEQ ID NO:5, wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, and often no more than about 50 kb in length. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0221]
  • In yet another embodiment, the invention provides an isolated nucleic acid comprising a sequence that hybridizes under moderate stringency conditions to a hybridization probe consisting of a nucleotide sequence that encodes SEQ ID NO:5, wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, and often no more than about 50 kb in length. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0222]
  • In an additional embodiment, the invention provides an isolated nucleic acid comprising a sequence that hybridizes under high stringency conditions to a hybridization probe the nucleotide sequence of which consists of SEQ ID NO:6 or the complement of SEQ ID NO:6, wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, and often no more than about 50 kb in length. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0223]
  • The invention further provides an isolated nucleic acid comprising a sequence that hybridizes under moderate stringency conditions to a hybridization probe the nucleotide sequence of which consists of SEQ ID NO:6 or the complement of SEQ ID NO:6, wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, and often no more than about 50 kb in length. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0224]
  • The invention also provides an isolated nucleic acid comprising a sequence that hybridizes under high stringency conditions to a hybridization probe the nucleotide sequence of which (i) encodes a polypeptide having the sequence of SEQ ID NO:7, (ii) encodes a polypeptide having the sequence of SEQ ID NO:7 with conservative amino acid substitutions, or [0225]
  • (iii) is the complement of (i) or (ii), wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, and often no more than about 50 kb in length. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0226]
  • Additionally, the invention provides an isolated nucleic acid comprising a sequence that hybridizes under moderate stringency conditions to a hybridization probe the nucleotide sequence of which (i) encodes a polypeptide having the sequence of SEQ ID NO:7, (ii) encodes a polypeptide having the sequence of SEQ ID NO:7 with conservative amino acid substitutions, or (iii) is the complement of (i) or (ii), wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, and often no more than about 50 kb in length. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0227]
  • In a further embodiment, the invention provides an isolated nucleic acid comprising a sequence that hybridizes under high stringency conditions to a probe the nucleotide sequence of which consists of SEQ ID NO:11 or the complement of SEQ ID NO:11, wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, and often no more than about 50 kb in length. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0228]
  • In another embodiment, the invention provides an isolated nucleic acid comprising a sequence that hybridizes under moderate stringency conditions to a probe the nucleotide sequence of which consists of SEQ ID NO:11 or the complement of SEQ ID NO:11, wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, and often no more than about 50 kb in length. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0229]
  • In a further embodiment, the invention provides an isolated nucleic acid comprising a sequence that hybridizes under high stringency conditions to a hybridization probe the nucleotide sequence of which (i) encodes a polypeptide having the sequence of SEQ ID NO:12, (ii) encodes a polypeptide having the sequence of SEQ ID NO:12 with conservative amino acid substitutions, or (iii) is the complement of (i) or (ii), wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, and often no more than about 50 kb in length. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0230]
  • In a yet further embodiment, the invention provides an isolated nucleic acid comprising a sequence that hybridizes under moderate stringency conditions to a hybridization probe the nucleotide sequence of which (i) encodes a polypeptide having the sequence of SEQ ID NO:12, (ii) encodes a polypeptide having the sequence of SEQ ID NO:12 with conservative amino acid substitutions, or (iii) is the complement of (i) or (ii), wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, and often no more than about 50 kb in length. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0231]
  • Preferred Nucleic Acids [0232]
  • Particularly preferred among the above-described nucleic acids are those that are expressed, or the complement of which are expressed, in placental tissue, preferably at a level greater than that in HeLa cells, typically at a level at least two-fold that in HeLa cells, often at least three-fold, four-fold, or even five-fold that in HeLa cells. [0233]
  • Also particularly preferred among the above-described nucleic acids are those that encode, or the complement of which encode, a polypeptide having metalloproteinase activity, particularly those having cleavage specificity for an IGF binding protein. [0234]
  • Other preferred embodiments of the nucleic acids above-described are those that encode, or the complement of which encode, a polypeptide having any or all of (i) at least one zinc binding domain, (ii) at least one notch domain, and (iii) tandemly repeated SCR domains. [0235]
  • Nucleic Acid Fragments [0236]
  • In another series of nucleic acid embodiments, the invention provides fragments of various of the isolated nucleic acids of the present invention which prove useful, inter alia, as nucleic acid probes, as amplification primers, and to direct expression or synthesis of epitopic or immunogenic protein fragments. [0237]
  • In a first such embodiment, the invention provides an isolated nucleic acid comprising at least 17 nucleotides, 18 nucleotides, 20 nucleotides, 24 nucleotides, or 25 nucleotides of (i) SEQ ID NO:4 or (ii) the complement of SEQ ID NO:4, wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, more typically no more than about 50 kb in length. SEQ ID NO:4 is the nucleotide sequence of the 5′ region of the PAPP-Ea cDNA absent from the PAPP-Ef cDNA; accordingly, these fragments can be used to identify PAPP-E isoforms other than PAPP-Ef. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0238]
  • In another embodiment, the invention provides an isolated nucleic acid comprising at least 17 nucleotides, 18 nucleotides, 20 nucleotides, 24 nucleotides, or 25 nucleotides of (i) SEQ ID NO:5 or (ii) the complement of SEQ ID NO:5, wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, more typically no more than about 50 kb in length. SEQ ID NO:5 is the nucleotide sequence of the 5′ UT of the PAPP-Ea cDNA, which is absent from the PAPP-Ef cDNA; accordingly, these fragments can be used to identify PAPP-E isoforms other than PAPP-Ef. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0239]
  • In a yet further embodiment, the invention provides an isolated nucleic acid comprising at least at least 17 nucleotides, 18 nucleotides, 20 nucleotides, 24 nucleotides, or 25 nucleotides of (i) SEQ ID NO:6, (ii) a degenerate variant of SEQ ID NO:6, or (ii) the complement of (i) or (ii), wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, more typically no more than about 50 kb in length. SEQ ID NO:6 is the nucleotide sequence encoding the N-terminal amino acids absent from the PAPP-Ef isoform; accordingly, these fragments can be used to identify PAPP-E isoforms other than PAPP-Ef. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0240]
  • In another embodiment, the invention provides an isolated nucleic acid comprising a nucleotide sequence that (i) encodes a polypeptide having the sequence of at least 8 contiguous amino acids of SEQ ID NO:7, (ii) encodes a polypeptide having the sequence of at least 8 contiguous amino acids of SEQ ID NO:7 with conservative amino acid substitutions, or (iii) is the complement of (i) or (ii), wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, more typically no more than about 50 kb in length. SEQ ID NO:7 is the amino acid sequence of the 19 N-terminal amino acids absent from the PAPP-Ef isoform. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0241]
  • In a further embodiment, the invention provides an isolated nucleic acid comprising at least 17 nucleotides, 18 nucleotides, 20 nucleotides, 24 nucleotides, or 25 nucleotides of (i) SEQ ID NO:11, (ii) a degenerate variant of SEQ ID NO:11, or (iii) the complement of (i) or (ii), wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, more typically no more than about 50 kb in length. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0242]
  • The invention also provides an isolated nucleic acid comprising (i) a nucleotide sequence that encodes a peptide of at least 8 contiguous amino acids of SEQ ID NO:12, or (ii) the complement of a nucleotide sequence that encodes a peptide of at least 8 contiguous amino acids of SEQ ID NO:12, wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, more typically no more than about 50 kb in length. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0243]
  • The invention also provides an isolated nucleic acid comprising a nucleotide sequence that (i) encodes a polypeptide having the sequence of at least 8 contiguous amino acids of SEQ ID NO:12, (ii) encodes a polypeptide having the sequence of at least 8 contiguous amino acids of SEQ ID NO:12 with conservative amino acid substitutions, or (iii) is the complement of (i) or (ii). [0244]
  • The structural chemical formulas of representative 17-mer nucleic acid fragments of the present invention as above-described are provided as SEQ ID NOs: [x-x′] and [z-z′] in the attached Sequence Listing, incorporated herein by reference in its entirety. The structural chemical formulas of representative 25-mer nucleic acid fragments of the present invention as above-described are reduced to drawings as SEQ ID Nos: [y-y′] and [*-*′] in the attached Sequence Listing, incorporated herein by reference in its entirety. [0245]
  • Single Exon Probes [0246]
  • The invention further provides genome-derived single exon probes having portions of no more than one exon of the PAPP-E gene. As further described in commonly owned and copending U.S. patent application Ser. No. 09/632,366 (“Methods and Apparatus for High Throughput Detection and Characterization of alternatively Spliced Genes”), the disclosure of which is incorporated herein by reference in its entirety, such single exon probes have particular utility in identifying and characterizing splice variants. In particular, such single exon probes are useful for identifying and discriminating the expression of PAPP-Ea, PAPP-Eb, and PAPP-Ec isoforms. [0247]
  • In a first embodiment, the invention provides an isolated nucleic acid comprising a nucleotide sequence of no more than one portion of SEQ ID NOs:19 to 41 or the complement of SEQ ID NOs: 19 to 41, wherein the portion comprises at least 17 contiguous nucleotides, 18 contiguous nucleotides, 20 contiguous nucleotides, 24 contiguous nucleotides, 25 contiguous nucleotides, or 50 contiguous nucleotides of any one of SEQ ID NOs: 19 to 41, or their complement, and hybridizes under high stringency conditions to a nucleic acid expressed in human placenta. In a further embodiment, the exonic portion comprises the entirety of the referenced SEQ ID NO: or its complement. [0248]
  • In other embodiments, the invention provides isolated single exon probes having the nucleotide sequence of any one of SEQ ID NOs: 42-65. [0249]
  • In a particular embodiment, the invention provides a single exon probe having a portion of SEQ ID NO:39. SEQ ID NO:39 presents the exon (exon 21) that is unique to the PAPP-Eb isoform; single exon probes, including genome-derived single exon probes, having a portion drawn from exon 21 can be used to identify and or measure expression of PAPP-Eb. [0250]
  • Transcription Control Nucleic Acids [0251]
  • In another aspect, the present invention provides genome-derived isolated nucleic acids that include nucleic acid sequence elements that control transcription of the PAPP-E gene and its various isoforms. These nucleic acids can be used, inter alia, to drive expression of heterologous coding regions in recombinant constructs, thus conferring upon such hetereologous coding regions the expression pattern of the native PAPP-E gene. These nucleic acids can also be used, conversely, to target heterologous transcription control elements to the PAPP-E genomic locus, altering the expression pattern of the PAPP-E gene itself. [0252]
  • In a first such embodiment, the invention provides an isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO:65 or its complement, wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, more typically no more than about 50 kb in length. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0253]
  • In another embodiment, the invention provides an isolated nucleic acid comprising at least 17, 18, 20, 24, or 25 nucleotides of the sequence of SEQ ID NO:65 or its complement, wherein wherein the isolated nucleic acid is no more than about 100 kb in length, typically no more than about 75 kb in length, more typically no more than about 50 kb in length. Often, the isolated nucleic acids of this embodiment are no more than about 25 kb in length, often no more than about 15 kb in length, and frequently no more than about 10 kb in length. [0254]
  • Vectors and Host Cells [0255]
  • In another aspect, the present invention provides vectors that comprise one or more of the isolated nucleic acids of the present invention, and host cells in which such vectors have been introduced. [0256]
  • The vectors can be used, inter alia, for propagating the nucleic acids of the present invention in host cells (cloning vectors), for shuttling the nucleic acids of the present invention between host cells derived from disparate organisms (shuttle vectors), for inserting the nucleic acids of the present invention into host cell chromosomes (insertion vectors), for expressing sense or antisense RNA transcripts of the nucleic acids of the present invention in vitro or within a host cell, and for expressing polypeptides encoded by the nucleic acids of the present invention, alone or as fusions to heterologous polypeptides. Vectors of the present invention will often be suitable for several such uses. [0257]
  • Vectors are by now well-known in the art, and are described, inter alia, in Jones et al. (eds.), [0258] Vectors: Cloning Applications: Essential Techniques (Essential Techniques Series), John Wiley & Son Ltd 1998 (ISBN: 047196266X); Jones et al. (eds.), Vectors: Expression Systems: Essential Techniques (Essential Techniques Series), John Wiley & Son Ltd, 1998 (ISBN:0471962678); Gacesa et al., Vectors: Essential Data, John Wiley & Sons, 1995 (ISBN: 0471948411); Cid-Arregui (eds.), Viral Vectors: Basic Science and Gene Therapy, Eaton Publishing Co., 2000 (ISBN: 188129935X); Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed.), Cold Spring Harbor Laboratory Press, 2001 (ISBN: 0879695773); Ausubel et al. (eds.), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology (4th ed.), John Wiley & Sons, 1999 (ISBN: 047132938X), the disclosures of which are incorporated herein by reference in their entireties. Furthermore, an enormous variety of vectors are available commercially. Use of existing vectors and modifications thereof being well within the skill in the art, only basic features need be described here.
  • Typically, vectors are derived from virus, plasmid, prokaryotic or eukaryotic chromosomal elements, or some combination thereof, and include at least one origin of replication, at least one site for insertion of heterologous nucleic acid, typically in the form of a polylinker with multiple, tightly clustered, single cutting restriction sites, and at least one selectable marker, although some integrative vectors will lack an origin that is functional in the host to be chromosomally modified, and some vectors will lack selectable markers. Vectors of the present invention will further include at least one nucleic acid of the present invention inserted into the vector in at least one location. [0259]
  • Where present, the origin of replication and selectable markers are chosen based upon the desired host cell or host cells; the host cells, in turn, are selected based upon the desired application. [0260]
  • For example, prokaryotic cells, typically [0261] E. coli, are typically chosen for cloning. In such case, vector replication is predicated on the replication strategies of coliform-infecting phage—such as phage lambda, M13, T7, T3 and P1—or on the replication origin of autonomously replicating episomes, notably the ColE1 plasmid and later derivatives, including pBR322 and the pUC series plasmids. Where E. coli is used as host, selectable markers are, analogously, chosen for selectivity in gram negative bacteria: e.g., typical markers confer resistance to antibiotics, such as ampicillin, tetracycline, chlorampenicol, kanamycin, streptomycin, zeocin; auxotrophic markers can also be used.
  • As another example, yeast cells, typically [0262] S. cerevisiae, are chosen, inter alia, for eukaryotic genetic studies, due to the ease of targeting genetic changes by homologous recombination and to the ready ability to complement genetic defects using recombinantly expressed proteins, for identification of interacting protein components, e.g. through use of a two-hybrid system, and for protein expression. Vectors of the present invention for use in yeast will typically, but not invariably, contain an origin of replication suitable for use in yeast and a selectable marker that is functional in yeast.
  • Integrative YIp vectors do not replicate autonomously, but integrate, typically in single copy, into the yeast genome at low frequencies and thus replicate as part of the host cell chromosome; these vectors lack an origin of replication that is functional in yeast, although they typically have at least one origin of replication suitable for progation of the vector in bacterial cells. YEp vectors, in contrast, replicate episomally and autonomously due to presence of the yeast 2 micron plasmid origin (2 μm ori). The YCp yeast centromere plasmid vectors are autonomously replicating vectors containing centromere sequences, CEN, and autonomously replicating sequences, ARS; the ARS sequences are believed to correspond to the natural replication origins of yeast chromosomes. YACs are based on yeast linear plasmids, denoted YLp, containing homologous or heterologous DNA sequences that function as telomeres (TEL) in vivo, as well as containing yeast ARS (origins of replication) and CEN (centromeres) segments. [0263]
  • Selectable markers in yeast vectors include a variety of auxotrophic markers, the most common of which are (in [0264] Saccharomyces cerevisiae) URA3, HIS3, LEU2, TRP1 and LYS2, which complement specific auxotrophic mutations, such as ura3-52, his3-D1, leu2-D1, trp1-D1 and lys2-201. The URA3 and LYS2 yeast genes further permit negative selection based on specific inhibitors, 5-fluoro-orotic acid (FOA) and α-aminoadipic acid (αAA), respectively, that prevent growth of the prototrophic strains but allows growth of the ura3 and lys2 mutants, respectively. Other selectable markers confer resistance to, e.g., zeocin.
  • As yet another example, insect cells are often chosen for high efficiency protein expression. Where the host cells are from [0265] Spodoptera frugiperda—e.g., Sf9 and Sf21 cell lines, and expresSF™ cells (Protein Sciences Corp., Meriden, Conn., USA)— the vector replicative strategy is typically based upon the baculovirus life cycle. Typically, baculovirus transfer vectors are used to replace the wild-type AcMNPV polyhedrin gene with a heterologous gene of interest. Sequences that flank the polyhedrin gene in the wild-type genome are positioned 5′ and 3′ of the expression cassette on the transfer vectors. Following cotransfection with AcMNPV DNA, a homologous recombination event occurs between these sequences resulting in a recombinant virus carrying the gene of interest and the polyhedrin or p10 promoter. Selection can be based upon visual screening for lacZ fusion activity.
  • As yet another example, mammalian cells are often chosen for expression of proteins intended as pharmaceutical agents, and are also chosen as host cells for screening of potential agonist and antagonists of a protein or a physiological pathway. [0266]
  • Where mammalian cells are chosen as host cells, vectors intended for autonomous extrachromosomal replication will typically include a viral origin, such as the SV40 origin (for replication in cell lines expressing the large T-antigen, such as COS1 and COS7 cells), the papillomavirus origin, or the EBV origin for long term episomal replication (for use, e.g., in 293-EBNA cells, which constitutively express the EBV EBNA-1 gene product and adenovirus E1A). Vectors intended for integration, and thus replication as part of the mammalian chromosome, can, but need not, include an origin of replication functional in mammalian cells, such as the SV40 origin. Vectors based upon viruses, such as adenovirus, adeno-associated virus, vaccinia virus, and various mammalian retroviruses, will typically replicate according to the viral replicative strategy. [0267]
  • Selectable markers for use in mammalian cells include resistance to neomycin (G418), blasticidin, hygromycin and to zeocin, and selection based upon the purine salvage pathway using HAT medium. [0268]
  • Vectors of the present invention will also often include elements that permit in vitro transcription of RNA from the inserted heterologous nucleic acid. Such vectors typically include a phage promoter, such as that from T7, T3, or SP6, flanking the nucleic acid insert. Often two different such promoters flank the inserted nucleic acid, permitting separate in vitro production of both sense and antisense strands. [0269]
  • Expression vectors of the present invention—that is, those vectors that will drive expression of polypeptides from the inserted heterologous nucleic acid—will often include a variety of other genetic elements operatively linked to the protein-encoding heterologous nucleic acid insert, typically genetic elements that drive transcription, such as promoters and enhancer elements, those that facilitate RNA processing, such as transcription termination and/or polyadenylation signals, and those that facilitate translation, such as ribosomal consensus sequences. [0270]
  • For example, vectors for expressing proteins of the present invention in prokaryotic cells, typically [0271] E. coli, will include a promoter, often a phage promoter, such as phage lambda pL promoter, the trc promoter, a hybrid derived from the trp and lac promoters, the bacteriophage T7 promoter (in E. coli cells engineered to express the T7 polymerase), or the araBAD operon. Often, such prokaryotic expression vectors will further include transcription terminators, such as the aspA terminator, and elements that facilitate translation, such as a consensus ribosome binding site and translation termination codon, Schomer et al., Proc. Natl. Acad. Sci. USA 83:8506-8510 (1986).
  • As another example, vectors for expressing proteins of the present invention in yeast cells, typically [0272] S. cerevisiae, will include a yeast promoter, such as the CYC1 promoter, the GAL1 promoter, ADH1 promoter, or the GPD promoter, and will typically have elements that facilitate transcription termination, such as the transcription termination signals from the CYC1 or ADH1 gene.
  • As another example, vectors for expressing proteins of the present invention in mammalian cells will include a promoter active in mammalian cells. Such promoters are often drawn from mammalian viruses—such as the enhancer-promoter sequences from the immediate early gene of the human cytomegalovirus (CMV), the enhancer-promoter sequences from the Rous sarcoma virus long terminal repeat (RSV LTR), and the enhancer-promoter from SV40. Often, expression is enhanced by incorporation of polyadenylation sites, such as the late SV40 polyadenylation site and the polyadenylation signal and transcription termination sequences from the bovine growth hormone (BGH) gene, and ribosome binding sites. Furthermore, vectors can include introns, such as intron II of rabbit β-globin gene and the SV40 splice elements. [0273]
  • Vector-drive protein expression can be constitutive or inducible. [0274]
  • Inducible vectors include either naturally inducible promoters, such as the trc promoter, which is regulated by the lac operon, and the pL promoter, which is regulated by tryptophan, the MMTV-LTR promoter, which is inducible by dexamethasone, or can contain synthetic promoters and/or additional elements that confer inducible control on adjacent promoters. Examples of inducible synthetic promoters are the hybrid Plac/ara-1 promoter and the PLtetO-1 promoter. The PltetO-1 promoter takes advantage of the high expression levels from the PL promoter of phage lambda, but replaces the lambda repressor sites with two copies of operator 2 of the Tn10 tetracycline resistance operon, causing this promoter to be tightly repressed by the Tet repressor protein and induced in response to tetracycline (Tc) and Tc derivatives such as anhydrotetracycline. [0275]
  • As another example of inducible elements, hormone response elements, such as the glucocorticoid response element (GRE) and the estrogen response element (ERE), can confer hormone inducibility where vectors are used for expression in cells having the respective hormone receptors. To reduce background levels of expression, elements responsive to ecdysone, an insect hormone, can be used instead, with coexpression of the ecdysone receptor. [0276]
  • Expression vectors can be designed to fuse the expressed polypeptide to small protein tags that facilitate purification and/or visualization. [0277]
  • For example, proteins can be expressed with a polyhistidine tag that facilitates purification of the fusion protein by immobilized metal affinity chromatography, for example using NiNTA resin (Qiagen Inc., Valencia, Calif., USA) or TALON resin (cobalt immobilized affinity chromatography medium, Clontech Labs, Palo Alto, Calif., USA). As another example, the fusion protein can include a chitin-binding tag and self-excising intein, permitting chitin-based purification with self-removal of the fused tag (IMPACT™ system, New England Biolabs, Inc., Beverley, Mass., USA). Alternatively, the fusion protein can include a calmodulin-binding peptide tag, permitting purification by calmodulin affinity resin (Stratagene, La Jolla, Calif., USA), or a specifically excisable fragment of the biotin carboxylase carrier protein, permitting purification of in vivo biotinylated protein using an avidin resin and subsequent tag removal (Promega, Madison, Wis., USA). [0278]
  • Other tags include, for example, the Xpress epitope, detectable by anti-Xpress antibody (Invitrogen, Carlsbad, Calif., USA), a myc tag, detectable by anti-myc tag antibody, the V5 epitope, detectable by anti-V5 antibody (Invitrogen, Carlsbad, Calif., USA), FLAG® epitope, detectable by anti-FLAG® antibody (Stratagene, La Jolla, Calif., USA), and the HA epitope. [0279]
  • For secretion of expressed proteins, vectors can include appropriate sequences that encode secretion signals, such as leader peptides. For example, the pSecTag2 vectors (Invitrogen, Carlsbad, Calif., USA) are 5.2 kb mammalian expression vectors that carry the secretion signal from the V-J2-C region of the mouse Ig kappa-chain for efficient secretion of recombinant proteins from a variety of mammalian cell lines. [0280]
  • Expression vectors can also be designed to fuse proteins encoded by the heterologous nucleic acid insert to polypeptides larger than purification and/or identification tags. Useful protein fusions include those that permit display of the encoded protein on the surface of a phage or cell, fusions to intrinsically fluorescent proteins, such as green fluorescent protein (GFP), fusions to the IgG Fc region, and fusions for use in two hybrid systems. [0281]
  • Vectors for phage display fuse the encoded polypeptide to, e.g., the gene III protein (pIII) or gene VIII protein (pVIII) for display on the surface of filamentous phage, such as M13. See Barbas et al., [0282] Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001) (ISBN 0-87969-546-3); Kay et al. (eds.), Phage Display of Peptides and Proteins: A Laboratory Manual, San Diego: Academic Press, Inc., 1996; Abelson et al. (eds.), Combinatorial Chemistry, Methods in Enzymology vol. 267, Academic Press (May 1996).
  • Vectors for yeast display, e.g. the pYD1 yeast display vector (Invitrogen, Carlsbad, Calif., USA), use the α-agglutinin yeast adhesion receptor to display recombinant protein on the surface of [0283] S. cerevisiae. Vectors for mammalian display, e.g., the pDisplay™ vector (Invitrogen, Carlsbad, Calif., USA), target recombinant proteins using an N-terminal cell surface targeting signal and a C-terminal transmembrane anchoring domain of platelet derived growth factor receptor.
  • A wide variety of vectors now exist that fuse proteins encoded by heterologous nucleic acids to the chromophore of the substrate-independent, intrinsically fluorescent green fluorescent protein from [0284] Aequorea victoria (“GFP”) and its variants. These proteins are intrinsically fluorescent: the GFP-like chromophore is entirely encoded by its amino acid sequence and can fluoresce without requirement for cofactor or substrate.
  • Structurally, the GFP-like chromophore comprises an 11-stranded β-barrel (β-can) with a central α-helix, the central α-helix having a conjugated n-resonance system that includes two aromatic ring systems and the bridge between them. The n-resonance system is created by autocatalytic cyclization among amino acids; cyclization proceeds through an imidazolinone intermediate, with subsequent dehydrogenation by molecular oxygen at the Cα-Cβ bond of a participating tyrosine. [0285]
  • The GFP-like chromophore can be selected from GFP-like chromophores found in naturally occurring proteins, such as [0286] A. victoria GFP (GenBank accession number AAA27721), Renilla reniformis GFP, FP583 (GenBank accession no. AF168419) (DsRed), FP593 (AF272711), FP483 (AF168420), FP484 (AF168424), FP595 (AF246709), FP486 (AF168421), FP538 (AF168423), and FP506 (AF168422), and need include only so much of the native protein as is needed to retain the chromophore's intrinsic fluorescence. Methods for determining the minimal domain required for fluorescence are known in the art. Li et al., “Deletions of the Aequorea victoria Green Fluorescent Protein Define the Minimal Domain Required for Fluorescence,” J. Biol. Chem. 272:28545-28549 (1997).
  • Alternatively, the GFP-like chromophore can be selected from GFP-like chromophores modified from those found in nature. Typically, such modifications are made to improve recombinant production in heterologous expression systems (with or without change in protein sequence), to alter the excitation and/or emission spectra of the native protein, to facilitate purification, to facilitate or as a consequence of cloning, or are a fortuitous consequence of research investigation. [0287]
  • The methods for engineering such modified GFP-like chromophores and testing them for fluorescence activity, both alone and as part of protein fusions, are well-known in the art. Early results of these efforts are reviewed in Heim et al., [0288] Curr. Biol. 6:178-182 (1996), incorporated herein by reference in its entirety; a more recent review, with tabulation of useful mutations, is found in Palm et al., “Spectral Variants of Green Fluorescent Protein,” in Green Fluorescent Proteins, Conn (ed.), Methods Enzymol. vol. 302, pp. 378-394 (1999), incorporated herein by reference in its entirety. A variety of such modified chromophores are now commercially available and can readily be used in the fusion proteins of the present invention.
  • For example, EGFP (“enhanced GFP”), Cormack et al., [0289] Gene 173:33-38 (1996); U.S. Pat. Nos. 6,090,919 and 5,804,387, is a red-shifted, human codon-optimized variant of GFP that has been engineered for brighter fluorescence, higher expression in mammalian cells, and for an excitation spectrum optimized for use in flow cytometers. EGFP can usefully contribute a GFP-like chromophore to the fusion proteins of the present invention. A variety of EGFP vectors, both plasmid and viral, are available commercially (Clontech Labs, Palo Alto, Calif., USA), including vectors for bacterial expression, vectors for N-terminal protein fusion expression, vectors for expression of C-terminal protein fusions, and for bicistronic expression.
  • Toward the other end of the emission spectrum, EBFP (“enhanced blue fluorescent protein”) and BFP2 contain four amino acid substitutions that shift the emission from green to blue, enhance the brightness of fluorescence and improve solubility of the protein, Heim et al., [0290] Curr. Biol. 6:178-182 (1996); Cormack et al., Gene 173:33-38 (1996). EBFP is optimized for expression in mammalian cells whereas BFP2, which retains the original jellyfish codons, can be expressed in bacteria; as is further discussed below, the host cell of production does not affect the utility of the resulting fusion protein. The GFP-like chromophores from EBFP and BFP2 can usefully be included in the fusion proteins of the present invention, and vectors containing these blue-shifted variants are available from Clontech Labs (Palo Alto, Calif., USA).
  • Analogously, EYFP (“enhanced yellow fluorescent protein”), also available from Clontech Labs, contains four amino acid substitutions, different from EBFP, Ormö et al., [0291] Science 273:1392-1395 (1996), that shift the emission from green to yellowish-green. Citrine, an improved yellow fluorescent protein mutant, is described in Heikal et al., Proc. Natl. Acad. Sci. USA 97:11996-12001 (2000). ECFP (“enhanced cyan fluorescent protein”) (Clontech Labs, Palo Alto, Calif., USA) contains six amino acid substitutions, one of which shifts the emission spectrum from green to cyan. Heim et al., Curr. Biol. 6:178-182 (1996); Miyawaki et al., Nature 388:882-887 (1997). The GFP-like chromophore of each of these GFP variants can usefully be included in the fusion proteins of the present invention.
  • The GFP-like chromophore can also be drawn from other modified GFPs, including those described in U.S. Pat. Nos. 6,124,128; 6,096,865; 6,090,919; 6,066,476; 6,054,321; 6,027,881; 5,968,750; 5,874,304; 5,804,387; 5,777,079; 5,741,668; and 5,625,048, the disclosures of which are incorporated herein by reference in their entireties. See also Conn (ed.), [0292] Green Fluorescent Protein, Methods in
  • Fusions to the IgG Fc region increase serum half life of protein pharmaceutical products through interaction with the FcRn receptor (also denominated the FcRp receptor and the Brambell receptor, FcRb), further described in international patent application nos. WO 97/43316, WO 97/34631, WO 96/32478, WO 96/18412. [0293]
  • The present invention further includes host cells comprising the vectors of the present invention, either present episomally within the cell or integrated, in whole or in part, into the host cell chromosome. [0294]
  • As noted earlier, host cells can be prokaryotic or eukaryotic. Representative examples of appropriate host cells include, but are not limited to, bacterial cells, such as [0295] E. coli, Caulobacter crescentus, Streptomyces species, and Salmonella typhimurium; yeast cells, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Pichia methanolica; insect cell lines, such as those from Spodoptera frugiperda—e.g., Sf9 and Sf21 cell lines, and expresSF™ cells (Protein Sciences Corp., Meriden, Conn., USA)—Drosophila S2 cells, and Trichoplusia ni High Five® Cells (Invitrogen, Carlsbad, Calif., USA); and mammalian cells. Typical mammalian cells include COS1 and COS7 cells, chinese hamster ovary (CHO) cells, NIH 3T3 cells, 293 cells, HEPG2 cells, HeLa cells, L cells, murine ES cell lines (e.g., from strains 129/SV, C57/BL6, DBA-1, 129/SVJ), K562, Jurkat cells, and BW5147. Other mammalian cell lines are well known and readily available from the American Type Culture Collection (ATCC) (Manassas, Va., USA) and the National Institute of General medical Sciences (NIGMS) Human Genetic Cell Repository at the Coriell Cell Repositories (Camden, N.J., USA).
  • Methods for introducing the vectors and nucleic acids of the present invention into the host cells are well known in the art; the choice of technique will depend primarily upon the specific vector to be introduced. [0296]
  • For example, phage lambda vectors will typically be packaged using a packaging extract (e.g., Gigapack® packaging extract, Stratagene, La Jolla, Calif., USA), and the packaged virus used to infect [0297] E. coli. Plasmid vectors will typically be introduced into chemically competent or electrocompetent bacterial cells.
  • [0298] E. coli cells can be rendered chemically competent by treatment, e.g., with CaCl2, or a solution of Mg2+, Mn2+, Ca2+, Rb+ or K+, dimethyl sulfoxide, dithiothreitol, and hexamine cobalt (III), Hanahan, J. Mol. Biol. 166(4):557-80 (1983), and vectors introduced by heat shock. A wide variety of chemically competent strains are also available commercially (e.g., Epicurian Coli® XL10-Gold® Ultracompetent Cells (Stratagene, La Jolla, Calif., USA); DH5α competent cells (Clontech Laboratories, Palo Alto, Calif., USA); TOP10 Chemically Competent E. coli Kit (Invitrogen, Carlsbad, Calif., USA)).
  • Bacterial cells can be rendered electrocompetent—that is, competent to take up exogenous DNA by electroporation—by various pre-pulse treatments; vectors are introduced by electroporation followed by subsequent outgrowth in selected media. An extensive series of protocols is provided online in Electroprotocols (BioRad, Richmond, Calif., USA) (http://www.bio-rad.com/LifeScience/pdf/New_Gene_Pulser.pdf). [0299]
  • Vectors can be introduced into yeast cells by spheroplasting, treatment with lithium salts, electroporation, or protoplast fusion. [0300]
  • Spheroplasts are prepared by the action of hydrolytic enzymes—a snail-gut extract, usually denoted Glusulase, or Zymolyase, an enzyme from [0301] Arthrobacter luteus—to remove portions of the cell wall in the presence of osmotic stabilizers, typically 1 M sorbitol. DNA is added to the spheroplasts, and the mixture is co-precipitated with a solution of polyethylene glycol (PEG) and Ca2+. Subsequently, the cells are resuspended in a solution of sorbitol, mixed with molten agar and then layered on the surface of a selective plate containing sorbitol. For lithium-mediated transformation, yeast cells are treated with lithium acetate, which apparently permeabilizes the cell wall, DNA is added and the cells are co-precipitated with PEG. The cells are exposed to a brief heat shock, washed free of PEG and lithium acetate, and subsequently spread on plates containing ordinary selective medium. Increased frequencies of transformation are obtained by using specially-prepared single-stranded carrier DNA and certain organic solvents. Schiestl et al., Curr. Genet. 16(5-6):339-46 (1989). For electroporation, freshly-grown yeast cultures are typically washed, suspended in an osmotic protectant, such as sorbitol, mixed with DNA, and the cell suspension pulsed in an electroporation device. Subsequently, the cells are spread on the surface of plates containing selective media. Becker et al., Methods Enzymol. 194:182-7 (1991). The efficiency of transformation by electroporation can be increased over 100-fold by using PEG, single-stranded carrier DNA and cells that are in late log-phase of growth. Larger constructs, such as YACs, can be introduced by protoplast fusion.
  • Mammalian and insect cells can be directly infected by packaged viral vectors, or transfected by chemical or electrical means. [0302]
  • For chemical transfection, DNA can be coprecipitated with CaPO[0303] 4 or introduced using liposomal and nonliposomal lipid-based agents. Commercial kits are available for CaPO4 transfection (CalPhos™ Mammalian Transfection Kit, Clontech Laboratories, Palo Alto, Calif., USA), and lipid-mediated transfection can be practiced using commercial reagents, such as LIPOFECTAMINE™ 2000, LIPOFECTAMINE™ Reagent, CELLFECTIN® Reagent, and LIPOFECTIN® Reagent (Invitrogen, Carlsbad, Calif., USA), DOTAP Liposomal Transfection Reagent, FuGENE 6, X-tremeGENE Q2, DOSPER, (Roche Molecular Biochemicals, Indianapolis, Ind. USA), Effectene™, PolyFect®, Superfect® (Qiagen, Inc., Valencia, Calif., USA). Protocols for electroporating mammalian cells can be found online in Electroprotocols (Bio-Rad, Richmond, Calif., USA) (http://www.bio-rad.com/LifeScience/pdf/New_Gene_Pulser.pdf). See also, Norton et al. (eds.), Gene Transfer Methods: Introducing DNA into Living Cells and Organisms, BioTechiques Books, Eaton Publishing Co. (2000) (ISBN 1-881299-34-1), incorporated herein by reference in its entirety.
  • Proteins [0304]
  • In another aspect, the present invention provides PAPP-E isoform proteins, various fragments thereof suitable for use as antigens (e.g., for epitope mapping) and for use as immunogens (e.g., for raising antibodies or as vaccines), fusions of PAPP-E isoform polypeptides and fragments to heterologous polypeptides, and conjugates of the proteins, fragments, and fusions of the present invention to other moieties (e.g., to carrier proteins, to fluorophores). [0305]
  • FIGS. 3, 4, and [0306] 5 present the predicted amino acid sequences encoded by PAPP-Ea, PAPP-Eb, and PAPP-Ec cDNA clones. The amino acid sequences are further presented, respectively, in SEQ ID Nos: 3 (full length PAPP-Ea isoform), 7 (PAPPE-Ea isoform from aa 1-19), 10 (full length PAPP-Eb isoform), 12 (amino acid sequence entirely within the novel exon of PAPP-Eb (aa 1735-1762)), 13 (amino acid sequence of PAPP-Eb resulting from the frame shift (aa 1763-1770)), 14 (amino acids present uniquely within PappE-b, due to exon insertion followed by frameshift (aa 1735-1770)), 16 (full length PAPP-Ec isoform), 18 (20 amino acids centered about deletion of exon 21 in PAPP-Ec (aa 298-317)).
  • Unless otherwise indicated, amino acid sequences of the proteins of the present invention were determined as a predicted translation from a nucleic acid sequence. Accordingly, any amino acid sequence presented herein may contain errors due to errors in the nucleic acid sequence, as described in detail above. Furthermore, single nucleotide polymorphisms (SNPs) occur frequently in eukaryotic genomes—more than 1.4 million SNPs have already identified in the human genome, International Human Genome Sequencing Consortium, [0307] Nature 409:860-921 (2001)—and the sequence determined from one individual of a species may differ from other allelic forms present within the population. Small deletions and insertions can often be found that do not alter the function of the protein.
  • Accordingly, it is an aspect of the present invention to provide proteins not only identical in sequence to those described with particularity herein, but also to provide isolated proteins at least about 90% identical in sequence to those described with particularity herein, typically at least about 91%, 92%, 93%, 94%, or 95% identical in sequence to those decribed with particularity herein, usefully at least about 96%, 97%, 98%, or 99% identical in sequence to those described with particularity herein, and, most conservatively, at least about 99.5%, 99.6%, 99.7%, 99.8% and 99.9% identical in sequence to those described with particularity herein. These sequence variants can be naturally occurring or can result from human intervention by way of random or directed mutagenesis. [0308]
  • For purposes herein, percent identity of two amino acid sequences is determined using the procedure of Tatiana et al., “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, [0309] FEMS Microbiol Lett. 174:247-250 (1999), which procedure is effectuated by the computer program BLAST 2 SEQUENCES, available online at
  • http://www.ncbi.nlm.nih.gov/blast/b12seq/b12.html, [0310]
  • To assess percent identity of amino acid sequences, the BLASTP module of BLAST 2 SEQUENCES is used with default values of (i) BLOSUM62 matrix, Henikoff et al., [0311] Proc. Natl. Acad. Sci USA 89(22):10915-9 (1992); (ii) open gap 11 and extension gap 1 penalties; and (iii) gap x_dropoff 50 expect 10 word size 3 filter, and both sequences are entered in their entireties.
  • As is well known, amino acid substitutions occur frequently among natural allelic variants, with conservative substitutions often occasioning only de minimis change in protein function. [0312]
  • Accordingly, it is an aspect of the present invention to provide proteins not only identical in sequence to those described with particularity herein, but also to provide isolated proteins having the sequence of PAPP-E proteins, or portions thereof, with conservative amino acid substitutions, and to provide isolated proteins having the sequence of PAPP-E proteins, and portions thereof, with moderately conservative amino acid substitutions. These conservatively-sustituted or moderately conservatively-substituted variants can be naturally occurring or can result from human intervention. [0313]
  • Although there are a variety of metrics for calling conservative amino acid substitutions, based primarily on either observed changes among evolutionarily related proteins or on predicted chemical similarity, for purposes herein a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix reproduced herein below (see Gonnet et al., [0314] Science 256(5062):1443-5 (1992)):
    A R N D C Q E G H I L K M F P S T W Y V
    A 2 −1 0 0 0 0 0 0 −1 −1 −1 0 −1 −2 0 1 1 −4 −2 0
    R −1 5 0 0 −2 2 0 −1 1 −2 −2 3 −2 −3 −1 0 0 −2 −2 −2
    N 0 0 4 2 −2 1 1 0 1 −3 −3 1 −2 −3 −1 1 0 −4 −1 −2
    D 0 0 2 5 −3 1 3 0 0 −4 −4 0 −3 −4 −1 0 0 −5 −3 −3
    C 0 −2 −2 −3 12 −2 −3 −2 −1 −1 −2 −3 −1 −1 −3 0 0 −1 0 0
    Q 0 2 1 1 −2 3 2 −1 1 −2 −2 2 −1 −3 0 0 0 −3 −2 −2
    E 0 0 1 3 −3 2 4 −1 0 −3 −3 1 −2 −4 0 0 0 −4 −3 −2
    G 0 −1 0 0 −2 −1 −1 7 −1 −4 −4 −1 −4 −5 −2 0 −1 −4 −4 −3
    H −1 1 1 0 −1 1 0 −1 6 −2 −2 1 −1 0 −1 0 0 −1 2 −2
    I −1 −2 −3 −4 −1 −2 −3 −4 −2 4 3 −2 2 1 −3 −2 −1 −2 −1 3
    L −1 −2 −3 −4 −2 −2 −3 −4 −2 3 4 −2 3 2 −2 −2 −1 −1 0 2
    K 0 3 1 0 −3 2 1 −1 1 −2 −2 3 −1 −3 −1 0 0 −4 −2 −2
    M −1 −2 −2 −3 −1 −1 −2 −4 −1 2 3 −1 4 2 −2 −1 −1 −1 0 2
    F −2 −3 −3 −4 −1 −3 −4 −5 0 1 2 −3 2 7 −4 −3 −2 4 5 0
    P 0 −1 −1 −1 −3 0 0 −2 −1 −3 −2 −1 −2 −4 8 0 0 −5 −3 2
    S 1 0 1 0 0 0 0 0 0 −2 −2 0 −1 −3 0 2 2 −3 −2 −1
    T 1 0 0 0 0 0 0 −1 0 −1 −1 0 −1 −2 0 2 2 −4 −2 0
    W −4 −2 −4 −5 −1 −3 −4 −4 −1 −2 −1 −4 −1 4 −5 −3 −4 14 4 −3
    Y −2 −2 −1 −3 0 −2 −3 −4 2 −1 0 −2 0 5 −3 −2 −2 4 8 −1
    V 0 −2 −2 −3 0 −2 −2 −3 −2 3 2 −2 2 0 −2 −1 0 −3 −1 3
  • For purposes herein, a “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix reproduced herein above. [0315]
  • As is also well known in the art, relatedness of proteins can also be characterized using a functional test, the ability of the encoding nucleic acids to base-pair to one another at defined hybridization stringencies. [0316]
  • It is, therefore, another aspect of the invention to provide isolated proteins not only identical in sequence to those described with particularity herein, but also to provide isolated proteins (“hybridization related proteins”) that are encoded by nucleic acids that hybridize under high stringency conditions (as defined herein above) to all or to a portion of various of the isolated PAPP-E nucleic acids of the present invention (“reference nucleic acids”). It is a further aspect of the invention to provide isolated proteins (“hybridization related proteins”) that are encoded by nucleic acids that hybridize under moderate sringency conditions (as defined herein above) to all or to a portion of various of the isolated PAPP-E nucleic acids of the present invention. [0317]
  • The hybridization related proteins can be alternative isoforms, homologues, paralogues, and orthologues of the PAPP-E proteins of the present invention. Particularly preferred orthologues are those from other primate species, such as chimpanzee, rhesus macaque, baboon, and gorilla, from rodents, such as rats, mice, guinea pigs, and from livestock, such as cow, pig, sheep, horse, goat. [0318]
  • Relatedness of proteins can also be characterized using a second functional test, the ability of a first protein competitively to inhibit the binding of a second protein to an antibody. [0319]
  • It is, therefore, another aspect of the present invention to provide isolated proteins not only identical in sequence to those described with particularity herein, but also to provide isolated proteins (“cross-reactive proteins”) that competitively inhibit the binding of antibodies to all or to a portion of various of the isolated PAPP-E proteins of the present invention (“reference proteins”). Such competitive inhibition can readily be determined using immunoassays well known in the art. [0320]
  • Among the proteins of the present invention that differ in amino acid sequence from those described with particularity herein—including those that have deletions and insertions causing up to 10% non-identity, those having conservative or moderately conservative substitutions, hybridization related proteins, and cross-reactive proteins—those that substantially retain one or more PAPP-E activities are preferred. As described above, those activities include metalloprotease activity, specifically an ability to cleave an IGFBF, ability to heteromultimerize with serum proteins, such as eosinophil major basic protein (proMBP), and the ability to control survival, growth, and/or differentiation of the dominant ovarian follicle. [0321]
  • Residues that are tolerant of change while retaining function can be identified by altering the protein at known residues using methods known in the art, such as alanine scanning mutagenesis, Cunningham et al., [0322] Science 244(4908):1081-5 (1989); transposon linker scanning mutagenesis, Chen et al., Gene 263(1−2):39-48 (2001); combinations of homolog- and alanine-scanning mutagenesis, Jin et al., J. Mol. Biol. 226(3):851-65 (1992); combinatorial alanine scanning, Weiss et al., Proc. Natl. Acad. Sci USA 97(16):8950-4 (2000), followed by functional assay. Transposon linker scanning kits are available commercially (New England Biolabs, Beverly, Mass., USA, catalog. no. E7-102S; EZ::TN™ In-Frame Linker Insertion Kit, catalogue no. EZI04KN, Epicentre Technologies Corporation, Madison, Wis., USA).
  • As further described below, the isolated proteins of the present invention can readily be used as specific immunogens to raise antibodies that specifically recognize PAPP-E proteins, their isoforms, homologues, paralogues, and/or orthologues. The antibodies, in turn, can be used, inter alia, specifically to assay for the PAPP-E proteins of the present invention—e.g. by ELISA for detection of protein fluid samples, such as serum, by immunohistochemistry or laser scanning cytometry, for detection of protein in tissue samples, or by flow cytometry, for detection of intracellular protein in cell suspensions—for specific antibody-mediated isolation and/or purification of PAPP-E proteins, as for example by immunoprecipitation, and for use as specific agonists or antagonists of PAPP-E action. [0323]
  • The isolated proteins of the present invention are also immediately available for use as specific standards in assays used to determine the concentration and/or amount specifically of the PAPP-E proteins of the present invention. For example, ELISA kits for detection and quantitation of protein analytes include purified protein of known concentration for use as a measurement standard (e.g., the human interferon-γ OptEIA kit, catalog no. 555142, Pharmingen, San Diego, Calif., USA includes human recombinant gamma interferon, baculovirus produced). [0324]
  • The isolated proteins of the present invention are also immediately available for use as specific biomolecule capture probes for surface-enhanced laser desorption ionization (SELDI) detection of protein-protein interactions, WO 98/59362; WO 98/59360; WO 98/59361; and Merchant et al., [0325] Electrophoresis 21(6):1164-77 (2000), the disclosures of which are incorporated herein by reference in their entireties. The isolated proteins of the present invention are also immediately available for use as specific biomolecule capture probes on BIACORE surface plasmon resonance probes.
  • The isolated proteins of the present invention are also useful as a therapeutic supplement in patients having a specific deficiency in PAPP-E production. [0326]
  • In another aspect, the invention also provides fragments of various of the proteins of the present invention. The protein fragments are useful, inter alia, as antigenic and immunogenic fragments of a PAPP-E isoform. [0327]
  • By “fragments” of a protein is here intended isolated proteins (equally, polypeptides, peptides, oligopeptides), however obtained, that have an amino acid sequence identical to a portion of the reference amino acid sequence, which portion is at least 6 amino acids and less than the entirety of the reference nucleic acid. As so defined, “fragments” need not be obtained by physical fragmentation of the reference protein, although such provenance is not thereby precluded. [0328]
  • Fragments of at least 6 contiguous amino acids are useful in mapping B cell and T cell epitopes of the reference protein. See, e.g., Geysen et al., “Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid,” [0329] Proc. Natl. Acad. Sci. USA 81:3998-4002 (1984) and U.S. Pat. Nos. 4,708,871 and 5,595,915, the disclosures of which are incorporated herein by reference in their entireties. Because the fragment need not itself be immunogenic, part of an immunodominant epitope, nor even recognized by native antibody, to be useful in such epitope mapping, all fragments of at least 6 amino acids of the proteins of the present invention have utility in such a study.
  • Fragments of at least 8 contiguous amino acids, often at least 15 contiguous amino acids, have utility as immunogens for raising antibodies that recognize the proteins of the present invention. See, e.g., Lerner, “Tapping the immunological repertoire to produce antibodies of predetermined specificity,” Nature 299:592-596 (1982); Shinnick et al., “Synthetic peptide immunogens as vaccines,” [0330] Annu. Rev. Microbiol. 37:425-46 (1983); Sutcliffe et al., “Antibodies that react with predetermined sites on proteins,” Science 219:660-6 (1983), the disclosures of which are incorporated herein by reference in their entireties. As further described in the above-cited references, virtually all 8-mers, conjugated to a carrier, such as a protein, prove immunogenic—that is, prove capable of eliciting antibody for the conjugated peptide; accordingly, all fragments of at least 8 amino acids of the proteins of the present invention have utility as immunogens.
  • Fragments of at least 8, 9, 10 or 12 contiguous amino acids are also useful as competitive inhibitors of binding of the entire protein, or a portion thereof, to antibodies (as in epitope mapping), and to natural binding partners, such as subunits in a multerimic complex or to receptors or ligands of the subject protein; this competitive inhibition permits identification and separation of molecules that bind specifically to the protein of interest, U.S. Pat. Nos. 5,539,084 and 5,783,674, incorporated herein by reference in their entireties. [0331]
  • The protein, or protein fragment, of the present invention is thus at least 6 amino acids in length, typically at least 8, 9, 10 or 12 amino acids in length, and often at least 15 amino acids in length. Often, the protein or the present invention, or fragment thereof, is at least 20 amino acids in length, even 25 amino acids, 30 amino acids, 35 amino acids, or 50 amino acids or more in length. Of course, larger fragments having at least 75 amino acids, 100 amino acids, or even 150 amino acids are also useful, and at times preferred. [0332]
  • The present invention further provides fusions of the proteins and protein fragments of the present invention to heterologous polypeptides. [0333]
  • By fusion is here intended that the protein or protein fragment of the present invention is linearly contiguous to the heterologous polypeptide in a peptide-bonded polymer of amino acids or amino acid analogues; by “heterologous polypeptide” is here intended a polypeptide that does not naturally occur in contiguity with the protein or protein fragment of the present invention. As so defined, the fusion can consist entirely of a plurality of fragments of the PAPP-E protein in altered arrangement; in such case, any of the PAPP-E fragments can be considered heterologous to the other PAPP-E fragments in the fusion protein. More typically, however, the heterologous polypeptide is not drawn from the PAPP-E protein itself. [0334]
  • The fusion proteins of the present invention will include at least one fragment of the protein of the present invention, which fragment is at least 6, typically at least 8, often at least 15, and usefully at least 16, 17, 18, 19, or 20 amino acids long. The fragment of the protein of the present to be included in the fusion can usefully be at least 25 amino acids long, at least 50 amino acids long, and can be at least 75, 100, or even 150 amino acids long. Fusions that include the entirety of the proteins of the present invention have particular utility. [0335]
  • The heterologous polypeptide included within the fusion protein of the present invention is at least 6 amino acids in length, often at least 8 amino acids in length, and usefully at least 15, 20, and 25 amino acids in length. Fusions that include larger polypeptides, such as the IgG Fc region, and even entire proteins (such as GFP chromophore-containing proteins), have particular utility. [0336]
  • As described above in the description of vectors and expression vectors of the present invention, which discussion is incorporated herein by reference in its entirety, heterologous polypeptides to be included in the fusion proteins of the present invention can usefully include those designed to facilitate purification and/or visualization of recombinantly-expressed proteins. Although purification tags can also be incorporated into fusions that are chemically synthesized, chemical synthesis typically provides sufficient purity that further purification by HPLC suffices; however, visualization tags as above described retain their utility even when the protein is produced by chemical synthesis, and when so included render the fusion proteins of the present invention useful as directly detectable markers of PAPP-E presence. [0337]
  • As also discussed above, heterologous polypeptides to be included in the fusion proteins of the present invention can usefully include those that facilitate secretion of recombinantly expressed proteins—into the periplasmic space or extracellular milieu for prokaryotic hosts, into the culture medium for eukaryotic cells—through incorporation of secretion signals and/or leader sequences. [0338]
  • Other useful protein fusions of the present invention include those that permit use of the protein of the present invention as bait in a yeast two-hybrid system. See Bartel et al. (eds.), [0339] The Yeast Two-Hybrid System, Oxford University Press (1997) (ISBN: 0195109384); Zhu et al., Yeast Hybrid Technologies, Eaton Publishing, (2000) (ISBN 1-881299-15-5); Fields et al., Trends Genet. 10(8):286-92 (1994); Mendelsohn et al., Curr. Opin. Biotechnol. 5(5):482-6 (1994); Luban et al., Curr. Opin. Biotechnol. 6(1):59-64 (1995);.Allen et al., Trends Biochem. Sci. 20(12):511-6 (1995); Drees, Curr. Opin. Chem. Biol. 3(1):64-70 (1999); Topcu et al., Pharm. Res. 17(9):1049-55 (2000); Fashena et al., Gene 250(1−2):1-14 (2000), the disclosures of which are incorporated herein by reference in their entireties. Typically, such fusion is to either E. coli LexA or yeast GAL4 DNA binding domains. Related bait plasmids are available that express the bait fused to a nuclear localization signal.
  • Other useful protein fusions include those that permit display of the encoded protein on the surface of a phage or cell, fusions to intrinsically fluorescent proteins, such as green fluorescent protein (GFP), and fusions to the IgG Fc region. [0340]
  • The proteins and protein fragments of the present invention can also usefully be fused to protein toxins, such as Pseudomonas exotoxin A, diphtheria toxin, shiga toxin A, anthrax toxin lethal factor, ricin, in order to effect ablation of cells that bind or take up the proteins of the present invention. [0341]
  • The isolated proteins, protein fragments, and protein fusions of the present invention can be composed of natural amino acids linked by native peptide bonds, or can contain any or all of nonnatural amino acid analogues, normative bonds, and post-synthetic (post translational) modifications, either throughout the length of the protein or localized to one or more portions thereof. [0342]
  • As is well known in the art, when the isolated protein is used, e.g., for epitope mapping, the range of such nonnatural analogues, normative inter-residue bonds, or post-synthesis modifications will be limited to those that permit binding of the peptide to antibodies. When used as an immunogen for the preparation of antibodies in a non-human host, such as a mouse, the range of such nonnatural analogues, normative inter-residue bonds, or post-synthesis modifications will be limited to those that do not interfere with the immunogenicity of the protein. When the isolated protein is used as a therapeutic agent, such as a vaccine or for replacement therapy, the range of such changes will be limited to those that do not confer toxicity upon the isolated protein. [0343]
  • Non-natural amino acids can be incorporated during solid phase chemical synthesis or by recombinant techniques, although the former is typically more common. [0344]
  • For example, D-enantiomers of natural amino acids can readily be incorporated during chemical peptide synthesis: peptides assembled from D-amino acids are more resistant to proteolytic attack; incorporation of D-enantiomers can also be used to confer specific three dimensional conformations on the peptide. Other amino acid analogues commonly added during chemical synthesis include ornithine, norleucine, phosphorylated amino acids (typically phosphoserine, phosphothreonine, phosphotyrosine), L-malonyltyrosine, a non-hydrolyzable analog of phosphotyrosine (Kole et al., [0345] Biochem. Biophys. Res. Com. 209:817-821 (1995)), and various halogenated phenylalanine derivatives.
  • Amino acid analogues having detectable labels are also usefully incorporated during synthesis to provide a labeled polypeptide. [0346]
  • Biotin, for example, can be added using biotinoyl—(9-fluorenylmethoxycarbonyl)-L-lysine (FMOC biocytin) (Molecular Probes, Eugene, Oreg., USA). The FMOC and tBOC derivatives of dabcyl-L-lysine (Molecular Probes, Inc., Eugene, Oreg., USA) can be used to incorporate the dabcyl chromophore at selected sites in the peptide sequence during synthesis. The aminonaphthalene derivative EDANS, the most common fluorophore for pairing with the dabcyl quencher in fluorescence resonance energy transfer (FRET) systems, can be introduced during automated synthesis of peptides by using EDANS—FMOC-L-glutamic acid or the corresponding tBOC derivative (both from Molecular Probes, Inc., Eugene, Oreg., USA). Tetramethylrhodamine fluorophores can be incorporated during automated FMOC synthesis of peptides using (FMOC)—TMR-L-lysine (Molecular Probes, Inc. Eugene, Oreg., USA). [0347]
  • Other useful amino acid analogues that can be incorporated during chemical synthesis include aspartic acid, glutamic acid, lysine, and tyrosine analogues having allyl side-chain protection (Applied Biosystems, Inc., Foster City, Calif., USA); the allyl side chain permits synthesis of cyclic, branched-chain, sulfonated, glycosylated, and phosphorylated peptides. [0348]
  • A large number of other FMOC-protected non-natural amino acid analogues capable of incorporation during chemical synthesis are available commercially, including, e.g., Fmoc-2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, Fmoc-3-endo-aminobicyclo[2.2.1]heptane-2-endo-carboxylic acid, Fmoc-3-exo-aminobicyclo[2.2.1]heptane-2-exo-carboxylic acid, Fmoc-3-endo-amino-bicyclo[2.2.1]hept-5-ene-2-endo-carboxylic acid, Fmoc-3-exo-amino-bicyclo[2.2.1]hept-5-ene-2-exo-carboxylic acid, Fmoc-cis-2-amino-1-cyclohexanecarboxylic acid, Fmoc-trans-2-amino-1-cyclohexanecarboxylic acid, Fmoc-1-amino-1-cyclopentanecarboxylic acid, Fmoc-cis-2-amino-1-cyclopentanecarboxylic acid, Fmoc-1-amino-1-cyclopropanecarboxylic acid, Fmoc-D-2-amino-4-(ethylthio)butyric acid, Fmoc-L-2-amino-4-(ethylthio)butyric acid, Fmoc-L-buthionine, Fmoc-S-methyl-L-Cysteine, Fmoc-2-aminobenzoic acid (anthranillic acid), Fmoc-3-aminobenzoic acid, Fmoc-4-aminobenzoic acid, Fmoc-2-aminobenzophenone-2′-carboxylic acid, Fmoc-N-(4-aminobenzoyl)-b-alanine, Fmoc-2-amino-4,5-dimethoxybenzoic acid, Fmoc-4-aminohippuric acid, Fmoc-2-amino-3-hydroxybenzoic acid, Fmoc-2-amino-5-hydroxybenzoic acid, Fmoc-3-amino-4-hydroxybenzoic acid, Fmoc-4-amino-3-hydroxybenzoic acid, Fmoc-4-amino-2-hydroxybenzoic acid, Fmoc-5-amino-2-hydroxybenzoic acid, Fmoc-2-amino-3-methoxybenzoic acid, Fmoc-4-amino-3-methoxybenzoic acid, Fmoc-2-amino-3-methylbenzoic acid, Fmoc-2-amino-5-methylbenzoic acid, Fmoc-2-amino-6-methylbenzoic acid, Fmoc-3-amino-2-methylbenzoic acid, Fmoc-3-amino-4-methylbenzoic acid, Fmoc-4-amino-3-methylbenzoic acid, Fmoc-3-amino-2-naphtoic acid, Fmoc-D,L-3-amino-3-phenylpropionic acid, Fmoc-L-Methyldopa, Fmoc-2-amino-4,6-dimethyl-3-pyridinecarboxylic acid, Fmoc-D,L-?-amino-2-thiophenacetic acid, Fmoc-4-(carboxymethyl)piperazine, Fmoc-4-carboxypiperazine, Fmoc-4-(carboxymethyl)homopiperazine, Fmoc-4-phenyl-4-piperidinecarboxylic acid, Fmoc-L-1,2,3,4-tetrahydronorharman-3-carboxylic acid, Fmoc-L-thiazolidine-4-carboxylic acid, all available from The Peptide Laboratory (Richmond, Calif., USA). [0349]
  • Non-natural residues can also be added biosynthetically by engineering a suppressor tRNA, typically one that recognizes the UAG stop codon, by chemical aminoacylation with the desired unnatural amino acid and. Conventional site-directed mutagenesis is used to introduce the chosen stop codon UAG at the site of interest in the protein gene. When the acylated suppressor tRNA and the mutant gene are combined in an in vitro transcription/translation system, the unnatural amino acid is incorporated in response to the UAG codon to give a protein containing that amino acid at the specified position. Liu et al., [0350] Proc. Natl. Acad. Sci. USA 96(9):4780-5 (1999).
  • The isolated proteins, protein fragments and fusion proteins of the present invention can also include normative inter-residue bonds, including bonds that lead to circular and branched forms. [0351]
  • The isolated proteins and protein fragments of the present invention can also include post-translational and post-synthetic modifications, either throughout the length of the protein or localized to one or more portions thereof. [0352]
  • For example, when produced by recombinant expression in eukaryotic cells, the isolated proteins, fragments, and fusion proteins of the present invention will typically include N-linked and/or O-linked glycosylation, the pattern of which will reflect both the availability of glycosylation sites on the protein sequence and the identity of the host cell. Further modification of glycosylation pattern can be performed enzymatically. [0353]
  • As another example, recombinant polypeptides of the invention may also include an initial modified methionine residue, in some cases resulting from host-mediated processes. [0354]
  • When the proteins, protein fragments, and protein fusions of the present invention are produced by chemical synthesis, post-synthetic modification can be performed before deprotection and cleavage from the resin or after deprotection and cleavage. Modification before deprotection and cleavage of the synthesized protein often allows greater control, e.g. by allowing targeting of the modifying moiety to the N-terminus of a resin-bound synthetic peptide. [0355]
  • Useful post-synthetic (and post-translational) modifications include conjugation to detectable labels, such as fluorophores. [0356]
  • A wide variety of amine-reactive and thiol-reactive fluorophore derivatives have been synthesized that react under nondenaturating conditions with N-terminal amino groups and epsilon amino groups of lysine residues, on the one hand, and with free thiol groups of cysteine residues, on the other. [0357]
  • Kits are available commercially that permit conjugation of proteins to a variety of amine-reactive or thiol-reactive fluorophores: Molecular Probes, Inc. (Eugene, Oreg., USA), e.g., offers kits for conjugating proteins to Alexa Fluor 350, Alexa Fluor 430, Fluorescein-EX, Alexa Fluor 488, Oregon Green 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, and Texas Red-X. [0358]
  • A wide variety of other amine-reactive and thiol-reactive fluorophores are available commercially (Molecular Probes, Inc., Eugene, Oreg., USA), including Alexa Fluor® 350, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (monoclonal antibody labeling kits available from Molecular Probes, Inc., Eugene, Oreg., USA), BODIPY dyes, such as BODIPY 493/503, BODIPY FL, BODIPY R6G, BODIPY 530/550, BODIPY TMR, BODIPY 558/568, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY TR, BODIPY 630/650, BODIPY 650/665, Cascade Blue, Cascade Yello, Dansyl, lissamine rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, Oreg., USA). [0359]
  • The polypeptides of the present invention can also be conjugated to fluorophores, other proteins, and other macromolecules, using bifunctional linking reagents. [0360]
  • Common homobifunctional reagents include, e.g., APG, AEDP, BASED, BMB, BMDB, BMH, BMOE, BM[PEO]3, BM[PEO]4, BS3, BSOCOES, DFDNB, DMA, DMP, DMS, DPDPB, DSG, DSP (Lomant's Reagent), DSS, DST, DTBP, DTME, DTSSP, EGS, HBVS, Sulfo-BSOCOES, Sulfo-DST, Sulfo-EGS (all available Pierce, Rockford, Ill., USA); common heterobifunctional cross-linkers include ABH, AMAS, ANB-NOS, APDP, ASBA, BMPA, BMPH, BMPS, EDC, EMCA, EMCH, EMCS, KMUA, KMUH, GMBS, LC-SMCC, LC-SPDP, MBS, M2C2H, MPBH, MSA, NHS-ASA, PDPH, PMPI, SADP, SAED, SAND, SANPAH, SASD, SATP, SBAP, SFAD, SIA, SIAB, SMCC, SMPB, SMPH, SMPT, SPDP, Sulfo-EMCS, Sulfo-GMBS, Sulfo-HSAB, Sulfo-KMUS, Sulfo-LC-SPDP, Sulfo-MBS, Sulfo-NHS-LC-ASA, Sulfo-SADP, Sulfo-SANPAH, Sulfo-SIAB, Sulfo-SMCC, Sulfo-SMPB, Sulfo-LC-SMPT, SVSB, TFCS (all available Pierce, Rockford, Ill., USA). [0361]
  • The proteins, protein fragments, and protein fusions of the present invention can be conjugated, using such cross-linking reagents, to fluorophores that are not amine- or thiol-reactive. [0362]
  • Other labels that usefully can be conjugated to the proteins, protein fragments, and fusion proteins of the present invention include radioactive labels, echosonographic contrast reagents, and MRI contrast agents. [0363]
  • The proteins, protein fragments, and protein fusions of the present invention can also usefully be conjugated using cross-linking agents to carrier proteins, such as KLH, bovine thyroglobulin, and even bovine serum albumin (BSA), to increase immunogenicity for raising anti-PAPP-E antibodies. [0364]
  • The proteins, protein fragments, and protein fusions of the present invention can also usefully be conjugated to polyethylene glycol (PEG); PEGylation increases the serum half life of proteins administered intravenously for replacement therapy. Delgado et al., Crit. Rev. Ther. Drug Carrier Syst. 9(3-4):249-304 (1992); Scott et al., Curr. Pharm. Des. 4(6):423-38 (1998); DeSantis et al., [0365] Curr. Opin. Biotechnol. 10(4):324-30 (1999), incorporated herein by reference in their entireties. PEG monomers can be attached to the protein directly or through a linker, with PEGylation using PEG monomers activated with tresyl chloride (2,2,2-trifluoroethanesulphonyl chloride) permitting direct attachment under mild conditions.
  • The isolated proteins of the present invention, including fusions thereof, can be produced by recombinant expression, typically using the expression vectors of the present invention as above-described or, if fewer than about 100 amino acids, by chemical synthesis (typically, solid phase synthesis), and, on occasion, by in vitro translation. [0366]
  • Production of the isolated proteins of the present invention can optionally be followed by purification. [0367]
  • Purification of recombinantly expressed proteins is now well within the skill in the art. See, e.g., Thorner et al. (eds.), [0368] Applications of Chimeric Genes and Hybrid Proteins, Part A: Gene Expression and Protein Purification (Methods in Enzymology, Volume 326), Academic Press (2000), (ISBN: 0121822273); Harbin (ed.), Cloning, Gene Expression and Protein Purification: Experimental Procedures and Process Rationale, Oxford Univ. Press (2001) (ISBN: 0195132947); Marshak et al., Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Cold Spring Harbor Laboratory Press (1996) (ISBN: 0-87969-385-1); and Roe (ed.), Protein Purification Applications, Oxford University Press (2001), the disclosures of which are incorporated herein by reference in their entireties, and thus need not be detailed here.
  • Briefly, however, if purification tags have been fused through use of an expression vector that appends such tag, purification can be effected, at least in part, by means appropriate to the tag, such as use of immobilized metal affinity chromatography for polyhistidine tags. Other techniques common in the art include ammonium sulfate fractionation, immunoprecipitation, fast protein liquid chromatography (FPLC), high performance liquid chromatography (HPLC), and preparative gel electrophoresis. [0369]
  • Purification of chemically-synthesized peptides can readily be effected, e.g., by HPLC. [0370]
  • Accordingly, it is an aspect of the present invention to provide the isolated proteins of the present invention in pure or substantially pure form. [0371]
  • A purified protein of the present invention is an isolated protein, as above described, that is present at a concentration of at least 95%, as measured on a mass basis with respect to total protein in a composition. Such purities can often be obtained during chemical synthesis without further purification, as, e.g., by HPLC. Purified proteins of the present invention can be present at a concentration (measured on a mass basis with respect to total protein in a composition) of 96%, 97%, 98%, and even 99%. The proteins of the present invention can even be present at levels of 99.5%, 99.6%, and even 99.7%, 99.8%, or even 99.9% following purification, as by HPLC. [0372]
  • Although high levels of purity are preferred when the isolated proteins of the present invention are used as therapeutic agents—such as vaccines, or for replacement therapy—the isolated proteins of the present invention are also useful at lower purity. For example, partially purified proteins of the present invention can be used as immunogens to raise antibodies in laboratory animals. [0373]
  • Thus, in another aspect, the present invention provides the isolated proteins of the present invention in substantially purified form. A “substantially purified protein” of the present invention is an isolated protein, as above described, present at a concentration of at least 70%, measured on a mass basis with respect to total protein in a composition. Usefully, the substantially purified protein is present at a concentration, measured on a mass basis with respect to total protein in a composition, of at least 75%, 80%, or even at least 85%, 90%, 91%, 92%, 93%, 94%, 94.5% or even at least 94.9%. [0374]
  • In preferred embodiments, the purified and substantially purified proteins of the present invention are in compositions that lack detectable ampholytes, acrylamide monomers, bis-acrylamide monomers, and polyacrylamide. [0375]
  • The proteins, fragments, and fusions of the present invention can usefully be attached to a substrate. The substrate can porous or solid, planar or non-planar; the bond can be covalent or noncovalent. [0376]
  • For example, the proteins, fragments, and fusions of the present invention can usefully be bound to a porous substrate, commonly a membrane, typically comprising nitrocellulose, polyvinylidene fluoride (PVDF), or cationically derivatized, hydrophilic PVDF; so bound, the proteins, fragments, and fusions of the present invention can be used to detect and quantify antibodies, e.g. in serum, that bind specifically to the immobilized protein of the present invention. [0377]
  • As another example, the proteins, fragments, and fusions of the present invention can usefully be bound to a substantially nonporous substrate, such as plastic, to detect and quantify antibodies, e.g. in serum, that bind specifically to the immobilized protein of the present invention. Such plastics include polymethylacrylic, polyethylene, polypropylene, polyacrylate, polymethylmethacrylate, polyvinylchloride, polytetrafluoroethylene, polystyrene, polycarbonate, polyacetal, polysulfone, celluloseacetate, cellulosenitrate, nitrocellulose, or mixtures thereof; when the assay is performed in standard microtiter dish, the plastic is typically polystyrene. [0378]
  • The proteins, fragments, and fusions of the present invention can also be attached to a substrate suitable for use as a surface enhanced laser desorption ionization source; so attached, the protein, fragment, or fusion of the present invention is useful for binding and then detecting secondary proteins that bind with sufficient affinity or avidity to the surface-bound protein to indicate biologic interaction therebetween. The proteins, fragments, and fusions of the present invention can also be attached to a substrate suitable for use in surface plasmon resonance detection; so attached, the protein, fragment, or fusion of the present ivnention is useful for binding and then detecting secondary proteins that bind with sufficient affinity or avidity to the surface-bound protein to indicate biological interaction therebetween. [0379]
  • PAPP-E Isoform Proteins [0380]
  • In a first series of protein embodiments, the invention provides an isolated PAPP-E polypeptide having an amino acid sequence encoded by the cDNA in ATCC Deposit No. ______, or the amino acid sequence in SEQ ID NO:3, which are full length human PAPP-Ea isoforms. The invention further provides isolated PAPP-E polypeptides having an amino acid sequence encoded by the cDNA in ATCC Deposit No. ______, or the amino acid sequence in SEQ ID NO:10, which are full length human PAPP-Eb isoforms. The invention also provides isolated PAPP-E polypeptides having an amino acid sequence encoded by the cDNA in ATCC Deposit No. ______, or the amino acid sequence in SEQ ID NO:16, which are full length human PAPP-Ec isoforms. [0381]
  • When used as immunogens, the full length proteins of the present invention can be used, inter alia, to elicit antibodies that bind to epitopes that are common to all known PAPP-E isoforms. Such epitopes are encoded by any of exons 2-20, and by that portion of exon 1 translated in the PAPP-Ef isoform. When such antibodies are used for analytical assay of PAPP-E—e.g., in an ELISA intended to report the presence and/or amount of all isoforms, without distinction thereamong—any of the full length proteins can be used as a standard. [0382]
  • When used as immunogens, the full length proteins of the present invention can be used, inter alia, to elicit antibodies that bind to an epitope that is shared by PAPP-Ea, PAPP-Eb, and PAPP-Ec but absent from PAPP-Ef: such epitopes are encoded by that portion of exon 1 not translated in PAPP-Ef. Such antibodies are identified by counterscreening using PAPP-Ef protein. When such antibodies are used for analytical assay of PAPP-E—e.g., an ELISA intended to report the amount of PAPP-Ea, PAPP-Eb, and PAPP-Ec, but not PAPP-Ef—any of the full length proteins can be used as a standard. [0383]
  • When used as an immunogen, the full length PAPP-Eb protein can be used, inter alia, as an immunogen to elicit antibodies that bind to an epitope unique to the PAPP-Eb isoform. Such epitopes are encoded by exon 21 and the translated portion of exon 22. Such antibodies are identified by counterscreening using PAPP-Ea, PAPP-Ec, and/or PAPP-Ef isoforms. When such antibodies are used for analytical assay of PAPP-E—e.g., an ELISA intended to report the presence and/or amount of PAPP-Eb—the full length PAPP-Eb protein can be used uniquely among the isoforms as a standard. [0384]
  • The invention further provides fragments of the above-described polypeptides, particularly fragments having at least 6 amino acids, typically at least 8 amino acids, often at least 15 amino acids, and even the entirety of the sequence given in SEQ ID NO:7. This fragment (amino acids 1-19 of PAPP-Ea, -Eb, and Ec) is common to PAPP-Ea, PAPP-Eb and PAPP-Ec isoforms but absent from PAPP-Ef. These protein fragments can thus be used to identify and/or generate antibodies that recognize PAPP-Ea, PAPP-Eb, and PAPP-Ec isoforms without distinction thereamong, but that do not recognize PAPP-Ef. [0385]
  • The invention further provides fragments of at least 6 amino acids, typically at least 8 amino acids, often at least 15 amino acids, and even the entirety of the sequence given in SEQ ID NO:12, which is encoded by the exon that is novel in PAPP-Eb. The fragments have particular utility in identifying and in generating antibodies that recognize epitopes unique to the PAPP-Eb isoform. [0386]
  • The invention further provides fragments of at least 6 amino acids, typically at least 8 amino acids, often at least 15 amino acids, and even the entirety of the sequence given in SEQ ID NO:13, the coding sequence of the PAPP-Eb isoform that results from frameshift relative to PAPP-Ea isoform. These fragments have particular utility in identifying and in generating antibodies that recognize epitopes unique to the PAPP-Eb isoform. [0387]
  • The invention further provides fragments of at least 6 amino acids, typically at least 8 amino acids, often at least 15 amino acids, and even the entirety of the sequence given in SEQ ID NO:14, the coding sequence present uniquely in the PAPP-Eb isoform. These fragments have particular utility in dientifying and in generating antibodies that recognize epitopes unique to the PAPP-Eb isoform. [0388]
  • The invention further provides fragments of at least 6 amino acids, typically at least 8 amino acids, often at least 15 amino acids, and even the entirety of the sequence given in SEQ ID NO:18, the twenty amino acids centered about the exon 21 deletion in PAPP-Ec. These fragments have particular utility in identifying and generating antibodies that recognize epitopes created in the PAPP-Ec isoform due to absence of exon 21. [0389]
  • As described above, the invention further provides proteins that differ in sequence from those described with particularity in the above-referenced SEQ ID NOs., whether by way of insertion or deletion, by way of conservative or moderately conservative substitutions, as hybridization related proteins, or as cross-hybridizing proteins, with those that substantially retain a PAPP-E activity preferred. [0390]
  • The invention further provides fusions of the proteins and protein fragments herein described to heterologous polypeptides. [0391]
  • Antibodies and Antibody-Producing Cells [0392]
  • In another aspect, the invention provides antibodies, including fragments and derivatives thereof, that bind specifically to one or more of the PAPP-E proteins and protein fragments of the present invention or to one or more of the proteins and protein fragments encoded by the isolated PAPP-E nucleic acids of the present invention. The antibodies of the present invention specifically recognize any or all of linear epitopes, discontinuous epitopes, or conformational epitopes of such proteins or protein fragments, either as present on the protein in its native conformation or, in some cases, as present on the proteins as denatured, as, e.g., by solubilization in SDS. [0393]
  • In other embodiments, the invention provides antibodies, including fragments and derivatives thereof, the binding of which can be competitively inhibited by one or more of the PAPP-E proteins and protein fragments of the present invention, or by one or more of the proteins and protein fragments encoded by the isolated PAPP-E nucleic acids of the present invention. [0394]
  • As used herein, the term “antibody” refers to a polypeptide, at least a portion of which is encoded by at least one immunoglobulin gene, which can bind specifically to a first molecular species, and to fragments or derivatives thereof that remain capable of such specific binding. [0395]
  • By “bind specifically” and “specific binding” is here intended the ability of the antibody to bind to a first molecular species in preference to binding to other molecular species with which the antibody and first molecular species are admixed. An antibody is said specifically to “recognize” a first molecular species when it can bind specifically to that first molecular species. [0396]
  • As is well known in the art, the degree to which an antibody can discriminate as among molecular species in a mixture will depend, in part, upon the conformational relatedness of the species in the mixture; typically, the antibodies of the present invention will discriminate over adventitious binding to non-PAPP-E proteins by at least two-fold, more typically by at least 5-fold, typically by more than 10-fold, 25-fold, 50-fold, 75-fold, and often by more than 100-fold, and on occasion by more than 500-fold or 1000-fold. When used to detect the proteins or protein fragments of the present invention, the antibody of the present invention is sufficiently specific when it can be used to determine the presence of the protein of the present invention in human serum. [0397]
  • Typically, the affinity or avidity of an antibody (or antibody multimer, as in the case of an IgM pentamer) of the present invention for a protein or protein fragment of the present invention will be at least about 1×10[0398] −6 molar (M), typically at least about 5×10−7 M, usefully at least about 1×10−7 M, with affinities and avidities of at least 1×10−8 M, 5×10−9 M, and 1×10−10 M proving especially useful.
  • The antibodies of the present invention can be naturally-occurring forms, such as IgG, IgM, IgD, IgE, and IgA, from any mammalian species. [0399]
  • Human antibodies can, but will infrequently, be drawn directly from human donors or human cells. In such case, antibodies to the proteins of the present invention will typically have resulted from fortuitous immunization, such as autoimmune immunization, with the protein or protein fragments of the present invention. Such antibodies will typically, but will not invariably, be polyclonal. [0400]
  • Human antibodies are more frequently obtained using transgenic animals that express human immunoglobulin genes, which transgenic animals can be affirmatively immunized with the protein immunogen of the present invention. Human Ig-transgenic mice capable of producing human antibodies and methods of producing human antibodies therefrom upon specific immunization are described, inter alia, in U.S. Pat. Nos. 6,162,963; 6,150,584; 6,114,598; 6,075,181; 5,939,598; 5,877,397; 5,874,299; 5,814,318; 5,789,650; 5,770,429; 5,661,016; 5,633,425; 5,625,126; 5,569,825; 5,545,807; 5,545,806, and 5,591,669, the disclosures of which are incorporated herein by reference in their entireties. Such antibodies are typically monoclonal, and are typically produced using techniques developed for production of murine antibodies. [0401]
  • Human antibodies are particularly useful, and often preferred, when the antibodies of the present invention are to be administered to human beings as in vivo diagnostic or therapeutic agents, since recipient immune response to the administered antibody will often be substantially less than that occasioned by administration of an antibody derived from another species, such as mouse. [0402]
  • IgG, IgM, IgD, IgE and IgA antibodies of the present invention are also usefully obtained from other mammalian species, including rodents—typically mouse, but also rat, guinea pig, and hamster—lagomorphs, typically rabbits, and also larger mammals, such as sheep, goats, cows, and horses. In such cases, as with the transgenic human-antibody-producing non-human mammals, fortuitous immunization is not required, and the non-human mammal is typically affirmatively immunized, according to standard immunization protocols, with the protein or protein fragment of the present invention. [0403]
  • As discussed above, virtually all fragments of 8 or more contiguous amino acids of the proteins of the present invention can be used effectively as immunogens when conjugated to a carrier, typically a protein such as bovine thryoglobulin, keyhole limpet hemocyanin, or bovine serum albumin, conveniently using a bifunctional linker such as those described elsewhere above, which discussion is incorporated by reference here. [0404]
  • Immunogenicity can also be conferred by fusion of the proteins and protein fragments of the present invention to other moieties. [0405]
  • For example, peptides of the present invention can be produced by solid phase synthesis on a branched polylysine core matrix; these multiple antigenic peptides (MAPs) provide high purity, increased avidity, accurate chemical definition and improved safety in vaccine development. Tam et al., Proc. Natl. Acad. Sci. USA 85:5409-5413 (1988); Posnett et al., [0406] J. Biol. Chem. 263, 1719-1725 (1988).
  • Protocols for immunizing non-human mammals are well-established in the art, Harlow et al. (eds.), [0407] Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1998) (ISBN: 0879693142); Coligan et al. (eds.), Current Protocols in Immunology, John Wiley & Sons, Inc. (2001) (ISBN: 0-471-52276-7); Zola, Monoclonal Antibodies: Preparation and Use of Monoclonal Antibodies and Engineered Antibody Derivatives (Basics: From Background to Bench), Springer Verlag (2000) (ISBN: 0387915907), the disclosures of which are incorporated herein by reference, and often include multiple immunizations, either with or without adjuvants such as Freund's complete adjuvant and Freund's incomplete adjuvant.
  • Antibodies from nonhuman mammals can be polyclonal or monoclonal, with polyclonal antibodies having certain advantages in immunohistochemical detection of the proteins of the present invention and monoclonal antibodies having advantages in identifying and distinguishing particular epitopes of the proteins of the present invention. [0408]
  • Following immunization, the antibodies of the present invention can be produced using any art-accepted technique. Such techniques are well known in the art, Coligan et al. (eds.), [0409] Current Protocols in Immunology, John Wiley & Sons, Inc. (2001) (ISBN: 0-471-52276-7); Zola, Monoclonal Antibodies: Preparation and Use of Monoclonal Antibodies and Engineered Antibody Derivatives (Basics: From Background to Bench), Springer Verlag (2000) (ISBN: 0387915907); Howard et al. (eds.), Basic Methods in Antibody Production and Characterization, CRC Press (2000) (ISBN: 0849394457); Harlow et al. (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1998) (ISBN: 0879693142); Davis (ed.), Monoclonal Antibody Protocols, Vol. 45, Humana Press (1995) (ISBN: 0896033082); Delves (ed.), Antibody Production: Essential Techniques, John Wiley & Son Ltd (1997) (ISBN: 0471970107); Kenney, Antibody Solution: An Antibody Methods Manual, Chapman & Hall (1997) (ISBN: 0412141914), incorporated herein by reference in their entireties, and thus need not be detailed here.
  • Briefly, however, such techniques include, inter alia, production of monoclonal antibodies by hybridomas and expression of antibodies or fragments or derivatives thereof from host cells engineered to express immunoglobulin genes or fragments thereof. These two methods of production are not mutually exclusive: genes encoding antibodies specific for the proteins or protein fragments of the present invention can be cloned from hybridomas and thereafter expressed in other host cells. Nor need the two necessarily be performed together: e.g., genes encoding antibodies specific for the proteins and protein fragments of the present invention can be cloned directly from B cells known to be specific for the desired protein, as further described in U.S. Pat. No. 5,627,052, the disclosure of which is incorporated herein by reference in its entirety, or from antibody-displaying phage. [0410]
  • Recombinant expression in host cells is particularly useful when fragments or derivatives of the antibodies of the present invention are desired. [0411]
  • Host cells for recombinant antibody production—either whole antibodies, antibody fragments, or antibody derivatives—can be prokaryotic or eukaryotic. [0412]
  • Prokaryotic hosts are particularly useful for producing phage displayed antibodies of the present invention. [0413]
  • The technology of phage-displayed antibodies, in which antibody variable region fragments are fused, for example, to the gene III protein (pIII) or gene VIII protein (pVIII) for display on the surface of filamentous phage, such as M13, is by now well-established, Sidhu, [0414] Curr. Opin. Biotechnol. 11(6):610-6 (2000); Griffiths et al., Curr. Opin. Biotechnol. 9(1):102-8 (1998); Hoogenboom et al., Immunotechnology, 4(1):1-20 (1998); Rader et al., Current Opinion in Biotechnology 8:503-508 (1997); Aujame et al., Human Antibodies 8:155-168 (1997); Hoogenboom, Trends in Biotechnol. 15:62-70 (1997); de Kruif et al., 17:453-455 (1996); Barbas et al., Trends in Biotechnol. 14:230-234 (1996); Winter et al., Ann. Rev. Immunol. 433-455 (1994), and techniques and protocols required to generate, propagate, screen (pan), and use the antibody fragments from such libraries have recently been compiled, Barbas et al., Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001) (ISBN 0-87969-546-3); Kay et al. (eds.), Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, Inc. (1996); Abelson et al. (eds.), Combinatorial Chemistry, Methods in Enzymology vol. 267, Academic Press (May 1996), the disclosures of which are incorporated herein by reference in their entireties.
  • Typically, phage-displayed antibody fragments are scFv fragments or Fab fragments; when desired, full length antibodies can be produced by cloning the variable regions from the displaying phage into a complete antibody and expressing the full length antibody in a further prokaryotic or a eukaryotic host cell. [0415]
  • Eukaryotic cells are also useful for expression of the antibodies, antibody fragments, and antibody derivatives of the present invention. [0416]
  • For example, antibody fragments of the present invention can be produced in [0417] Pichia pastoris, Takahashi et al., Biosci. Biotechnol. Biochem. 64(10):2138-44 (2000); Freyre et al., J. Biotechnol. 76(2-3):157-63 (2000); Fischer et al., Biotechnol. Appl. Biochem. 30 (Pt 2):117-20 (1999); Pennell et al., Res. Immunol. 149(6):599-603 (1998); Eldin et al., J. Immunol. Methods. 201(1):67-75 (1997); and in Saccharomyces cerevisiae, Frenken et al., Res. Immunol. 149(6):589-99 (1998); Shusta et al., Nature Biotechnol. 16(8):773-7 (1998), the disclosures of which are incorporated herein by reference in their entireties.
  • Antibodies, including antibody fragments and derivatives, of the present invention can also be produced in insect cells, Li et al., [0418] Protein Expr. Purif. 21(1):121-8 (2001); Ailor et al., Biotechnol. Bioeng. 58(2-3):196-203 (1998); Hsu et al., Biotechnol. Prog. 13(1):96-104 (1997); Edelman et al., Immunology 91(1):13-9 (1997); and Nesbit et al., J. Immunol. Methods. 151(1−2):201-8 (1992), the disclosures of which are incorporated herein by reference in their entireties.
  • Antibodies and fragments and derivatives thereof of the present invention can also be produced in plant cells, Giddings et al., [0419] Nature Biotechnol. 18(11):1151-5 (2000); Gavilondo et al., Biotechniques 29(1):128-38 (2000); Fischer et al., J. Biol. Regul. Homeost. Agents 14(2):83-92 (2000); Fischer et al., Biotechnol. Appl. Biochem. 30 (Pt 2):113-6 (1999); Fischer et al., Biol. Chem. 380(7-8):825-39 (1999); Russell, Curr. Top. Microbiol. Immunol. 240:119-38 (1999); and Ma et al., Plant Physiol. 109(2):341-6 (1995), the disclosures of which are incorporated herein by reference in their entireties.
  • Mammalian cells useful for recombinant expression of antibodies, antibody fragments, and antibody derivatives of the present invention include CHO cells, COS cells, 293 cells, and myeloma cells. [0420]
  • Verma et al., [0421] J. Immunol. Methods 216(1−2):165-81 (1998), review and compare bacterial, yeast, insect and mammalian expression systems for expression of antibodies.
  • Antibodies of the present invention can also be prepared by cell free translation, as further described in Merk et al., J. Biochem. (Tokyo). 125(2):328-33 (1999) and Ryabova et al., [0422] Nature Biotechnol. 15(1):79-84 (1997), and in the milk of transgenic animals, as further described in Pollock et al., J. Immunol. Methods 231(1−2):147-57 (1999), the disclosures of which are incorporated herein by reference in their entireties.
  • The invention further provides antibody fragments that bind specifically to one or more of the proteins and protein fragments of the present invention, to one or more of the proteins and protein fragments encoded by the isolated nucleic acids of the present invention, or the binding of which can be competitively inhibited by one or more of the proteins and protein fragments of the present invention or one or more of the proteins and protein fragments encoded by the isolated nucleic acids of the present invention. [0423]
  • Among such useful fragments are Fab, Fab′, Fv, F(ab)′[0424] 2, and single chain Fv (scFv) fragments. Other useful fragments are described in Hudson, Curr. Opin. Biotechnol. 9(4):395-402 (1998).
  • It is also an aspect of the present invention to provide antibody derivatives that bind specifically to one or more of the proteins and protein fragments of the present invention, to one or more of the proteins and protein fragments encoded by the isolated nucleic acids of the present invention, or the binding of which can be competitively inhibited by one or more of the proteins and protein fragments of the present invention or one or more of the proteins and protein fragments encoded by the isolated nucleic acids of the present invention. [0425]
  • Among such useful derivatives are chimeric, primatized, and humanized antibodies; such derivatives are less immunogenic in human beings, and thus more suitable for in vivo administration, than are unmodified antibodies from non-human mammalian species. [0426]
  • Chimeric antibodies typically include heavy and/or light chain variable regions (including both CDR and framework residues) of immunoglobulins of one species, typically mouse, fused to constant regions of another species, typically human. See, e.g., U.S. Pat. No. 5,807,715; Morrison et al., [0427] Proc. Natl. Acad. Sci USA. 81(21):6851-5 (1984); Sharon et al., Nature 309(5966):364-7 (1984); Takeda et al., Nature 314(6010):452-4 (1985), the disclosures of which are incorporated herein by reference in their entireties. Primatized and humanized antibodies typically include heavy and/or light chain CDRs from a murine antibody grafted into a non-human primate or human antibody V region framework, usually further comprising a human constant region, Riechmann et al., Nature 332(6162):323-7 (1988); Co et al., Nature 351(6326):501-2 (1991); U.S. Pat. Nos. 6,054,297; 5,821,337; 5,770,196; 5,766,886; 5,821,123; 5,869,619; 6,180,377; 6,013,256; 5,693,761; and 6,180,370, the disclosures of which are incorporated herein by reference in their entireties.
  • Other useful antibody derivatives of the invention include heteromeric antibody complexes and antibody fusions, such as diabodies (bispecific antibodies), single-chain diabodies, and intrabodies. [0428]
  • The antibodies of the present invention, including fragments and derivatives thereof, can usefully be labeled. It is, therefore, another aspect of the present invention to provide labeled antibodies that bind specifically to one or more of the proteins and protein fragments of the present invention, to one or more of the proteins and protein fragments encoded by the isolated nucleic acids of the present invention, or the binding of which can be competitively inhibited by one or more of the proteins and protein fragments of the present invention or one or more of the proteins and protein fragments encoded by the isolated nucleic acids of the present invention. [0429]
  • The choice of label depends, in part, upon the desired use. [0430]
  • For example, when the antibodies of the present invention are used for immunohistochemical staining of tissue samples, the label can usefully be an enzyme that catalyzes production and local deposition of a detectable product. [0431]
  • Enzymes typically conjugated to antibodies to permit their immunohistochemical visualization are well known, and include alkaline phosphatase, β-galactosidase, glucose oxidase, horseradish peroxidase (HRP), and urease. Typical substrates for production and deposition of visually detectable products include o-Nitrophenyl-beta-D-galactopyranoside (ONPG); o-Phenylenediamine Dihydrochloride (OPD); p-Nitrophenyl Phosphate (PNPP); p-Nitrophenyl-beta-D-galactopryanoside (PNPG); 3′,3′Diaminobenzidine (DAB); 3-Amino-9-ethylcarbazole (AEC); 4-Chloro-1-naphthol (CN); 5-Bromo-4-chloro-3-indolyl-phosphate (BCIP); ABTS®; BluoGal; iodonitrotetrazolium (INT); nitroblue tetrazolium chloride (NBT); phenazine methosulfate (PMS); phenolphthalein monophosphate (PMP); tetramethyl benzidine (TMB); tetranitroblue tetrazolium (TNBT); X-Gal; X-Gluc; and X-Glucoside. [0432]
  • Other substrates can be used to produce products for local deposition that are luminescent. For example, in the presence of hydrogen peroxide (H[0433] 2O2), horseradish peroxidase (HRP) can catalyze the oxidation of cyclic diacylhydrazides, such as luminol. Immediately following the oxidation, the luminol is in an excited state (intermediate reaction product), which decays to the ground state by emitting light. Strong enhancement of the light emission is produced by enhancers, such as phenolic compounds. Advantages include high sensitivity, high resolution, and rapid detection without radioactivity and requiring only small amounts of antibody. See, e.g., Thorpe et al., Methods Enzymol. 133:331-53 (1986); Kricka et al., J. Immunoassay 17(1):67-83 (1996); and Lundqvist et al., J. Biolumin. Chemilumin. 10(6):353-9 (1995), the disclosures of which are incorporated herein by reference in their entireties. Kits for such enhanced chemiluminescent detection (ECL) are available commercially.
  • The antibodies can also be labeled using colloidal gold. [0434]
  • As another example, when the antibodies of the present invention are used, e.g., for flow cytometric detection, for scanning laser cytometric detection, or for fluorescent immunoassay, they can usefully be labeled with fluorophores. [0435]
  • There are a wide variety of fluorophore labels that can usefully be attached to the antibodies of the present invention. [0436]
  • For flow cytometric applications, both for extracellular detection and for intracellular detection, common useful fluorophores can be fluorescein isothiocyanate (FITC), allophycocyanin (APC), R-phycoerythrin (PE), peridinin chlorophyll protein (PerCP), Texas Red, Cy3, Cy5, fluorescence resonance energy tandem fluorophores such as PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, and APC-Cy7. [0437]
  • Other fluorophores include, inter alia, Alexa Fluor® 350, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (monoclonal antibody labeling kits available from Molecular Probes, Inc., Eugene, Oreg., USA), BODIPY dyes, such as BODIPY 493/503, BODIPY FL, BODIPY R6G, BODIPY 530/550, BODIPY TMR, BODIPY 558/568, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY TR, BODIPY 630/650, BODIPY 650/665, Cascade Blue, Cascade Yello, Dansyl, lissamine rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, Oreg., USA), and Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, all of which are also useful for fluorescently labeling the antibodies of the present invention. [0438]
  • For secondary detection using labeled avidin, streptavidin, captavidin or neutravidin, the antibodies of the present invention can usefully be labeled with biotin. [0439]
  • When the antibodies of the present invention are used, e.g., for western blotting applications, they can usefully be labeled with radioisotopes, such as [0440] 33P, 32P, 35S, 3H, and 125I.
  • As another example, when the antibodies of the present invention are used for radioimmunotherapy, the label can usefully be [0441] 228Th, 227Ac, 225Ac, 223Ra, 213Bi, 212Pb, 212Bi, 211At, 203Pb, 194Os, 188Re, 186Re, 153Sm, 149Tb, 131I, 125I, 111In, 105Rh, 99mTc, 97Ru, 90Y, 90Sr, 88Y, 72Se, 67Cu, or 47Sc.
  • As another example, when the antibodies of the present invention are to be used for in vivo diagnostic use, they can be rendered detectable by conjugation to MRI contrast agents, such as gadolinium diethylenetriaminepentaacetic acid (DTPA), Lauffer et al., [0442] Radiology 207(2):529-38 (1998), or by radioisotopic labeling
  • As would be understood, use of the labels described above is not restricted to the application as for which they were mentioned. [0443]
  • The antibodies of the present invention, including fragments and derivatives thereof, can also be conjugated to toxins, in order to target the toxin's ablative action to cells that display and/or express the proteins of the present invention. Commonly, the antibody in such immunotoxins is conjugated to Pseudomonas exotoxin A, diphtheria toxin, shiga toxin A, anthrax toxin lethal factor, or ricin. See Hall (ed.), [0444] Immunotoxin Methods and Protocols (Methods in Molecular Biology, Vol 166), Humana Press (2000) (ISBN:0896037754); and Frankel et al. (eds.), Clinical Applications of Immunotoxins, Springer-Verlag New York, Incorporated (1998) (ISBN:3540640975), the disclosures of which are incorporated herein by reference in their entireties, for review.
  • The antibodies of the present invention can usefully be attached to a substrate, and it is, therefore, another aspect of the invention to provide antibodies that bind specifically to one or more of the proteins and protein fragments of the present invention, to one or more of the proteins and protein fragments encoded by the isolated nucleic acids of the present invention, or the binding of which can be competitively inhibited by one or more of the proteins and protein fragments of the present invention or one or more of the proteins and protein fragments encoded by the isolated nucleic acids of the present invention, attached to a substrate. [0445]
  • Substrates can be porous or nonporous, planar or nonplanar. [0446]
  • For example, the antibodies of the present invention can usefully be conjugated to filtration media, such as NHS-activated Sepharose or CNBr-activated Sepharose for purposes of immunoaffinity chromatography. [0447]
  • For example, the antibodies of the present invention can usefully be attached to paramagnetic microspheres, typically by biotin-streptavidin interaction, which microsphere can then be used for isolation of cells that express or display the proteins of the present invention. As another example, the antibodies of the present invention can usefully be attached to the surface of a microtiter plate for ELISA. [0448]
  • As noted above, the antibodies of the present invention can be produced in prokaryotic and eukaryotic cells. It is, therefore, another aspect of the present invention to provide cells that express the antibodies of the present invention, including hybridoma cells, B cells, plasma cells, and host cells recombinantly modified to express the antibodies of the present invention. [0449]
  • In yet a further aspect, the present invention provides aptamers evolved to bind specifically to one or more of the proteins and protein fragments of the present invention, to one or more of the proteins and protein fragments encoded by the isolated nucleic acids of the present invention, or the binding of which can be competitively inhibited by one or more of the proteins and protein fragments of the present invention or one or more of the proteins and protein fragments encoded by the isolated nucleic acids of the present invention. [0450]
  • PAPP-E Antibodies [0451]
  • In a first series of antibody embodiments, the invention provides antibodies, both polyclonal and monoclonal, and fragments and derivatives thereof, that bind specifically to polypeptides comprising an amino acid sequence as provided in SEQ ID NO:7—the N-terminal portion of PAPP-Ea, PAPP-Eb, and PAPP-Ec that is absent from the PAPP-Ef isoform—and the binding of which can be competitively inhibited by a polypeptide the sequence of which is given in SEQ ID NO:7. Such antibodies can be used to discriminate the novel isoforms described herein from the PAPP-Ef isoform, but will not be able to discriminate as among PAPP-Ea, -Eb, and -Ec isoforms. [0452]
  • Such antibodies are useful in in vitro immunoassays, such as ELISA of maternal serum, western blot of maternal serum, or immunohistochemical assay of chorionic villus samples, in which the collective concentration and/or quantity of PAPP-Ea, -Eb, and -Ec protein isoforms provides diagnostic and/or prognostic information on pregnancy status. Such antibodies are also useful in isolating and purifying PAPP-E isoforms other than PAPP-Ef by immunoprecipitation, immunoaffinity chromatography, or magnetic bead-mediated purification. [0453]
  • In another series of embodiments, the invention provides antibodies, both polyclonal and monoclonal, and fragments and derivatives thereof, that bind specifically to polypeptides comprising an amino acid sequence as provided in SEQ ID NO:12—the region encoded by exon 21, unique to the PAPP-Eb isoform—and the binding of which can be competitively inhibited by a polypeptide the sequence of which is given in SEQ ID NO:12. In a further series of embodiments, the invention provides antibodies, both polyclonal and monoclonal, and fragments and derivatives thereof, that bind specifically to polypeptides comprising an amino acid sequence as provided in SEQ ID NO:13—the region C-terminal to exon 21 that is unique to PAPP-Eb isoform due to frameshift relative to the reading frame in PAPP-Ea, PAPP-Ec, and PAPP-Ef—and the binding of which can be competitively inhibited by a polypeptide the sequence of which is given in SEQ ID NO:13. In yet another series of embodiments, the invention provides antibodies, both polyclonal and monoclonal, and fragments and derivatives thereof, that bind specifically to polypeptides comprising an amino acid sequence as provided in SEQ ID NO:14—the region that is uniquely found in PAPP-Eb—and the binding of which can be competitively inhibited by a polypeptide the sequence of which is given in SEQ ID NO:14. [0454]
  • All of the antibodies of these latter three series of embodiments can be used to discriminate the PAPP-Eb isoform from all other isoforms. Such antibodies are useful in in vitro immunoassays, such as ELISA of maternal serum, western blot of maternal serum, or immunohistochemical assay of chorionic villus samples, in which the collective concentration and/or quantity of the PAPP-Eb isoform provides diagnostic and/or prognostic information on pregnancy status. Such antibodies are also useful in isolating and purifying the PAPP-Eb isoform by immunoprecipitation, immunoaffinity chromatography, or magnetic bead-mediated purification. [0455]
  • In yet a further series of embodiments, the invention provides antibodies, both polyclonal and monoclonal, and fragments and derivatives thereof, that bind specifically to polypeptides comprising an amino acid sequence as provided in SEQ ID NO:18—a 20 amino acid region of PAPP-Ec centered about the deletion of exons 2 and 3—and the binding of which can be competitively inhibited by a polypeptide the amino acid sequence of which is given in SEQ ID NO:18 and cannot be competitively inhibited by a polypeptide having the amino acid sequence of SEQ ID NO:1 (the full-length PAPP-Ea protein). [0456]
  • Such antibodies can be used to discriminate the PAPP-Ec isoform from the other known isoforms, and are useful in in vitro immunoassays, such as ELISA of maternal serum, western blot of maternal serum, or immunohistochemical assay of chorionic villus samples, in which the concentration and/or quantity of PAPP-Ec isoform provides diagnostic and/or prognostic information on pregnancy status. Such antibodies are also useful in isolating and purifying the PAPP-Ec isoform by immunoprecipitation, immunoaffinity chromatography, or magnetic bead-mediated purification. [0457]
  • In other embodiments, the invention further provides the above-described antibodies detectably labeled, and in yet other embodiments, provides the above-described antibodies attached to a substrate. [0458]
  • Pharmaceutical Compositions [0459]
  • PAPP-E isoforms are important for maintenance of pregnancy and maturation of ovarian follicles. Thus, compositions comprising nucleic acids and proteins of the present invention can be administered as contraceptive vaccines and antibodies of the present invention can be administered for passive immunization, and thus reversible contraception. Alternatively, proteins of the present invention can be administered by nonimmunogenic routes as replacement therapy in patients with decreased levels of PAPP-E isoforms, thus supporting at-risk pregnancies. [0460]
  • Accordingly, in another aspect, the invention provides pharmaceutical compositions comprising the nucleic acids, nucleic acid fragments, proteins, protein fusions, protein fragments, antibodies, antibody derivatives, and antibody fragments of the present invention. [0461]
  • Such a composition typically contains from about 0.1 to 90% by weight (such as 1 to 20% or 1 to 10%) of a therapeutic agent of the invention in a pharmaceutically accepted carrier. Solid formulations of the compositions for oral administration can contain suitable carriers or excipients, such as corn starch, gelatin, lactose, acacia, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, calcium carbonate, sodium chloride, or alginic acid. Disintegrators that can be used include, without limitation, microcrystalline cellulose, corn starch, sodium starch glycolate, and alginic acid. Tablet binders that can be used include acacia, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone(Povidone™), hydroxypropyl methylcellulose, sucrose, starch and ethylcellulose. Lubricants that can be used include magnesium stearates, stearic acid, silicone fluid, talc, waxes, oils, and colloidal silica. [0462]
  • Liquid formulations of the compositions for oral administration prepared in water or other aqueous vehicles can contain various suspending agents such as methylcellulose, alginates, tragacanth, pectin, kelgin, carrageenan, acacia, polyvinylpyrrolidone, and polyvinyl alcohol. The liquid formulations can also include solutions, emulsions, syrups and elixirs containing, together with the active compound(s), wetting agents, sweetners, and coloring and flavoring agents. Various liquid and powder formulations can be prepared by conventional methods for inhalation into the lungs of the mammal to be treated. [0463]
  • Injectable formulations of the compositions can contain various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like). For intravenous injections, water soluble versions of the compounds can be administered by the drip method, whereby a pharmaceutical formulation containing the antifungal agent and a physiologically acceptable excipient is infused. Physiologically acceptable excipients can include, for example, 5% dextrose, 0.9% saline, Ringer's solution or other suitable excipients. Intramuscular preparations, e.g., a sterile formulation f a suitable soluble salt form of the compounds, can e dissolved and administered in a pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or 5% glucose solution. A suitable insoluble form of the compound can be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, such as an ester of a long chain fatty acid (e.g., ethyl oleate). [0464]
  • A topical semi-solid ointment formulation typically contains a concentration of the active ingredient from about 1 to 20%, e.g., 5 to 10%, in a carrier such as a pharmaceutical cream base. Various formulations for topical use include drops, tinctures, lotions, creams, solutions, and ointments containing the active ingredient and various supports and vehicles. The optimal percentage of the therapeutic agent in each pharmaceutical formulation varies according to the formulation itself and the therapeutic effect desired in the specific pathologies and correlated therapeutic regimens. [0465]
  • Inhalation and transdermal formulations can also readily be prepared. [0466]
  • Pharmaceutical formulation is a well-established art, and is further described in Gennaro (ed.), [0467] Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins (2000) (ISBN: 0683306472); and Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippincott Williams & Wilkins Publishers (1999) (ISBN: 0683305727), the disclosures of which are incorporated herein by reference in their entireties.
  • Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the pharmaceutical formulation(s) to the patient. [0468]
  • Typically, the pharmaceutical formulation will be administered to the patient by applying to the skin of the patient a transdermal patch containing the pharmaceutical formulation., and leaving the patch in contact with the patient's skin (generally for 1 to 5 hours per patch). Other transdermal routes of administration (e.g., through use of a topically applied cream, ointment, or the like) can be used by applying conventional techniques. The pharmaceutical formulation(s) can also be administered via other conventional routes (e.g., enteral, subcutaneous, intrapulmonary, transmucosal, intraperitoneal, intrauterine, sublingual, intrathecal, or intramuscular routes) by using standard methods. In addition, the pharmaceutical formulations can be administered to the patient via injectable depot routes of administration such as by using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods. [0469]
  • Regardless of the route of administration, the therapeutic protein or antibody agent typically is administered at a daily dosage of 0.01 mg to 30 mg/kg of body weight of the patient (e.g., 1 mg/kg to 5 mg/kg). The pharmaceutical formulation can be administered in multiple doses per day, if desired, to achieve the total desired daily dose. [0470]
  • The effectiveness of the method of treatment can be assessed by monitoring the patient for known signs or symptoms of a disorder. [0471]
  • Transgenic Animals and Cells [0472]
  • In another aspect, the invention provides transgenic cells and non-human organisms comprising human PAPP-E isoform nucleic acids, and transgenic cells and non-human organisms with targeted disruption of the endogenous orthologue of the human PAPP-E gene. [0473]
  • The cells can be embryonic stem cells or somatic cells. The transgenic non-human organisms can be chimeric, nonchimeric heterozygotes, and nonchimeric homozygotes. [0474]
  • Diagnostic Methods [0475]
  • The nucleic acids of the present invention can be used as nucleic acid probes to assess the levels of PAPP-E isoform mRNA in chorionic villus samples, and antibodies of the present invention can be used to assess the expression levels of PAPP-E isoform proteins in chorionic villus samples, to diagnose dysgenetic pregnancies antenatally. [0476]
  • EXAMPLE 1 Identification and Characterization of cDNAs Encoding Multiple Isoforms of Human PAPP-E
  • Predicating our gene discovery efforts on use of genome-derived single exon probes and hybridization to genome-derived single exon microarrays—an approach that we have previously demonstrated will readily identify novel genes that have proven refractory to mRNA-based identification efforts—we identified an exon in raw human genomic sequence that is particularly expressed in human placenta. [0477]
  • Briefly, bioinformatic algorithms were applied to human genomic sequence data to identify putative exons. Each of the predicted exons was amplified from genomic DNA, typically centering the putative coding sequence within a larger amplicon that included flanking noncoding sequence. These genome-derived single exon probes were arrayed on a support and expression of the bioinformatically predicted exons assessed through a series of simultaneous two-color hybridizations to the genome-derived single exon microarrays. [0478]
  • The approach and procedures are further described in detail in Penn et al., “Mining the Human Genome using Microarrays of Open Reading Frames,” [0479] Nature Genetics 26:315-318 (2000); commonly owned and copending U.S. patent application Ser. No. 09/774,203, filed Jan. 29, 2001 (“Methods and Apparatus for Predicting, Confirming, and Displaying Functional Information Derived from Genomic Sequence”) and 09/632,366, filed Aug. 3, 2000 (“Methods and Apparatus for High-throughput Detection and Characterization of Alternatively Spliced Genes”), and commonly owned and copending U.S. provisional patent application No. 60/207,456, filed May 26, 2000 (“Human Genome-derived Single Exon Nucleic Acid Probes Useful for Gene Expression Analysis by Microarray”), the disclosures of which are incorporated herein by reference in their entireties.
  • Using a graphical display particularly designed to facilitate computerized query of the resulting exon-specific expression data, as further described in commonly owned and copending U.S. patent application Ser. No. 09/774,203, filed Jan. 29, 2001 (“Methods and Apparatus for Predicting, Confirming, and Displaying Functional Information Derived from Genomic Sequence”), two exons were identified that are expressed at high levels in human placenta, but that are expressed, if at all, at low levels in human heart, brain, adult liver, HeLa cells, lung, fetal liver, HBL100 cells, bone marrow, and BT474 cells; subsequent analysis revealed that the two exons belong to the same gene. Further details of procedures and results are set forth in commonly owned and copending U.S. provisional patent application No. 60/207,456, filed May 26, 2000 (“Human Genome-derived Single Exon Nucleic Acid Probes Useful for Gene Expression Analysis by Microarray”). [0480]
  • Table 1 summarizes the microarray expression data obtained using genome-derived single exon probes corresponding to exons 1 and 2. Each probe was completely sequenced on both strands prior to its use on a genome-derived single exon microarray; sequencing confirmed the exact chemical structure of each probe. An added benefit of sequencing is that it placed us in possession of a set of single base-incremented fragments of the sequenced nucleic acid, starting from the sequencing primer's 3′ OH. (Since the single exon probes were first obtained by PCR amplification from genomic DNA, we were of course additionally in possession of an even larger set of single base incremented fragments of each of the single exon probes, each fragment corresponding to an extension product from one of the two amplification primers.) [0481]
  • Signals and expression ratios are normalized values measured and calculated as further described in commonly owned and copending U.S. patent application Ser. No. 09/774,203, filed Jan. 29, 2001 (“Methods and Apparatus for Predicting, Confirming, and Displaying Functional Information Derived from Genomic Sequence”), and U.S. provisional patent application No. 60/207,456, filed May 26, 2000 (“Human Genome-derived Probes Useful for Gene Expression Analysis by Microarray”). [0482]
    TABLE 1
    Expression Analysis
    Genome-Derived Single Exon Microarray
    Amplicon 7403 Amplicon 7409
    (exon 1) (exon 2)
    Expression Expression
    Signal Ratio Signal Ratio
    Heart 1.01 4.64
    Brain 0.79 1.11 −7.46
    Adult 0.80 −12.73 1.40
    Liver
    HeLa 0.89 −7.18 1.29
    Lung 0.90 1.81
    Fetal 0.82 1.56 −8.27
    Liver
    Bone 1.02 1.86
    Marrow
    Placenta 74.34 8.08 43.62 6.05
  • As shown in Table 1, significant expression of exons 1 and 2 was seen only in placenta. Placenta-specific expression was further confirmed by northern blot analysis (see below). [0483]
  • Marathon-Ready™ placenta cDNA (Clontech Laboratories, Palo Alto, Calif., USA, catalogue no. 7411-1) was used as a substrate for standard RACE (rapid amplification of cDNA ends) to obtain a cDNA clone that spans 6.3 kilobases and appears to contain the entire coding region of the gene to which the two exons contribute; for reasons described below, we termed this cDNA PAPP-Ea. Marathon-Ready™ cDNAs are adaptor-ligated double stranded cDNAs suitable for 3′ and 5′ RACE. Chenchik et al., [0484] BioTechniques 21:526-532 (1996); Chenchik et al., CLONTECHniques X(1):5-8 (January 1995). RACE techniques are described, inter alia, in the Marathon-Ready™ cDNA User Manual (Clontech Labs., Palo Alto, Calif., USA, Mar. 30, 2000, Part No. PT1156-1 (PRO3517)), Ausubel et al. (eds.), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th edition (April 1999), John Wiley & Sons (ISBN: 047132938X) and Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual (3rd ed.), Cold Spring Harbor Laboratory Press (2000) (ISBN: 0879695773), the disclosures of which are incorporated herein by reference in their entireties.
  • The PAPP-Ea cDNA was sequenced on both strands using a MegaBace™ sequencer (Molecular Dynamics, Inc., Sunnyvale, Calif., USA). Sequencing both strands provided us with the exact chemical structure of the cDNA, which is shown in FIG. 3 and further presented in the SEQUENCE LISTING as SEQ ID NO:1, and placed us in actual physical possession of the entire set of single-base incremented fragments of the sequenced clone, starting at the 5′ and 3′ termini. [0485]
  • A 398 bp fragment of PAPP-Ea cDNA (nt 2314 to 2711, including part of exon 4, the whole of exon 5, and part of exon 6) was prepared by PCR using PAPP-Ea cDNA as template; the fragment was thereafter labeled by random priming and used to probe a northern blot of 12 tissues (leukocyte, lung, placenta, small intestine, liver, kidney, spleen, thymus, colon, skeletal muscle, heart and brain). Blot (not shown) confirmed the placenta-specific expression pattern. In addition, the random priming placed us in possession of a near complete set of fragments of the 398 bp PAPP-Ea cDNA fragment. [0486]
  • Further cDNA clones were obtained from the same cDNA library using primers designed to capture the entire PAPP-Ea ORF; these efforts identified two splice variants that we designated PAPP-Eb and PAPP-Ec, respectively. [0487]
  • The PAPP-Eb and PAPP-Ec cDNAs were sequenced on both strands using a MegaBace™ sequencer (Molecular Dynamics, Inc., Sunnyvale, Calif., USA). Sequencing both strands provided us with the exact chemical structure of the PAPP-Eb cDNA, shown in FIG. 4 and further presented in the SEQUENCE LISTING as SEQ ID NO:8, and of the PAPP-Ec cDNA, shown in FIG. 5 and further presented in the SEQUENCE LISTING as SEQ ID NO:10. Sequencing further placed us in actual physical possession of the entire set of single-base incremented fragments of the sequenced clones, starting at the 5′ and 3′ termini. [0488]
  • PAPP-Ea, PAPP-Eb, and PAPP-Ec cDNAs were deposited at the American Type Culture Collection on ______, 2001, under accession numbers ______, ______, and ______, respectively. [0489]
  • As shown in FIG. 3, the PAPP-Ea cDNA spans 6719 nucleotides and contains an open reading frame from nucleotide 767 through and including nt 6142 (inclusive of termination codon), predicting a protein of 1791 amino acids with a (posttranslationally unmodified) molecular weight of 198.6 kD. The clone appears full length, with the reading frame opening with a methionine and terminating with a stop codon before a 3′ poly-A tail. [0490]
  • As further shown in FIGS. 4 and 5, respectively, splice variants PAPP-Eb and PAPP-Ec are 5461 and 4158 nt, respectively. Because the two clones were obtained using a 5′ primer designed to amplify only the PAPP-Ea coding region, the clones lack 5′ untranslated region (5′ UT); we presume that the 5′ UT of these two clones, both of which start with the same exon as PAPP-Ea, should be identical to that for the PAPP-Ea clone. The PAPP-Eb and PAPP-Ec clones encode proteins of 1770 (PAPP-Eb) and 1385 (PAPP-Ec) amino acids, respectively, with predicted (post-translationally unmodified) molecular weights of 196 kD and 152 kD, respectively. [0491]
  • BLAST query of genomic sequence identified four BACs, spanning 265 kb, that constitute the minimum set of clones encompassing the three cDNA sequences. Based upon the known origin of the four BACs (GenBank accession numbers AL031734, AC027620, AL139282, and AL031290), the PAPP-E gene can be mapped to human chromosome 1q24.1-1q25.2. [0492]
  • Comparison of the cDNA and genomic sequences identified 23 exons. Exon organization is listed in Table 2. [0493]
    TABLE 2
    hPAPP-E Exon Structure
    Exon cDNA range BAC
    no (PAPP-Ea) genomic range accession
     1 1-1685 102055-103564 AL031734.9
     2 1686-2757 140847-141918
     3 2758-2903 122946-123091 AC027620.4
     4 2904-3197 172521-172228
     5 3198-3390 170533-170341
     6 3391-3512 166922-166801
     7 3513-4002 163562-163073
     8 4003-4131 160055-159927
     9 4132-4223 156303-156212
    10 4224-4417 16422-16229
    11 4418-4564 115036-114890 AL139282.4
    12 4565-4700 87245-87110
    13 4701-4917 86891-86675
    14 4918-5089 60806-60635
    15 5090-5267 56865-56688
    16 5268-5481 55505-55292
    17 5482-5650 10208-10376 AL031290.1
    18 5651-5786 11746-11881
    19 5787-5896 13959-14068
    20 5897-5968 20460-20531
    21 85bp* 23336-23420
    (PAPP-Eb
    only)
    22 5969-6067 60572-60670
    23 6068-6707 62779-63418
  • FIG. 2 schematizes the exon organization of the PAPP-Ea, Eb, and Ec clones. [0494]
  • Insertion of the 85 bp exon 21 uniquely in PAPP-Eb leads to a downstream frame shift, shown by shading of exon 21, with earlier termination of translation. PAPP-Ec lacks exons 2, 3 and 21. [0495]
  • The sequence of the PAPP-Ea cDNA was used as a BLAST query into the GenBank nr and dbEst databases. The nr database includes all non-redundant GenBank coding sequence translations, sequences derived from the 3-dimensional structures in the Brookhaven Protein Data Bank (PDB), sequences from SwissProt, sequences from the protein information resource (PIR), and sequences from protein research foundation (PRF). The dbEst (database of expressed sequence tags) includes ESTs, short, single pass read cDNA (mRNA) sequences, and cDNA sequences from differential display experiments and RACE experiments. [0496]
  • BLAST search identified multiple human ESTs, mainly from placental sources, one EST from mouse (AI157031), one from rat (AW916144), and one from cow (AW660476) as having sequence closely related to PAPP-Ea. BLAST search also identified as closely related a newly described human gene, termed PAPP-E, further described in Farr et al., [0497] Biochim. Biophys. Acta 1493:356-362 (October 2000), and human pregnancy-associated protein-A (PAPP-A).
  • Because the PAPP-E clone described by Farr et al. appears to be either another isoform, or an incomplete clone, of the gene that we have identified, we have named our gene PAPP-E and termed the Farr et al. clone PAPP-Ef (“f” in deference to the authors). [0498]
  • As shown in FIG. 2, the PAPP-Ef cDNA includes only a portion of exon 1, and is thereafter identical (with a single nucleotide change) to PAPP-Ea, including all of exons 2-20, 22 and 23. The exact start of the PAPP-Ef and PAPP-Ef translations are shown on each of FIGS. 3, 4, and [0499] 5. We detect only a single nucleotide difference between PAPP-Ea and PAPP-Ef in the region in which they are coextensive.
  • Globally, PAPP-Ea resembles human (44% amino acid identity and 63% amino acid similarity) and mouse (46% amino acid identity and 63% amino acid similarity) PAPP-A protein. [0500]
  • Motif searches using Pfam (http://pfam.wustl.edu), SMART (http://smart.embl-heidelberg.de), and PROSITE pattern and profile databases (http://www.expasy.ch/prosite), identified several known domains shared with PAPP-A. [0501]
  • FIG. 1 shows the domain structure of PAPP-A and all known isoforms of the PAPP-E protein. [0502]
  • As schematized in FIG. 1, our newly isolated isoforms—PAPP-Ea, PAPP-Eb, and PAPP-Ec—share certain protein domains and an overall structural organization with PAPP-A; in conjunction with a pattern of expression strikingly similar to that of PAPP-A, with high level expression in placenta, the shared structural features strongly imply that the three PAPP-E isoforms play a similar role in regulating the activity of a plasma borne growth factor(s), possibly IGF, which in turn is important for maintenance of pregnancy and/or normal fetal development, making the PAPP-E isoforms clinically useful diagnostic markers and potential therapeutic agents. [0503]
  • Like PAPP-A, all three novel isoforms have the zinc-binding domain (“zinc”) characteristic of metzincin superfamily metalloproteases, defined by the degenerate motif “[0504] HEXXHXXGXXH”, where invariant residues are shown underlined and variable residues are shown as “X”. In PAPP-Ea, the longest isoform, the zinc binding domain occurs at residues 733-743 with sequence HEVGHVLGLYH; the sequence is underlined in FIG. 3.
  • In common with PAPP-A, all three novel isoforms of PAPP-E have an at least four-fold repetition near the C-terminus of the short consensus repeat (“SCR”; alternatively denominated “sushi” domain) (residues 1396-1459, 1464-1521, 1525-1590, and 1595-1646, numbered as in PAPP-Ea). Relaxation of certain bioinformatic parameters suggests the presence of a fifth SCR domain. [0505]
  • In common with PAPP-A, all three novel isoforms of PAPP-E also have at least one “NL” (notch-lin, also termed lin notch repeat, or “LNR”) domain, so-called due to its presence in Notch and Lin-12 proteins, both of which proteins regulate early tissue differentiation. As shown in FIG. 1, PAPP-Ea possesses three NL domains in the same general spaced relationship to the zinc domain as is found in PAPP-A. PAPP-Eb, in contrast, lacks the C-terminal NL domain, whereas PAPPE-c, the shortest of the novel isoforms, lacks the two NL domains on the N-terminal side of the zinc-binding domain. [0506]
  • The four-fold repetition of SCR (“sushi”) domains is characteristic of complement proteins and selectins. Five-fold repetition of SCR domains with further presence of at least one NL domain has been previously identified in complement decay-accelerating factor and P-selectin. [0507]
  • In contrast to PAPP-A, two of the novel isoforms of PAPP-E—PAPP-Ea and PAPP-Eb—have a laminin G domain. Laminin G domains are found in a number of extracellular and receptor proteins, and are implicated in interactions with cellular receptors (integrins, alpha-dystroglycan), sulfated carbohydrates and other extracellular ligands. [0508]
  • In contrast to PAPP-A, all three novel isoforms of PAPP-E contain nuclear localization signals (“NLS”); with concurrent presence of a leader sequence (not shown), these signals suggest that all three PAPP-E isoforms can be secreted and also localize to the cell nucleus. [0509]
  • Possession of the genomic sequence permitted search for promoter and other control sequences for the hPAPP-E gene. [0510]
  • A putative transcriptional control region, inclusive of promoter and downstream elements, was defined as 1 kb around the transcription start site, itself defined as the first nucleotide of the PAPP-Ea cDNA clone. The region, drawn from sequence of BAC AL031734.9, has the following sequence, where nucleotide number 1001 is the transcription start site: [0511]
    [SEQ ID NO:65]
    tcttccccatcctttccatccatttcaaatcaattggaaa 40
    catggttccttgggtctagctgttcatttttgtaaattac 80
    ttattttgaacatctcattgtttatttgctcactcagcat 120
    atggtgacttttagtaacttcagattgagaaacttctgag 160
    ataaaaaggagacctatgtagtatgaattcatggcatttc 200
    catttagtacttctcacagcaggatacttgatttctcctt 240
    tctcccatgtccgatttaaagtgaatttaagatattgttc 280
    ttttaaatccccaatgattgaacaaagtaagaaaaaatac 320
    tttgttttgtttgtgacaaaacaaaagaaaaatacaaggg 360
    atccctaaaaggttagtgtgggcttattaggcagtaggta 400
    gatctgttcacagtaagtgtgtgtgtgtgtgtgtgtgtgt 440
    gtgtgagagagagagagaqaqagagagggagaatacacac 480
    agagaagagtactccaaaacactattgattttttgctatt 520
    gattgtgtaggctgcggctgctgaaagagaaagcccgaga 560
    tgtttactggggaaaccaagagtagcgtctgtcccctgtg 600
    ccttggtgaggtgggtaggttttcaggaggaaggagggga 640
    cagggaggagtaggtggagtgatgcattgaacttactagc 680
    tttgacatcatcattgtctttaaatgaaaacaaaaacaaa 720
    aacaaaaacaaaaaacaagaagatatttacaggcagacag 760
    aaagggagccaaggggagcaggagagactggagagaacag 800
    gtcccctgaagtgtatgctcttctttttgctcttttcccg 840
    atcttcccaggaacccacaagactcccagaaggtgaagtt 880
    aagagctcccagactcataaggttattagaacagcaaact 920
    ggcaccccaaagaactttacggagacttgcaacctatcaa 960
    caagttggatgagggattaaaagccttcaacaaccaacaa. 1000
  • Using PROSCAN, (http://bimas.dcrt.nih.gov/molbio/proscan/), no significant promoter was identified in the putative promoter region. However, transcription factor binding sites were identified using a web site at http://motif.genome.ad.jp/, including a group of SRY (sex-determining region Y gene product) binding sites (726.732; 714.720; 339.345 bp, with numbering according to SEQ ID NO:65). [0512]
  • We have thus identified three novel isoforms of a newly described human gene, PAPP-E, which share certain protein domains and an overall structural organization with PAPP-A; in conjunction with a pattern of expression strikingly similar to that of PAPP-A, with high level expression in placenta, the shared structural features strongly imply that the three PAPP-E isoforms play a role similar to PAPP-A, regulating the activity of a plasma-borne growth factor(s), possibly IGF, which in turn is important for maintenance of pregnancy and/or normal fetal development, making the PAPP-E isoforms clinically useful diagnostic markers and potential therapeutic agents. [0513]
  • EXAMPLE 2 Preparation and Labeling of Useful Fragments of Human PAPP-E Isoforms
  • Useful fragments of PAPP-Ea, PAPP-Eb, and PAPP-Ec clones are produced by PCR, using standard techniques, or solid phase chemical synthesis using an automated nucleic acid synthesizer. Each fragment is sequenced, confirming the exact chemical structure thereof. [0514]
  • The exact chemical structure of preferred fragments is provided in the attached SEQUENCE LISTING, the disclosure of which is incorporated herein by reference in its entirety. The following summary identifies the fragments whose structures are more fully described in the SEQUENCE LISTING: [0515]
    SEQ ID NO:1 nt full length PAPP-Ea cDNA
    SEQ ID NO:2 nt coding region of PAPP-Ea
    SEQ ID NO:3 aa full length coding sequence of
    PAPP-Ea
    SEQ ID NO:4 nt 5′ sequence absent from PAPP-
    Ef clone
    SEQ ID NO:5 nt 5′ UT absent from PAPP-Ef
    clone (nt 1-766)
    SEQ ID NO:6 nt N-terminal coding region
    absent from PAPP-Ef clone
    SEQ ID NO:7 aa coding region of PAPPE-Ea from
    aa 1-19 [N terminal coding
    region absent from PAPP-Ef
    clone]
    SEQ ID NO:8 nt full length PAPP-Eb cDNA
    SEQ ID NO:9 nt coding region of PAPP-Eb cDNA
    SEQ ID NO:10 aa full length coding sequence of
    PAPP-Eb (aa 1-1770)
    SEQ ID NO:11 nt exon novel in PAPP-Eb (nt
    5203-5287)
    SEQ ID NO:12 aa CDS entirely within novel exon
    (aa 1735-1762)
    SEQ ID NO:13 aa CDS due to frame shift (aa
    1763-1770)
    SEQ ID NO:14 aa CDS novel within PappE-b (aa
    1735-1770) (inclusive of old
    nt)
    SEQ ID NO:15 nt coding region of PAPP-Ec cDNA
    SEQ ID NO:16 aa full length coding sequence of
    PAPP-Ec
    SEQ ID NO:17 nt nt 892-951 (around splice
    junction)
    SEQ ID NO:18 aa 20 amino acids centered about
    deletion: aa 298-317
    SEQ ID NO:19 nt exon 1 (from genomic sequence)
    SEQ ID NO:20 nt exon 2
    SEQ ID NO:21 nt exon 3
    SEQ ID NO:22 nt exon 4
    SEQ ID NO:23 nt exon 5
    SEQ ID NO:24 nt exon 6
    SEQ ID NO:25 nt exon 7
    SEQ ID NO:26 nt exon 8
    SEQ ID NO:27 nt exon 9
    SEQ ID NO:28 nt exon 10
    SEQ ID NO:29 nt exon 11
    SEQ ID NO:30 nt exon 12
    SEQ ID NO:31 nt exon 13
    SEQ ID NO:32 nt exon 14
    SEQ ID NO:33 nt exon 15
    SEQ ID NO:34 nt exon 16
    SEQ ID NO:35 nt exon 17
    SEQ ID NO:36 nt exon 18
    SEQ ID NO:37 nt exon 19
    SEQ ID NO:38 nt exon 20
    SEQ ID NO:39 nt exon 21
    SEQ ID NO:40 nt exon 22
    SEQ ID NO:41 nt exon 23
    SEQ ID NO:42 nt 500 bp genomic amplicon
    centered about exon 1
    SEQ ID NO:43 nt 500 bp genomic amplicon
    centered about exon 2
    SEQ ID NO:44 nt 500 bp genomic amplicon
    centered about exon 3
    SEQ ID NO:45 nt 500 bp genomic amplicon
    centered about exon 4
    SEQ ID NO:46 nt 500 bp genomic amplicon
    centered about exon 5
    SEQ ID NO:47 nt 500 bp genomic amplicon
    centered about exon 6
    SEQ ID NO:48 nt 500 bp genomic amplicon
    centered about exon 7
    SEQ ID NO:49 nt 500 bp genomic amplicon
    centered about exon 8
    SEQ ID NO:50 nt 500 bp genomic amplicon
    centered about exon 9
    SEQ ID NO:51 nt 500 bp genomic amplicon
    centered about exon 10
    SEQ ID NO:52 nt 500 bp genomic amplicon
    centered about exon 11
    SEQ ID NO:53 nt 500 bp genomic amplicon
    centered about exon 12
    SEQ ID NO:54 nt 500 bp genomic amplicon
    centered about exon 13
    SEQ ID NO:55 nt 500 bp genomic amplicon
    centered about exon 14
    SEQ ID NO:56 nt 500 bp genomic amplicon
    centered about exon 15
    SEQ ID NO:57 nt 500 bp genomic amplicon
    centered about exon 16
    SEQ ID NO:58 nt 500 bp genomic amplicon
    centered about exon 17
    SEQ ID NO:59 nt 500 bp genomic amplicon
    centered about exon 18
    SEQ ID NO:60 nt 500 bp genomic amplicon
    centered about exon 19
    SEQ ID NO:61 nt 500 bp genomic amplicon
    centered about exon 20
    SEQ ID NO:62 nt 500 bp genomic amplicon
    centered about exon 21
    SEQ ID NO:63 nt 500 bp genomic amplicon
    centered about exon 22
    SEQ ID NO:64 nt 500 bp genomic amplicon
    centered about exon 23
    SEQ ID NO:65 nt 1000 bp putative promoter
    SEQ ID NOs:66-888 nt 17-mers scanning nt 1-823 of
    PAPP-Ea
    SEQ ID NOs: 889-1711 nt 25-mers scanning nt 1-823 of
    PAPP-Ea
    SEQ ID NOs: 1712-1796 nt 17-mers scanning SEQ ID NO:11
    SEQ ID NOs: 1787-1881 nt 25-mers scanning SEQ ID NO:25
  • Upon confirmation of the exact structure, each of the above-described nucleic acids of confirmed structure is recognized to be immediately useful as a PAPP-E-specific probe. [0516]
  • For use as labeled nucleic acid probes, the above-described PAPP-E nucleic acids are separately labeled by random priming. As is well known in the art of molecular biology, random priming places the investigator in possession of a near-complete set of labeled fragments of the template of varying length and varying starting nucleotide. [0517]
  • The labeled probes are used to identify the PAPP-E gene on a Southern blot, and are used to measure expression of PAPP-E isoforms on a northern blot and by RT-PCR, using standard techniques. [0518]
  • EXAMPLE 3 Production of PAPP-E Protein
  • In parallel, each of the full length PAPP-Ea, PAPP-Eb, and PAPP-Ec cDNA clones is separately cloned into the mammalian expression vector pcDNA3.1/HISA (Invitrogen, Carlsbad, Calif., USA), transfected into COS7 cells, transfectants selected with G418, and protein expression in transfectants confirmed by detection of the anti-Xpress™ epitope according to manufacturer's instructions. Protein is purified using immobilized metal affinity chromatography and vector-encoded protein sequence is then removed with enterokinase, per manufacturer's instructions, followed by gel filtration and/or HPLC. [0519]
  • Following epitope tag removal, each of PAPP-Ea, PAPP-Eb, and PAPP-Ec proteins is present at a concentration of at least 70%, measured on a weight basis with respect to total protein, and is free of acrylamide monomers, bis acrylamide monomers, polyacrylamide and ampholytes. Further HPLC purification provides PAPP-Ea, PAPP-Eb, and PAPP-Ec proteins at concentrations, respectively, of at least 95%, measured on a weight basis with respect to total protein. [0520]
  • EXAMPLE 4 Production of Anti-PAPP-E Antibody
  • Purified proteins prepared as in Example 3 are conjugated to carrier proteins and used to prepare murine monoclonal antibodies by standard techniques. Initial screening with the unconjugated purified proteins, followed by competitive inhibition screening using peptide fragments of the PAPP-E isoforms, identifies monoclonal antibodies with specificities in each of the following categories: antibodies that recognize all PAPP-E isoforms, antibodies that recognize PAPP-Eb alone, and antibodies that recognize PAPP-Ec alone. [0521]
  • EXAMPLE 5 Use of hPAPP-E Probes and Antibodies for Antenatal Diagnosis of Aneuploidy
  • After informed consent is obtained, peripheral blood samples are drawn from pregnant women at 14 weeks gestation and tested for PAPP-A levels by standard techniques and tested additionally for PAPP-E levels using anti-PAPP-E antibodies in a standard ELISA. [0522]
  • After pregnancy outcome is fully determined for all patients, tabulated results demonstrate a statistically significant decrease in the circulating maternal level of PAPP-E correlated with adverse outcome, and specifically with presence of either trisomy 18 or trisomy 21. Results further demonstrate that determination of concurrent PAPP-A and PAPP-E levels provides greater predictive value than either marker alone. [0523]
  • In a second series of experiments, chorionic villus samples obtained for purpose of antenatal fetal karyotyping are further examined for expression of PAPP-E isoforms. [0524]
  • In a first series of tests, total RNA is separately extracted from each CVS using a commercial kit and poly-A[0525] + RNA isolated with a commercial kit. A northern blot is prepared and probed with a radiolabeled PAPP-E nucleic acid probe. Specific hybridization is quantitated using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, Calif., USA). RNA levels are normalized to expression levels of β-actin.
  • After pregnancy outcome is fully determined for all patients, tabulated results demonstrate that a statistically significant decrease in chorionic villus PAPP-E expression is correlated with adverse outcome, and specifically with presence of either trisomy 18 or trisomy 21. Results further demonstrate that determination of maternal serum PAPP-E levels in conjunction with fetal chorionic villus expression provides greater predictive value than either marker alone. [0526]
  • In a second series of tests, chorionic villus samples are prepared for immunohistochemical analysis by standard techniques. Anti-PAPP-E antibodies labeled with alkaline phosphatase are used to visualize PAPP-E protein in the CVS samples by enhanced chemiluminescence. [0527]
  • After pregnancy outcome is fully determined for all patients, tabulated results demonstrate a statistically significant decrease in chorionic villus PAPP-E protein expression is correlated with adverse outcome, and specifically with presence of either trisomy 18 or trisomy 21. Results further demonstrate that determination of maternal serum PAPP-E levels in conjunction with fetal chorionic villus expression provides greater predictive value than either marker alone. [0528]
  • All patents, patent publications, and other published references mentioned herein are hereby incorporated by reference in their entireties as if each had been individually and specifically incorporated by reference herein. While preferred illustrative embodiments of the present invention are described, one skilled in the art will appreciate that the present invention can be practiced by other than the described embodiments, which are presented for purposes of illustration only and not by way of limitation. The present invention is limited only by the claims that follow. [0529]
  • 1 1881 1 6719 DNA Homo sapiens 1 ccccaagcat caaactgaag gaaacattct aaccttcaca gacagactgg aggctggatg 60 gggacctggc tgaagacatc tggagaatga aagttaagta ccagcttgca tttttgtgcc 120 cctagattat ttttgcattt taaaataaga agcatcaaat tgcgtgtctc tgtgtaaaag 180 ttctagcaat ttgttttaag gtgaacttat tttggcttag ggactacaaa aagagaaggt 240 aattcctagg gaaggaagaa gagaaagaaa tgaaaattag agaataagat tattttgaat 300 gacttcaggt agcgaggagt gtgtgtttgt gagtgtgtat ttgagagact tggctcatgc 360 ctgtgggtct tctcttctag tatcagtgag gggagggatt actgaagaag aaggggggaa 420 aaaaaaagaa agaaatctga gctttctggg aggaaattca aaggaaccaa gagaaattaa 480 cttcgttctg caaggactaa agtacagcaa gaggagagag gtcaagcgag aagcgtgcgg 540 gaagcacatg ccctggggag gcatagaagc cacactggca gagcggccag cacaggtagc 600 cagcagaggc attcttgggg ctatttgaaa aagtttggtc tgtgaacaaa acagtttccc 660 tggtgactgc aaatccattg ctagctgcct ctttctcgtc tgcccatcac tctggtgtgg 720 tacccagaag ttgacttctg gttctgtaga aagagctagg ggaggtatga tgtgcttaaa 780 gatcctaaga ataagcctgg cgattttggc tgggtgggca ctctgttctg ccaactctga 840 gctgggctgg acacgcaaga aatccttggt tgagagggaa cacctgaatc aggtgctgtt 900 ggaaggagaa cgttgttggc tgggggccaa ggttcgaaga cccagagctt ctccacagca 960 tcacctcttt ggagtctacc ccagcagggc tgggaactac ctaaggccct accccgtggg 1020 ggagcaagaa atccatcata caggacgcag caaaccagac actgaaggaa atgctgtgag 1080 ccttgttccc ccagacctga ctgaaaatcc agcaggactg aggggtgcag ttgaagagcc 1140 ggctgcccca tgggtagggg atagtcctat tgggcaatct gagctgctgg gagatgatga 1200 cgcttatctc ggcaatcaaa gatccaagga gtctctaggt gaggccggga ttcagaaagg 1260 ctcagccatg gctgccacta ctaccaccgc cattttcaca accctgaacg aacccaaacc 1320 agagacccaa aggaggggct gggccaagtc caggcagcgt cgccaagtgt ggaagaggcg 1380 ggcggaagat gggcagggag actccggtat ctcttcacat ttccaacctt ggcccaagca 1440 ttcccttaaa cacagggtca aaaagagtcc accggaggaa agcaaccaaa atggtggaga 1500 gggctcctac cgagaagcag agacctttaa ctcccaagta ggactgccca tcttatactt 1560 ctctgggagg cgggagcggc tgctgctgcg tccagaagtg ctggctgaga ttccccggga 1620 ggcgttcaca gtggaagcct gggttaaacc ggagggagga cagaacaacc cagccatcat 1680 cgcaggtgtg tttgataact gctcccacac tgtcagtgac aaaggctggg ccctggggat 1740 ccgctcaggg aaggacaagg gaaagcggga tgctcgcttc ttcttctccc tctgcaccga 1800 ccgcgtgaag aaagccacca tcttgattag ccacagtcgc taccaaccag gcacatggac 1860 ccatgtggca gccacttacg atggacggca catggccctg tatgtggatg gcactcaggt 1920 ggctagcagt ctagaccagt ctggtcccct gaacagcccc ttcatggcat cttgccgctc 1980 tttgctcctg gggggagaca gctctgagga tgggcactat ttccgtggac acctgggcac 2040 actggttttc tggtcgaccg ccctgccaca aagccatttt cagcacagtt ctcagcattc 2100 aagtgaggag gaggaagcga ctgacttggt cctgacagcg agctttgagc ctgtgaacac 2160 agagtgggtt ccctttagag atgagaagta cccacgactt gaggttctcc agggctttga 2220 gccagagcct gagattctgt cgcctttgca gcccccactc tgtgggcaaa cagtctgtga 2280 caatgtggaa ttgatctccc agtacaatgg atactggccc cttcggggag agaaggtgat 2340 acgctaccag gtggtgaaca tctgtgatga tgagggccta aaccccattg tgagtgagga 2400 gcagattcgt ctgcagcacg aggcactgaa tgaggccttc agccgctaca acatcagctg 2460 gcagctgagc gtccaccagg tccacaattc caccctgcga caccgggttg tgcttgtgaa 2520 ctgtgagccc agcaagattg gcaatgacca ttgtgacccc gagtgtgagc acccactcac 2580 aggctatgat gggggtgact gccgcctgca gggccgctgc tactcctgga accgcaggga 2640 tgggctctgt cacgtggagt gtaacaacat gctgaacgac tttgacgacg gagactgctg 2700 cgacccccag gtggctgatg tgcgcaagac ctgctttgac cctgactcac ccaagagggc 2760 atacatgagt gtgaaggagc tgaaggaggc cctgcagctg aacagtactc acttcctcaa 2820 catctacttt gccagctcag tgcgggaaga ccttgcaggt gctgccacct ggccttggga 2880 caaggacgct gtcactcacc tgggtggcat tgtcctcagc ccagcatatt atgggatgcc 2940 tggccacacc gacaccatga tccatgaagt gggacatgtt ctgggactct accatgtctt 3000 taaaggagtc agtgaaagag aatcctgcaa tgacccctgc aaggagacag tgccatccat 3060 ggaaacggga gacctctgtg ccgacaccgc ccccactccc aagagtgagc tgtgccggga 3120 accagagccc actagtgaca cctgtggctt cactcgcttc ccaggggctc cgttcaccaa 3180 ctacatgagc tacacggatg ataactgcac tgacaacttc actcctaacc aagtggcccg 3240 aatgcattgc tatttggacc tagtctatca gcagtggact gaaagcagaa agcccacccc 3300 catccccatt ccacctatgg tcatcggaca gaccaacaag tccctcacta tccactggct 3360 gcctcctatt agtggagttg tatatgacag ggcctcaggc agcttgtgtg gcgcttgcac 3420 tgaagatggg acctttcgtc agtatgtgca cacagcttcc tcccggcggg tgtgtgactc 3480 ctcaggttat tggaccccag aggaggctgt ggggcctcct gatgtggatc agccctgcga 3540 gccaagctta caggcctgga gccctgaggt ccacctgtac cacatgaaca tgacggtccc 3600 ctgccccaca gaaggctgta gcttggagct gctcttccaa cacccggtcc aagccgacac 3660 cctcaccctg tgggtcactt ccttcttcat ggagtcctcg caggtcctct ttgacacaga 3720 gatcttgctg gaaaacaagg agtcagtgca cctgggcccc ttagacactt tctgtgacat 3780 cccactcacc atcaaactgc acgtggatgg gaaggtgtcg ggggtgaaag tctacacctt 3840 tgatgagagg atagagattg atgcagcact cctgacttct cagccccaca gtcccttgtg 3900 ctctggctgc aggcctgtga ggtaccaggt tctccgcgat cccccatttg ccagtggttt 3960 gcccgtggtg gtgacacatt ctcacaggaa gttcacggac gtggaggtca cacctggaca 4020 gatgtatcag taccaagttc tagctgaagc tggaggagaa ctgggagaag cttcgcctcc 4080 tctgaaccac attcatggag ctccttattg tggagatggg aaggtgtcag agagactggg 4140 agaagagtgt gatgatggag accttgtgag cggagatggc tgctccaagg tgtgtgagct 4200 ggaggaaggt ttcaactgtg taggagagcc aagcctttgc tacatgtatg agggagatgg 4260 catatgtgaa ccttttgaga gaaaaaccag cattgtagac tgtggcatct acactcccaa 4320 aggatacttg gatcaatggg ctacccgggc ttactcctct catgaagaca agaagaagtg 4380 tcctgtttcc ttggtaactg gagaacctca ttccctaatt tgcacatcat accatccaga 4440 tttacccaac caccgtcccc taactggctg gtttccctgt gttgccagtg aaaatgaaac 4500 tcaggatgac aggagtgaac agccagaagg tagcctgaag aaagaggatg aggtttggct 4560 caaagtgtgt ttcaatagac caggagaggc cagagcaatt tttatttttt tgacaactga 4620 tggcctagtt cccggagagc atcagcagcc gacagtgact ctctacctga ccgatgtccg 4680 tggaagcaac cactctcttg gaacctatgg actgtcatgc cagcataatc cactgattat 4740 caatgtgacc catcaccaga atgtcctttt ccaccatacc acctcagtgc tgccgaattt 4800 ctcatcccca cgggtcggca tctcagctgt ggctctaagg acatcctccc gcattggtct 4860 ttcggctccc agtaactgca tctcagagga cgaggggcag aatcatcagg gacagagctg 4920 tatccatcgg ccctgtggga agcaggacag ctgtccgtca ttgctgcttg atcatgctga 4980 tgtggtgaac tgtacctcta taggcccagg tctcatgaag tgtgctatca cttgtcaaag 5040 gggatttgcc cttcaggcca gcagtgggca gtacatcagg cccatgcaga aggaaattct 5100 gctcacatgt tcttctgggc actgggacca gaatgtgagc tgccttcccg tggactgcgg 5160 tgttcccgac ccgtctttgg tgaactatgc aaacttctcc tgctcagagg gaaccaaatt 5220 tctgaaacgc tgctcaatct cttgtgtccc accagccaag ctgcaaggac tgagcccatg 5280 gctgacatgt cttgaagatg gtctctggtc tctccctgaa gtctactgca agttggagtg 5340 tgatgctccc cctattattc tgaatgccaa cttgctcctg cctcactgcc tccaggacaa 5400 ccacgacgtg ggcaccatct gcaaatatga atgcaaacca gggtactatg tggcagaaag 5460 tgcagagggt aaagtcagga acaagctcct gaagatacaa tgcctggaag gtggaatctg 5520 ggagcaaggc agctgcattc ctgtggtgtg tgagccaccc cctcctgtgt ttgaaggcat 5580 gtatgaatgt accaatggct tcagcctgga cagccagtgt gtgctcaact gtaaccagga 5640 acgtgaaaag cttcccatcc tctgcactaa agagggcctg tggacccagg agtttaagtt 5700 gtgtgagaat ctgcaaggag aatgcccacc acccccctca gagctgaatt ctgtggagta 5760 caaatgtgaa caaggatatg ggattggtgc agtgtgttcc ccattgtgtg taatcccccc 5820 cagtgacccc gtgatgctac ctgagaatat cactgctgac actctggagc actggatgga 5880 acctgtcaaa gtccagagca ttgtgtgcac tggccggcgt caatggcacc cagaccccgt 5940 cttagtccac tgcatccagt catgtgagcc cttccaagca aatggttggt gtgacactat 6000 caacaaccga gcctactgcc actatgacgg gggagactgc tgctcttcca cactctcctc 6060 caagaaggtc attccatttg ctgctgactg tgacctggat gagtgcacct gccgggaccc 6120 caaggcagaa gaaaatcagt aactgtggga acaagcccct ccctccactg cctcagaggc 6180 agtaagaaag agaggccgac ccaggaggaa acaaagggtg aatgaagaag aacaatcatg 6240 aaatggaaga aggaggaaga gcatgaagga tcttataaga aatgcaagag gatattgata 6300 ggtgtgaact agttcatcaa gtagcccaag taggagagaa tcataggcaa aagtttcttt 6360 aaagtggcag ttgattaaca tggaagggga aatatgatag atatataagg accctcctcc 6420 ctcacttata ttctattaaa tcctatcctc aactcttgcc ctgctctccg ctccaccccc 6480 tgccaactac tcagtcccac ccaacttgta aaccaatacc aaaatactag aggagaagtt 6540 ggcagggata ctgttaatac ccattttgaa tggattgcca tctttcagag cttgtctgct 6600 ctcaactggc tctttttctt tttgtgtagt ttccaatgaa taatgaagtt agttattaat 6660 tctttataag tatttaaaca taattatata aatatattat atatattaaa aaaaaaaaa 6719 2 5376 DNA Homo sapiens 2 atgatgtgct taaagatcct aagaataagc ctggcgattt tggctgggtg ggcactctgt 60 tctgccaact ctgagctggg ctggacacgc aagaaatcct tggttgagag ggaacacctg 120 aatcaggtgc tgttggaagg agaacgttgt tggctggggg ccaaggttcg aagacccaga 180 gcttctccac agcatcacct ctttggagtc taccccagca gggctgggaa ctacctaagg 240 ccctaccccg tgggggagca agaaatccat catacaggac gcagcaaacc agacactgaa 300 ggaaatgctg tgagccttgt tcccccagac ctgactgaaa atccagcagg actgaggggt 360 gcagttgaag agccggctgc cccatgggta ggggatagtc ctattgggca atctgagctg 420 ctgggagatg atgacgctta tctcggcaat caaagatcca aggagtctct aggtgaggcc 480 gggattcaga aaggctcagc catggctgcc actactacca ccgccatttt cacaaccctg 540 aacgaaccca aaccagagac ccaaaggagg ggctgggcca agtccaggca gcgtcgccaa 600 gtgtggaaga ggcgggcgga agatgggcag ggagactccg gtatctcttc acatttccaa 660 ccttggccca agcattccct taaacacagg gtcaaaaaga gtccaccgga ggaaagcaac 720 caaaatggtg gagagggctc ctaccgagaa gcagagacct ttaactccca agtaggactg 780 cccatcttat acttctctgg gaggcgggag cggctgctgc tgcgtccaga agtgctggct 840 gagattcccc gggaggcgtt cacagtggaa gcctgggtta aaccggaggg aggacagaac 900 aacccagcca tcatcgcagg tgtgtttgat aactgctccc acactgtcag tgacaaaggc 960 tgggccctgg ggatccgctc agggaaggac aagggaaagc gggatgctcg cttcttcttc 1020 tccctctgca ccgaccgcgt gaagaaagcc accatcttga ttagccacag tcgctaccaa 1080 ccaggcacat ggacccatgt ggcagccact tacgatggac ggcacatggc cctgtatgtg 1140 gatggcactc aggtggctag cagtctagac cagtctggtc ccctgaacag ccccttcatg 1200 gcatcttgcc gctctttgct cctgggggga gacagctctg aggatgggca ctatttccgt 1260 ggacacctgg gcacactggt tttctggtcg accgccctgc cacaaagcca ttttcagcac 1320 agttctcagc attcaagtga ggaggaggaa gcgactgact tggtcctgac agcgagcttt 1380 gagcctgtga acacagagtg ggttcccttt agagatgaga agtacccacg acttgaggtt 1440 ctccagggct ttgagccaga gcctgagatt ctgtcgcctt tgcagccccc actctgtggg 1500 caaacagtct gtgacaatgt ggaattgatc tcccagtaca atggatactg gccccttcgg 1560 ggagagaagg tgatacgcta ccaggtggtg aacatctgtg atgatgaggg cctaaacccc 1620 attgtgagtg aggagcagat tcgtctgcag cacgaggcac tgaatgaggc cttcagccgc 1680 tacaacatca gctggcagct gagcgtccac caggtccaca attccaccct gcgacaccgg 1740 gttgtgcttg tgaactgtga gcccagcaag attggcaatg accattgtga ccccgagtgt 1800 gagcacccac tcacaggcta tgatgggggt gactgccgcc tgcagggccg ctgctactcc 1860 tggaaccgca gggatgggct ctgtcacgtg gagtgtaaca acatgctgaa cgactttgac 1920 gacggagact gctgcgaccc ccaggtggct gatgtgcgca agacctgctt tgaccctgac 1980 tcacccaaga gggcatacat gagtgtgaag gagctgaagg aggccctgca gctgaacagt 2040 actcacttcc tcaacatcta ctttgccagc tcagtgcggg aagaccttgc aggtgctgcc 2100 acctggcctt gggacaagga cgctgtcact cacctgggtg gcattgtcct cagcccagca 2160 tattatggga tgcctggcca caccgacacc atgatccatg aagtgggaca tgttctggga 2220 ctctaccatg tctttaaagg agtcagtgaa agagaatcct gcaatgaccc ctgcaaggag 2280 acagtgccat ccatggaaac gggagacctc tgtgccgaca ccgcccccac tcccaagagt 2340 gagctgtgcc gggaaccaga gcccactagt gacacctgtg gcttcactcg cttcccaggg 2400 gctccgttca ccaactacat gagctacacg gatgataact gcactgacaa cttcactcct 2460 aaccaagtgg cccgaatgca ttgctatttg gacctagtct atcagcagtg gactgaaagc 2520 agaaagccca cccccatccc cattccacct atggtcatcg gacagaccaa caagtccctc 2580 actatccact ggctgcctcc tattagtgga gttgtatatg acagggcctc aggcagcttg 2640 tgtggcgctt gcactgaaga tgggaccttt cgtcagtatg tgcacacagc ttcctcccgg 2700 cgggtgtgtg actcctcagg ttattggacc ccagaggagg ctgtggggcc tcctgatgtg 2760 gatcagccct gcgagccaag cttacaggcc tggagccctg aggtccacct gtaccacatg 2820 aacatgacgg tcccctgccc cacagaaggc tgtagcttgg agctgctctt ccaacacccg 2880 gtccaagccg acaccctcac cctgtgggtc acttccttct tcatggagtc ctcgcaggtc 2940 ctctttgaca cagagatctt gctggaaaac aaggagtcag tgcacctggg ccccttagac 3000 actttctgtg acatcccact caccatcaaa ctgcacgtgg atgggaaggt gtcgggggtg 3060 aaagtctaca cctttgatga gaggatagag attgatgcag cactcctgac ttctcagccc 3120 cacagtccct tgtgctctgg ctgcaggcct gtgaggtacc aggttctccg cgatccccca 3180 tttgccagtg gtttgcccgt ggtggtgaca cattctcaca ggaagttcac ggacgtggag 3240 gtcacacctg gacagatgta tcagtaccaa gttctagctg aagctggagg agaactggga 3300 gaagcttcgc ctcctctgaa ccacattcat ggagctcctt attgtggaga tgggaaggtg 3360 tcagagagac tgggagaaga gtgtgatgat ggagaccttg tgagcggaga tggctgctcc 3420 aaggtgtgtg agctggagga aggtttcaac tgtgtaggag agccaagcct ttgctacatg 3480 tatgagggag atggcatatg tgaacctttt gagagaaaaa ccagcattgt agactgtggc 3540 atctacactc ccaaaggata cttggatcaa tgggctaccc gggcttactc ctctcatgaa 3600 gacaagaaga agtgtcctgt ttccttggta actggagaac ctcattccct aatttgcaca 3660 tcataccatc cagatttacc caaccaccgt cccctaactg gctggtttcc ctgtgttgcc 3720 agtgaaaatg aaactcagga tgacaggagt gaacagccag aaggtagcct gaagaaagag 3780 gatgaggttt ggctcaaagt gtgtttcaat agaccaggag aggccagagc aatttttatt 3840 tttttgacaa ctgatggcct agttcccgga gagcatcagc agccgacagt gactctctac 3900 ctgaccgatg tccgtggaag caaccactct cttggaacct atggactgtc atgccagcat 3960 aatccactga ttatcaatgt gacccatcac cagaatgtcc ttttccacca taccacctca 4020 gtgctgccga atttctcatc cccacgggtc ggcatctcag ctgtggctct aaggacatcc 4080 tcccgcattg gtctttcggc tcccagtaac tgcatctcag aggacgaggg gcagaatcat 4140 cagggacaga gctgtatcca tcggccctgt gggaagcagg acagctgtcc gtcattgctg 4200 cttgatcatg ctgatgtggt gaactgtacc tctataggcc caggtctcat gaagtgtgct 4260 atcacttgtc aaaggggatt tgcccttcag gccagcagtg ggcagtacat caggcccatg 4320 cagaaggaaa ttctgctcac atgttcttct gggcactggg accagaatgt gagctgcctt 4380 cccgtggact gcggtgttcc cgacccgtct ttggtgaact atgcaaactt ctcctgctca 4440 gagggaacca aatttctgaa acgctgctca atctcttgtg tcccaccagc caagctgcaa 4500 ggactgagcc catggctgac atgtcttgaa gatggtctct ggtctctccc tgaagtctac 4560 tgcaagttgg agtgtgatgc tccccctatt attctgaatg ccaacttgct cctgcctcac 4620 tgcctccagg acaaccacga cgtgggcacc atctgcaaat atgaatgcaa accagggtac 4680 tatgtggcag aaagtgcaga gggtaaagtc aggaacaagc tcctgaagat acaatgcctg 4740 gaaggtggaa tctgggagca aggcagctgc attcctgtgg tgtgtgagcc accccctcct 4800 gtgtttgaag gcatgtatga atgtaccaat ggcttcagcc tggacagcca gtgtgtgctc 4860 aactgtaacc aggaacgtga aaagcttccc atcctctgca ctaaagaggg cctgtggacc 4920 caggagttta agttgtgtga gaatctgcaa ggagaatgcc caccaccccc ctcagagctg 4980 aattctgtgg agtacaaatg tgaacaagga tatgggattg gtgcagtgtg ttccccattg 5040 tgtgtaatcc cccccagtga ccccgtgatg ctacctgaga atatcactgc tgacactctg 5100 gagcactgga tggaacctgt caaagtccag agcattgtgt gcactggccg gcgtcaatgg 5160 cacccagacc ccgtcttagt ccactgcatc cagtcatgtg agcccttcca agcaaatggt 5220 tggtgtgaca ctatcaacaa ccgagcctac tgccactatg acgggggaga ctgctgctct 5280 tccacactct cctccaagaa ggtcattcca tttgctgctg actgtgacct ggatgagtgc 5340 acctgccggg accccaaggc agaagaaaat cagtaa 5376 3 1791 PRT Homo sapiens 3 Met Met Cys Leu Lys Ile Leu Arg Ile Ser Leu Ala Ile Leu Ala Gly 1 5 10 15 Trp Ala Leu Cys Ser Ala Asn Ser Glu Leu Gly Trp Thr Arg Lys Lys 20 25 30 Ser Leu Val Glu Arg Glu His Leu Asn Gln Val Leu Leu Glu Gly Glu 35 40 45 Arg Cys Trp Leu Gly Ala Lys Val Arg Arg Pro Arg Ala Ser Pro Gln 50 55 60 His His Leu Phe Gly Val Tyr Pro Ser Arg Ala Gly Asn Tyr Leu Arg 65 70 75 80 Pro Tyr Pro Val Gly Glu Gln Glu Ile His His Thr Gly Arg Ser Lys 85 90 95 Pro Asp Thr Glu Gly Asn Ala Val Ser Leu Val Pro Pro Asp Leu Thr 100 105 110 Glu Asn Pro Ala Gly Leu Arg Gly Ala Val Glu Glu Pro Ala Ala Pro 115 120 125 Trp Val Gly Asp Ser Pro Ile Gly Gln Ser Glu Leu Leu Gly Asp Asp 130 135 140 Asp Ala Tyr Leu Gly Asn Gln Arg Ser Lys Glu Ser Leu Gly Glu Ala 145 150 155 160 Gly Ile Gln Lys Gly Ser Ala Met Ala Ala Thr Thr Thr Thr Ala Ile 165 170 175 Phe Thr Thr Leu Asn Glu Pro Lys Pro Glu Thr Gln Arg Arg Gly Trp 180 185 190 Ala Lys Ser Arg Gln Arg Arg Gln Val Trp Lys Arg Arg Ala Glu Asp 195 200 205 Gly Gln Gly Asp Ser Gly Ile Ser Ser His Phe Gln Pro Trp Pro Lys 210 215 220 His Ser Leu Lys His Arg Val Lys Lys Ser Pro Pro Glu Glu Ser Asn 225 230 235 240 Gln Asn Gly Gly Glu Gly Ser Tyr Arg Glu Ala Glu Thr Phe Asn Ser 245 250 255 Gln Val Gly Leu Pro Ile Leu Tyr Phe Ser Gly Arg Arg Glu Arg Leu 260 265 270 Leu Leu Arg Pro Glu Val Leu Ala Glu Ile Pro Arg Glu Ala Phe Thr 275 280 285 Val Glu Ala Trp Val Lys Pro Glu Gly Gly Gln Asn Asn Pro Ala Ile 290 295 300 Ile Ala Gly Val Phe Asp Asn Cys Ser His Thr Val Ser Asp Lys Gly 305 310 315 320 Trp Ala Leu Gly Ile Arg Ser Gly Lys Asp Lys Gly Lys Arg Asp Ala 325 330 335 Arg Phe Phe Phe Ser Leu Cys Thr Asp Arg Val Lys Lys Ala Thr Ile 340 345 350 Leu Ile Ser His Ser Arg Tyr Gln Pro Gly Thr Trp Thr His Val Ala 355 360 365 Ala Thr Tyr Asp Gly Arg His Met Ala Leu Tyr Val Asp Gly Thr Gln 370 375 380 Val Ala Ser Ser Leu Asp Gln Ser Gly Pro Leu Asn Ser Pro Phe Met 385 390 395 400 Ala Ser Cys Arg Ser Leu Leu Leu Gly Gly Asp Ser Ser Glu Asp Gly 405 410 415 His Tyr Phe Arg Gly His Leu Gly Thr Leu Val Phe Trp Ser Thr Ala 420 425 430 Leu Pro Gln Ser His Phe Gln His Ser Ser Gln His Ser Ser Glu Glu 435 440 445 Glu Glu Ala Thr Asp Leu Val Leu Thr Ala Ser Phe Glu Pro Val Asn 450 455 460 Thr Glu Trp Val Pro Phe Arg Asp Glu Lys Tyr Pro Arg Leu Glu Val 465 470 475 480 Leu Gln Gly Phe Glu Pro Glu Pro Glu Ile Leu Ser Pro Leu Gln Pro 485 490 495 Pro Leu Cys Gly Gln Thr Val Cys Asp Asn Val Glu Leu Ile Ser Gln 500 505 510 Tyr Asn Gly Tyr Trp Pro Leu Arg Gly Glu Lys Val Ile Arg Tyr Gln 515 520 525 Val Val Asn Ile Cys Asp Asp Glu Gly Leu Asn Pro Ile Val Ser Glu 530 535 540 Glu Gln Ile Arg Leu Gln His Glu Ala Leu Asn Glu Ala Phe Ser Arg 545 550 555 560 Tyr Asn Ile Ser Trp Gln Leu Ser Val His Gln Val His Asn Ser Thr 565 570 575 Leu Arg His Arg Val Val Leu Val Asn Cys Glu Pro Ser Lys Ile Gly 580 585 590 Asn Asp His Cys Asp Pro Glu Cys Glu His Pro Leu Thr Gly Tyr Asp 595 600 605 Gly Gly Asp Cys Arg Leu Gln Gly Arg Cys Tyr Ser Trp Asn Arg Arg 610 615 620 Asp Gly Leu Cys His Val Glu Cys Asn Asn Met Leu Asn Asp Phe Asp 625 630 635 640 Asp Gly Asp Cys Cys Asp Pro Gln Val Ala Asp Val Arg Lys Thr Cys 645 650 655 Phe Asp Pro Asp Ser Pro Lys Arg Ala Tyr Met Ser Val Lys Glu Leu 660 665 670 Lys Glu Ala Leu Gln Leu Asn Ser Thr His Phe Leu Asn Ile Tyr Phe 675 680 685 Ala Ser Ser Val Arg Glu Asp Leu Ala Gly Ala Ala Thr Trp Pro Trp 690 695 700 Asp Lys Asp Ala Val Thr His Leu Gly Gly Ile Val Leu Ser Pro Ala 705 710 715 720 Tyr Tyr Gly Met Pro Gly His Thr Asp Thr Met Ile His Glu Val Gly 725 730 735 His Val Leu Gly Leu Tyr His Val Phe Lys Gly Val Ser Glu Arg Glu 740 745 750 Ser Cys Asn Asp Pro Cys Lys Glu Thr Val Pro Ser Met Glu Thr Gly 755 760 765 Asp Leu Cys Ala Asp Thr Ala Pro Thr Pro Lys Ser Glu Leu Cys Arg 770 775 780 Glu Pro Glu Pro Thr Ser Asp Thr Cys Gly Phe Thr Arg Phe Pro Gly 785 790 795 800 Ala Pro Phe Thr Asn Tyr Met Ser Tyr Thr Asp Asp Asn Cys Thr Asp 805 810 815 Asn Phe Thr Pro Asn Gln Val Ala Arg Met His Cys Tyr Leu Asp Leu 820 825 830 Val Tyr Gln Gln Trp Thr Glu Ser Arg Lys Pro Thr Pro Ile Pro Ile 835 840 845 Pro Pro Met Val Ile Gly Gln Thr Asn Lys Ser Leu Thr Ile His Trp 850 855 860 Leu Pro Pro Ile Ser Gly Val Val Tyr Asp Arg Ala Ser Gly Ser Leu 865 870 875 880 Cys Gly Ala Cys Thr Glu Asp Gly Thr Phe Arg Gln Tyr Val His Thr 885 890 895 Ala Ser Ser Arg Arg Val Cys Asp Ser Ser Gly Tyr Trp Thr Pro Glu 900 905 910 Glu Ala Val Gly Pro Pro Asp Val Asp Gln Pro Cys Glu Pro Ser Leu 915 920 925 Gln Ala Trp Ser Pro Glu Val His Leu Tyr His Met Asn Met Thr Val 930 935 940 Pro Cys Pro Thr Glu Gly Cys Ser Leu Glu Leu Leu Phe Gln His Pro 945 950 955 960 Val Gln Ala Asp Thr Leu Thr Leu Trp Val Thr Ser Phe Phe Met Glu 965 970 975 Ser Ser Gln Val Leu Phe Asp Thr Glu Ile Leu Leu Glu Asn Lys Glu 980 985 990 Ser Val His Leu Gly Pro Leu Asp Thr Phe Cys Asp Ile Pro Leu Thr 995 1000 1005 Ile Lys Leu His Val Asp Gly Lys Val Ser Gly Val Lys Val Tyr Thr 1010 1015 1020 Phe Asp Glu Arg Ile Glu Ile Asp Ala Ala Leu Leu Thr Ser Gln Pro 1025 1030 1035 1040 His Ser Pro Leu Cys Ser Gly Cys Arg Pro Val Arg Tyr Gln Val Leu 1045 1050 1055 Arg Asp Pro Pro Phe Ala Ser Gly Leu Pro Val Val Val Thr His Ser 1060 1065 1070 His Arg Lys Phe Thr Asp Val Glu Val Thr Pro Gly Gln Met Tyr Gln 1075 1080 1085 Tyr Gln Val Leu Ala Glu Ala Gly Gly Glu Leu Gly Glu Ala Ser Pro 1090 1095 1100 Pro Leu Asn His Ile His Gly Ala Pro Tyr Cys Gly Asp Gly Lys Val 1105 1110 1115 1120 Ser Glu Arg Leu Gly Glu Glu Cys Asp Asp Gly Asp Leu Val Ser Gly 1125 1130 1135 Asp Gly Cys Ser Lys Val Cys Glu Leu Glu Glu Gly Phe Asn Cys Val 1140 1145 1150 Gly Glu Pro Ser Leu Cys Tyr Met Tyr Glu Gly Asp Gly Ile Cys Glu 1155 1160 1165 Pro Phe Glu Arg Lys Thr Ser Ile Val Asp Cys Gly Ile Tyr Thr Pro 1170 1175 1180 Lys Gly Tyr Leu Asp Gln Trp Ala Thr Arg Ala Tyr Ser Ser His Glu 1185 1190 1195 1200 Asp Lys Lys Lys Cys Pro Val Ser Leu Val Thr Gly Glu Pro His Ser 1205 1210 1215 Leu Ile Cys Thr Ser Tyr His Pro Asp Leu Pro Asn His Arg Pro Leu 1220 1225 1230 Thr Gly Trp Phe Pro Cys Val Ala Ser Glu Asn Glu Thr Gln Asp Asp 1235 1240 1245 Arg Ser Glu Gln Pro Glu Gly Ser Leu Lys Lys Glu Asp Glu Val Trp 1250 1255 1260 Leu Lys Val Cys Phe Asn Arg Pro Gly Glu Ala Arg Ala Ile Phe Ile 1265 1270 1275 1280 Phe Leu Thr Thr Asp Gly Leu Val Pro Gly Glu His Gln Gln Pro Thr 1285 1290 1295 Val Thr Leu Tyr Leu Thr Asp Val Arg Gly Ser Asn His Ser Leu Gly 1300 1305 1310 Thr Tyr Gly Leu Ser Cys Gln His Asn Pro Leu Ile Ile Asn Val Thr 1315 1320 1325 His His Gln Asn Val Leu Phe His His Thr Thr Ser Val Leu Pro Asn 1330 1335 1340 Phe Ser Ser Pro Arg Val Gly Ile Ser Ala Val Ala Leu Arg Thr Ser 1345 1350 1355 1360 Ser Arg Ile Gly Leu Ser Ala Pro Ser Asn Cys Ile Ser Glu Asp Glu 1365 1370 1375 Gly Gln Asn His Gln Gly Gln Ser Cys Ile His Arg Pro Cys Gly Lys 1380 1385 1390 Gln Asp Ser Cys Pro Ser Leu Leu Leu Asp His Ala Asp Val Val Asn 1395 1400 1405 Cys Thr Ser Ile Gly Pro Gly Leu Met Lys Cys Ala Ile Thr Cys Gln 1410 1415 1420 Arg Gly Phe Ala Leu Gln Ala Ser Ser Gly Gln Tyr Ile Arg Pro Met 1425 1430 1435 1440 Gln Lys Glu Ile Leu Leu Thr Cys Ser Ser Gly His Trp Asp Gln Asn 1445 1450 1455 Val Ser Cys Leu Pro Val Asp Cys Gly Val Pro Asp Pro Ser Leu Val 1460 1465 1470 Asn Tyr Ala Asn Phe Ser Cys Ser Glu Gly Thr Lys Phe Leu Lys Arg 1475 1480 1485 Cys Ser Ile Ser Cys Val Pro Pro Ala Lys Leu Gln Gly Leu Ser Pro 1490 1495 1500 Trp Leu Thr Cys Leu Glu Asp Gly Leu Trp Ser Leu Pro Glu Val Tyr 1505 1510 1515 1520 Cys Lys Leu Glu Cys Asp Ala Pro Pro Ile Ile Leu Asn Ala Asn Leu 1525 1530 1535 Leu Leu Pro His Cys Leu Gln Asp Asn His Asp Val Gly Thr Ile Cys 1540 1545 1550 Lys Tyr Glu Cys Lys Pro Gly Tyr Tyr Val Ala Glu Ser Ala Glu Gly 1555 1560 1565 Lys Val Arg Asn Lys Leu Leu Lys Ile Gln Cys Leu Glu Gly Gly Ile 1570 1575 1580 Trp Glu Gln Gly Ser Cys Ile Pro Val Val Cys Glu Pro Pro Pro Pro 1585 1590 1595 1600 Val Phe Glu Gly Met Tyr Glu Cys Thr Asn Gly Phe Ser Leu Asp Ser 1605 1610 1615 Gln Cys Val Leu Asn Cys Asn Gln Glu Arg Glu Lys Leu Pro Ile Leu 1620 1625 1630 Cys Thr Lys Glu Gly Leu Trp Thr Gln Glu Phe Lys Leu Cys Glu Asn 1635 1640 1645 Leu Gln Gly Glu Cys Pro Pro Pro Pro Ser Glu Leu Asn Ser Val Glu 1650 1655 1660 Tyr Lys Cys Glu Gln Gly Tyr Gly Ile Gly Ala Val Cys Ser Pro Leu 1665 1670 1675 1680 Cys Val Ile Pro Pro Ser Asp Pro Val Met Leu Pro Glu Asn Ile Thr 1685 1690 1695 Ala Asp Thr Leu Glu His Trp Met Glu Pro Val Lys Val Gln Ser Ile 1700 1705 1710 Val Cys Thr Gly Arg Arg Gln Trp His Pro Asp Pro Val Leu Val His 1715 1720 1725 Cys Ile Gln Ser Cys Glu Pro Phe Gln Ala Asn Gly Trp Cys Asp Thr 1730 1735 1740 Ile Asn Asn Arg Ala Tyr Cys His Tyr Asp Gly Gly Asp Cys Cys Ser 1745 1750 1755 1760 Ser Thr Leu Ser Ser Lys Lys Val Ile Pro Phe Ala Ala Asp Cys Asp 1765 1770 1775 Leu Asp Glu Cys Thr Cys Arg Asp Pro Lys Ala Glu Glu Asn Gln 1780 1785 1790 4 823 DNA Homo sapiens 4 ccccaagcat caaactgaag gaaacattct aaccttcaca gacagactgg aggctggatg 60 gggacctggc tgaagacatc tggagaatga aagttaagta ccagcttgca tttttgtgcc 120 cctagattat ttttgcattt taaaataaga agcatcaaat tgcgtgtctc tgtgtaaaag 180 ttctagcaat ttgttttaag gtgaacttat tttggcttag ggactacaaa aagagaaggt 240 aattcctagg gaaggaagaa gagaaagaaa tgaaaattag agaataagat tattttgaat 300 gacttcaggt agcgaggagt gtgtgtttgt gagtgtgtat ttgagagact tggctcatgc 360 ctgtgggtct tctcttctag tatcagtgag gggagggatt actgaagaag aaggggggaa 420 aaaaaaagaa agaaatctga gctttctggg aggaaattca aaggaaccaa gagaaattaa 480 cttcgttctg caaggactaa agtacagcaa gaggagagag gtcaagcgag aagcgtgcgg 540 gaagcacatg ccctggggag gcatagaagc cacactggca gagcggccag cacaggtagc 600 cagcagaggc attcttgggg ctatttgaaa aagtttggtc tgtgaacaaa acagtttccc 660 tggtgactgc aaatccattg ctagctgcct ctttctcgtc tgcccatcac tctggtgtgg 720 tacccagaag ttgacttctg gttctgtaga aagagctagg ggaggtatga tgtgcttaaa 780 gatcctaaga ataagcctgg cgattttggc tgggtgggca ctc 823 5 766 DNA Homo sapiens 5 ccccaagcat caaactgaag gaaacattct aaccttcaca gacagactgg aggctggatg 60 gggacctggc tgaagacatc tggagaatga aagttaagta ccagcttgca tttttgtgcc 120 cctagattat ttttgcattt taaaataaga agcatcaaat tgcgtgtctc tgtgtaaaag 180 ttctagcaat ttgttttaag gtgaacttat tttggcttag ggactacaaa aagagaaggt 240 aattcctagg gaaggaagaa gagaaagaaa tgaaaattag agaataagat tattttgaat 300 gacttcaggt agcgaggagt gtgtgtttgt gagtgtgtat ttgagagact tggctcatgc 360 ctgtgggtct tctcttctag tatcagtgag gggagggatt actgaagaag aaggggggaa 420 aaaaaaagaa agaaatctga gctttctggg aggaaattca aaggaaccaa gagaaattaa 480 cttcgttctg caaggactaa agtacagcaa gaggagagag gtcaagcgag aagcgtgcgg 540 gaagcacatg ccctggggag gcatagaagc cacactggca gagcggccag cacaggtagc 600 cagcagaggc attcttgggg ctatttgaaa aagtttggtc tgtgaacaaa acagtttccc 660 tggtgactgc aaatccattg ctagctgcct ctttctcgtc tgcccatcac tctggtgtgg 720 tacccagaag ttgacttctg gttctgtaga aagagctagg ggaggt 766 6 57 DNA Homo sapiens 6 atgatgtgct taaagatcct aagaataagc ctggcgattt tggctgggtg ggcactc 57 7 19 PRT Homo sapiens 7 Met Met Cys Leu Lys Ile Leu Arg Ile Ser Leu Ala Ile Leu Ala Gly 1 5 10 15 Trp Ala Leu 8 5461 DNA Homo sapiens 8 atgatgtgct taaagatcct aagaataagc ctggcgattt tggctgggtg ggcactctgt 60 tctgccaact ctgagctggg ctggacacgc aagaaatcct tggttgagag ggaacacctg 120 aatcaggtgc tgttggaagg agaacgttgt tggctggggg ccaaggttcg aagacccaga 180 gcttctccac agcatcacct ctttggagtc taccccagca gggctgggaa ctacctaagg 240 ccctaccccg tgggggagca agaaatccat catacaggac gcagcaaacc agacactgaa 300 ggaaatgctg tgagccttgt tcccccagac ctgactgaaa atccagcagg actgaggggt 360 gcagttgaag agccggctgc cccatgggta ggggatagtc ctattgggca atctgagctg 420 ctgggagatg atgacgctta tctcggcaat caaagatcca aggagtctct aggtgaggcc 480 gggattcaga aaggctcagc catggctgcc actactacca ccgccatttt cacaaccctg 540 aacgaaccca aaccagagac ccaaaggagg ggctgggcca agtccaggca gcgtcgccaa 600 gtgtggaaga ggcgggcgga agatgggcag ggagactccg gtatctcttc acatttccaa 660 ccttggccca agcattccct taaacacagg gtcaaaaaga gtccaccgga ggaaagcaac 720 caaaatggtg gagagggctc ctaccgagaa gcagagacct ttaactccca agtaggactg 780 cccatcttat acttctctgg gaggcgggag cggctgctgc tgcgtccaga agtgctggct 840 gagattcccc gggaggcgtt cacagtggaa gcctgggtta aaccggaggg aggacagaac 900 aacccagcca tcatcgcagg tgtgtttgat aactgctccc acactgtcag tgacaaaggc 960 tgggccctgg ggatccgctc agggaaggac aagggaaagc gggatgctcg cttcttcttc 1020 tccctctgca ccgaccgcgt gaagaaagcc accatcttga ttagccacag tcgctaccaa 1080 ccaggcacat ggacccatgt ggcagccact tacgatggac ggcacatggc cctgtatgtg 1140 gatggcactc aggtggctag cagtctagac cagtctggtc ccctgaacag ccccttcatg 1200 gcatcttgcc gctctttgct cctgggggga gacagctctg aggatgggca ctatttccgt 1260 ggacacctgg gcacactggt tttctggtcg accgccctgc cacaaagcca ttttcagcac 1320 agttctcagc attcaagtga ggaggaggaa gcgactgact tggtcctgac agcgagcttt 1380 gagcctgtga acacagagtg ggttcccttt agagatgaga agtacccacg acttgaggtt 1440 ctccagggct ttgagccaga gcctgagatt ctgtcgcctt tgcagccccc actctgtggg 1500 caaacagtct gtgacaatgt ggaattgatc tcccagtaca atggatactg gccccttcgg 1560 ggagagaagg tgatacgcta ccaggtggtg aacatctgtg atgatgaggg cctaaacccc 1620 attgtgagtg aggagcagat tcgtctgcag cacgaggcac tgaatgaggc cttcagccgc 1680 tacaacatca gctggcagct gagcgtccac caggtccaca attccaccct gcgacaccgg 1740 gttgtgcttg tgaactgtga gcccagcaag attggcaatg accattgtga ccccgagtgt 1800 gagcacccac tcacaggcta tgatgggggt gactgccgcc tgcagggccg ctgctactcc 1860 tggaaccgca gggatgggct ctgtcacgtg gagtgtaaca acatgctgaa cgactttgac 1920 gacggagact gctgcgaccc ccaggtggct gatgtgcgca agacctgctt tgaccctgac 1980 tcacccaaga gggcatacat gagtgtgaag gagctgaagg aggccctgca gctgaacagt 2040 actcacttcc tcaacatcta ctttgccagc tcagtgcggg aagaccttgc aggtgctgcc 2100 acctggcctt gggacaagga cgctgtcact cacctgggtg gcattgtcct cagcccagca 2160 tattatggga tgcctggcca caccgacacc atgatccatg aagtgggaca tgttctggga 2220 ctctaccatg tctttaaagg agtcagtgaa agagaatcct gcaatgaccc ctgcaaggag 2280 acagtgccat ccatggaaac gggagacctc tgtgccgaca ccgcccccac tcccaagagt 2340 gagctgtgcc gggaaccaga gcccactagt gacacctgtg gcttcactcg cttcccaggg 2400 gctccgttca ccaactacat gagctacacg gatgataact gcactgacaa cttcactcct 2460 aaccaagtgg cccgaatgca ttgctatttg gacctagtct atcagcagtg gactgaaagc 2520 agaaagccca cccccatccc cattccacct atggtcatcg gacagaccaa caagtccctc 2580 actatccact ggctgcctcc tattagtgga gttgtatatg acagggcctc aggcagcttg 2640 tgtggcgctt gcactgaaga tgggaccttt cgtcagtatg tgcacacagc ttcctcccgg 2700 cgggtgtgtg actcctcagg ttattggacc ccagaggagg ctgtggggcc tcctgatgtg 2760 gatcagccct gcgagccaag cttacaggcc tggagccctg aggtccacct gtaccacatg 2820 aacatgacgg tcccctgccc cacagaaggc tgtagcttgg agctgctctt ccaacacccg 2880 gtccaagccg acaccctcac cctgtgggtc acttccttct tcatggagtc ctcgcaggtc 2940 ctctttgaca cagagatctt gctggaaaac aaggagtcag tgcacctggg ccccttagac 3000 actttctgtg acatcccact caccatcaaa ctgcacgtgg atgggaaggt gtcgggggtg 3060 aaagtctaca cctttgatga gaggatagag attgatgcag cactcctgac ttctcagccc 3120 cacagtccct tgtgctctgg ctgcaggcct gtgaggtacc aggttctccg cgatccccca 3180 tttgccagtg gtttgcccgt ggtggtgaca cattctcaca ggaagttcac ggacgtggag 3240 gtcacacctg gacagatgta tcagtaccaa gttctagctg aagctggagg agaactggga 3300 gaagcttcgc ctcctctgaa ccacattcat ggagctcctt attgtggaga tgggaaggtg 3360 tcagagagac tgggagaaga gtgtgatgat ggagaccttg tgagcggaga tggctgctcc 3420 aaggtgtgtg agctggagga aggtttcaac tgtgtaggag agccaagcct ttgctacatg 3480 tatgagggag atggcatatg tgaacctttt gagagaaaaa ccagcattgt agactgtggc 3540 atctacactc ccaaaggata cttggatcaa tgggctaccc gggcttactc ctctcatgaa 3600 gacaagaaga agtgtcctgt ttccttggta actggagaac ctcattccct aatttgcaca 3660 tcataccatc cagatttacc caaccaccgt cccctaactg gctggtttcc ctgtgttgcc 3720 agtgaaaatg aaactcagga tgacaggagt gaacagccag aaggtagcct gaagaaagag 3780 gatgaggttt ggctcaaagt gtgtttcaat agaccaggag aggccagagc aatttttatt 3840 tttttgacaa ctgatggcct agttcccgga gagcatcagc agccgacagt gactctctac 3900 ctgaccgatg tccgtggaag caaccactct cttggaacct atggactgtc atgccagcat 3960 aatccactga ttatcaatgt gacccatcac cagaatgtcc ttttccacca taccacctca 4020 gtgctgccga atttctcatc cccacgggtc ggcatctcag ctgtggctct aaggacatcc 4080 tcccgcattg gtctttcggc tcccagtaac tgcatctcag aggacgaggg gcagaatcat 4140 cagggacaga gctgtatcca tcggccctgt gggaagcagg acagctgtcc gtcattgctg 4200 cttgatcatg ctgatgtggt gaactgtacc tctataggcc caggtctcat gaagtgtgct 4260 atcacttgtc aaaggggatt tgcccttcag gccagcagtg ggcagtacat caggcccatg 4320 cagaaggaaa ttctgctcac atgttcttct gggcactggg accagaatgt gagctgcctt 4380 cccgtggact gcggtgttcc cgacccgtct ttggtgaact atgcaaactt ctcctgctca 4440 gagggaacca aatttctgaa acgctgctca atctcttgtg tcccaccagc caagctgcaa 4500 ggactgagcc catggctgac atgtcttgaa gatggtctct ggtctctccc tgaagtctac 4560 tgcaagttgg agtgtgatgc tccccctatt attctgaatg ccaacttgct cctgcctcac 4620 tgcctccagg acaaccacga cgtgggcacc atctgcaaat atgaatgcaa accagggtac 4680 tatgtggcag aaagtgcaga gggtaaagtc aggaacaagc tcctgaagat acaatgcctg 4740 gaaggtggaa tctgggagca aggcagctgc attcctgtgg tgtgtgagcc accccctcct 4800 gtgtttgaag gcatgtatga atgtaccaat ggcttcagcc tggacagcca gtgtgtgctc 4860 aactgtaacc aggaacgtga aaagcttccc atcctctgca ctaaagaggg cctgtggacc 4920 caggagttta agttgtgtga gaatctgcaa ggagaatgcc caccaccccc ctcagagctg 4980 aattctgtgg agtacaaatg tgarcaagga tatgggattg gtgcagtgtg ttccccattg 5040 tgtgtaatcc cccccagtga ccccgtgatg ctacctgaga atatcactgc tgacactctg 5100 gagcactgga tggaacctgt caaagtccag agcattgtgt gcactggccg gcgtcaatgg 5160 cacccagacc ccgtcttagt ccactgcatc cagtcatgtg aggtcataag ccagttgttg 5220 ctgcttgtgt tcccattgtc ccagcaagaa cacacgtatg ctacatatct gcaatccaaa 5280 attgttgccc ttccaagcag atggttggtg tgacactatc aacaaccgag cctactgcca 5340 ctatgacggg ggagactgct gctcttccac actctcctcc aagaaggtca ttccatttgc 5400 tgctgactgt gacctggatg agtgcacctg ccgggacccc aaggcagaag aaaatcagta 5460 a 5461 9 5313 DNA Homo sapiens 9 atgatgtgct taaagatcct aagaataagc ctggcgattt tggctgggtg ggcactctgt 60 tctgccaact ctgagctggg ctggacacgc aagaaatcct tggttgagag ggaacacctg 120 aatcaggtgc tgttggaagg agaacgttgt tggctggggg ccaaggttcg aagacccaga 180 gcttctccac agcatcacct ctttggagtc taccccagca gggctgggaa ctacctaagg 240 ccctaccccg tgggggagca agaaatccat catacaggac gcagcaaacc agacactgaa 300 ggaaatgctg tgagccttgt tcccccagac ctgactgaaa atccagcagg actgaggggt 360 gcagttgaag agccggctgc cccatgggta ggggatagtc ctattgggca atctgagctg 420 ctgggagatg atgacgctta tctcggcaat caaagatcca aggagtctct aggtgaggcc 480 gggattcaga aaggctcagc catggctgcc actactacca ccgccatttt cacaaccctg 540 aacgaaccca aaccagagac ccaaaggagg ggctgggcca agtccaggca gcgtcgccaa 600 gtgtggaaga ggcgggcgga agatgggcag ggagactccg gtatctcttc acatttccaa 660 ccttggccca agcattccct taaacacagg gtcaaaaaga gtccaccgga ggaaagcaac 720 caaaatggtg gagagggctc ctaccgagaa gcagagacct ttaactccca agtaggactg 780 cccatcttat acttctctgg gaggcgggag cggctgctgc tgcgtccaga agtgctggct 840 gagattcccc gggaggcgtt cacagtggaa gcctgggtta aaccggaggg aggacagaac 900 aacccagcca tcatcgcagg tgtgtttgat aactgctccc acactgtcag tgacaaaggc 960 tgggccctgg ggatccgctc agggaaggac aagggaaagc gggatgctcg cttcttcttc 1020 tccctctgca ccgaccgcgt gaagaaagcc accatcttga ttagccacag tcgctaccaa 1080 ccaggcacat ggacccatgt ggcagccact tacgatggac ggcacatggc cctgtatgtg 1140 gatggcactc aggtggctag cagtctagac cagtctggtc ccctgaacag ccccttcatg 1200 gcatcttgcc gctctttgct cctgggggga gacagctctg aggatgggca ctatttccgt 1260 ggacacctgg gcacactggt tttctggtcg accgccctgc cacaaagcca ttttcagcac 1320 agttctcagc attcaagtga ggaggaggaa gcgactgact tggtcctgac agcgagcttt 1380 gagcctgtga acacagagtg ggttcccttt agagatgaga agtacccacg acttgaggtt 1440 ctccagggct ttgagccaga gcctgagatt ctgtcgcctt tgcagccccc actctgtggg 1500 caaacagtct gtgacaatgt ggaattgatc tcccagtaca atggatactg gccccttcgg 1560 ggagagaagg tgatacgcta ccaggtggtg aacatctgtg atgatgaggg cctaaacccc 1620 attgtgagtg aggagcagat tcgtctgcag cacgaggcac tgaatgaggc cttcagccgc 1680 tacaacatca gctggcagct gagcgtccac caggtccaca attccaccct gcgacaccgg 1740 gttgtgcttg tgaactgtga gcccagcaag attggcaatg accattgtga ccccgagtgt 1800 gagcacccac tcacaggcta tgatgggggt gactgccgcc tgcagggccg ctgctactcc 1860 tggaaccgca gggatgggct ctgtcacgtg gagtgtaaca acatgctgaa cgactttgac 1920 gacggagact gctgcgaccc ccaggtggct gatgtgcgca agacctgctt tgaccctgac 1980 tcacccaaga gggcatacat gagtgtgaag gagctgaagg aggccctgca gctgaacagt 2040 actcacttcc tcaacatcta ctttgccagc tcagtgcggg aagaccttgc aggtgctgcc 2100 acctggcctt gggacaagga cgctgtcact cacctgggtg gcattgtcct cagcccagca 2160 tattatggga tgcctggcca caccgacacc atgatccatg aagtgggaca tgttctggga 2220 ctctaccatg tctttaaagg agtcagtgaa agagaatcct gcaatgaccc ctgcaaggag 2280 acagtgccat ccatggaaac gggagacctc tgtgccgaca ccgcccccac tcccaagagt 2340 gagctgtgcc gggaaccaga gcccactagt gacacctgtg gcttcactcg cttcccaggg 2400 gctccgttca ccaactacat gagctacacg gatgataact gcactgacaa cttcactcct 2460 aaccaagtgg cccgaatgca ttgctatttg gacctagtct atcagcagtg gactgaaagc 2520 agaaagccca cccccatccc cattccacct atggtcatcg gacagaccaa caagtccctc 2580 actatccact ggctgcctcc tattagtgga gttgtatatg acagggcctc aggcagcttg 2640 tgtggcgctt gcactgaaga tgggaccttt cgtcagtatg tgcacacagc ttcctcccgg 2700 cgggtgtgtg actcctcagg ttattggacc ccagaggagg ctgtggggcc tcctgatgtg 2760 gatcagccct gcgagccaag cttacaggcc tggagccctg aggtccacct gtaccacatg 2820 aacatgacgg tcccctgccc cacagaaggc tgtagcttgg agctgctctt ccaacacccg 2880 gtccaagccg acaccctcac cctgtgggtc acttccttct tcatggagtc ctcgcaggtc 2940 ctctttgaca cagagatctt gctggaaaac aaggagtcag tgcacctggg ccccttagac 3000 actttctgtg acatcccact caccatcaaa ctgcacgtgg atgggaaggt gtcgggggtg 3060 aaagtctaca cctttgatga gaggatagag attgatgcag cactcctgac ttctcagccc 3120 cacagtccct tgtgctctgg ctgcaggcct gtgaggtacc aggttctccg cgatccccca 3180 tttgccagtg gtttgcccgt ggtggtgaca cattctcaca ggaagttcac ggacgtggag 3240 gtcacacctg gacagatgta tcagtaccaa gttctagctg aagctggagg agaactggga 3300 gaagcttcgc ctcctctgaa ccacattcat ggagctcctt attgtggaga tgggaaggtg 3360 tcagagagac tgggagaaga gtgtgatgat ggagaccttg tgagcggaga tggctgctcc 3420 aaggtgtgtg agctggagga aggtttcaac tgtgtaggag agccaagcct ttgctacatg 3480 tatgagggag atggcatatg tgaacctttt gagagaaaaa ccagcattgt agactgtggc 3540 atctacactc ccaaaggata cttggatcaa tgggctaccc gggcttactc ctctcatgaa 3600 gacaagaaga agtgtcctgt ttccttggta actggagaac ctcattccct aatttgcaca 3660 tcataccatc cagatttacc caaccaccgt cccctaactg gctggtttcc ctgtgttgcc 3720 agtgaaaatg aaactcagga tgacaggagt gaacagccag aaggtagcct gaagaaagag 3780 gatgaggttt ggctcaaagt gtgtttcaat agaccaggag aggccagagc aatttttatt 3840 tttttgacaa ctgatggcct agttcccgga gagcatcagc agccgacagt gactctctac 3900 ctgaccgatg tccgtggaag caaccactct cttggaacct atggactgtc atgccagcat 3960 aatccactga ttatcaatgt gacccatcac cagaatgtcc ttttccacca taccacctca 4020 gtgctgccga atttctcatc cccacgggtc ggcatctcag ctgtggctct aaggacatcc 4080 tcccgcattg gtctttcggc tcccagtaac tgcatctcag aggacgaggg gcagaatcat 4140 cagggacaga gctgtatcca tcggccctgt gggaagcagg acagctgtcc gtcattgctg 4200 cttgatcatg ctgatgtggt gaactgtacc tctataggcc caggtctcat gaagtgtgct 4260 atcacttgtc aaaggggatt tgcccttcag gccagcagtg ggcagtacat caggcccatg 4320 cagaaggaaa ttctgctcac atgttcttct gggcactggg accagaatgt gagctgcctt 4380 cccgtggact gcggtgttcc cgacccgtct ttggtgaact atgcaaactt ctcctgctca 4440 gagggaacca aatttctgaa acgctgctca atctcttgtg tcccaccagc caagctgcaa 4500 ggactgagcc catggctgac atgtcttgaa gatggtctct ggtctctccc tgaagtctac 4560 tgcaagttgg agtgtgatgc tccccctatt attctgaatg ccaacttgct cctgcctcac 4620 tgcctccagg acaaccacga cgtgggcacc atctgcaaat atgaatgcaa accagggtac 4680 tatgtggcag aaagtgcaga gggtaaagtc aggaacaagc tcctgaagat acaatgcctg 4740 gaaggtggaa tctgggagca aggcagctgc attcctgtgg tgtgtgagcc accccctcct 4800 gtgtttgaag gcatgtatga atgtaccaat ggcttcagcc tggacagcca gtgtgtgctc 4860 aactgtaacc aggaacgtga aaagcttccc atcctctgca ctaaagaggg cctgtggacc 4920 caggagttta agttgtgtga gaatctgcaa ggagaatgcc caccaccccc ctcagagctg 4980 aattctgtgg agtacaaatg tgarcaagga tatgggattg gtgcagtgtg ttccccattg 5040 tgtgtaatcc cccccagtga ccccgtgatg ctacctgaga atatcactgc tgacactctg 5100 gagcactgga tggaacctgt caaagtccag agcattgtgt gcactggccg gcgtcaatgg 5160 cacccagacc ccgtcttagt ccactgcatc cagtcatgtg aggtcataag ccagttgttg 5220 ctgcttgtgt tcccattgtc ccagcaagaa cacacgtatg ctacatatct gcaatccaaa 5280 attgttgccc ttccaagcag atggttggtg tga 5313 10 1770 PRT Homo sapiens 10 Met Met Cys Leu Lys Ile Leu Arg Ile Ser Leu Ala Ile Leu Ala Gly 1 5 10 15 Trp Ala Leu Cys Ser Ala Asn Ser Glu Leu Gly Trp Thr Arg Lys Lys 20 25 30 Ser Leu Val Glu Arg Glu His Leu Asn Gln Val Leu Leu Glu Gly Glu 35 40 45 Arg Cys Trp Leu Gly Ala Lys Val Arg Arg Pro Arg Ala Ser Pro Gln 50 55 60 His His Leu Phe Gly Val Tyr Pro Ser Arg Ala Gly Asn Tyr Leu Arg 65 70 75 80 Pro Tyr Pro Val Gly Glu Gln Glu Ile His His Thr Gly Arg Ser Lys 85 90 95 Pro Asp Thr Glu Gly Asn Ala Val Ser Leu Val Pro Pro Asp Leu Thr 100 105 110 Glu Asn Pro Ala Gly Leu Arg Gly Ala Val Glu Glu Pro Ala Ala Pro 115 120 125 Trp Val Gly Asp Ser Pro Ile Gly Gln Ser Glu Leu Leu Gly Asp Asp 130 135 140 Asp Ala Tyr Leu Gly Asn Gln Arg Ser Lys Glu Ser Leu Gly Glu Ala 145 150 155 160 Gly Ile Gln Lys Gly Ser Ala Met Ala Ala Thr Thr Thr Thr Ala Ile 165 170 175 Phe Thr Thr Leu Asn Glu Pro Lys Pro Glu Thr Gln Arg Arg Gly Trp 180 185 190 Ala Lys Ser Arg Gln Arg Arg Gln Val Trp Lys Arg Arg Ala Glu Asp 195 200 205 Gly Gln Gly Asp Ser Gly Ile Ser Ser His Phe Gln Pro Trp Pro Lys 210 215 220 His Ser Leu Lys His Arg Val Lys Lys Ser Pro Pro Glu Glu Ser Asn 225 230 235 240 Gln Asn Gly Gly Glu Gly Ser Tyr Arg Glu Ala Glu Thr Phe Asn Ser 245 250 255 Gln Val Gly Leu Pro Ile Leu Tyr Phe Ser Gly Arg Arg Glu Arg Leu 260 265 270 Leu Leu Arg Pro Glu Val Leu Ala Glu Ile Pro Arg Glu Ala Phe Thr 275 280 285 Val Glu Ala Trp Val Lys Pro Glu Gly Gly Gln Asn Asn Pro Ala Ile 290 295 300 Ile Ala Gly Val Phe Asp Asn Cys Ser His Thr Val Ser Asp Lys Gly 305 310 315 320 Trp Ala Leu Gly Ile Arg Ser Gly Lys Asp Lys Gly Lys Arg Asp Ala 325 330 335 Arg Phe Phe Phe Ser Leu Cys Thr Asp Arg Val Lys Lys Ala Thr Ile 340 345 350 Leu Ile Ser His Ser Arg Tyr Gln Pro Gly Thr Trp Thr His Val Ala 355 360 365 Ala Thr Tyr Asp Gly Arg His Met Ala Leu Tyr Val Asp Gly Thr Gln 370 375 380 Val Ala Ser Ser Leu Asp Gln Ser Gly Pro Leu Asn Ser Pro Phe Met 385 390 395 400 Ala Ser Cys Arg Ser Leu Leu Leu Gly Gly Asp Ser Ser Glu Asp Gly 405 410 415 His Tyr Phe Arg Gly His Leu Gly Thr Leu Val Phe Trp Ser Thr Ala 420 425 430 Leu Pro Gln Ser His Phe Gln His Ser Ser Gln His Ser Ser Glu Glu 435 440 445 Glu Glu Ala Thr Asp Leu Val Leu Thr Ala Ser Phe Glu Pro Val Asn 450 455 460 Thr Glu Trp Val Pro Phe Arg Asp Glu Lys Tyr Pro Arg Leu Glu Val 465 470 475 480 Leu Gln Gly Phe Glu Pro Glu Pro Glu Ile Leu Ser Pro Leu Gln Pro 485 490 495 Pro Leu Cys Gly Gln Thr Val Cys Asp Asn Val Glu Leu Ile Ser Gln 500 505 510 Tyr Asn Gly Tyr Trp Pro Leu Arg Gly Glu Lys Val Ile Arg Tyr Gln 515 520 525 Val Val Asn Ile Cys Asp Asp Glu Gly Leu Asn Pro Ile Val Ser Glu 530 535 540 Glu Gln Ile Arg Leu Gln His Glu Ala Leu Asn Glu Ala Phe Ser Arg 545 550 555 560 Tyr Asn Ile Ser Trp Gln Leu Ser Val His Gln Val His Asn Ser Thr 565 570 575 Leu Arg His Arg Val Val Leu Val Asn Cys Glu Pro Ser Lys Ile Gly 580 585 590 Asn Asp His Cys Asp Pro Glu Cys Glu His Pro Leu Thr Gly Tyr Asp 595 600 605 Gly Gly Asp Cys Arg Leu Gln Gly Arg Cys Tyr Ser Trp Asn Arg Arg 610 615 620 Asp Gly Leu Cys His Val Glu Cys Asn Asn Met Leu Asn Asp Phe Asp 625 630 635 640 Asp Gly Asp Cys Cys Asp Pro Gln Val Ala Asp Val Arg Lys Thr Cys 645 650 655 Phe Asp Pro Asp Ser Pro Lys Arg Ala Tyr Met Ser Val Lys Glu Leu 660 665 670 Lys Glu Ala Leu Gln Leu Asn Ser Thr His Phe Leu Asn Ile Tyr Phe 675 680 685 Ala Ser Ser Val Arg Glu Asp Leu Ala Gly Ala Ala Thr Trp Pro Trp 690 695 700 Asp Lys Asp Ala Val Thr His Leu Gly Gly Ile Val Leu Ser Pro Ala 705 710 715 720 Tyr Tyr Gly Met Pro Gly His Thr Asp Thr Met Ile His Glu Val Gly 725 730 735 His Val Leu Gly Leu Tyr His Val Phe Lys Gly Val Ser Glu Arg Glu 740 745 750 Ser Cys Asn Asp Pro Cys Lys Glu Thr Val Pro Ser Met Glu Thr Gly 755 760 765 Asp Leu Cys Ala Asp Thr Ala Pro Thr Pro Lys Ser Glu Leu Cys Arg 770 775 780 Glu Pro Glu Pro Thr Ser Asp Thr Cys Gly Phe Thr Arg Phe Pro Gly 785 790 795 800 Ala Pro Phe Thr Asn Tyr Met Ser Tyr Thr Asp Asp Asn Cys Thr Asp 805 810 815 Asn Phe Thr Pro Asn Gln Val Ala Arg Met His Cys Tyr Leu Asp Leu 820 825 830 Val Tyr Gln Gln Trp Thr Glu Ser Arg Lys Pro Thr Pro Ile Pro Ile 835 840 845 Pro Pro Met Val Ile Gly Gln Thr Asn Lys Ser Leu Thr Ile His Trp 850 855 860 Leu Pro Pro Ile Ser Gly Val Val Tyr Asp Arg Ala Ser Gly Ser Leu 865 870 875 880 Cys Gly Ala Cys Thr Glu Asp Gly Thr Phe Arg Gln Tyr Val His Thr 885 890 895 Ala Ser Ser Arg Arg Val Cys Asp Ser Ser Gly Tyr Trp Thr Pro Glu 900 905 910 Glu Ala Val Gly Pro Pro Asp Val Asp Gln Pro Cys Glu Pro Ser Leu 915 920 925 Gln Ala Trp Ser Pro Glu Val His Leu Tyr His Met Asn Met Thr Val 930 935 940 Pro Cys Pro Thr Glu Gly Cys Ser Leu Glu Leu Leu Phe Gln His Pro 945 950 955 960 Val Gln Ala Asp Thr Leu Thr Leu Trp Val Thr Ser Phe Phe Met Glu 965 970 975 Ser Ser Gln Val Leu Phe Asp Thr Glu Ile Leu Leu Glu Asn Lys Glu 980 985 990 Ser Val His Leu Gly Pro Leu Asp Thr Phe Cys Asp Ile Pro Leu Thr 995 1000 1005 Ile Lys Leu His Val Asp Gly Lys Val Ser Gly Val Lys Val Tyr Thr 1010 1015 1020 Phe Asp Glu Arg Ile Glu Ile Asp Ala Ala Leu Leu Thr Ser Gln Pro 1025 1030 1035 1040 His Ser Pro Leu Cys Ser Gly Cys Arg Pro Val Arg Tyr Gln Val Leu 1045 1050 1055 Arg Asp Pro Pro Phe Ala Ser Gly Leu Pro Val Val Val Thr His Ser 1060 1065 1070 His Arg Lys Phe Thr Asp Val Glu Val Thr Pro Gly Gln Met Tyr Gln 1075 1080 1085 Tyr Gln Val Leu Ala Glu Ala Gly Gly Glu Leu Gly Glu Ala Ser Pro 1090 1095 1100 Pro Leu Asn His Ile His Gly Ala Pro Tyr Cys Gly Asp Gly Lys Val 1105 1110 1115 1120 Ser Glu Arg Leu Gly Glu Glu Cys Asp Asp Gly Asp Leu Val Ser Gly 1125 1130 1135 Asp Gly Cys Ser Lys Val Cys Glu Leu Glu Glu Gly Phe Asn Cys Val 1140 1145 1150 Gly Glu Pro Ser Leu Cys Tyr Met Tyr Glu Gly Asp Gly Ile Cys Glu 1155 1160 1165 Pro Phe Glu Arg Lys Thr Ser Ile Val Asp Cys Gly Ile Tyr Thr Pro 1170 1175 1180 Lys Gly Tyr Leu Asp Gln Trp Ala Thr Arg Ala Tyr Ser Ser His Glu 1185 1190 1195 1200 Asp Lys Lys Lys Cys Pro Val Ser Leu Val Thr Gly Glu Pro His Ser 1205 1210 1215 Leu Ile Cys Thr Ser Tyr His Pro Asp Leu Pro Asn His Arg Pro Leu 1220 1225 1230 Thr Gly Trp Phe Pro Cys Val Ala Ser Glu Asn Glu Thr Gln Asp Asp 1235 1240 1245 Arg Ser Glu Gln Pro Glu Gly Ser Leu Lys Lys Glu Asp Glu Val Trp 1250 1255 1260 Leu Lys Val Cys Phe Asn Arg Pro Gly Glu Ala Arg Ala Ile Phe Ile 1265 1270 1275 1280 Phe Leu Thr Thr Asp Gly Leu Val Pro Gly Glu His Gln Gln Pro Thr 1285 1290 1295 Val Thr Leu Tyr Leu Thr Asp Val Arg Gly Ser Asn His Ser Leu Gly 1300 1305 1310 Thr Tyr Gly Leu Ser Cys Gln His Asn Pro Leu Ile Ile Asn Val Thr 1315 1320 1325 His His Gln Asn Val Leu Phe His His Thr Thr Ser Val Leu Pro Asn 1330 1335 1340 Phe Ser Ser Pro Arg Val Gly Ile Ser Ala Val Ala Leu Arg Thr Ser 1345 1350 1355 1360 Ser Arg Ile Gly Leu Ser Ala Pro Ser Asn Cys Ile Ser Glu Asp Glu 1365 1370 1375 Gly Gln Asn His Gln Gly Gln Ser Cys Ile His Arg Pro Cys Gly Lys 1380 1385 1390 Gln Asp Ser Cys Pro Ser Leu Leu Leu Asp His Ala Asp Val Val Asn 1395 1400 1405 Cys Thr Ser Ile Gly Pro Gly Leu Met Lys Cys Ala Ile Thr Cys Gln 1410 1415 1420 Arg Gly Phe Ala Leu Gln Ala Ser Ser Gly Gln Tyr Ile Arg Pro Met 1425 1430 1435 1440 Gln Lys Glu Ile Leu Leu Thr Cys Ser Ser Gly His Trp Asp Gln Asn 1445 1450 1455 Val Ser Cys Leu Pro Val Asp Cys Gly Val Pro Asp Pro Ser Leu Val 1460 1465 1470 Asn Tyr Ala Asn Phe Ser Cys Ser Glu Gly Thr Lys Phe Leu Lys Arg 1475 1480 1485 Cys Ser Ile Ser Cys Val Pro Pro Ala Lys Leu Gln Gly Leu Ser Pro 1490 1495 1500 Trp Leu Thr Cys Leu Glu Asp Gly Leu Trp Ser Leu Pro Glu Val Tyr 1505 1510 1515 1520 Cys Lys Leu Glu Cys Asp Ala Pro Pro Ile Ile Leu Asn Ala Asn Leu 1525 1530 1535 Leu Leu Pro His Cys Leu Gln Asp Asn His Asp Val Gly Thr Ile Cys 1540 1545 1550 Lys Tyr Glu Cys Lys Pro Gly Tyr Tyr Val Ala Glu Ser Ala Glu Gly 1555 1560 1565 Lys Val Arg Asn Lys Leu Leu Lys Ile Gln Cys Leu Glu Gly Gly Ile 1570 1575 1580 Trp Glu Gln Gly Ser Cys Ile Pro Val Val Cys Glu Pro Pro Pro Pro 1585 1590 1595 1600 Val Phe Glu Gly Met Tyr Glu Cys Thr Asn Gly Phe Ser Leu Asp Ser 1605 1610 1615 Gln Cys Val Leu Asn Cys Asn Gln Glu Arg Glu Lys Leu Pro Ile Leu 1620 1625 1630 Cys Thr Lys Glu Gly Leu Trp Thr Gln Glu Phe Lys Leu Cys Glu Asn 1635 1640 1645 Leu Gln Gly Glu Cys Pro Pro Pro Pro Ser Glu Leu Asn Ser Val Glu 1650 1655 1660 Tyr Lys Cys Glu Gln Gly Tyr Gly Ile Gly Ala Val Cys Ser Pro Leu 1665 1670 1675 1680 Cys Val Ile Pro Pro Ser Asp Pro Val Met Leu Pro Glu Asn Ile Thr 1685 1690 1695 Ala Asp Thr Leu Glu His Trp Met Glu Pro Val Lys Val Gln Ser Ile 1700 1705 1710 Val Cys Thr Gly Arg Arg Gln Trp His Pro Asp Pro Val Leu Val His 1715 1720 1725 Cys Ile Gln Ser Cys Glu Val Ile Ser Gln Leu Leu Leu Leu Val Phe 1730 1735 1740 Pro Leu Ser Gln Gln Glu His Thr Tyr Ala Thr Tyr Leu Gln Ser Lys 1745 1750 1755 1760 Ile Val Ala Leu Pro Ser Arg Trp Leu Val 1765 1770 11 85 DNA Homo sapiens 11 gtcataagcc agttgttgct gcttgtgttc ccattgtccc agcaagaaca cacgtatgct 60 acatatctgc aatccaaaat tgttg 85 12 28 PRT Homo sapiens 12 Val Ile Ser Gln Leu Leu Leu Leu Val Phe Pro Leu Ser Gln Gln Glu 1 5 10 15 His Thr Tyr Ala Thr Tyr Leu Gln Ser Lys Ile Val 20 25 13 8 PRT Homo sapiens 13 Ala Leu Pro Ser Arg Trp Leu Val 1 5 14 36 PRT Homo sapiens 14 Val Ile Ser Gln Leu Leu Leu Leu Val Phe Pro Leu Ser Gln Gln Glu 1 5 10 15 His Thr Tyr Ala Thr Tyr Leu Gln Ser Lys Ile Val Ala Leu Pro Ser 20 25 30 Arg Trp Leu Val 35 15 4158 DNA Homo sapiens 15 atgatgtgct taaagatcct aagaataagc ctggcgattt tggctgggtg ggcactctgt 60 tctgccaact ctgagctggg ctggacacgc aagaaatcct tggttgagag ggaacacctg 120 aatcaggtgc tgttggaagg agaacgttgt tggctggggg ccaaggttcg aagacccaga 180 gcttctccac agcatcacct ctttggagtc taccccagca gggctgggaa ctacctaagg 240 ccctaccccg tgggggagca agaaatccat catacaggac gcagcaaacc agacactgaa 300 ggaaatgctg tgagccttgt tcccccagac ctgactgaaa atccagcagg actgaggggt 360 gcagttgaag agccggctgc cccatgggta ggggatagtc ctattgggca atctgagctg 420 ctgggagatg atgacgctta tctcggcaat caaagatcca aggagtctct aggtgaggcc 480 gggattcaga aaggctcagc catggctgcc actactacca ccgccatttt cacaaccctg 540 aacgaaccca aaccagagac ccaaaggagg ggctgggcca agtccaggca gcgtcgccaa 600 gtgtggaaga ggcgggcgga agatgggcag ggagactccg gtatctcttc acatttccaa 660 ccttggccca agcattccct taaacacggg gtcaaaaaga gtccaccgga ggaaagcaac 720 caaaatggtg gagagggctc ctaccgagaa gcagagacct ttaactccca agtaggactg 780 cccatcttat acttctctgg gaggcgggag cggctgctgc tgcgtccaga agtgctggct 840 gagattcccc gggaggcgtt cacagtggaa gcctgggtta aaccggaggg aggacagaac 900 aacccagcca tcatcgcagg tggcattgtc ctcagcccag catattatgg gatgcctggc 960 cacaccgaca ccatgatcca tgaagtggga catgttctgg gactctacca tgtctttaaa 1020 ggagtcagtg aaagagaatc ctgcaatgac ccctgcaagg agacagtgcc atccatggaa 1080 acgggagacc tctgtgccga caccgccccc actcccaaga gtgagctgtg ccgggaacca 1140 gagcccacta gtgacacctg tggcttcact cgcttcccag gggctccgtt caccaactac 1200 atgagctaca cggatgataa ctgcactgac aacttcactc ctaaccaagt ggcccgaatg 1260 cattgctatt tggacctagt ctatcagcag tggactgaaa gcagaaagcc cacccccatc 1320 cccattccac ctatggtcat cggacagacc aacaagtccc tcactatcca ctggctgcct 1380 cctattagtg gagttgtata tgacagggcc tcaggcagct tgtgtggcgc ttgcactgaa 1440 gatgggacct ttcgtcagta tgtgcacaca gcttcctccc ggcgggtgtg tgactcctca 1500 ggttattgga ccccagagga ggctgtgggg cctcctgatg tggatcagcc ctgcgagcca 1560 agcttacagg cctggagccc tgaggtccac ctgtaccaca tgaacatgac ggtcccctgc 1620 cccacagaag gctgtagctt ggagctgctc ttccaacacc cggtccaagc cgacaccctc 1680 accctgtggg tcacttcctt cttcatggag tcctcgcagg tcctctttga cacagagatc 1740 ttgctggaaa acaaggagtc agtgcacctg ggccccttag acactttctg tgacatccca 1800 ctcaccatca aactgcacgt ggatgggaag gtgtcggggg tgaaagtcta cacctttgat 1860 gagaggatag agattgatgc agcactcctg acttctcagc cccacagtcc cttgtgctct 1920 ggctgcaggc ctgtgaggta ccaggttctc cgcgatcccc catttgccag tggtttgccc 1980 gtggtggtga cacattctca caggaagttc acggacgtgg aggtcacacc tggacagatg 2040 tatcagtacc aagttctagc tgaagctgga ggagaactgg gagaagcttc gcctcctctg 2100 aaccacattc atggagctcc ttattgtgga gatgggaagg tgtcagagag actgggagaa 2160 gagtgtgatg atggagacct tgtgagcgga gatggctgct ccaaggtgtg tgagctggag 2220 gaaggtttca actgtgtagg agagccaagc ctttgctaca tgtatgaggg agatggcata 2280 tgtgaacctt ttgagagaaa aaccagcatt gtagactgtg gcatctacac tcccaaagga 2340 tacttggatc aatgggctac ccgggcttac tcctctcatg aagacaagaa gaagtgtcct 2400 gtttccttgg taactggaga acctcattcc ctaattcgca catcatacca tccagattta 2460 cccaaccacc gtcccctaac tggctggttt ccctgtgttg ccagtgaaaa tgaaactcag 2520 gatgacagga gtgaacagcc agaaggtagc ctgaagaaag aggatgaggt ttggctcaaa 2580 gtgtgtttca atagaccagg agaggccaga gcaattttta tttttttgac aactgatggc 2640 ctagttcccg gagagcatca gcagccgaca gtgactctct acctgaccga tgtccgtgga 2700 agcaaccact ctcttggaac ctatggactg tcatgccagc acaatccact gattatcaat 2760 gtgacccatc accagaatgt ccttttccgc cataccacct cagtgctgct gaatttctca 2820 tccccacggg tcggcatctc agctgtggct ctaaggacat cctcccgcat tggtctctcg 2880 gctcccagta actgcatctc agaggacgag gggcagaatc atcagggaca gagctgtatc 2940 catcggccct gtgggaagca ggacagctgt ccgtcattgc tgcttgatca tgctgatgtg 3000 gtgaactgta cctctatagg cccaggtctc atgaagtgtg ctaccacttg tcaaagggga 3060 tttgcccttc aggccagcag tgagcagtac atcaggctca tgcagaagga gattctgctc 3120 acatgttctt ctgggcactg ggaccagaat gtgagctgcc ttcccgtgga ctgcggtgtt 3180 cccgacccgt ctttggtgaa ctatgcaaac ttctcctgct cagagggaac caaatttctg 3240 aaacgctgct caatctcttg tgtcccacca gccaagctgc aaggactgag cccatggctg 3300 acatgtcttg aagatggtct ctggtctctc cctgaagtct actgcaagtt ggagtgtgat 3360 gctcccccta ttattctgaa tgccaacttg ctcctgcctc actgcctcca ggacaaccac 3420 gacgtgggca ccatctgcaa atatgaatgc aaaccagggt actatgtggc agaaagtgca 3480 gagggtaaag tcaggaacaa gctcctgaag atacaatgcc tggaaggtgg aatctgggag 3540 caaggcagct gcattcctgt ggtgtgtgag ccaccccctc ctgtgtttga aggcatgtat 3600 gaatgtacca atggcttcag cctggacagc cagtgtgtgc tcaactgtaa ccaggaacgt 3660 gaaaagcttc ccatcctctg cactaaagag ggcctgtgga cccaggagtt taagttgtgt 3720 gagaatctgc aaggagaatg cccgccaccc ccctcagagc tgaattctgt ggagtacaaa 3780 tgtgaacaag gatatgggat tggtgcagtg tgttccccat tgtgtgtaat cccccccagt 3840 gaccccgtga tgctacctga gaatatcact gctgacactc tggagcactg gatggaacct 3900 gtcaaagtcc agagcattgt gtgcactggc cggcgtcaat ggcacccaga ccccgtctta 3960 gtccactgca tccagtcatg tgagcccttc caagcagatg gttggtgtga cactatcaac 4020 aaccgagcct actgccacta tgacggggga gactgctgct cttccacact ctcctccaag 4080 aaggtcattc catttgctgc tgactgtgac ctggatgagt gcacctgccg ggaccccaag 4140 gcagaagaaa atcagtaa 4158 16 1385 PRT Homo sapiens 16 Met Met Cys Leu Lys Ile Leu Arg Ile Ser Leu Ala Ile Leu Ala Gly 1 5 10 15 Trp Ala Leu Cys Ser Ala Asn Ser Glu Leu Gly Trp Thr Arg Lys Lys 20 25 30 Ser Leu Val Glu Arg Glu His Leu Asn Gln Val Leu Leu Glu Gly Glu 35 40 45 Arg Cys Trp Leu Gly Ala Lys Val Arg Arg Pro Arg Ala Ser Pro Gln 50 55 60 His His Leu Phe Gly Val Tyr Pro Ser Arg Ala Gly Asn Tyr Leu Arg 65 70 75 80 Pro Tyr Pro Val Gly Glu Gln Glu Ile His His Thr Gly Arg Ser Lys 85 90 95 Pro Asp Thr Glu Gly Asn Ala Val Ser Leu Val Pro Pro Asp Leu Thr 100 105 110 Glu Asn Pro Ala Gly Leu Arg Gly Ala Val Glu Glu Pro Ala Ala Pro 115 120 125 Trp Val Gly Asp Ser Pro Ile Gly Gln Ser Glu Leu Leu Gly Asp Asp 130 135 140 Asp Ala Tyr Leu Gly Asn Gln Arg Ser Lys Glu Ser Leu Gly Glu Ala 145 150 155 160 Gly Ile Gln Lys Gly Ser Ala Met Ala Ala Thr Thr Thr Thr Ala Ile 165 170 175 Phe Thr Thr Leu Asn Glu Pro Lys Pro Glu Thr Gln Arg Arg Gly Trp 180 185 190 Ala Lys Ser Arg Gln Arg Arg Gln Val Trp Lys Arg Arg Ala Glu Asp 195 200 205 Gly Gln Gly Asp Ser Gly Ile Ser Ser His Phe Gln Pro Trp Pro Lys 210 215 220 His Ser Leu Lys His Gly Val Lys Lys Ser Pro Pro Glu Glu Ser Asn 225 230 235 240 Gln Asn Gly Gly Glu Gly Ser Tyr Arg Glu Ala Glu Thr Phe Asn Ser 245 250 255 Gln Val Gly Leu Pro Ile Leu Tyr Phe Ser Gly Arg Arg Glu Arg Leu 260 265 270 Leu Leu Arg Pro Glu Val Leu Ala Glu Ile Pro Arg Glu Ala Phe Thr 275 280 285 Val Glu Ala Trp Val Lys Pro Glu Gly Gly Gln Asn Asn Pro Ala Ile 290 295 300 Ile Ala Gly Gly Ile Val Leu Ser Pro Ala Tyr Tyr Gly Met Pro Gly 305 310 315 320 His Thr Asp Thr Met Ile His Glu Val Gly His Val Leu Gly Leu Tyr 325 330 335 His Val Phe Lys Gly Val Ser Glu Arg Glu Ser Cys Asn Asp Pro Cys 340 345 350 Lys Glu Thr Val Pro Ser Met Glu Thr Gly Asp Leu Cys Ala Asp Thr 355 360 365 Ala Pro Thr Pro Lys Ser Glu Leu Cys Arg Glu Pro Glu Pro Thr Ser 370 375 380 Asp Thr Cys Gly Phe Thr Arg Phe Pro Gly Ala Pro Phe Thr Asn Tyr 385 390 395 400 Met Ser Tyr Thr Asp Asp Asn Cys Thr Asp Asn Phe Thr Pro Asn Gln 405 410 415 Val Ala Arg Met His Cys Tyr Leu Asp Leu Val Tyr Gln Gln Trp Thr 420 425 430 Glu Ser Arg Lys Pro Thr Pro Ile Pro Ile Pro Pro Met Val Ile Gly 435 440 445 Gln Thr Asn Lys Ser Leu Thr Ile His Trp Leu Pro Pro Ile Ser Gly 450 455 460 Val Val Tyr Asp Arg Ala Ser Gly Ser Leu Cys Gly Ala Cys Thr Glu 465 470 475 480 Asp Gly Thr Phe Arg Gln Tyr Val His Thr Ala Ser Ser Arg Arg Val 485 490 495 Cys Asp Ser Ser Gly Tyr Trp Thr Pro Glu Glu Ala Val Gly Pro Pro 500 505 510 Asp Val Asp Gln Pro Cys Glu Pro Ser Leu Gln Ala Trp Ser Pro Glu 515 520 525 Val His Leu Tyr His Met Asn Met Thr Val Pro Cys Pro Thr Glu Gly 530 535 540 Cys Ser Leu Glu Leu Leu Phe Gln His Pro Val Gln Ala Asp Thr Leu 545 550 555 560 Thr Leu Trp Val Thr Ser Phe Phe Met Glu Ser Ser Gln Val Leu Phe 565 570 575 Asp Thr Glu Ile Leu Leu Glu Asn Lys Glu Ser Val His Leu Gly Pro 580 585 590 Leu Asp Thr Phe Cys Asp Ile Pro Leu Thr Ile Lys Leu His Val Asp 595 600 605 Gly Lys Val Ser Gly Val Lys Val Tyr Thr Phe Asp Glu Arg Ile Glu 610 615 620 Ile Asp Ala Ala Leu Leu Thr Ser Gln Pro His Ser Pro Leu Cys Ser 625 630 635 640 Gly Cys Arg Pro Val Arg Tyr Gln Val Leu Arg Asp Pro Pro Phe Ala 645 650 655 Ser Gly Leu Pro Val Val Val Thr His Ser His Arg Lys Phe Thr Asp 660 665 670 Val Glu Val Thr Pro Gly Gln Met Tyr Gln Tyr Gln Val Leu Ala Glu 675 680 685 Ala Gly Gly Glu Leu Gly Glu Ala Ser Pro Pro Leu Asn His Ile His 690 695 700 Gly Ala Pro Tyr Cys Gly Asp Gly Lys Val Ser Glu Arg Leu Gly Glu 705 710 715 720 Glu Cys Asp Asp Gly Asp Leu Val Ser Gly Asp Gly Cys Ser Lys Val 725 730 735 Cys Glu Leu Glu Glu Gly Phe Asn Cys Val Gly Glu Pro Ser Leu Cys 740 745 750 Tyr Met Tyr Glu Gly Asp Gly Ile Cys Glu Pro Phe Glu Arg Lys Thr 755 760 765 Ser Ile Val Asp Cys Gly Ile Tyr Thr Pro Lys Gly Tyr Leu Asp Gln 770 775 780 Trp Ala Thr Arg Ala Tyr Ser Ser His Glu Asp Lys Lys Lys Cys Pro 785 790 795 800 Val Ser Leu Val Thr Gly Glu Pro His Ser Leu Ile Arg Thr Ser Tyr 805 810 815 His Pro Asp Leu Pro Asn His Arg Pro Leu Thr Gly Trp Phe Pro Cys 820 825 830 Val Ala Ser Glu Asn Glu Thr Gln Asp Asp Arg Ser Glu Gln Pro Glu 835 840 845 Gly Ser Leu Lys Lys Glu Asp Glu Val Trp Leu Lys Val Cys Phe Asn 850 855 860 Arg Pro Gly Glu Ala Arg Ala Ile Phe Ile Phe Leu Thr Thr Asp Gly 865 870 875 880 Leu Val Pro Gly Glu His Gln Gln Pro Thr Val Thr Leu Tyr Leu Thr 885 890 895 Asp Val Arg Gly Ser Asn His Ser Leu Gly Thr Tyr Gly Leu Ser Cys 900 905 910 Gln His Asn Pro Leu Ile Ile Asn Val Thr His His Gln Asn Val Leu 915 920 925 Phe Arg His Thr Thr Ser Val Leu Leu Asn Phe Ser Ser Pro Arg Val 930 935 940 Gly Ile Ser Ala Val Ala Leu Arg Thr Ser Ser Arg Ile Gly Leu Ser 945 950 955 960 Ala Pro Ser Asn Cys Ile Ser Glu Asp Glu Gly Gln Asn His Gln Gly 965 970 975 Gln Ser Cys Ile His Arg Pro Cys Gly Lys Gln Asp Ser Cys Pro Ser 980 985 990 Leu Leu Leu Asp His Ala Asp Val Val Asn Cys Thr Ser Ile Gly Pro 995 1000 1005 Gly Leu Met Lys Cys Ala Thr Thr Cys Gln Arg Gly Phe Ala Leu Gln 1010 1015 1020 Ala Ser Ser Glu Gln Tyr Ile Arg Leu Met Gln Lys Glu Ile Leu Leu 1025 1030 1035 1040 Thr Cys Ser Ser Gly His Trp Asp Gln Asn Val Ser Cys Leu Pro Val 1045 1050 1055 Asp Cys Gly Val Pro Asp Pro Ser Leu Val Asn Tyr Ala Asn Phe Ser 1060 1065 1070 Cys Ser Glu Gly Thr Lys Phe Leu Lys Arg Cys Ser Ile Ser Cys Val 1075 1080 1085 Pro Pro Ala Lys Leu Gln Gly Leu Ser Pro Trp Leu Thr Cys Leu Glu 1090 1095 1100 Asp Gly Leu Trp Ser Leu Pro Glu Val Tyr Cys Lys Leu Glu Cys Asp 1105 1110 1115 1120 Ala Pro Pro Ile Ile Leu Asn Ala Asn Leu Leu Leu Pro His Cys Leu 1125 1130 1135 Gln Asp Asn His Asp Val Gly Thr Ile Cys Lys Tyr Glu Cys Lys Pro 1140 1145 1150 Gly Tyr Tyr Val Ala Glu Ser Ala Glu Gly Lys Val Arg Asn Lys Leu 1155 1160 1165 Leu Lys Ile Gln Cys Leu Glu Gly Gly Ile Trp Glu Gln Gly Ser Cys 1170 1175 1180 Ile Pro Val Val Cys Glu Pro Pro Pro Pro Val Phe Glu Gly Met Tyr 1185 1190 1195 1200 Glu Cys Thr Asn Gly Phe Ser Leu Asp Ser Gln Cys Val Leu Asn Cys 1205 1210 1215 Asn Gln Glu Arg Glu Lys Leu Pro Ile Leu Cys Thr Lys Glu Gly Leu 1220 1225 1230 Trp Thr Gln Glu Phe Lys Leu Cys Glu Asn Leu Gln Gly Glu Cys Pro 1235 1240 1245 Pro Pro Pro Ser Glu Leu Asn Ser Val Glu Tyr Lys Cys Glu Gln Gly 1250 1255 1260 Tyr Gly Ile Gly Ala Val Cys Ser Pro Leu Cys Val Ile Pro Pro Ser 1265 1270 1275 1280 Asp Pro Val Met Leu Pro Glu Asn Ile Thr Ala Asp Thr Leu Glu His 1285 1290 1295 Trp Met Glu Pro Val Lys Val Gln Ser Ile Val Cys Thr Gly Arg Arg 1300 1305 1310 Gln Trp His Pro Asp Pro Val Leu Val His Cys Ile Gln Ser Cys Glu 1315 1320 1325 Pro Phe Gln Ala Asp Gly Trp Cys Asp Thr Ile Asn Asn Arg Ala Tyr 1330 1335 1340 Cys His Tyr Asp Gly Gly Asp Cys Cys Ser Ser Thr Leu Ser Ser Lys 1345 1350 1355 1360 Lys Val Ile Pro Phe Ala Ala Asp Cys Asp Leu Asp Glu Cys Thr Cys 1365 1370 1375 Arg Asp Pro Lys Ala Glu Glu Asn Gln 1380 1385 17 60 DNA Homo sapiens 17 ggacagaaca acccagccat catcgcaggt ggcattgtcc tcagcccagc atattatggg 60 18 20 PRT Homo sapiens 18 Gly Gln Asn Asn Pro Ala Ile Ile Ala Gly Gly Ile Val Leu Ser Pro 1 5 10 15 Ala Tyr Tyr Gly 20 19 1685 DNA Homo sapiens 19 ccccaagcat caaactgaag gaaacattct aaccttcaca gacagactgg aggctggatg 60 gggacctggc tgaagacatc tggagaatga aagttaagta ccagcttgca tttttgtgcc 120 cctagattat ttttgcattt taaaataaga agcatcaaat tgcgtgtctc tgtgtaaaag 180 ttctagcaat ttgttttaag gtgaacttat tttggcttag ggactacaaa aagagaaggt 240 aattcctagg gaaggaagaa gagaaagaaa tgaaaattag agaataagat tattttgaat 300 gacttcaggt agcgaggagt gtgtgtttgt gagtgtgtat ttgagagact tggctcatgc 360 ctgtgggtct tctcttctag tatcagtgag gggagggatt actgaagaag aaggggggaa 420 aaaaaaagaa agaaatctga gctttctggg aggaaattca aaggaaccaa gagaaattaa 480 cttcgttctg caaggactaa agtacagcaa gaggagagag gtcaagcgag aagcgtgcgg 540 gaagcacatg ccctggggag gcatagaagc cacactggca gagcggccag cacaggtagc 600 cagcagaggc attcttgggg ctatttgaaa aagtttggtc tgtgaacaaa acagtttccc 660 tggtgactgc aaatccattg ctagctgcct ctttctcgtc tgcccatcac tctggtgtgg 720 tacccagaag ttgacttctg gttctgtaga aagagctagg ggaggtatga tgtgcttaaa 780 gatcctaaga ataagcctgg cgattttggc tgggtgggca ctctgttctg ccaactctga 840 gctgggctgg acacgcaaga aatccttggt tgagagggaa cacctgaatc aggtgctgtt 900 ggaaggagaa cgttgttggc tgggggccaa ggttcgaaga cccagagctt ctccacagca 960 tcacctcttt ggagtctacc ccagcagggc tgggaactac ctaaggccct accccgtggg 1020 ggagcaagaa atccatcata caggacgcag caaaccagac actgaaggaa atgctgtgag 1080 ccttgttccc ccagacctga ctgaaaatcc agcaggactg aggggtgcag ttgaagagcc 1140 ggctgcccca tgggtagggg atagtcctat tgggcaatct gagctgctgg gagatgatga 1200 cgcttatctc ggcaatcaaa gatccaagga gtctctaggt gaggccggga ttcagaaagg 1260 ctcagccatg gctgccacta ctaccaccgc cattttcaca accctgaacg aacccaaacc 1320 agagacccaa aggaggggct gggccaagtc caggcagcgt cgccaagtgt ggaagaggcg 1380 ggcggaagat gggcagggag actccggtat ctcttcacat ttccaacctt ggcccaagca 1440 ttcccttaaa cacagggtca aaaagagtcc accggaggaa agcaaccaaa atggtggaga 1500 gggctcctac cgagaagcag agacctttaa ctcccaagta ggactgccca tcttatactt 1560 ctctgggagg cgggagcggc tgctgctgcg tccagaagtg ctggctgaga ttccccggga 1620 ggcgttcaca gtggaagcct gggttaaacc ggagggagga cagaacaacc cagccatcat 1680 cgcag 1685 20 1072 DNA Homo sapiens 20 gtgtgtttga taactgctcc cacactgtca gtgacaaagg ctgggccctg gggatccgct 60 cagggaagga caagggaaag cgggatgctc gcttcttctt ctccctctgc accgaccgcg 120 tgaagaaagc caccatcttg attagccaca gtcgctacca accaggcaca tggacccatg 180 tggcagccac ttacgatgga cggcacatgg ccctgtatgt ggatggcact caggtggcta 240 gcagtctaga ccagtctggt cccctgaaca gccccttcat ggcatcttgc cgctctttgc 300 tcctgggggg agacagctct gaggatgggc actatttccg tggacacctg ggcacactgg 360 ttttctggtc gaccgccctg ccacaaagcc attttcagca cagttctcag cattcaagtg 420 aggaggagga agcgactgac ttggtcctga cagcgagctt tgagcctgtg aacacagagt 480 gggttccctt tagagatgag aagtacccac gacttgaggt tctccagggc tttgagccag 540 agcctgagat tctgtcgcct ttgcagcccc cactctgtgg gcaaacagtc tgtgacaatg 600 tggaattgat ctcccagtac aatggatact ggccccttcg gggagagaag gtgatacgct 660 accaggtggt gaacatctgt gatgatgagg gcctaaaccc cattgtgagt gaggagcaga 720 ttcgtctgca gcacgaggca ctgaatgagg ccttcagccg ctacaacatc agctggcagc 780 tgagcgtcca ccaggtccac aattccaccc tgcgacaccg ggttgtgctt gtgaactgtg 840 agcccagcaa gattggcaat gaccattgtg accccgagtg tgagcaccca ctcacaggct 900 atgatggggg tgactgccgc ctgcagggcc gctgctactc ctggaaccgc agggatgggc 960 tctgtcacgt ggagtgtaac aacatgctga acgactttga cgacggagac tgctgcgacc 1020 cccaggtggc tgatgtgcgc aagacctgct ttgaccctga ctcacccaag ag 1072 21 146 DNA Homo sapiens 21 ggcatacatg agtgtgaagg agctgaagga ggccctgcag ctgaacagta ctcacttcct 60 caacatctac tttgccagct cagtgcggga agaccttgca ggtgctgcca cctggccttg 120 ggacaaggac gctgtcactc acctgg 146 22 294 DNA Homo sapiens 22 gtggcattgt cctcagccca gcatattatg ggatgcctgg ccacaccgac accatgatcc 60 atgaagtggg acatgttctg ggactctacc atgtctttaa aggagtcagt gaaagagaat 120 cctgcaatga cccctgcaag gagacagtgc catccatgga aacgggagac ctctgtgccg 180 acaccgcccc cactcccaag agtgagctgt gccgggaacc agagcccact agtgacacct 240 gtggcttcac tcgcttccca ggggctccgt tcaccaacta catgagctac acgg 294 23 193 DNA Homo sapiens 23 atgataactg cactgacaac ttcactccta accaagtggc ccgaatgcat tgctatttgg 60 acctagtcta tcagcagtgg actgaaagca gaaagcccac ccccatcccc attccaccta 120 tggtcatcgg acagaccaac aagtccctca ctatccactg gctgcctcct attagtggag 180 ttgtatatga cag 193 24 122 DNA Homo sapiens 24 ggcctcaggc agcttgtgtg gcgcttgcac tgaagatggg acctttcgtc agtatgtgca 60 cacagcttcc tcccggcggg tgtgtgactc ctcaggttat tggaccccag aggaggctgt 120 gg 122 25 490 DNA Homo sapiens 25 ggcctcctga tgtggatcag ccctgcgagc caagcttaca ggcctggagc cctgaggtcc 60 acctgtacca catgaacatg acggtcccct gccccacaga aggctgtagc ttggagctgc 120 tcttccaaca cccggtccaa gccgacaccc tcaccctgtg ggtcacttcc ttcttcatgg 180 agtcctcgca ggtcctcttt gacacagaga tcttgctgga aaacaaggag tcagtgcacc 240 tgggcccctt agacactttc tgtgacatcc cactcaccat caaactgcac gtggatggga 300 aggtgtcggg ggtgaaagtc tacacctttg atgagaggat agagattgat gcagcactcc 360 tgacttctca gccccacagt cccttgtgct ctggctgcag gcctgtgagg taccaggttc 420 tccgcgatcc cccatttgcc agtggtttgc ccgtggtggt gacacattct cacaggaagt 480 tcacggacgt 490 26 129 DNA Homo sapiens 26 ggaggtcaca cctggacaga tgtatcagta ccaagttcta gctgaagctg gaggagaact 60 gggagaagct tcgcctcctc tgaaccacat tcatggagct ccttattgtg gagatgggaa 120 ggtgtcaga 129 27 92 DNA Homo sapiens 27 gagactggga gaagagtgtg atgatggaga ccttgtgagc ggagatggct gctccaaggt 60 gtgtgagctg gaggaaggtt tcaactgtgt ag 92 28 194 DNA Homo sapiens 28 gagagccaag cctttgctac atgtatgagg gagatggcat atgtgaacct tttgagagaa 60 aaaccagcat tgtagactgt ggcatctaca ctcccaaagg atacttggat caatgggcta 120 cccgggctta ctcctctcat gaagacaaga agaagtgtcc tgtttccttg gtaactggag 180 aacctcattc ccta 194 29 147 DNA Homo sapiens 29 atttgcacat cataccatcc agatttaccc aaccaccgtc ccctaactgg ctggtttccc 60 tgtgttgcca gtgaaaatga aactcaggat gacaggagtg aacagccaga aggtagcctg 120 aagaaagagg atgaggtttg gctcaaa 147 30 136 DNA Homo sapiens 30 gtgtgtttca atagaccagg agaggccaga gcaattttta tttttttgac aactgatggc 60 ctagttcccg gagagcatca gcagccgaca gtgactctct acctgaccga tgtccgtgga 120 agcaaccact ctcttg 136 31 217 DNA Homo sapiens 31 gaacctatgg actgtcatgc cagcataatc cactgattat caatgtgacc catcaccaga 60 atgtcctttt ccaccatacc acctcagtgc tgccgaattt ctcatcccca cgggtcggca 120 tctcagctgt ggctctaagg acatcctccc gcattggtct ttcggctccc agtaactgca 180 tctcagagga cgaggggcag aatcatcagg gacagag 217 32 172 DNA Homo sapiens 32 ctgtatccat cggccctgtg ggaagcagga cagctgtccg tcattgctgc ttgatcatgc 60 tgatgtggtg aactgtacct ctataggccc aggtctcatg aagtgtgcta tcacttgtca 120 aaggggattt gcccttcagg ccagcagtgg gcagtacatc aggcccatgc ag 172 33 178 DNA Homo sapiens 33 aaggaaattc tgctcacatg ttcttctggg cactgggacc agaatgtgag ctgccttccc 60 gtggactgcg gtgttcccga cccgtctttg gtgaactatg caaacttctc ctgctcagag 120 ggaaccaaat ttctgaaacg ctgctcaatc tcttgtgtcc caccagccaa gctgcaag 178 34 214 DNA Homo sapiens 34 gactgagccc atggctgaca tgtcttgaag atggtctctg gtctctccct gaagtctact 60 gcaagttgga gtgtgatgct ccccctatta ttctgaatgc caacttgctc ctgcctcact 120 gcctccagga caaccacgac gtgggcacca tctgcaaata tgaatgcaaa ccagggtact 180 atgtggcaga aagtgcagag ggtaaagtca ggaa 214 35 169 DNA Homo sapiens 35 caagctcctg aagatacaat gcctggaagg tggaatctgg gagcaaggca gctgcattcc 60 tgtggtgtgt gagccacccc ctcctgtgtt tgaaggcatg tatgaatgta ccaatggctt 120 cagcctggac agccagtgtg tgctcaactg taaccaggaa cgtgaaaag 169 36 136 DNA Homo sapiens 36 cttcccatcc tctgcactaa agagggcctg tggacccagg agtttaagtt gtgtgagaat 60 ctgcaaggag aatgcccacc acccccctca gagctgaatt ctgtggagta caaatgtgaa 120 caaggatatg ggattg 136 37 110 DNA Homo sapiens 37 gtgcagtgtg ttccccattg tgtgtaatcc cccccagtga ccccgtgatg ctacctgaga 60 atatcactgc tgacactctg gagcactgga tggaacctgt caaagtccag 110 38 72 DNA Homo sapiens 38 agcattgtgt gcactggccg gcgtcaatgg cacccagacc ccgtcttagt ccactgcatc 60 cagtcatgtg ag 72 39 85 DNA Homo sapiens 39 gtcataagcc agttgttgct gcttgtgttc ccattgtccc agcaagaaca cacgtatgct 60 acatatctgc aatccaaaat tgttg 85 40 99 DNA Homo sapiens 40 cccttccaag caaatggttg gtgtgacact atcaacaacc gagcctactg ccactatgac 60 gggggagact gctgctcttc cacactctcc tccaagaag 99 41 640 DNA Homo sapiens 41 gtcattccat ttgctgctga ctgtgacctg gatgagtgca cctgccggga ccccaaggca 60 gaagaaaatc agtaactgtg ggaacaagcc cctccctcca ctgcctcaga ggcagtaaga 120 aagagaggcc gacccaggag gaaacaaagg gtgaatgaag aagaacaatc atgaaatgga 180 agaaggagga agagcatgaa ggatcttata agaaatgcaa gaggatattg ataggtgtga 240 actagttcat caagtagccc aagtaggaga gaatcatagg caaaagtttc tttaaagtgg 300 cagttgatta acatggaagg ggaaatatga tagatatata aggaccctcc tccctcactt 360 atattctatt aaatcctatc ctcaactctt gccctgctct ccgctccacc ccctgccaac 420 tactcagtcc cacccaactt gtaaaccaat accaaaatac tagaggagaa gttggcaggg 480 atactgttaa tacccatttt gaatggattg ccatctttca gagcttgtct gctctcaact 540 ggctcttttt ctttttgtgt agtttccaat gaataatgaa gttagttatt aattctttat 600 aagtatttaa acataattat ataaatatat tatatatatt 640 42 500 DNA Homo sapiens 42 tatttgaaaa agtttggtct gtgaacaaaa cagtttccct ggtgactgca aatccattgc 60 tagctgcctc tttctcgtct gcccatcact ctggtgtggt acccagaagt tgacttctgg 120 ttctgtagaa agagctaggg gaggtatgat gtgcttaaag atcctaagaa taagcctggc 180 gattttggct gggtgggcac tctgttctgc caactctgag ctgggctgga cacgcaagaa 240 atccttggtt gagagggaac acctgaatca ggtgctgttg gaaggagaac gttgttggct 300 gggggccaag gttcgaagac ccagagcttc tccacagcat cacctctttg gagtctaccc 360 cagcagggct gggaactacc taaggcccta ccccgtgggg gagcaagaaa tccatcatac 420 aggacgcagc aaaccagaca ctgaaggaaa tgctgtgagc cttgttcccc cagacctgac 480 tgaaaatcca gcaggactga 500 43 500 DNA Homo sapiens 43 gaacagcccc ttcatggcat cttgccgctc tttgctcctg gggggagaca gctctgagga 60 tgggcactat ttccgtggac acctgggcac actggttttc tggtcgaccg ccctgccaca 120 aagccatttt cagcacagtt ctcagcattc aagtgaggag gaggaagcga ctgacttggt 180 cctgacagcg agctttgagc ctgtgaacac agagtgggtt ccctttagag atgagaagta 240 cccacgactt gaggttctcc agggctttga gccagagcct gagattctgt cgcctttgca 300 gcccccactc tgtgggcaaa cagtctgtga caatgtggaa ttgatctccc agtacaatgg 360 atactggccc cttcggggag agaaggtgat acgctaccag gtggtgaaca tctgtgatga 420 tgagggccta aaccccattg tgagtgagga gcagattcgt ctgcagcacg aggcactgaa 480 tgaggccttc agccgctaca 500 44 500 DNA Homo sapiens 44 aatgtgtatt agtgcctttt cttctggtct tcatgacagg ctggtctctg ccccatgccc 60 ctccaaaagg taatgctggc tgggagtgcc attagaatct gattaggtct ggctctgcta 120 ttctctaagt accaattctt tgctgtgaca ttttttcatc ttgcatgctc tctagggcat 180 acatgagtgt gaaggagctg aaggaggccc tgcagctgaa cagtactcac ttcctcaaca 240 tctactttgc cagctcagtg cgggaagacc ttgcaggtgc tgccacctgg ccttgggaca 300 aggacgctgt cactcacctg ggtaagtgaa atgaagacca aacatagtag gaaaaaaaca 360 aagaaggctg aaggaagctt gcgaaagtaa gtttggggaa aaaagaaaga cagagaaaaa 420 gtgaatttac acagtgaatt aactgctttg tgctgagaat ggcacttagt agagcctgtc 480 aaaatgtttc aatatagaca 500 45 500 DNA Homo sapiens 45 gtttatcaga aaacataatt aaggattgga gagctattca ttctgtgctc tgaaaggctt 60 ttcaaaactt tcattgcagg tggcattgtc ctcagcccag catattatgg gatgcctggc 120 cacaccgaca ccatgatcca tgaagtggga catgttctgg gactctacca tgtctttaaa 180 ggagtcagtg aaagagaatc ctgcaatgac ccctgcaagg agacagtgcc atccatggaa 240 acgggagacc tctgtgccga caccgccccc actcccaaga gtgagctgtg ccgggaacca 300 gagcccacta gtgacacctg tggcttcact cgcttcccag gggctccgtt caccaactac 360 atgagctaca cgggtatcac cactgtcttg ttttgttttc tgttaagaat acatgggggc 420 ctttgagagc tgggagggtg gaggtgtggg agctgatggg agaatgatta agtggtcatt 480 tgtgtcggag agttgaagtg 500 46 500 DNA Homo sapiens 46 agaccctttc tggaacagat tgcatttgtt cattcattca acaagtaagt gctcattgag 60 cctaataagt taactcagcc atgaacaaga cacaaattta tccctgcctt ggtggacttg 120 atgggttaaa atctccatct cttgtgccac cttttttgtc tccacagatg ataactgcac 180 tgacaacttc actcctaacc aagtggcccg aatgcattgc tatttggacc tagtctatca 240 gcagtggact gaaagcagaa agcccacccc catccccatt ccacctatgg tcatcggaca 300 gaccaacaag tccctcacta tccactggct gcctcctatt agtggagttg tatatgacag 360 gtgagagagg cactggctgt gggtgagctg gcttatttct ctgtggcatt tcatatgaat 420 gaggggaaga atatgaatcc aggggaactc caatttggaa gtataaatgt gtgcaccact 480 gctcagagct gctgctccag 500 47 500 DNA Homo sapiens 47 caaagatgag gtccacacaa gaaaaaacga ttgagataca gttgttctta gttactctct 60 aggtccttac taaccatcaa cccctgccct ccccacacaa aggtctttct aattttctta 120 tcccaactca tctgatggac tctcctcatc tcccatctcc atccctttat ctccccaggg 180 cctcaggcag cttgtgtggc gcttgcactg aagatgggac ctttcgtcag tatgtgcaca 240 cagcttcctc ccggcgggtg tgtgactcct caggttattg gaccccagag gaggctgtgg 300 gtaaagtacc atgacatttt ttctttatac cctggtgacc actgaggatg ggggtggagg 360 taaagagtgg agtagtgaca tggtgacaaa aaggcaatgt atgtgtatgt gtgtgtgtgt 420 gtgtgtgtgt gtgtgtgtgt gtgtgttttg ctggatatct tttggagctg cctggtgccg 480 ctgtgtgcag aatgttccat 500 48 500 DNA Homo sapiens 48 ccccccaggg cctcctgatg tggatcagcc ctgcgagcca agcttacagg cctggagccc 60 tgaggtccac ctgtaccaca tgaacatgac ggtcccctgc cccacagaag gctgtagctt 120 ggagctgctc ttccaacacc cggtccaagc cgacaccctc accctgtggg tcacttcctt 180 cttcatggag tcctcgcagg tcctctttga cacagagatc ttgctggaaa acaaggagtc 240 agtgcacctg ggccccttag acactttctg tgacatccca ctcaccatca aactgcacgt 300 ggatgggaag gtgtcggggg tgaaagtcta cacctttgat gagaggatag agattgatgc 360 agcactcctg acttctcagc cccacagtcc cttgtgctct ggctgcaggc ctgtgaggta 420 ccaggttctc cgcgatcccc catttgccag tggtttgccc gtggtggtga cacattctca 480 caggaagttc acggacgtgt 500 49 500 DNA Homo sapiens 49 atctttgttt tagacaatgc agcgtatgtt taacctttat gtttcccata atatttccct 60 tcaacttctg tgaaccatca acaaaggacc cagggcatgc ctcactttgt ggctgtagtg 120 gtcacaaacc ctttggtttc cacagggagg tcacacctgg acagatgtat cagtaccaag 180 ttctagctga agctggagga gaactgggag aagcttcgcc tcctctgaac cacattcatg 240 gagctcctta ttgtggagat gggaaggtgt cagagtgagt attttgtgtg tgtgtgtgtg 300 tgtgtgtgtg tgtgtgtgag agagagagag agagagagag agagggaggg agagagagca 360 gggacactgt tctctaaaca gaagtttcag gagtttgact tgttttggaa atgtagggag 420 caaggcacca tttgattcta gtgttgaaat tctaaaatct gggacttggt tgtgtcaggt 480 cttgagatct ggcatctcta 500 50 500 DNA Homo sapiens 50 attgcttaaa ttgtatctac tttatatatc catatgtttg tttctatttt ctgtaaaagc 60 attcttctag ctcctacttt tttccaactt gcacttttta atatatactg agaaattgta 120 agaattttaa atgatggtag ctaaacaaga aaatttgtgt gtatgtgtta tatatgcata 180 tatattttac cctctaggag actgggagaa gagtgtgatg atggagacct tgtgagcgga 240 gatggctgct ccaaggtgtg tgagctggag gaaggtttca actgtgtagg taagttcaag 300 agtttcagtc taagattgtg tcctactttt agaggtgtat tattttgtga gttcttgata 360 tcctgtaaca tctagcttag tgattataat aataacctct gatgtgtccc ttgtatgcca 420 ggcatgaatt gtttcacagg tattatctca gtcaatcctt agaataactg gggaagacaa 480 agtgaggcac agagcttaaa 500 51 500 DNA Homo sapiens 51 gtaggcatta gactggcagg caattgcaga attctcttca gtgggctgga gaagaagtgt 60 tgtggacatt cccatgttca tttttttcat gacatttatg aaaacacttt ttctgtgtca 120 cacaatattt cttggcagga gagccaagcc tttgctacat gtatgaggga gatggcatat 180 gtgaaccttt tgagagaaaa accagcattg tagactgtgg catctacact cccaaaggat 240 acttggatca atgggctacc cgggcttact cctctcatga agacaagaag aagtgtcctg 300 tttccttggt aactggagaa cctcattccc tagtaagtta agccagatga atagagtcga 360 gcctgcgcaa aattgtaaag tgactccccc tatgcttaca cttggggtct atgtttggat 420 aattaaaaga agaagtaagg agttagaagc cctagggaac attactggat ctgccagcat 480 tgctattaca attacatgat 500 52 500 DNA Homo sapiens 52 gcactgagaa attcttcaat gttgagaaaa gttttctgcc aataatgtga tcattaggca 60 ttcagaaaga gagaacaact ttgctttgag ctggagagtt tcattgtcct tacttatatc 120 ttcacactcc acacttcaaa ttgttggttg aaattgtatt tcagatttgc acatcatacc 180 atccagattt acccaaccac cgtcccctaa ctggctggtt tccctgtgtt gccagtgaaa 240 atgaaactca ggatgacagg agtgaacagc cagaaggtag cctgaagaaa gaggatgagg 300 tttggctcaa agtaagtggc ccaaatgttt cttttgtgca tgtgaaaggt gtaagcatat 360 gtgtgtgtgt gtgtgtgtgt gtgtgtgtaa atggtgcatt tggatgactt gtcagaaaat 420 tttcttttgt tatctcaaag tgttaatatt ttgagtttat tttcacctct ctgtattgtg 480 gacttctttc tgttaataca 500 53 500 DNA Homo sapiens 53 tttctgtgtc tactccatat ttttagcatt tataaaaaaa atggaagctc ttactttata 60 aatagtgagc tctctaagaa gaataaaaat tgtatagcac atgtcttgat agctcaatgg 120 aaataatgac ttatacataa atgtgcttat gcttaggtgt gtttcaatag accaggagag 180 gccagagcaa tttttatttt tttgacaact gatggcctag ttcccggaga gcatcagcag 240 ccgacagtga ctctctacct gaccgatgtc cgtggaagca accactctct tggtgagtct 300 gacaaatatc cctttagggt cactagagga caacccggta gacccagaag tcagcttgat 360 gtctgtcttt tatgttgtct gggatcttgc ctacatcata catctaggtt ttggcatcat 420 acacctatta agaggataaa atgaatacaa gatatggaaa catggtacta acaatggctg 480 aaaatatgat tcatctccgt 500 54 500 DNA Homo sapiens 54 atctaggttt tggcatcata cacctattaa gaggataaaa tgaatacaag atatggaaac 60 atggtactaa caatggctga aaatatgatt catctccgtt tatcttcagg aacctatgga 120 ctgtcatgcc agcataatcc actgattatc aatgtgaccc atcaccagaa tgtccttttc 180 caccatacca cctcagtgct gctgaatttc tcatccccac gggtcggcat ctcagctgtg 240 gctctaagga catcctcccg cattggtctt tcggctccca gtaactgcat ctcagaggac 300 gaggggcaga atcatcaggg acagaggtac aaacttccct ttctttcttt tgtttccttt 360 tcttgtggct ctaatgattg gctcacattt acatagtgtt atgtatagag tgtttgctca 420 atcactaact gaaaaatgaa tttaggaact actaaaaaga cataggaagc aagcaaagaa 480 taatttgtgg aaggcatact 500 55 500 DNA Homo sapiens 55 aaaaaaggtg atgagttaaa tatcagtttt ggagaaaatt gttctctctg gtttaggagc 60 cctcccagat ggagctgcaa agtcattgct cctccttact ggttctcaac cagtcctaag 120 atggttctat ttctcttatc ccagctgtat ccatcggccc tgtgggaagc aggacagctg 180 tccgtcattg ctgcttgatc atgctgatgt ggtgaactgt acctctatag gcccaggtct 240 catgaagtgt gctatcactt gtcaaagggg atttgccctt caggccagca gtgggcagta 300 catcaggccc atgcaggtga gttgaaagaa cactatcacc aggaccaagt tcctgggaag 360 gggaggtatt cacactcttc tctctggctc cacagggaaa gagcagatgt ttttaaacca 420 tttgaaagca aaacatgggg gctctaacaa gcagagagaa tgatggctta atagaaatgc 480 aaggtatata aaagcatgta 500 56 500 DNA Homo sapiens 56 gagggagaaa ggcaaaataa tttgtcccgt tttaaatgtt tagacagata atcaccaaga 60 ctcttctaaa gcctggaaac tactctgata cgccttttaa tgtgactttg acagaagcaa 120 tttctctgtt tctagaagga aattctgctc acatgttctt ctgggcactg ggaccagaat 180 gtgagctgcc ttcccgtgga ctgcggtgtt cccgacccgt ctttggtgaa ctatgcaaac 240 ttctcctgct cagagggaac caaatttctg aaacgctgct caatctcttg tgtcccacca 300 gccaagctgc aaggtattgt ctggtcaacc aggaactgta tgcaagttct ctgccatccc 360 tcgctcttga gggtactttg ggactctttt cgttacccca acacctttcc tatttgctcc 420 actgtgtcac catcttctga taactccaga aaagtcccaa aaggcaacac cactctttgt 480 ccccaactac ttgaaaggat 500 57 500 DNA Homo sapiens 57 tgggattttt gagatgctac tcaagaaaga ataatatgac ttttgaaagg caccagtaag 60 ttcagaggtt tttatttcat ctgctatgat tatctttctg ggctctctgt tctgagcatc 120 ttgtgcttct ctttcctgga gtcaggactg agcccatggc tgacatgtct tgaagatggt 180 ctctggtctc tccctgaagt ctactgcaag ttggagtgtg atgctccccc tattattctg 240 aatgccaact tgctcctgcc tcactgcctc caggacaacc acgacgtggg caccatctgc 300 aaatatgaat gcaaaccagg gtactatgtg gcagaaagtg cagagggtaa agtcaggaag 360 taagttgaat gttcctggtc tttggagttc tacctacctc gctgcttgtt atgtttgttt 420 ttctgtataa tctctcttta cctgcagggc tatattctcc agtcatgaca ggcaggcaac 480 ctgctgtgct ttctgtaaat 500 58 500 DNA Homo sapiens 58 atttactact actttgaata ataaatgaac attactattt ttagttccat gatttagaaa 60 ttagcctagg acagtgggac atatggctca caagaaccat ctgtccctgc ctatttgcta 120 attcttatgc catattgctg aggatcaagt ctttcatgac cttcttgaaa gattctgatg 180 agctattttt gttttatgtt ttatcagcaa gctcctgaag atacaatgcc tggaaggtgg 240 aatctgggag caaggcagct gcattcctgt ggtgtgtgag ccaccccctc ctgtgtttga 300 aggcatgtat gaatgtacca atggcttcag cctggacagc cagtgtgtgc tcaactgtaa 360 ccaggaacgt gaaaaggtaa ggaacatttt ttgaacttat tttcatcagg ctgtgctcta 420 atcttgatgc ccaatccaag aaatttgaga aatgaaattt ctaaagaatt ccaacaggca 480 gggacttggg attctaatcg 500 59 500 DNA Homo sapiens 59 tttttttttt tttttttttt ggccagtaac aatatgttgt ttcaatagtg tcttttggtc 60 caggttctag aactggagaa tactaccact taatcagacc acttttttca actctcaata 120 aagttaacaa aacaataatt aagatgttta cttttgatat tctagcttcc catcctctgc 180 actaaagagg gcctgtggac ccaggagttt aagttgtgtg agaatctgca aggagaatgc 240 ccaccacccc cctcagagct gaattctgtg gagtacaaat gtgaacaagg atatgggatt 300 ggtaaggata ggagtgaatc ttaaagtcaa tttctatgtc attcttttat taagcacagc 360 tgaatttttt ttaattttat ttatttttgt cagacggagt cttgctctgt cgcccaggct 420 ggggtgcagt ggcacagtct cagctcactg caacctccat ttcccgggtt caagcaattc 480 tcctgcctca gcctcccaag 500 60 500 DNA Homo sapiens 60 agttgagtta ggttggggca gagtctcttt accaattagt gtgaaggtat tcctacagga 60 acacagtaga tgaatatcag ccctgcctac aacgtcataa tatggtgacc catgatgaaa 120 gcagttgttc tttaaaaact caaagctatt gattcctgaa agaaaaagaa tgagatctgg 180 gaagttcaag tctctgctgt aaacttctgt tctttcaggt gcagtgtgtt ccccattgtg 240 tgtaatcccc cccagtgacc ccgtgatgct acctgagaat atcactgctg acactctgga 300 gcactggatg gaacctgtca aagtccaggt gaggaaaggg acattgttat gtgccaaaga 360 catgagtctg ctgagcaaca cttggagcat tattttttgg agtaatttca gaggctttca 420 aagcatcctc aaagcaaaca accacatgga ttattcctag aaagaaggat taaacattga 480 aaagaaaaaa gagggaagct 500 61 500 DNA Homo sapiens 61 tgagacttcc tgcctgaacc acatctgtca gggtgggcag tgtgcacggg aaaggctaga 60 tagccccaat ccactttatg ggacacaatt agaaactagc tgcttgagaa atggagttgg 120 actcctgtgg tgagctcagg atttgcccct tctcacacac aacaaatgtc tttaattttg 180 ttttctgttt ttcccgtctt tccccttaga gcattgtgtg cactggccgg cgtcaatggc 240 acccagaccc cgtcttagtc cactgcatcc agtcatgtga ggtaagatag cctccccttc 300 cccaactcag actagagaac tcaggtggat ttaacttatg gagcttgaat ccttctaatt 360 taggacctgg tccctctcct attcctctgt cctttgttta acttcttaaa ttaagttggt 420 tccacgatct taaatttaca gaaattagga gttctgattt ttgttttgtt ttagcaaaat 480 ctttggagat ccactttaaa 500 62 500 DNA Homo sapiens 62 gagtgcctca ttcattattt ctcccaataa gcccaaggca gtaaatccac cagctcagta 60 tcctcagccc atattgccct tcatgttact tcagcacttt tgtttgcatc atattctctt 120 ggcataagga tttgctttca attatccact attctgtgct gcttatgttt tctgaggtgc 180 cactaagact ttctttaaaa actcttatct aaataattcc atcttcttat gttaggtcat 240 aagccagttg ttgctgcttg tgttcccatt gtcccagcaa gaacacacgt atgctacata 300 tctgcaatcc aaaattgttg gtacgttcaa ttctattttt ctagttgttg cttgcatttg 360 gcctttgaga cctgaatact ccttgtatct tctgtttctt tcgtaaactg catatgggca 420 aggaatggca acttagttaa tggaaaactt ttgaagtcaa acgaccctag attccaatcc 480 tgggtcagct gagtagtgtg 500 63 500 DNA Homo sapiens 63 ggagcagtgc tgccagtgaa atgtgacaat tccttggacc tagtggtctt gagatttttt 60 caacttcttg caaatattag gtcaatttgt gtcctgcacc ttgggtgctt ttctgtaata 120 tctctggagc tgtatggatt acgctgggat gggaggagtg gcagagtcaa acaagacata 180 ataaggctat actgagatgt tgccttctaa cctccctaca gcccttccaa gcagatggtt 240 ggtgtgacac tatcaacaac cgagcctact gccactatga cgggggagac tgctgctctt 300 ccacactctc ctccaagaag gtgagtgaga gaacctgggg atgggggagg cagtggcttc 360 aggaataaag gcagggtctt cagctagctc tcattcatgg tgtgtaataa tgggtttgta 420 ttcaacaatt aggagggaat tataatgaac aaacattgga gcttctaaag tgacagggtt 480 aagggtagca taggttctta 500 64 500 DNA Homo sapiens 64 gtgacctgga tgagtgcacc tgccgggacc ccaaggcaga agaaaatcag taactgtggg 60 aacaagcccc tccctccact gcctcagagg cagtaagaaa gagaggccga cccaggagga 120 aacaaagggt gaatgaagaa gaacaatcat gaaatggaag aaggaggaag agcatgaagg 180 atcttataag aaatgcaaga ggatattgat aggtgtgaac tagttcatca agtagcccaa 240 gtaggagaga atcataggca aaagtttctt taaagtggca gttgattaac atggaagggg 300 aaatatgata gatatataag gaccctcctc cctcacttat attctattaa atcctatcct 360 caactcttgc cctgctctcc gctccacccc ctgccaacta ctcagtccca cccaacttgt 420 aaaccaatac caaaatacta gaggagaagt tggcagggat actgttaata cccattttga 480 atggattgcc atctttcaga 500 65 1000 DNA Homo sapiens 65 tcttccccat cctttccatc catttcaaat caattggaaa catggttcct tgggtctagc 60 tgttcatttt tgtaaattac ttattttgaa catctcattg tttatttgct cactcagcat 120 atggtgactt ttagtaactt cagattgaga aacttctgag ataaaaagga gacctatgta 180 gtatgaattc atggcatttc catttagtac ttctcacagc aggatacttg atttctcctt 240 tctcccatgt ccgatttaaa gtgaatttaa gatattgttc ttttaaatcc ccaatgattg 300 aacaaagtaa gaaaaaatac tttgttttgt ttgtgacaaa acaaaagaaa aatacaaggg 360 atccctaaaa ggttagtgtg ggcttattag gcagtaggta gatctgttca cagtaagtgt 420 gtgtgtgtgt gtgtgtgtgt gtgtgagaga gagagagaga gagagaggga gaatacacac 480 agagaagagt actccaaaac actattgatt ttttgctatt gattgtgtag gctgcggctg 540 ctgaaagaga aagcccgaga tgtttactgg ggaaaccaag agtagcgtct gtcccctgtg 600 ccttggtgag gtgggtaggt tttcaggagg aaggagggga cagggaggag taggtggagt 660 gatgcattga acttactagc tttgacatca tcattgtctt taaatgaaaa caaaaacaaa 720 aacaaaaaca aaaaacaaga agatatttac aggcagacag aaagggagcc aaggggagca 780 ggagagactg gagagaacag gtcccctgaa gtgtatgctc ttctttttgc tcttttcccg 840 atcttcccag gaacccacaa gactcccaga aggtgaagtt aagagctccc agactcataa 900 ggttattaga acagcaaact ggcaccccaa agaactttac ggagacttgc aacctatcaa 960 caagttggat gagggattaa aagccttcaa caaccaacaa 1000 66 17 DNA Homo sapiens 66 ccccaagcat caaactg 17 67 17 DNA Homo sapiens 67 cccaagcatc aaactga 17 68 17 DNA Homo sapiens 68 ccaagcatca aactgaa 17 69 17 DNA Homo sapiens 69 caagcatcaa actgaag 17 70 17 DNA Homo sapiens 70 aagcatcaaa ctgaagg 17 71 17 DNA Homo sapiens 71 agcatcaaac tgaagga 17 72 17 DNA Homo sapiens 72 gcatcaaact gaaggaa 17 73 17 DNA Homo sapiens 73 catcaaactg aaggaaa 17 74 17 DNA Homo sapiens 74 atcaaactga aggaaac 17 75 17 DNA Homo sapiens 75 tcaaactgaa ggaaaca 17 76 17 DNA Homo sapiens 76 caaactgaag gaaacat 17 77 17 DNA Homo sapiens 77 aaactgaagg aaacatt 17 78 17 DNA Homo sapiens 78 aactgaagga aacattc 17 79 17 DNA Homo sapiens 79 actgaaggaa acattct 17 80 17 DNA Homo sapiens 80 ctgaaggaaa cattcta 17 81 17 DNA Homo sapiens 81 tgaaggaaac attctaa 17 82 17 DNA Homo sapiens 82 gaaggaaaca ttctaac 17 83 17 DNA Homo sapiens 83 aaggaaacat tctaacc 17 84 17 DNA Homo sapiens 84 aggaaacatt ctaacct 17 85 17 DNA Homo sapiens 85 ggaaacattc taacctt 17 86 17 DNA Homo sapiens 86 gaaacattct aaccttc 17 87 17 DNA Homo sapiens 87 aaacattcta accttca 17 88 17 DNA Homo sapiens 88 aacattctaa ccttcac 17 89 17 DNA Homo sapiens 89 acattctaac cttcaca 17 90 17 DNA Homo sapiens 90 cattctaacc ttcacag 17 91 17 DNA Homo sapiens 91 attctaacct tcacaga 17 92 17 DNA Homo sapiens 92 ttctaacctt cacagac 17 93 17 DNA Homo sapiens 93 tctaaccttc acagaca 17 94 17 DNA Homo sapiens 94 ctaaccttca cagacag 17 95 17 DNA Homo sapiens 95 taaccttcac agacaga 17 96 17 DNA Homo sapiens 96 aaccttcaca gacagac 17 97 17 DNA Homo sapiens 97 accttcacag acagact 17 98 17 DNA Homo sapiens 98 ccttcacaga cagactg 17 99 17 DNA Homo sapiens 99 cttcacagac agactgg 17 100 17 DNA Homo sapiens 100 ttcacagaca gactgga 17 101 17 DNA Homo sapiens 101 tcacagacag actggag 17 102 17 DNA Homo sapiens 102 cacagacaga ctggagg 17 103 17 DNA Homo sapiens 103 acagacagac tggaggc 17 104 17 DNA Homo sapiens 104 cagacagact ggaggct 17 105 17 DNA Homo sapiens 105 agacagactg gaggctg 17 106 17 DNA Homo sapiens 106 gacagactgg aggctgg 17 107 17 DNA Homo sapiens 107 acagactgga ggctgga 17 108 17 DNA Homo sapiens 108 cagactggag gctggat 17 109 17 DNA Homo sapiens 109 agactggagg ctggatg 17 110 17 DNA Homo sapiens 110 gactggaggc tggatgg 17 111 17 DNA Homo sapiens 111 actggaggct ggatggg 17 112 17 DNA Homo sapiens 112 ctggaggctg gatgggg 17 113 17 DNA Homo sapiens 113 tggaggctgg atgggga 17 114 17 DNA Homo sapiens 114 ggaggctgga tggggac 17 115 17 DNA Homo sapiens 115 gaggctggat ggggacc 17 116 17 DNA Homo sapiens 116 aggctggatg gggacct 17 117 17 DNA Homo sapiens 117 ggctggatgg ggacctg 17 118 17 DNA Homo sapiens 118 gctggatggg gacctgg 17 119 17 DNA Homo sapiens 119 ctggatgggg acctggc 17 120 17 DNA Homo sapiens 120 tggatgggga cctggct 17 121 17 DNA Homo sapiens 121 ggatggggac ctggctg 17 122 17 DNA Homo sapiens 122 gatggggacc tggctga 17 123 17 DNA Homo sapiens 123 atggggacct ggctgaa 17 124 17 DNA Homo sapiens 124 tggggacctg gctgaag 17 125 17 DNA Homo sapiens 125 ggggacctgg ctgaaga 17 126 17 DNA Homo sapiens 126 gggacctggc tgaagac 17 127 17 DNA Homo sapiens 127 ggacctggct gaagaca 17 128 17 DNA Homo sapiens 128 gacctggctg aagacat 17 129 17 DNA Homo sapiens 129 acctggctga agacatc 17 130 17 DNA Homo sapiens 130 cctggctgaa gacatct 17 131 17 DNA Homo sapiens 131 ctggctgaag acatctg 17 132 17 DNA Homo sapiens 132 tggctgaaga catctgg 17 133 17 DNA Homo sapiens 133 ggctgaagac atctgga 17 134 17 DNA Homo sapiens 134 gctgaagaca tctggag 17 135 17 DNA Homo sapiens 135 ctgaagacat ctggaga 17 136 17 DNA Homo sapiens 136 tgaagacatc tggagaa 17 137 17 DNA Homo sapiens 137 gaagacatct ggagaat 17 138 17 DNA Homo sapiens 138 aagacatctg gagaatg 17 139 17 DNA Homo sapiens 139 agacatctgg agaatga 17 140 17 DNA Homo sapiens 140 gacatctgga gaatgaa 17 141 17 DNA Homo sapiens 141 acatctggag aatgaaa 17 142 17 DNA Homo sapiens 142 catctggaga atgaaag 17 143 17 DNA Homo sapiens 143 atctggagaa tgaaagt 17 144 17 DNA Homo sapiens 144 tctggagaat gaaagtt 17 145 17 DNA Homo sapiens 145 ctggagaatg aaagtta 17 146 17 DNA Homo sapiens 146 tggagaatga aagttaa 17 147 17 DNA Homo sapiens 147 ggagaatgaa agttaag 17 148 17 DNA Homo sapiens 148 gagaatgaaa gttaagt 17 149 17 DNA Homo sapiens 149 agaatgaaag ttaagta 17 150 17 DNA Homo sapiens 150 gaatgaaagt taagtac 17 151 17 DNA Homo sapiens 151 aatgaaagtt aagtacc 17 152 17 DNA Homo sapiens 152 atgaaagtta agtacca 17 153 17 DNA Homo sapiens 153 tgaaagttaa gtaccag 17 154 17 DNA Homo sapiens 154 gaaagttaag taccagc 17 155 17 DNA Homo sapiens 155 aaagttaagt accagct 17 156 17 DNA Homo sapiens 156 aagttaagta ccagctt 17 157 17 DNA Homo sapiens 157 agttaagtac cagcttg 17 158 17 DNA Homo sapiens 158 gttaagtacc agcttgc 17 159 17 DNA Homo sapiens 159 ttaagtacca gcttgca 17 160 17 DNA Homo sapiens 160 taagtaccag cttgcat 17 161 17 DNA Homo sapiens 161 aagtaccagc ttgcatt 17 162 17 DNA Homo sapiens 162 agtaccagct tgcattt 17 163 17 DNA Homo sapiens 163 gtaccagctt gcatttt 17 164 17 DNA Homo sapiens 164 taccagcttg cattttt 17 165 17 DNA Homo sapiens 165 accagcttgc atttttg 17 166 17 DNA Homo sapiens 166 ccagcttgca tttttgt 17 167 17 DNA Homo sapiens 167 cagcttgcat ttttgtg 17 168 17 DNA Homo sapiens 168 agcttgcatt tttgtgc 17 169 17 DNA Homo sapiens 169 gcttgcattt ttgtgcc 17 170 17 DNA Homo sapiens 170 cttgcatttt tgtgccc 17 171 17 DNA Homo sapiens 171 ttgcattttt gtgcccc 17 172 17 DNA Homo sapiens 172 tgcatttttg tgcccct 17 173 17 DNA Homo sapiens 173 gcatttttgt gccccta 17 174 17 DNA Homo sapiens 174 catttttgtg cccctag 17 175 17 DNA Homo sapiens 175 atttttgtgc ccctaga 17 176 17 DNA Homo sapiens 176 tttttgtgcc cctagat 17 177 17 DNA Homo sapiens 177 ttttgtgccc ctagatt 17 178 17 DNA Homo sapiens 178 tttgtgcccc tagatta 17 179 17 DNA Homo sapiens 179 ttgtgcccct agattat 17 180 17 DNA Homo sapiens 180 tgtgccccta gattatt 17 181 17 DNA Homo sapiens 181 gtgcccctag attattt 17 182 17 DNA Homo sapiens 182 tgcccctaga ttatttt 17 183 17 DNA Homo sapiens 183 gcccctagat tattttt 17 184 17 DNA Homo sapiens 184 cccctagatt atttttg 17 185 17 DNA Homo sapiens 185 ccctagatta tttttgc 17 186 17 DNA Homo sapiens 186 cctagattat ttttgca 17 187 17 DNA Homo sapiens 187 ctagattatt tttgcat 17 188 17 DNA Homo sapiens 188 tagattattt ttgcatt 17 189 17 DNA Homo sapiens 189 agattatttt tgcattt 17 190 17 DNA Homo sapiens 190 gattattttt gcatttt 17 191 17 DNA Homo sapiens 191 attatttttg catttta 17 192 17 DNA Homo sapiens 192 ttatttttgc attttaa 17 193 17 DNA Homo sapiens 193 tatttttgca ttttaaa 17 194 17 DNA Homo sapiens 194 atttttgcat tttaaaa 17 195 17 DNA Homo sapiens 195 tttttgcatt ttaaaat 17 196 17 DNA Homo sapiens 196 ttttgcattt taaaata 17 197 17 DNA Homo sapiens 197 tttgcatttt aaaataa 17 198 17 DNA Homo sapiens 198 ttgcatttta aaataag 17 199 17 DNA Homo sapiens 199 tgcattttaa aataaga 17 200 17 DNA Homo sapiens 200 gcattttaaa ataagaa 17 201 17 DNA Homo sapiens 201 cattttaaaa taagaag 17 202 17 DNA Homo sapiens 202 attttaaaat aagaagc 17 203 17 DNA Homo sapiens 203 ttttaaaata agaagca 17 204 17 DNA Homo sapiens 204 tttaaaataa gaagcat 17 205 17 DNA Homo sapiens 205 ttaaaataag aagcatc 17 206 17 DNA Homo sapiens 206 taaaataaga agcatca 17 207 17 DNA Homo sapiens 207 aaaataagaa gcatcaa 17 208 17 DNA Homo sapiens 208 aaataagaag catcaaa 17 209 17 DNA Homo sapiens 209 aataagaagc atcaaat 17 210 17 DNA Homo sapiens 210 ataagaagca tcaaatt 17 211 17 DNA Homo sapiens 211 taagaagcat caaattg 17 212 17 DNA Homo sapiens 212 aagaagcatc aaattgc 17 213 17 DNA Homo sapiens 213 agaagcatca aattgcg 17 214 17 DNA Homo sapiens 214 gaagcatcaa attgcgt 17 215 17 DNA Homo sapiens 215 aagcatcaaa ttgcgtg 17 216 17 DNA Homo sapiens 216 agcatcaaat tgcgtgt 17 217 17 DNA Homo sapiens 217 gcatcaaatt gcgtgtc 17 218 17 DNA Homo sapiens 218 catcaaattg cgtgtct 17 219 17 DNA Homo sapiens 219 atcaaattgc gtgtctc 17 220 17 DNA Homo sapiens 220 tcaaattgcg tgtctct 17 221 17 DNA Homo sapiens 221 caaattgcgt gtctctg 17 222 17 DNA Homo sapiens 222 aaattgcgtg tctctgt 17 223 17 DNA Homo sapiens 223 aattgcgtgt ctctgtg 17 224 17 DNA Homo sapiens 224 attgcgtgtc tctgtgt 17 225 17 DNA Homo sapiens 225 ttgcgtgtct ctgtgta 17 226 17 DNA Homo sapiens 226 tgcgtgtctc tgtgtaa 17 227 17 DNA Homo sapiens 227 gcgtgtctct gtgtaaa 17 228 17 DNA Homo sapiens 228 cgtgtctctg tgtaaaa 17 229 17 DNA Homo sapiens 229 gtgtctctgt gtaaaag 17 230 17 DNA Homo sapiens 230 tgtctctgtg taaaagt 17 231 17 DNA Homo sapiens 231 gtctctgtgt aaaagtt 17 232 17 DNA Homo sapiens 232 tctctgtgta aaagttc 17 233 17 DNA Homo sapiens 233 ctctgtgtaa aagttct 17 234 17 DNA Homo sapiens 234 tctgtgtaaa agttcta 17 235 17 DNA Homo sapiens 235 ctgtgtaaaa gttctag 17 236 17 DNA Homo sapiens 236 tgtgtaaaag ttctagc 17 237 17 DNA Homo sapiens 237 gtgtaaaagt tctagca 17 238 17 DNA Homo sapiens 238 tgtaaaagtt ctagcaa 17 239 17 DNA Homo sapiens 239 gtaaaagttc tagcaat 17 240 17 DNA Homo sapiens 240 taaaagttct agcaatt 17 241 17 DNA Homo sapiens 241 aaaagttcta gcaattt 17 242 17 DNA Homo sapiens 242 aaagttctag caatttg 17 243 17 DNA Homo sapiens 243 aagttctagc aatttgt 17 244 17 DNA Homo sapiens 244 agttctagca atttgtt 17 245 17 DNA Homo sapiens 245 gttctagcaa tttgttt 17 246 17 DNA Homo sapiens 246 ttctagcaat ttgtttt 17 247 17 DNA Homo sapiens 247 tctagcaatt tgtttta 17 248 17 DNA Homo sapiens 248 ctagcaattt gttttaa 17 249 17 DNA Homo sapiens 249 tagcaatttg ttttaag 17 250 17 DNA Homo sapiens 250 agcaatttgt tttaagg 17 251 17 DNA Homo sapiens 251 gcaatttgtt ttaaggt 17 252 17 DNA Homo sapiens 252 caatttgttt taaggtg 17 253 17 DNA Homo sapiens 253 aatttgtttt aaggtga 17 254 17 DNA Homo sapiens 254 atttgtttta aggtgaa 17 255 17 DNA Homo sapiens 255 tttgttttaa ggtgaac 17 256 17 DNA Homo sapiens 256 ttgttttaag gtgaact 17 257 17 DNA Homo sapiens 257 tgttttaagg tgaactt 17 258 17 DNA Homo sapiens 258 gttttaaggt gaactta 17 259 17 DNA Homo sapiens 259 ttttaaggtg aacttat 17 260 17 DNA Homo sapiens 260 tttaaggtga acttatt 17 261 17 DNA Homo sapiens 261 ttaaggtgaa cttattt 17 262 17 DNA Homo sapiens 262 taaggtgaac ttatttt 17 263 17 DNA Homo sapiens 263 aaggtgaact tattttg 17 264 17 DNA Homo sapiens 264 aggtgaactt attttgg 17 265 17 DNA Homo sapiens 265 ggtgaactta ttttggc 17 266 17 DNA Homo sapiens 266 gtgaacttat tttggct 17 267 17 DNA Homo sapiens 267 tgaacttatt ttggctt 17 268 17 DNA Homo sapiens 268 gaacttattt tggctta 17 269 17 DNA Homo sapiens 269 aacttatttt ggcttag 17 270 17 DNA Homo sapiens 270 acttattttg gcttagg 17 271 17 DNA Homo sapiens 271 cttattttgg cttaggg 17 272 17 DNA Homo sapiens 272 ttattttggc ttaggga 17 273 17 DNA Homo sapiens 273 tattttggct tagggac 17 274 17 DNA Homo sapiens 274 attttggctt agggact 17 275 17 DNA Homo sapiens 275 ttttggctta gggacta 17 276 17 DNA Homo sapiens 276 tttggcttag ggactac 17 277 17 DNA Homo sapiens 277 ttggcttagg gactaca 17 278 17 DNA Homo sapiens 278 tggcttaggg actacaa 17 279 17 DNA Homo sapiens 279 ggcttaggga ctacaaa 17 280 17 DNA Homo sapiens 280 gcttagggac tacaaaa 17 281 17 DNA Homo sapiens 281 cttagggact acaaaaa 17 282 17 DNA Homo sapiens 282 ttagggacta caaaaag 17 283 17 DNA Homo sapiens 283 tagggactac aaaaaga 17 284 17 DNA Homo sapiens 284 agggactaca aaaagag 17 285 17 DNA Homo sapiens 285 gggactacaa aaagaga 17 286 17 DNA Homo sapiens 286 ggactacaaa aagagaa 17 287 17 DNA Homo sapiens 287 gactacaaaa agagaag 17 288 17 DNA Homo sapiens 288 actacaaaaa gagaagg 17 289 17 DNA Homo sapiens 289 ctacaaaaag agaaggt 17 290 17 DNA Homo sapiens 290 tacaaaaaga gaaggta 17 291 17 DNA Homo sapiens 291 acaaaaagag aaggtaa 17 292 17 DNA Homo sapiens 292 caaaaagaga aggtaat 17 293 17 DNA Homo sapiens 293 aaaaagagaa ggtaatt 17 294 17 DNA Homo sapiens 294 aaaagagaag gtaattc 17 295 17 DNA Homo sapiens 295 aaagagaagg taattcc 17 296 17 DNA Homo sapiens 296 aagagaaggt aattcct 17 297 17 DNA Homo sapiens 297 agagaaggta attccta 17 298 17 DNA Homo sapiens 298 gagaaggtaa ttcctag 17 299 17 DNA Homo sapiens 299 agaaggtaat tcctagg 17 300 17 DNA Homo sapiens 300 gaaggtaatt cctaggg 17 301 17 DNA Homo sapiens 301 aaggtaattc ctaggga 17 302 17 DNA Homo sapiens 302 aggtaattcc tagggaa 17 303 17 DNA Homo sapiens 303 ggtaattcct agggaag 17 304 17 DNA Homo sapiens 304 gtaattccta gggaagg 17 305 17 DNA Homo sapiens 305 taattcctag ggaagga 17 306 17 DNA Homo sapiens 306 aattcctagg gaaggaa 17 307 17 DNA Homo sapiens 307 attcctaggg aaggaag 17 308 17 DNA Homo sapiens 308 ttcctaggga aggaaga 17 309 17 DNA Homo sapiens 309 tcctagggaa ggaagaa 17 310 17 DNA Homo sapiens 310 cctagggaag gaagaag 17 311 17 DNA Homo sapiens 311 ctagggaagg aagaaga 17 312 17 DNA Homo sapiens 312 tagggaagga agaagag 17 313 17 DNA Homo sapiens 313 agggaaggaa gaagaga 17 314 17 DNA Homo sapiens 314 gggaaggaag aagagaa 17 315 17 DNA Homo sapiens 315 ggaaggaaga agagaaa 17 316 17 DNA Homo sapiens 316 gaaggaagaa gagaaag 17 317 17 DNA Homo sapiens 317 aaggaagaag agaaaga 17 318 17 DNA Homo sapiens 318 aggaagaaga gaaagaa 17 319 17 DNA Homo sapiens 319 ggaagaagag aaagaaa 17 320 17 DNA Homo sapiens 320 gaagaagaga aagaaat 17 321 17 DNA Homo sapiens 321 aagaagagaa agaaatg 17 322 17 DNA Homo sapiens 322 agaagagaaa gaaatga 17 323 17 DNA Homo sapiens 323 gaagagaaag aaatgaa 17 324 17 DNA Homo sapiens 324 aagagaaaga aatgaaa 17 325 17 DNA Homo sapiens 325 agagaaagaa atgaaaa 17 326 17 DNA Homo sapiens 326 gagaaagaaa tgaaaat 17 327 17 DNA Homo sapiens 327 agaaagaaat gaaaatt 17 328 17 DNA Homo sapiens 328 gaaagaaatg aaaatta 17 329 17 DNA Homo sapiens 329 aaagaaatga aaattag 17 330 17 DNA Homo sapiens 330 aagaaatgaa aattaga 17 331 17 DNA Homo sapiens 331 agaaatgaaa attagag 17 332 17 DNA Homo sapiens 332 gaaatgaaaa ttagaga 17 333 17 DNA Homo sapiens 333 aaatgaaaat tagagaa 17 334 17 DNA Homo sapiens 334 aatgaaaatt agagaat 17 335 17 DNA Homo sapiens 335 atgaaaatta gagaata 17 336 17 DNA Homo sapiens 336 tgaaaattag agaataa 17 337 17 DNA Homo sapiens 337 gaaaattaga gaataag 17 338 17 DNA Homo sapiens 338 aaaattagag aataaga 17 339 17 DNA Homo sapiens 339 aaattagaga ataagat 17 340 17 DNA Homo sapiens 340 aattagagaa taagatt 17 341 17 DNA Homo sapiens 341 attagagaat aagatta 17 342 17 DNA Homo sapiens 342 ttagagaata agattat 17 343 17 DNA Homo sapiens 343 tagagaataa gattatt 17 344 17 DNA Homo sapiens 344 agagaataag attattt 17 345 17 DNA Homo sapiens 345 gagaataaga ttatttt 17 346 17 DNA Homo sapiens 346 agaataagat tattttg 17 347 17 DNA Homo sapiens 347 gaataagatt attttga 17 348 17 DNA Homo sapiens 348 aataagatta ttttgaa 17 349 17 DNA Homo sapiens 349 ataagattat tttgaat 17 350 17 DNA Homo sapiens 350 taagattatt ttgaatg 17 351 17 DNA Homo sapiens 351 aagattattt tgaatga 17 352 17 DNA Homo sapiens 352 agattatttt gaatgac 17 353 17 DNA Homo sapiens 353 gattattttg aatgact 17 354 17 DNA Homo sapiens 354 attattttga atgactt 17 355 17 DNA Homo sapiens 355 ttattttgaa tgacttc 17 356 17 DNA Homo sapiens 356 tattttgaat gacttca 17 357 17 DNA Homo sapiens 357 attttgaatg acttcag 17 358 17 DNA Homo sapiens 358 ttttgaatga cttcagg 17 359 17 DNA Homo sapiens 359 tttgaatgac ttcaggt 17 360 17 DNA Homo sapiens 360 ttgaatgact tcaggta 17 361 17 DNA Homo sapiens 361 tgaatgactt caggtag 17 362 17 DNA Homo sapiens 362 gaatgacttc aggtagc 17 363 17 DNA Homo sapiens 363 aatgacttca ggtagcg 17 364 17 DNA Homo sapiens 364 atgacttcag gtagcga 17 365 17 DNA Homo sapiens 365 tgacttcagg tagcgag 17 366 17 DNA Homo sapiens 366 gacttcaggt agcgagg 17 367 17 DNA Homo sapiens 367 acttcaggta gcgagga 17 368 17 DNA Homo sapiens 368 cttcaggtag cgaggag 17 369 17 DNA Homo sapiens 369 ttcaggtagc gaggagt 17 370 17 DNA Homo sapiens 370 tcaggtagcg aggagtg 17 371 17 DNA Homo sapiens 371 caggtagcga ggagtgt 17 372 17 DNA Homo sapiens 372 aggtagcgag gagtgtg 17 373 17 DNA Homo sapiens 373 ggtagcgagg agtgtgt 17 374 17 DNA Homo sapiens 374 gtagcgagga gtgtgtg 17 375 17 DNA Homo sapiens 375 tagcgaggag tgtgtgt 17 376 17 DNA Homo sapiens 376 agcgaggagt gtgtgtt 17 377 17 DNA Homo sapiens 377 gcgaggagtg tgtgttt 17 378 17 DNA Homo sapiens 378 cgaggagtgt gtgtttg 17 379 17 DNA Homo sapiens 379 gaggagtgtg tgtttgt 17 380 17 DNA Homo sapiens 380 aggagtgtgt gtttgtg 17 381 17 DNA Homo sapiens 381 ggagtgtgtg tttgtga 17 382 17 DNA Homo sapiens 382 gagtgtgtgt ttgtgag 17 383 17 DNA Homo sapiens 383 agtgtgtgtt tgtgagt 17 384 17 DNA Homo sapiens 384 gtgtgtgttt gtgagtg 17 385 17 DNA Homo sapiens 385 tgtgtgtttg tgagtgt 17 386 17 DNA Homo sapiens 386 gtgtgtttgt gagtgtg 17 387 17 DNA Homo sapiens 387 tgtgtttgtg agtgtgt 17 388 17 DNA Homo sapiens 388 gtgtttgtga gtgtgta 17 389 17 DNA Homo sapiens 389 tgtttgtgag tgtgtat 17 390 17 DNA Homo sapiens 390 gtttgtgagt gtgtatt 17 391 17 DNA Homo sapiens 391 tttgtgagtg tgtattt 17 392 17 DNA Homo sapiens 392 ttgtgagtgt gtatttg 17 393 17 DNA Homo sapiens 393 tgtgagtgtg tatttga 17 394 17 DNA Homo sapiens 394 gtgagtgtgt atttgag 17 395 17 DNA Homo sapiens 395 tgagtgtgta tttgaga 17 396 17 DNA Homo sapiens 396 gagtgtgtat ttgagag 17 397 17 DNA Homo sapiens 397 agtgtgtatt tgagaga 17 398 17 DNA Homo sapiens 398 gtgtgtattt gagagac 17 399 17 DNA Homo sapiens 399 tgtgtatttg agagact 17 400 17 DNA Homo sapiens 400 gtgtatttga gagactt 17 401 17 DNA Homo sapiens 401 tgtatttgag agacttg 17 402 17 DNA Homo sapiens 402 gtatttgaga gacttgg 17 403 17 DNA Homo sapiens 403 tatttgagag acttggc 17 404 17 DNA Homo sapiens 404 atttgagaga cttggct 17 405 17 DNA Homo sapiens 405 tttgagagac ttggctc 17 406 17 DNA Homo sapiens 406 ttgagagact tggctca 17 407 17 DNA Homo sapiens 407 tgagagactt ggctcat 17 408 17 DNA Homo sapiens 408 gagagacttg gctcatg 17 409 17 DNA Homo sapiens 409 agagacttgg ctcatgc 17 410 17 DNA Homo sapiens 410 gagacttggc tcatgcc 17 411 17 DNA Homo sapiens 411 agacttggct catgcct 17 412 17 DNA Homo sapiens 412 gacttggctc atgcctg 17 413 17 DNA Homo sapiens 413 acttggctca tgcctgt 17 414 17 DNA Homo sapiens 414 cttggctcat gcctgtg 17 415 17 DNA Homo sapiens 415 ttggctcatg cctgtgg 17 416 17 DNA Homo sapiens 416 tggctcatgc ctgtggg 17 417 17 DNA Homo sapiens 417 ggctcatgcc tgtgggt 17 418 17 DNA Homo sapiens 418 gctcatgcct gtgggtc 17 419 17 DNA Homo sapiens 419 ctcatgcctg tgggtct 17 420 17 DNA Homo sapiens 420 tcatgcctgt gggtctt 17 421 17 DNA Homo sapiens 421 catgcctgtg ggtcttc 17 422 17 DNA Homo sapiens 422 atgcctgtgg gtcttct 17 423 17 DNA Homo sapiens 423 tgcctgtggg tcttctc 17 424 17 DNA Homo sapiens 424 gcctgtgggt cttctct 17 425 17 DNA Homo sapiens 425 cctgtgggtc ttctctt 17 426 17 DNA Homo sapiens 426 ctgtgggtct tctcttc 17 427 17 DNA Homo sapiens 427 tgtgggtctt ctcttct 17 428 17 DNA Homo sapiens 428 gtgggtcttc tcttcta 17 429 17 DNA Homo sapiens 429 tgggtcttct cttctag 17 430 17 DNA Homo sapiens 430 gggtcttctc ttctagt 17 431 17 DNA Homo sapiens 431 ggtcttctct tctagta 17 432 17 DNA Homo sapiens 432 gtcttctctt ctagtat 17 433 17 DNA Homo sapiens 433 tcttctcttc tagtatc 17 434 17 DNA Homo sapiens 434 cttctcttct agtatca 17 435 17 DNA Homo sapiens 435 ttctcttcta gtatcag 17 436 17 DNA Homo sapiens 436 tctcttctag tatcagt 17 437 17 DNA Homo sapiens 437 ctcttctagt atcagtg 17 438 17 DNA Homo sapiens 438 tcttctagta tcagtga 17 439 17 DNA Homo sapiens 439 cttctagtat cagtgag 17 440 17 DNA Homo sapiens 440 ttctagtatc agtgagg 17 441 17 DNA Homo sapiens 441 tctagtatca gtgaggg 17 442 17 DNA Homo sapiens 442 ctagtatcag tgagggg 17 443 17 DNA Homo sapiens 443 tagtatcagt gagggga 17 444 17 DNA Homo sapiens 444 agtatcagtg aggggag 17 445 17 DNA Homo sapiens 445 gtatcagtga ggggagg 17 446 17 DNA Homo sapiens 446 tatcagtgag gggaggg 17 447 17 DNA Homo sapiens 447 atcagtgagg ggaggga 17 448 17 DNA Homo sapiens 448 tcagtgaggg gagggat 17 449 17 DNA Homo sapiens 449 cagtgagggg agggatt 17 450 17 DNA Homo sapiens 450 agtgagggga gggatta 17 451 17 DNA Homo sapiens 451 gtgaggggag ggattac 17 452 17 DNA Homo sapiens 452 tgaggggagg gattact 17 453 17 DNA Homo sapiens 453 gaggggaggg attactg 17 454 17 DNA Homo sapiens 454 aggggaggga ttactga 17 455 17 DNA Homo sapiens 455 ggggagggat tactgaa 17 456 17 DNA Homo sapiens 456 gggagggatt actgaag 17 457 17 DNA Homo sapiens 457 ggagggatta ctgaaga 17 458 17 DNA Homo sapiens 458 gagggattac tgaagaa 17 459 17 DNA Homo sapiens 459 agggattact gaagaag 17 460 17 DNA Homo sapiens 460 gggattactg aagaaga 17 461 17 DNA Homo sapiens 461 ggattactga agaagaa 17 462 17 DNA Homo sapiens 462 gattactgaa gaagaag 17 463 17 DNA Homo sapiens 463 attactgaag aagaagg 17 464 17 DNA Homo sapiens 464 ttactgaaga agaaggg 17 465 17 DNA Homo sapiens 465 tactgaagaa gaagggg 17 466 17 DNA Homo sapiens 466 actgaagaag aaggggg 17 467 17 DNA Homo sapiens 467 ctgaagaaga agggggg 17 468 17 DNA Homo sapiens 468 tgaagaagaa gggggga 17 469 17 DNA Homo sapiens 469 gaagaagaag gggggaa 17 470 17 DNA Homo sapiens 470 aagaagaagg ggggaaa 17 471 17 DNA Homo sapiens 471 agaagaaggg gggaaaa 17 472 17 DNA Homo sapiens 472 gaagaagggg ggaaaaa 17 473 17 DNA Homo sapiens 473 aagaaggggg gaaaaaa 17 474 17 DNA Homo sapiens 474 agaagggggg aaaaaaa 17 475 17 DNA Homo sapiens 475 gaagggggga aaaaaaa 17 476 17 DNA Homo sapiens 476 aaggggggaa aaaaaaa 17 477 17 DNA Homo sapiens 477 aggggggaaa aaaaaag 17 478 17 DNA Homo sapiens 478 ggggggaaaa aaaaaga 17 479 17 DNA Homo sapiens 479 gggggaaaaa aaaagaa 17 480 17 DNA Homo sapiens 480 ggggaaaaaa aaagaaa 17 481 17 DNA Homo sapiens 481 gggaaaaaaa aagaaag 17 482 17 DNA Homo sapiens 482 ggaaaaaaaa agaaaga 17 483 17 DNA Homo sapiens 483 gaaaaaaaaa gaaagaa 17 484 17 DNA Homo sapiens 484 aaaaaaaaag aaagaaa 17 485 17 DNA Homo sapiens 485 aaaaaaaaga aagaaat 17 486 17 DNA Homo sapiens 486 aaaaaaagaa agaaatc 17 487 17 DNA Homo sapiens 487 aaaaaagaaa gaaatct 17 488 17 DNA Homo sapiens 488 aaaaagaaag aaatctg 17 489 17 DNA Homo sapiens 489 aaaagaaaga aatctga 17 490 17 DNA Homo sapiens 490 aaagaaagaa atctgag 17 491 17 DNA Homo sapiens 491 aagaaagaaa tctgagc 17 492 17 DNA Homo sapiens 492 agaaagaaat ctgagct 17 493 17 DNA Homo sapiens 493 gaaagaaatc tgagctt 17 494 17 DNA Homo sapiens 494 aaagaaatct gagcttt 17 495 17 DNA Homo sapiens 495 aagaaatctg agctttc 17 496 17 DNA Homo sapiens 496 agaaatctga gctttct 17 497 17 DNA Homo sapiens 497 gaaatctgag ctttctg 17 498 17 DNA Homo sapiens 498 aaatctgagc tttctgg 17 499 17 DNA Homo sapiens 499 aatctgagct ttctggg 17 500 17 DNA Homo sapiens 500 atctgagctt tctggga 17 501 17 DNA Homo sapiens 501 tctgagcttt ctgggag 17 502 17 DNA Homo sapiens 502 ctgagctttc tgggagg 17 503 17 DNA Homo sapiens 503 tgagctttct gggagga 17 504 17 DNA Homo sapiens 504 gagctttctg ggaggaa 17 505 17 DNA Homo sapiens 505 agctttctgg gaggaaa 17 506 17 DNA Homo sapiens 506 gctttctggg aggaaat 17 507 17 DNA Homo sapiens 507 ctttctggga ggaaatt 17 508 17 DNA Homo sapiens 508 tttctgggag gaaattc 17 509 17 DNA Homo sapiens 509 ttctgggagg aaattca 17 510 17 DNA Homo sapiens 510 tctgggagga aattcaa 17 511 17 DNA Homo sapiens 511 ctgggaggaa attcaaa 17 512 17 DNA Homo sapiens 512 tgggaggaaa ttcaaag 17 513 17 DNA Homo sapiens 513 gggaggaaat tcaaagg 17 514 17 DNA Homo sapiens 514 ggaggaaatt caaagga 17 515 17 DNA Homo sapiens 515 gaggaaattc aaaggaa 17 516 17 DNA Homo sapiens 516 aggaaattca aaggaac 17 517 17 DNA Homo sapiens 517 ggaaattcaa aggaacc 17 518 17 DNA Homo sapiens 518 gaaattcaaa ggaacca 17 519 17 DNA Homo sapiens 519 aaattcaaag gaaccaa 17 520 17 DNA Homo sapiens 520 aattcaaagg aaccaag 17 521 17 DNA Homo sapiens 521 attcaaagga accaaga 17 522 17 DNA Homo sapiens 522 ttcaaaggaa ccaagag 17 523 17 DNA Homo sapiens 523 tcaaaggaac caagaga 17 524 17 DNA Homo sapiens 524 caaaggaacc aagagaa 17 525 17 DNA Homo sapiens 525 aaaggaacca agagaaa 17 526 17 DNA Homo sapiens 526 aaggaaccaa gagaaat 17 527 17 DNA Homo sapiens 527 aggaaccaag agaaatt 17 528 17 DNA Homo sapiens 528 ggaaccaaga gaaatta 17 529 17 DNA Homo sapiens 529 gaaccaagag aaattaa 17 530 17 DNA Homo sapiens 530 aaccaagaga aattaac 17 531 17 DNA Homo sapiens 531 accaagagaa attaact 17 532 17 DNA Homo sapiens 532 ccaagagaaa ttaactt 17 533 17 DNA Homo sapiens 533 caagagaaat taacttc 17 534 17 DNA Homo sapiens 534 aagagaaatt aacttcg 17 535 17 DNA Homo sapiens 535 agagaaatta acttcgt 17 536 17 DNA Homo sapiens 536 gagaaattaa cttcgtt 17 537 17 DNA Homo sapiens 537 agaaattaac ttcgttc 17 538 17 DNA Homo sapiens 538 gaaattaact tcgttct 17 539 17 DNA Homo sapiens 539 aaattaactt cgttctg 17 540 17 DNA Homo sapiens 540 aattaacttc gttctgc 17 541 17 DNA Homo sapiens 541 attaacttcg ttctgca 17 542 17 DNA Homo sapiens 542 ttaacttcgt tctgcaa 17 543 17 DNA Homo sapiens 543 taacttcgtt ctgcaag 17 544 17 DNA Homo sapiens 544 aacttcgttc tgcaagg 17 545 17 DNA Homo sapiens 545 acttcgttct gcaagga 17 546 17 DNA Homo sapiens 546 cttcgttctg caaggac 17 547 17 DNA Homo sapiens 547 ttcgttctgc aaggact 17 548 17 DNA Homo sapiens 548 tcgttctgca aggacta 17 549 17 DNA Homo sapiens 549 cgttctgcaa ggactaa 17 550 17 DNA Homo sapiens 550 gttctgcaag gactaaa 17 551 17 DNA Homo sapiens 551 ttctgcaagg actaaag 17 552 17 DNA Homo sapiens 552 tctgcaagga ctaaagt 17 553 17 DNA Homo sapiens 553 ctgcaaggac taaagta 17 554 17 DNA Homo sapiens 554 tgcaaggact aaagtac 17 555 17 DNA Homo sapiens 555 gcaaggacta aagtaca 17 556 17 DNA Homo sapiens 556 caaggactaa agtacag 17 557 17 DNA Homo sapiens 557 aaggactaaa gtacagc 17 558 17 DNA Homo sapiens 558 aggactaaag tacagca 17 559 17 DNA Homo sapiens 559 ggactaaagt acagcaa 17 560 17 DNA Homo sapiens 560 gactaaagta cagcaag 17 561 17 DNA Homo sapiens 561 actaaagtac agcaaga 17 562 17 DNA Homo sapiens 562 ctaaagtaca gcaagag 17 563 17 DNA Homo sapiens 563 taaagtacag caagagg 17 564 17 DNA Homo sapiens 564 aaagtacagc aagagga 17 565 17 DNA Homo sapiens 565 aagtacagca agaggag 17 566 17 DNA Homo sapiens 566 agtacagcaa gaggaga 17 567 17 DNA Homo sapiens 567 gtacagcaag aggagag 17 568 17 DNA Homo sapiens 568 tacagcaaga ggagaga 17 569 17 DNA Homo sapiens 569 acagcaagag gagagag 17 570 17 DNA Homo sapiens 570 cagcaagagg agagagg 17 571 17 DNA Homo sapiens 571 agcaagagga gagaggt 17 572 17 DNA Homo sapiens 572 gcaagaggag agaggtc 17 573 17 DNA Homo sapiens 573 caagaggaga gaggtca 17 574 17 DNA Homo sapiens 574 aagaggagag aggtcaa 17 575 17 DNA Homo sapiens 575 agaggagaga ggtcaag 17 576 17 DNA Homo sapiens 576 gaggagagag gtcaagc 17 577 17 DNA Homo sapiens 577 aggagagagg tcaagcg 17 578 17 DNA Homo sapiens 578 ggagagaggt caagcga 17 579 17 DNA Homo sapiens 579 gagagaggtc aagcgag 17 580 17 DNA Homo sapiens 580 agagaggtca agcgaga 17 581 17 DNA Homo sapiens 581 gagaggtcaa gcgagaa 17 582 17 DNA Homo sapiens 582 agaggtcaag cgagaag 17 583 17 DNA Homo sapiens 583 gaggtcaagc gagaagc 17 584 17 DNA Homo sapiens 584 aggtcaagcg agaagcg 17 585 17 DNA Homo sapiens 585 ggtcaagcga gaagcgt 17 586 17 DNA Homo sapiens 586 gtcaagcgag aagcgtg 17 587 17 DNA Homo sapiens 587 tcaagcgaga agcgtgc 17 588 17 DNA Homo sapiens 588 caagcgagaa gcgtgcg 17 589 17 DNA Homo sapiens 589 aagcgagaag cgtgcgg 17 590 17 DNA Homo sapiens 590 agcgagaagc gtgcggg 17 591 17 DNA Homo sapiens 591 gcgagaagcg tgcggga 17 592 17 DNA Homo sapiens 592 cgagaagcgt gcgggaa 17 593 17 DNA Homo sapiens 593 gagaagcgtg cgggaag 17 594 17 DNA Homo sapiens 594 agaagcgtgc gggaagc 17 595 17 DNA Homo sapiens 595 gaagcgtgcg ggaagca 17 596 17 DNA Homo sapiens 596 aagcgtgcgg gaagcac 17 597 17 DNA Homo sapiens 597 agcgtgcggg aagcaca 17 598 17 DNA Homo sapiens 598 gcgtgcggga agcacat 17 599 17 DNA Homo sapiens 599 cgtgcgggaa gcacatg 17 600 17 DNA Homo sapiens 600 gtgcgggaag cacatgc 17 601 17 DNA Homo sapiens 601 tgcgggaagc acatgcc 17 602 17 DNA Homo sapiens 602 gcgggaagca catgccc 17 603 17 DNA Homo sapiens 603 cgggaagcac atgccct 17 604 17 DNA Homo sapiens 604 gggaagcaca tgccctg 17 605 17 DNA Homo sapiens 605 ggaagcacat gccctgg 17 606 17 DNA Homo sapiens 606 gaagcacatg ccctggg 17 607 17 DNA Homo sapiens 607 aagcacatgc cctgggg 17 608 17 DNA Homo sapiens 608 agcacatgcc ctgggga 17 609 17 DNA Homo sapiens 609 gcacatgccc tggggag 17 610 17 DNA Homo sapiens 610 cacatgccct ggggagg 17 611 17 DNA Homo sapiens 611 acatgccctg gggaggc 17 612 17 DNA Homo sapiens 612 catgccctgg ggaggca 17 613 17 DNA Homo sapiens 613 atgccctggg gaggcat 17 614 17 DNA Homo sapiens 614 tgccctgggg aggcata 17 615 17 DNA Homo sapiens 615 gccctgggga ggcatag 17 616 17 DNA Homo sapiens 616 ccctggggag gcataga 17 617 17 DNA Homo sapiens 617 cctggggagg catagaa 17 618 17 DNA Homo sapiens 618 ctggggaggc atagaag 17 619 17 DNA Homo sapiens 619 tggggaggca tagaagc 17 620 17 DNA Homo sapiens 620 ggggaggcat agaagcc 17 621 17 DNA Homo sapiens 621 gggaggcata gaagcca 17 622 17 DNA Homo sapiens 622 ggaggcatag aagccac 17 623 17 DNA Homo sapiens 623 gaggcataga agccaca 17 624 17 DNA Homo sapiens 624 aggcatagaa gccacac 17 625 17 DNA Homo sapiens 625 ggcatagaag ccacact 17 626 17 DNA Homo sapiens 626 gcatagaagc cacactg 17 627 17 DNA Homo sapiens 627 catagaagcc acactgg 17 628 17 DNA Homo sapiens 628 atagaagcca cactggc 17 629 17 DNA Homo sapiens 629 tagaagccac actggca 17 630 17 DNA Homo sapiens 630 agaagccaca ctggcag 17 631 17 DNA Homo sapiens 631 gaagccacac tggcaga 17 632 17 DNA Homo sapiens 632 aagccacact ggcagag 17 633 17 DNA Homo sapiens 633 agccacactg gcagagc 17 634 17 DNA Homo sapiens 634 gccacactgg cagagcg 17 635 17 DNA Homo sapiens 635 ccacactggc agagcgg 17 636 17 DNA Homo sapiens 636 cacactggca gagcggc 17 637 17 DNA Homo sapiens 637 acactggcag agcggcc 17 638 17 DNA Homo sapiens 638 cactggcaga gcggcca 17 639 17 DNA Homo sapiens 639 actggcagag cggccag 17 640 17 DNA Homo sapiens 640 ctggcagagc ggccagc 17 641 17 DNA Homo sapiens 641 tggcagagcg gccagca 17 642 17 DNA Homo sapiens 642 ggcagagcgg ccagcac 17 643 17 DNA Homo sapiens 643 gcagagcggc cagcaca 17 644 17 DNA Homo sapiens 644 cagagcggcc agcacag 17 645 17 DNA Homo sapiens 645 agagcggcca gcacagg 17 646 17 DNA Homo sapiens 646 gagcggccag cacaggt 17 647 17 DNA Homo sapiens 647 agcggccagc acaggta 17 648 17 DNA Homo sapiens 648 gcggccagca caggtag 17 649 17 DNA Homo sapiens 649 cggccagcac aggtagc 17 650 17 DNA Homo sapiens 650 ggccagcaca ggtagcc 17 651 17 DNA Homo sapiens 651 gccagcacag gtagcca 17 652 17 DNA Homo sapiens 652 ccagcacagg tagccag 17 653 17 DNA Homo sapiens 653 cagcacaggt agccagc 17 654 17 DNA Homo sapiens 654 agcacaggta gccagca 17 655 17 DNA Homo sapiens 655 gcacaggtag ccagcag 17 656 17 DNA Homo sapiens 656 cacaggtagc cagcaga 17 657 17 DNA Homo sapiens 657 acaggtagcc agcagag 17 658 17 DNA Homo sapiens 658 caggtagcca gcagagg 17 659 17 DNA Homo sapiens 659 aggtagccag cagaggc 17 660 17 DNA Homo sapiens 660 ggtagccagc agaggca 17 661 17 DNA Homo sapiens 661 gtagccagca gaggcat 17 662 17 DNA Homo sapiens 662 tagccagcag aggcatt 17 663 17 DNA Homo sapiens 663 agccagcaga ggcattc 17 664 17 DNA Homo sapiens 664 gccagcagag gcattct 17 665 17 DNA Homo sapiens 665 ccagcagagg cattctt 17 666 17 DNA Homo sapiens 666 cagcagaggc attcttg 17 667 17 DNA Homo sapiens 667 agcagaggca ttcttgg 17 668 17 DNA Homo sapiens 668 gcagaggcat tcttggg 17 669 17 DNA Homo sapiens 669 cagaggcatt cttgggg 17 670 17 DNA Homo sapiens 670 agaggcattc ttggggc 17 671 17 DNA Homo sapiens 671 gaggcattct tggggct 17 672 17 DNA Homo sapiens 672 aggcattctt ggggcta 17 673 17 DNA Homo sapiens 673 ggcattcttg gggctat 17 674 17 DNA Homo sapiens 674 gcattcttgg ggctatt 17 675 17 DNA Homo sapiens 675 cattcttggg gctattt 17 676 17 DNA Homo sapiens 676 attcttgggg ctatttg 17 677 17 DNA Homo sapiens 677 ttcttggggc tatttga 17 678 17 DNA Homo sapiens 678 tcttggggct atttgaa 17 679 17 DNA Homo sapiens 679 cttggggcta tttgaaa 17 680 17 DNA Homo sapiens 680 ttggggctat ttgaaaa 17 681 17 DNA Homo sapiens 681 tggggctatt tgaaaaa 17 682 17 DNA Homo sapiens 682 ggggctattt gaaaaag 17 683 17 DNA Homo sapiens 683 gggctatttg aaaaagt 17 684 17 DNA Homo sapiens 684 ggctatttga aaaagtt 17 685 17 DNA Homo sapiens 685 gctatttgaa aaagttt 17 686 17 DNA Homo sapiens 686 ctatttgaaa aagtttg 17 687 17 DNA Homo sapiens 687 tatttgaaaa agtttgg 17 688 17 DNA Homo sapiens 688 atttgaaaaa gtttggt 17 689 17 DNA Homo sapiens 689 tttgaaaaag tttggtc 17 690 17 DNA Homo sapiens 690 ttgaaaaagt ttggtct 17 691 17 DNA Homo sapiens 691 tgaaaaagtt tggtctg 17 692 17 DNA Homo sapiens 692 gaaaaagttt ggtctgt 17 693 17 DNA Homo sapiens 693 aaaaagtttg gtctgtg 17 694 17 DNA Homo sapiens 694 aaaagtttgg tctgtga 17 695 17 DNA Homo sapiens 695 aaagtttggt ctgtgaa 17 696 17 DNA Homo sapiens 696 aagtttggtc tgtgaac 17 697 17 DNA Homo sapiens 697 agtttggtct gtgaaca 17 698 17 DNA Homo sapiens 698 gtttggtctg tgaacaa 17 699 17 DNA Homo sapiens 699 tttggtctgt gaacaaa 17 700 17 DNA Homo sapiens 700 ttggtctgtg aacaaaa 17 701 17 DNA Homo sapiens 701 tggtctgtga acaaaac 17 702 17 DNA Homo sapiens 702 ggtctgtgaa caaaaca 17 703 17 DNA Homo sapiens 703 gtctgtgaac aaaacag 17 704 17 DNA Homo sapiens 704 tctgtgaaca aaacagt 17 705 17 DNA Homo sapiens 705 ctgtgaacaa aacagtt 17 706 17 DNA Homo sapiens 706 tgtgaacaaa acagttt 17 707 17 DNA Homo sapiens 707 gtgaacaaaa cagtttc 17 708 17 DNA Homo sapiens 708 tgaacaaaac agtttcc 17 709 17 DNA Homo sapiens 709 gaacaaaaca gtttccc 17 710 17 DNA Homo sapiens 710 aacaaaacag tttccct 17 711 17 DNA Homo sapiens 711 acaaaacagt ttccctg 17 712 17 DNA Homo sapiens 712 caaaacagtt tccctgg 17 713 17 DNA Homo sapiens 713 aaaacagttt ccctggt 17 714 17 DNA Homo sapiens 714 aaacagtttc cctggtg 17 715 17 DNA Homo sapiens 715 aacagtttcc ctggtga 17 716 17 DNA Homo sapiens 716 acagtttccc tggtgac 17 717 17 DNA Homo sapiens 717 cagtttccct ggtgact 17 718 17 DNA Homo sapiens 718 agtttccctg gtgactg 17 719 17 DNA Homo sapiens 719 gtttccctgg tgactgc 17 720 17 DNA Homo sapiens 720 tttccctggt gactgca 17 721 17 DNA Homo sapiens 721 ttccctggtg actgcaa 17 722 17 DNA Homo sapiens 722 tccctggtga ctgcaaa 17 723 17 DNA Homo sapiens 723 ccctggtgac tgcaaat 17 724 17 DNA Homo sapiens 724 cctggtgact gcaaatc 17 725 17 DNA Homo sapiens 725 ctggtgactg caaatcc 17 726 17 DNA Homo sapiens 726 tggtgactgc aaatcca 17 727 17 DNA Homo sapiens 727 ggtgactgca aatccat 17 728 17 DNA Homo sapiens 728 gtgactgcaa atccatt 17 729 17 DNA Homo sapiens 729 tgactgcaaa tccattg 17 730 17 DNA Homo sapiens 730 gactgcaaat ccattgc 17 731 17 DNA Homo sapiens 731 actgcaaatc cattgct 17 732 17 DNA Homo sapiens 732 ctgcaaatcc attgcta 17 733 17 DNA Homo sapiens 733 tgcaaatcca ttgctag 17 734 17 DNA Homo sapiens 734 gcaaatccat tgctagc 17 735 17 DNA Homo sapiens 735 caaatccatt gctagct 17 736 17 DNA Homo sapiens 736 aaatccattg ctagctg 17 737 17 DNA Homo sapiens 737 aatccattgc tagctgc 17 738 17 DNA Homo sapiens 738 atccattgct agctgcc 17 739 17 DNA Homo sapiens 739 tccattgcta gctgcct 17 740 17 DNA Homo sapiens 740 ccattgctag ctgcctc 17 741 17 DNA Homo sapiens 741 cattgctagc tgcctct 17 742 17 DNA Homo sapiens 742 attgctagct gcctctt 17 743 17 DNA Homo sapiens 743 ttgctagctg cctcttt 17 744 17 DNA Homo sapiens 744 tgctagctgc ctctttc 17 745 17 DNA Homo sapiens 745 gctagctgcc tctttct 17 746 17 DNA Homo sapiens 746 ctagctgcct ctttctc 17 747 17 DNA Homo sapiens 747 tagctgcctc tttctcg 17 748 17 DNA Homo sapiens 748 agctgcctct ttctcgt 17 749 17 DNA Homo sapiens 749 gctgcctctt tctcgtc 17 750 17 DNA Homo sapiens 750 ctgcctcttt ctcgtct 17 751 17 DNA Homo sapiens 751 tgcctctttc tcgtctg 17 752 17 DNA Homo sapiens 752 gcctctttct cgtctgc 17 753 17 DNA Homo sapiens 753 cctctttctc gtctgcc 17 754 17 DNA Homo sapiens 754 ctctttctcg tctgccc 17 755 17 DNA Homo sapiens 755 tctttctcgt ctgccca 17 756 17 DNA Homo sapiens 756 ctttctcgtc tgcccat 17 757 17 DNA Homo sapiens 757 tttctcgtct gcccatc 17 758 17 DNA Homo sapiens 758 ttctcgtctg cccatca 17 759 17 DNA Homo sapiens 759 tctcgtctgc ccatcac 17 760 17 DNA Homo sapiens 760 ctcgtctgcc catcact 17 761 17 DNA Homo sapiens 761 tcgtctgccc atcactc 17 762 17 DNA Homo sapiens 762 cgtctgccca tcactct 17 763 17 DNA Homo sapiens 763 gtctgcccat cactctg 17 764 17 DNA Homo sapiens 764 tctgcccatc actctgg 17 765 17 DNA Homo sapiens 765 ctgcccatca ctctggt 17 766 17 DNA Homo sapiens 766 tgcccatcac tctggtg 17 767 17 DNA Homo sapiens 767 gcccatcact ctggtgt 17 768 17 DNA Homo sapiens 768 cccatcactc tggtgtg 17 769 17 DNA Homo sapiens 769 ccatcactct ggtgtgg 17 770 17 DNA Homo sapiens 770 catcactctg gtgtggt 17 771 17 DNA Homo sapiens 771 atcactctgg tgtggta 17 772 17 DNA Homo sapiens 772 tcactctggt gtggtac 17 773 17 DNA Homo sapiens 773 cactctggtg tggtacc 17 774 17 DNA Homo sapiens 774 actctggtgt ggtaccc 17 775 17 DNA Homo sapiens 775 ctctggtgtg gtaccca 17 776 17 DNA Homo sapiens 776 tctggtgtgg tacccag 17 777 17 DNA Homo sapiens 777 ctggtgtggt acccaga 17 778 17 DNA Homo sapiens 778 tggtgtggta cccagaa 17 779 17 DNA Homo sapiens 779 ggtgtggtac ccagaag 17 780 17 DNA Homo sapiens 780 gtgtggtacc cagaagt 17 781 17 DNA Homo sapiens 781 tgtggtaccc agaagtt 17 782 17 DNA Homo sapiens 782 gtggtaccca gaagttg 17 783 17 DNA Homo sapiens 783 tggtacccag aagttga 17 784 17 DNA Homo sapiens 784 ggtacccaga agttgac 17 785 17 DNA Homo sapiens 785 gtacccagaa gttgact 17 786 17 DNA Homo sapiens 786 tacccagaag ttgactt 17 787 17 DNA Homo sapiens 787 acccagaagt tgacttc 17 788 17 DNA Homo sapiens 788 cccagaagtt gacttct 17 789 17 DNA Homo sapiens 789 ccagaagttg acttctg 17 790 17 DNA Homo sapiens 790 cagaagttga cttctgg 17 791 17 DNA Homo sapiens 791 agaagttgac ttctggt 17 792 17 DNA Homo sapiens 792 gaagttgact tctggtt 17 793 17 DNA Homo sapiens 793 aagttgactt ctggttc 17 794 17 DNA Homo sapiens 794 agttgacttc tggttct 17 795 17 DNA Homo sapiens 795 gttgacttct ggttctg 17 796 17 DNA Homo sapiens 796 ttgacttctg gttctgt 17 797 17 DNA Homo sapiens 797 tgacttctgg ttctgta 17 798 17 DNA Homo sapiens 798 gacttctggt tctgtag 17 799 17 DNA Homo sapiens 799 acttctggtt ctgtaga 17 800 17 DNA Homo sapiens 800 cttctggttc tgtagaa 17 801 17 DNA Homo sapiens 801 ttctggttct gtagaaa 17 802 17 DNA Homo sapiens 802 tctggttctg tagaaag 17 803 17 DNA Homo sapiens 803 ctggttctgt agaaaga 17 804 17 DNA Homo sapiens 804 tggttctgta gaaagag 17 805 17 DNA Homo sapiens 805 ggttctgtag aaagagc 17 806 17 DNA Homo sapiens 806 gttctgtaga aagagct 17 807 17 DNA Homo sapiens 807 ttctgtagaa agagcta 17 808 17 DNA Homo sapiens 808 tctgtagaaa gagctag 17 809 17 DNA Homo sapiens 809 ctgtagaaag agctagg 17 810 17 DNA Homo sapiens 810 tgtagaaaga gctaggg 17 811 17 DNA Homo sapiens 811 gtagaaagag ctagggg 17 812 17 DNA Homo sapiens 812 tagaaagagc tagggga 17 813 17 DNA Homo sapiens 813 agaaagagct aggggag 17 814 17 DNA Homo sapiens 814 gaaagagcta ggggagg 17 815 17 DNA Homo sapiens 815 aaagagctag gggaggt 17 816 17 DNA Homo sapiens 816 aagagctagg ggaggta 17 817 17 DNA Homo sapiens 817 agagctaggg gaggtat 17 818 17 DNA Homo sapiens 818 gagctagggg aggtatg 17 819 17 DNA Homo sapiens 819 agctagggga ggtatga 17 820 17 DNA Homo sapiens 820 gctaggggag gtatgat 17 821 17 DNA Homo sapiens 821 ctaggggagg tatgatg 17 822 17 DNA Homo sapiens 822 taggggaggt atgatgt 17 823 17 DNA Homo sapiens 823 aggggaggta tgatgtg 17 824 17 DNA Homo sapiens 824 ggggaggtat gatgtgc 17 825 17 DNA Homo sapiens 825 gggaggtatg atgtgct 17 826 17 DNA Homo sapiens 826 ggaggtatga tgtgctt 17 827 17 DNA Homo sapiens 827 gaggtatgat gtgctta 17 828 17 DNA Homo sapiens 828 aggtatgatg tgcttaa 17 829 17 DNA Homo sapiens 829 ggtatgatgt gcttaaa 17 830 17 DNA Homo sapiens 830 gtatgatgtg cttaaag 17 831 17 DNA Homo sapiens 831 tatgatgtgc ttaaaga 17 832 17 DNA Homo sapiens 832 atgatgtgct taaagat 17 833 17 DNA Homo sapiens 833 tgatgtgctt aaagatc 17 834 17 DNA Homo sapiens 834 gatgtgctta aagatcc 17 835 17 DNA Homo sapiens 835 atgtgcttaa agatcct 17 836 17 DNA Homo sapiens 836 tgtgcttaaa gatccta 17 837 17 DNA Homo sapiens 837 gtgcttaaag atcctaa 17 838 17 DNA Homo sapiens 838 tgcttaaaga tcctaag 17 839 17 DNA Homo sapiens 839 gcttaaagat cctaaga 17 840 17 DNA Homo sapiens 840 cttaaagatc ctaagaa 17 841 17 DNA Homo sapiens 841 ttaaagatcc taagaat 17 842 17 DNA Homo sapiens 842 taaagatcct aagaata 17 843 17 DNA Homo sapiens 843 aaagatccta agaataa 17 844 17 DNA Homo sapiens 844 aagatcctaa gaataag 17 845 17 DNA Homo sapiens 845 agatcctaag aataagc 17 846 17 DNA Homo sapiens 846 gatcctaaga ataagcc 17 847 17 DNA Homo sapiens 847 atcctaagaa taagcct 17 848 17 DNA Homo sapiens 848 tcctaagaat aagcctg 17 849 17 DNA Homo sapiens 849 cctaagaata agcctgg 17 850 17 DNA Homo sapiens 850 ctaagaataa gcctggc 17 851 17 DNA Homo sapiens 851 taagaataag cctggcg 17 852 17 DNA Homo sapiens 852 aagaataagc ctggcga 17 853 17 DNA Homo sapiens 853 agaataagcc tggcgat 17 854 17 DNA Homo sapiens 854 gaataagcct ggcgatt 17 855 17 DNA Homo sapiens 855 aataagcctg gcgattt 17 856 17 DNA Homo sapiens 856 ataagcctgg cgatttt 17 857 17 DNA Homo sapiens 857 taagcctggc gattttg 17 858 17 DNA Homo sapiens 858 aagcctggcg attttgg 17 859 17 DNA Homo sapiens 859 agcctggcga ttttggc 17 860 17 DNA Homo sapiens 860 gcctggcgat tttggct 17 861 17 DNA Homo sapiens 861 cctggcgatt ttggctg 17 862 17 DNA Homo sapiens 862 ctggcgattt tggctgg 17 863 17 DNA Homo sapiens 863 tggcgatttt ggctggg 17 864 17 DNA Homo sapiens 864 ggcgattttg gctgggt 17 865 17 DNA Homo sapiens 865 gcgattttgg ctgggtg 17 866 17 DNA Homo sapiens 866 cgattttggc tgggtgg 17 867 17 DNA Homo sapiens 867 gattttggct gggtggg 17 868 17 DNA Homo sapiens 868 attttggctg ggtgggc 17 869 17 DNA Homo sapiens 869 ttttggctgg gtgggca 17 870 17 DNA Homo sapiens 870 tttggctggg tgggcac 17 871 17 DNA Homo sapiens 871 ttggctgggt gggcact 17 872 17 DNA Homo sapiens 872 tggctgggtg ggcactc 17 873 17 DNA Homo sapiens 873 ggctgggtgg gcactct 17 874 17 DNA Homo sapiens 874 gctgggtggg cactctg 17 875 17 DNA Homo sapiens 875 ctgggtgggc actctgt 17 876 17 DNA Homo sapiens 876 tgggtgggca ctctgtt 17 877 17 DNA Homo sapiens 877 gggtgggcac tctgttc 17 878 17 DNA Homo sapiens 878 ggtgggcact ctgttct 17 879 17 DNA Homo sapiens 879 gtgggcactc tgttctg 17 880 17 DNA Homo sapiens 880 tgggcactct gttctgc 17 881 17 DNA Homo sapiens 881 gggcactctg ttctgcc 17 882 17 DNA Homo sapiens 882 ggcactctgt tctgcca 17 883 17 DNA Homo sapiens 883 gcactctgtt ctgccaa 17 884 17 DNA Homo sapiens 884 cactctgttc tgccaac 17 885 17 DNA Homo sapiens 885 actctgttct gccaact 17 886 17 DNA Homo sapiens 886 ctctgttctg ccaactc 17 887 17 DNA Homo sapiens 887 tctgttctgc caactct 17 888 17 DNA Homo sapiens 888 ctgttctgcc aactctg 17 889 25 DNA Homo sapiens 889 ccccaagcat caaactgaag gaaac 25 890 25 DNA Homo sapiens 890 cccaagcatc aaactgaagg aaaca 25 891 25 DNA Homo sapiens 891 ccaagcatca aactgaagga aacat 25 892 25 DNA Homo sapiens 892 caagcatcaa actgaaggaa acatt 25 893 25 DNA Homo sapiens 893 aagcatcaaa ctgaaggaaa cattc 25 894 25 DNA Homo sapiens 894 agcatcaaac tgaaggaaac attct 25 895 25 DNA Homo sapiens 895 gcatcaaact gaaggaaaca ttcta 25 896 25 DNA Homo sapiens 896 catcaaactg aaggaaacat tctaa 25 897 25 DNA Homo sapiens 897 atcaaactga aggaaacatt ctaac 25 898 25 DNA Homo sapiens 898 tcaaactgaa ggaaacattc taacc 25 899 25 DNA Homo sapiens 899 caaactgaag gaaacattct aacct 25 900 25 DNA Homo sapiens 900 aaactgaagg aaacattcta acctt 25 901 25 DNA Homo sapiens 901 aactgaagga aacattctaa ccttc 25 902 25 DNA Homo sapiens 902 actgaaggaa acattctaac cttca 25 903 25 DNA Homo sapiens 903 ctgaaggaaa cattctaacc ttcac 25 904 25 DNA Homo sapiens 904 tgaaggaaac attctaacct tcaca 25 905 25 DNA Homo sapiens 905 gaaggaaaca ttctaacctt cacag 25 906 25 DNA Homo sapiens 906 aaggaaacat tctaaccttc acaga 25 907 25 DNA Homo sapiens 907 aggaaacatt ctaaccttca cagac 25 908 25 DNA Homo sapiens 908 ggaaacattc taaccttcac agaca 25 909 25 DNA Homo sapiens 909 gaaacattct aaccttcaca gacag 25 910 25 DNA Homo sapiens 910 aaacattcta accttcacag acaga 25 911 25 DNA Homo sapiens 911 aacattctaa ccttcacaga cagac 25 912 25 DNA Homo sapiens 912 acattctaac cttcacagac agact 25 913 25 DNA Homo sapiens 913 cattctaacc ttcacagaca gactg 25 914 25 DNA Homo sapiens 914 attctaacct tcacagacag actgg 25 915 25 DNA Homo sapiens 915 ttctaacctt cacagacaga ctgga 25 916 25 DNA Homo sapiens 916 tctaaccttc acagacagac tggag 25 917 25 DNA Homo sapiens 917 ctaaccttca cagacagact ggagg 25 918 25 DNA Homo sapiens 918 taaccttcac agacagactg gaggc 25 919 25 DNA Homo sapiens 919 aaccttcaca gacagactgg aggct 25 920 25 DNA Homo sapiens 920 accttcacag acagactgga ggctg 25 921 25 DNA Homo sapiens 921 ccttcacaga cagactggag gctgg 25 922 25 DNA Homo sapiens 922 cttcacagac agactggagg ctgga 25 923 25 DNA Homo sapiens 923 ttcacagaca gactggaggc tggat 25 924 25 DNA Homo sapiens 924 tcacagacag actggaggct ggatg 25 925 25 DNA Homo sapiens 925 cacagacaga ctggaggctg gatgg 25 926 25 DNA Homo sapiens 926 acagacagac tggaggctgg atggg 25 927 25 DNA Homo sapiens 927 cagacagact ggaggctgga tgggg 25 928 25 DNA Homo sapiens 928 agacagactg gaggctggat gggga 25 929 25 DNA Homo sapiens 929 gacagactgg aggctggatg gggac 25 930 25 DNA Homo sapiens 930 acagactgga ggctggatgg ggacc 25 931 25 DNA Homo sapiens 931 cagactggag gctggatggg gacct 25 932 25 DNA Homo sapiens 932 agactggagg ctggatgggg acctg 25 933 25 DNA Homo sapiens 933 gactggaggc tggatgggga cctgg 25 934 25 DNA Homo sapiens 934 actggaggct ggatggggac ctggc 25 935 25 DNA Homo sapiens 935 ctggaggctg gatggggacc tggct 25 936 25 DNA Homo sapiens 936 tggaggctgg atggggacct ggctg 25 937 25 DNA Homo sapiens 937 ggaggctgga tggggacctg gctga 25 938 25 DNA Homo sapiens 938 gaggctggat ggggacctgg ctgaa 25 939 25 DNA Homo sapiens 939 aggctggatg gggacctggc tgaag 25 940 25 DNA Homo sapiens 940 ggctggatgg ggacctggct gaaga 25 941 25 DNA Homo sapiens 941 gctggatggg gacctggctg aagac 25 942 25 DNA Homo sapiens 942 ctggatgggg acctggctga agaca 25 943 25 DNA Homo sapiens 943 tggatgggga cctggctgaa gacat 25 944 25 DNA Homo sapiens 944 ggatggggac ctggctgaag acatc 25 945 25 DNA Homo sapiens 945 gatggggacc tggctgaaga catct 25 946 25 DNA Homo sapiens 946 atggggacct ggctgaagac atctg 25 947 25 DNA Homo sapiens 947 tggggacctg gctgaagaca tctgg 25 948 25 DNA Homo sapiens 948 ggggacctgg ctgaagacat ctgga 25 949 25 DNA Homo sapiens 949 gggacctggc tgaagacatc tggag 25 950 25 DNA Homo sapiens 950 ggacctggct gaagacatct ggaga 25 951 25 DNA Homo sapiens 951 gacctggctg aagacatctg gagaa 25 952 25 DNA Homo sapiens 952 acctggctga agacatctgg agaat 25 953 25 DNA Homo sapiens 953 cctggctgaa gacatctgga gaatg 25 954 25 DNA Homo sapiens 954 ctggctgaag acatctggag aatga 25 955 25 DNA Homo sapiens 955 tggctgaaga catctggaga atgaa 25 956 25 DNA Homo sapiens 956 ggctgaagac atctggagaa tgaaa 25 957 25 DNA Homo sapiens 957 gctgaagaca tctggagaat gaaag 25 958 25 DNA Homo sapiens 958 ctgaagacat ctggagaatg aaagt 25 959 25 DNA Homo sapiens 959 tgaagacatc tggagaatga aagtt 25 960 25 DNA Homo sapiens 960 gaagacatct ggagaatgaa agtta 25 961 25 DNA Homo sapiens 961 aagacatctg gagaatgaaa gttaa 25 962 25 DNA Homo sapiens 962 agacatctgg agaatgaaag ttaag 25 963 25 DNA Homo sapiens 963 gacatctgga gaatgaaagt taagt 25 964 25 DNA Homo sapiens 964 acatctggag aatgaaagtt aagta 25 965 25 DNA Homo sapiens 965 catctggaga atgaaagtta agtac 25 966 25 DNA Homo sapiens 966 atctggagaa tgaaagttaa gtacc 25 967 25 DNA Homo sapiens 967 tctggagaat gaaagttaag tacca 25 968 25 DNA Homo sapiens 968 ctggagaatg aaagttaagt accag 25 969 25 DNA Homo sapiens 969 tggagaatga aagttaagta ccagc 25 970 25 DNA Homo sapiens 970 ggagaatgaa agttaagtac cagct 25 971 25 DNA Homo sapiens 971 gagaatgaaa gttaagtacc agctt 25 972 25 DNA Homo sapiens 972 agaatgaaag ttaagtacca gcttg 25 973 25 DNA Homo sapiens 973 gaatgaaagt taagtaccag cttgc 25 974 25 DNA Homo sapiens 974 aatgaaagtt aagtaccagc ttgca 25 975 25 DNA Homo sapiens 975 atgaaagtta agtaccagct tgcat 25 976 25 DNA Homo sapiens 976 tgaaagttaa gtaccagctt gcatt 25 977 25 DNA Homo sapiens 977 gaaagttaag taccagcttg cattt 25 978 25 DNA Homo sapiens 978 aaagttaagt accagcttgc atttt 25 979 25 DNA Homo sapiens 979 aagttaagta ccagcttgca ttttt 25 980 25 DNA Homo sapiens 980 agttaagtac cagcttgcat ttttg 25 981 25 DNA Homo sapiens 981 gttaagtacc agcttgcatt tttgt 25 982 25 DNA Homo sapiens 982 ttaagtacca gcttgcattt ttgtg 25 983 25 DNA Homo sapiens 983 taagtaccag cttgcatttt tgtgc 25 984 25 DNA Homo sapiens 984 aagtaccagc ttgcattttt gtgcc 25 985 25 DNA Homo sapiens 985 agtaccagct tgcatttttg tgccc 25 986 25 DNA Homo sapiens 986 gtaccagctt gcatttttgt gcccc 25 987 25 DNA Homo sapiens 987 taccagcttg catttttgtg cccct 25 988 25 DNA Homo sapiens 988 accagcttgc atttttgtgc cccta 25 989 25 DNA Homo sapiens 989 ccagcttgca tttttgtgcc cctag 25 990 25 DNA Homo sapiens 990 cagcttgcat ttttgtgccc ctaga 25 991 25 DNA Homo sapiens 991 agcttgcatt tttgtgcccc tagat 25 992 25 DNA Homo sapiens 992 gcttgcattt ttgtgcccct agatt 25 993 25 DNA Homo sapiens 993 cttgcatttt tgtgccccta gatta 25 994 25 DNA Homo sapiens 994 ttgcattttt gtgcccctag attat 25 995 25 DNA Homo sapiens 995 tgcatttttg tgcccctaga ttatt 25 996 25 DNA Homo sapiens 996 gcatttttgt gcccctagat tattt 25 997 25 DNA Homo sapiens 997 catttttgtg cccctagatt atttt 25 998 25 DNA Homo sapiens 998 atttttgtgc ccctagatta ttttt 25 999 25 DNA Homo sapiens 999 tttttgtgcc cctagattat ttttg 25 1000 25 DNA Homo sapiens 1000 ttttgtgccc ctagattatt tttgc 25 1001 25 DNA Homo sapiens 1001 tttgtgcccc tagattattt ttgca 25 1002 25 DNA Homo sapiens 1002 ttgtgcccct agattatttt tgcat 25 1003 25 DNA Homo sapiens 1003 tgtgccccta gattattttt gcatt 25 1004 25 DNA Homo sapiens 1004 gtgcccctag attatttttg cattt 25 1005 25 DNA Homo sapiens 1005 tgcccctaga ttatttttgc atttt 25 1006 25 DNA Homo sapiens 1006 gcccctagat tatttttgca tttta 25 1007 25 DNA Homo sapiens 1007 cccctagatt atttttgcat tttaa 25 1008 25 DNA Homo sapiens 1008 ccctagatta tttttgcatt ttaaa 25 1009 25 DNA Homo sapiens 1009 cctagattat ttttgcattt taaaa 25 1010 25 DNA Homo sapiens 1010 ctagattatt tttgcatttt aaaat 25 1011 25 DNA Homo sapiens 1011 tagattattt ttgcatttta aaata 25 1012 25 DNA Homo sapiens 1012 agattatttt tgcattttaa aataa 25 1013 25 DNA Homo sapiens 1013 gattattttt gcattttaaa ataag 25 1014 25 DNA Homo sapiens 1014 attatttttg cattttaaaa taaga 25 1015 25 DNA Homo sapiens 1015 ttatttttgc attttaaaat aagaa 25 1016 25 DNA Homo sapiens 1016 tatttttgca ttttaaaata agaag 25 1017 25 DNA Homo sapiens 1017 atttttgcat tttaaaataa gaagc 25 1018 25 DNA Homo sapiens 1018 tttttgcatt ttaaaataag aagca 25 1019 25 DNA Homo sapiens 1019 ttttgcattt taaaataaga agcat 25 1020 25 DNA Homo sapiens 1020 tttgcatttt aaaataagaa gcatc 25 1021 25 DNA Homo sapiens 1021 ttgcatttta aaataagaag catca 25 1022 25 DNA Homo sapiens 1022 tgcattttaa aataagaagc atcaa 25 1023 25 DNA Homo sapiens 1023 gcattttaaa ataagaagca tcaaa 25 1024 25 DNA Homo sapiens 1024 cattttaaaa taagaagcat caaat 25 1025 25 DNA Homo sapiens 1025 attttaaaat aagaagcatc aaatt 25 1026 25 DNA Homo sapiens 1026 ttttaaaata agaagcatca aattg 25 1027 25 DNA Homo sapiens 1027 tttaaaataa gaagcatcaa attgc 25 1028 25 DNA Homo sapiens 1028 ttaaaataag aagcatcaaa ttgcg 25 1029 25 DNA Homo sapiens 1029 taaaataaga agcatcaaat tgcgt 25 1030 25 DNA Homo sapiens 1030 aaaataagaa gcatcaaatt gcgtg 25 1031 25 DNA Homo sapiens 1031 aaataagaag catcaaattg cgtgt 25 1032 25 DNA Homo sapiens 1032 aataagaagc atcaaattgc gtgtc 25 1033 25 DNA Homo sapiens 1033 ataagaagca tcaaattgcg tgtct 25 1034 25 DNA Homo sapiens 1034 taagaagcat caaattgcgt gtctc 25 1035 25 DNA Homo sapiens 1035 aagaagcatc aaattgcgtg tctct 25 1036 25 DNA Homo sapiens 1036 agaagcatca aattgcgtgt ctctg 25 1037 25 DNA Homo sapiens 1037 gaagcatcaa attgcgtgtc tctgt 25 1038 25 DNA Homo sapiens 1038 aagcatcaaa ttgcgtgtct ctgtg 25 1039 25 DNA Homo sapiens 1039 agcatcaaat tgcgtgtctc tgtgt 25 1040 25 DNA Homo sapiens 1040 gcatcaaatt gcgtgtctct gtgta 25 1041 25 DNA Homo sapiens 1041 catcaaattg cgtgtctctg tgtaa 25 1042 25 DNA Homo sapiens 1042 atcaaattgc gtgtctctgt gtaaa 25 1043 25 DNA Homo sapiens 1043 tcaaattgcg tgtctctgtg taaaa 25 1044 25 DNA Homo sapiens 1044 caaattgcgt gtctctgtgt aaaag 25 1045 25 DNA Homo sapiens 1045 aaattgcgtg tctctgtgta aaagt 25 1046 25 DNA Homo sapiens 1046 aattgcgtgt ctctgtgtaa aagtt 25 1047 25 DNA Homo sapiens 1047 attgcgtgtc tctgtgtaaa agttc 25 1048 25 DNA Homo sapiens 1048 ttgcgtgtct ctgtgtaaaa gttct 25 1049 25 DNA Homo sapiens 1049 tgcgtgtctc tgtgtaaaag ttcta 25 1050 25 DNA Homo sapiens 1050 gcgtgtctct gtgtaaaagt tctag 25 1051 25 DNA Homo sapiens 1051 cgtgtctctg tgtaaaagtt ctagc 25 1052 25 DNA Homo sapiens 1052 gtgtctctgt gtaaaagttc tagca 25 1053 25 DNA Homo sapiens 1053 tgtctctgtg taaaagttct agcaa 25 1054 25 DNA Homo sapiens 1054 gtctctgtgt aaaagttcta gcaat 25 1055 25 DNA Homo sapiens 1055 tctctgtgta aaagttctag caatt 25 1056 25 DNA Homo sapiens 1056 ctctgtgtaa aagttctagc aattt 25 1057 25 DNA Homo sapiens 1057 tctgtgtaaa agttctagca atttg 25 1058 25 DNA Homo sapiens 1058 ctgtgtaaaa gttctagcaa tttgt 25 1059 25 DNA Homo sapiens 1059 tgtgtaaaag ttctagcaat ttgtt 25 1060 25 DNA Homo sapiens 1060 gtgtaaaagt tctagcaatt tgttt 25 1061 25 DNA Homo sapiens 1061 tgtaaaagtt ctagcaattt gtttt 25 1062 25 DNA Homo sapiens 1062 gtaaaagttc tagcaatttg tttta 25 1063 25 DNA Homo sapiens 1063 taaaagttct agcaatttgt tttaa 25 1064 25 DNA Homo sapiens 1064 aaaagttcta gcaatttgtt ttaag 25 1065 25 DNA Homo sapiens 1065 aaagttctag caatttgttt taagg 25 1066 25 DNA Homo sapiens 1066 aagttctagc aatttgtttt aaggt 25 1067 25 DNA Homo sapiens 1067 agttctagca atttgtttta aggtg 25 1068 25 DNA Homo sapiens 1068 gttctagcaa tttgttttaa ggtga 25 1069 25 DNA Homo sapiens 1069 ttctagcaat ttgttttaag gtgaa 25 1070 25 DNA Homo sapiens 1070 tctagcaatt tgttttaagg tgaac 25 1071 25 DNA Homo sapiens 1071 ctagcaattt gttttaaggt gaact 25 1072 25 DNA Homo sapiens 1072 tagcaatttg ttttaaggtg aactt 25 1073 25 DNA Homo sapiens 1073 agcaatttgt tttaaggtga actta 25 1074 25 DNA Homo sapiens 1074 gcaatttgtt ttaaggtgaa cttat 25 1075 25 DNA Homo sapiens 1075 caatttgttt taaggtgaac ttatt 25 1076 25 DNA Homo sapiens 1076 aatttgtttt aaggtgaact tattt 25 1077 25 DNA Homo sapiens 1077 atttgtttta aggtgaactt atttt 25 1078 25 DNA Homo sapiens 1078 tttgttttaa ggtgaactta ttttg 25 1079 25 DNA Homo sapiens 1079 ttgttttaag gtgaacttat tttgg 25 1080 25 DNA Homo sapiens 1080 tgttttaagg tgaacttatt ttggc 25 1081 25 DNA Homo sapiens 1081 gttttaaggt gaacttattt tggct 25 1082 25 DNA Homo sapiens 1082 ttttaaggtg aacttatttt ggctt 25 1083 25 DNA Homo sapiens 1083 tttaaggtga acttattttg gctta 25 1084 25 DNA Homo sapiens 1084 ttaaggtgaa cttattttgg cttag 25 1085 25 DNA Homo sapiens 1085 taaggtgaac ttattttggc ttagg 25 1086 25 DNA Homo sapiens 1086 aaggtgaact tattttggct taggg 25 1087 25 DNA Homo sapiens 1087 aggtgaactt attttggctt aggga 25 1088 25 DNA Homo sapiens 1088 ggtgaactta ttttggctta gggac 25 1089 25 DNA Homo sapiens 1089 gtgaacttat tttggcttag ggact 25 1090 25 DNA Homo sapiens 1090 tgaacttatt ttggcttagg gacta 25 1091 25 DNA Homo sapiens 1091 gaacttattt tggcttaggg actac 25 1092 25 DNA Homo sapiens 1092 aacttatttt ggcttaggga ctaca 25 1093 25 DNA Homo sapiens 1093 acttattttg gcttagggac tacaa 25 1094 25 DNA Homo sapiens 1094 cttattttgg cttagggact acaaa 25 1095 25 DNA Homo sapiens 1095 ttattttggc ttagggacta caaaa 25 1096 25 DNA Homo sapiens 1096 tattttggct tagggactac aaaaa 25 1097 25 DNA Homo sapiens 1097 attttggctt agggactaca aaaag 25 1098 25 DNA Homo sapiens 1098 ttttggctta gggactacaa aaaga 25 1099 25 DNA Homo sapiens 1099 tttggcttag ggactacaaa aagag 25 1100 25 DNA Homo sapiens 1100 ttggcttagg gactacaaaa agaga 25 1101 25 DNA Homo sapiens 1101 tggcttaggg actacaaaaa gagaa 25 1102 25 DNA Homo sapiens 1102 ggcttaggga ctacaaaaag agaag 25 1103 25 DNA Homo sapiens 1103 gcttagggac tacaaaaaga gaagg 25 1104 25 DNA Homo sapiens 1104 cttagggact acaaaaagag aaggt 25 1105 25 DNA Homo sapiens 1105 ttagggacta caaaaagaga aggta 25 1106 25 DNA Homo sapiens 1106 tagggactac aaaaagagaa ggtaa 25 1107 25 DNA Homo sapiens 1107 agggactaca aaaagagaag gtaat 25 1108 25 DNA Homo sapiens 1108 gggactacaa aaagagaagg taatt 25 1109 25 DNA Homo sapiens 1109 ggactacaaa aagagaaggt aattc 25 1110 25 DNA Homo sapiens 1110 gactacaaaa agagaaggta attcc 25 1111 25 DNA Homo sapiens 1111 actacaaaaa gagaaggtaa ttcct 25 1112 25 DNA Homo sapiens 1112 ctacaaaaag agaaggtaat tccta 25 1113 25 DNA Homo sapiens 1113 tacaaaaaga gaaggtaatt cctag 25 1114 25 DNA Homo sapiens 1114 acaaaaagag aaggtaattc ctagg 25 1115 25 DNA Homo sapiens 1115 caaaaagaga aggtaattcc taggg 25 1116 25 DNA Homo sapiens 1116 aaaaagagaa ggtaattcct aggga 25 1117 25 DNA Homo sapiens 1117 aaaagagaag gtaattccta gggaa 25 1118 25 DNA Homo sapiens 1118 aaagagaagg taattcctag ggaag 25 1119 25 DNA Homo sapiens 1119 aagagaaggt aattcctagg gaagg 25 1120 25 DNA Homo sapiens 1120 agagaaggta attcctaggg aagga 25 1121 25 DNA Homo sapiens 1121 gagaaggtaa ttcctaggga aggaa 25 1122 25 DNA Homo sapiens 1122 agaaggtaat tcctagggaa ggaag 25 1123 25 DNA Homo sapiens 1123 gaaggtaatt cctagggaag gaaga 25 1124 25 DNA Homo sapiens 1124 aaggtaattc ctagggaagg aagaa 25 1125 25 DNA Homo sapiens 1125 aggtaattcc tagggaagga agaag 25 1126 25 DNA Homo sapiens 1126 ggtaattcct agggaaggaa gaaga 25 1127 25 DNA Homo sapiens 1127 gtaattccta gggaaggaag aagag 25 1128 25 DNA Homo sapiens 1128 taattcctag ggaaggaaga agaga 25 1129 25 DNA Homo sapiens 1129 aattcctagg gaaggaagaa gagaa 25 1130 25 DNA Homo sapiens 1130 attcctaggg aaggaagaag agaaa 25 1131 25 DNA Homo sapiens 1131 ttcctaggga aggaagaaga gaaag 25 1132 25 DNA Homo sapiens 1132 tcctagggaa ggaagaagag aaaga 25 1133 25 DNA Homo sapiens 1133 cctagggaag gaagaagaga aagaa 25 1134 25 DNA Homo sapiens 1134 ctagggaagg aagaagagaa agaaa 25 1135 25 DNA Homo sapiens 1135 tagggaagga agaagagaaa gaaat 25 1136 25 DNA Homo sapiens 1136 agggaaggaa gaagagaaag aaatg 25 1137 25 DNA Homo sapiens 1137 gggaaggaag aagagaaaga aatga 25 1138 25 DNA Homo sapiens 1138 ggaaggaaga agagaaagaa atgaa 25 1139 25 DNA Homo sapiens 1139 gaaggaagaa gagaaagaaa tgaaa 25 1140 25 DNA Homo sapiens 1140 aaggaagaag agaaagaaat gaaaa 25 1141 25 DNA Homo sapiens 1141 aggaagaaga gaaagaaatg aaaat 25 1142 25 DNA Homo sapiens 1142 ggaagaagag aaagaaatga aaatt 25 1143 25 DNA Homo sapiens 1143 gaagaagaga aagaaatgaa aatta 25 1144 25 DNA Homo sapiens 1144 aagaagagaa agaaatgaaa attag 25 1145 25 DNA Homo sapiens 1145 agaagagaaa gaaatgaaaa ttaga 25 1146 25 DNA Homo sapiens 1146 gaagagaaag aaatgaaaat tagag 25 1147 25 DNA Homo sapiens 1147 aagagaaaga aatgaaaatt agaga 25 1148 25 DNA Homo sapiens 1148 agagaaagaa atgaaaatta gagaa 25 1149 25 DNA Homo sapiens 1149 gagaaagaaa tgaaaattag agaat 25 1150 25 DNA Homo sapiens 1150 agaaagaaat gaaaattaga gaata 25 1151 25 DNA Homo sapiens 1151 gaaagaaatg aaaattagag aataa 25 1152 25 DNA Homo sapiens 1152 aaagaaatga aaattagaga ataag 25 1153 25 DNA Homo sapiens 1153 aagaaatgaa aattagagaa taaga 25 1154 25 DNA Homo sapiens 1154 agaaatgaaa attagagaat aagat 25 1155 25 DNA Homo sapiens 1155 gaaatgaaaa ttagagaata agatt 25 1156 25 DNA Homo sapiens 1156 aaatgaaaat tagagaataa gatta 25 1157 25 DNA Homo sapiens 1157 aatgaaaatt agagaataag attat 25 1158 25 DNA Homo sapiens 1158 atgaaaatta gagaataaga ttatt 25 1159 25 DNA Homo sapiens 1159 tgaaaattag agaataagat tattt 25 1160 25 DNA Homo sapiens 1160 gaaaattaga gaataagatt atttt 25 1161 25 DNA Homo sapiens 1161 aaaattagag aataagatta ttttg 25 1162 25 DNA Homo sapiens 1162 aaattagaga ataagattat tttga 25 1163 25 DNA Homo sapiens 1163 aattagagaa taagattatt ttgaa 25 1164 25 DNA Homo sapiens 1164 attagagaat aagattattt tgaat 25 1165 25 DNA Homo sapiens 1165 ttagagaata agattatttt gaatg 25 1166 25 DNA Homo sapiens 1166 tagagaataa gattattttg aatga 25 1167 25 DNA Homo sapiens 1167 agagaataag attattttga atgac 25 1168 25 DNA Homo sapiens 1168 gagaataaga ttattttgaa tgact 25 1169 25 DNA Homo sapiens 1169 agaataagat tattttgaat gactt 25 1170 25 DNA Homo sapiens 1170 gaataagatt attttgaatg acttc 25 1171 25 DNA Homo sapiens 1171 aataagatta ttttgaatga cttca 25 1172 25 DNA Homo sapiens 1172 ataagattat tttgaatgac ttcag 25 1173 25 DNA Homo sapiens 1173 taagattatt ttgaatgact tcagg 25 1174 25 DNA Homo sapiens 1174 aagattattt tgaatgactt caggt 25 1175 25 DNA Homo sapiens 1175 agattatttt gaatgacttc aggta 25 1176 25 DNA Homo sapiens 1176 gattattttg aatgacttca ggtag 25 1177 25 DNA Homo sapiens 1177 attattttga atgacttcag gtagc 25 1178 25 DNA Homo sapiens 1178 ttattttgaa tgacttcagg tagcg 25 1179 25 DNA Homo sapiens 1179 tattttgaat gacttcaggt agcga 25 1180 25 DNA Homo sapiens 1180 attttgaatg acttcaggta gcgag 25 1181 25 DNA Homo sapiens 1181 ttttgaatga cttcaggtag cgagg 25 1182 25 DNA Homo sapiens 1182 tttgaatgac ttcaggtagc gagga 25 1183 25 DNA Homo sapiens 1183 ttgaatgact tcaggtagcg aggag 25 1184 25 DNA Homo sapiens 1184 tgaatgactt caggtagcga ggagt 25 1185 25 DNA Homo sapiens 1185 gaatgacttc aggtagcgag gagtg 25 1186 25 DNA Homo sapiens 1186 aatgacttca ggtagcgagg agtgt 25 1187 25 DNA Homo sapiens 1187 atgacttcag gtagcgagga gtgtg 25 1188 25 DNA Homo sapiens 1188 tgacttcagg tagcgaggag tgtgt 25 1189 25 DNA Homo sapiens 1189 gacttcaggt agcgaggagt gtgtg 25 1190 25 DNA Homo sapiens 1190 acttcaggta gcgaggagtg tgtgt 25 1191 25 DNA Homo sapiens 1191 cttcaggtag cgaggagtgt gtgtt 25 1192 25 DNA Homo sapiens 1192 ttcaggtagc gaggagtgtg tgttt 25 1193 25 DNA Homo sapiens 1193 tcaggtagcg aggagtgtgt gtttg 25 1194 25 DNA Homo sapiens 1194 caggtagcga ggagtgtgtg tttgt 25 1195 25 DNA Homo sapiens 1195 aggtagcgag gagtgtgtgt ttgtg 25 1196 25 DNA Homo sapiens 1196 ggtagcgagg agtgtgtgtt tgtga 25 1197 25 DNA Homo sapiens 1197 gtagcgagga gtgtgtgttt gtgag 25 1198 25 DNA Homo sapiens 1198 tagcgaggag tgtgtgtttg tgagt 25 1199 25 DNA Homo sapiens 1199 agcgaggagt gtgtgtttgt gagtg 25 1200 25 DNA Homo sapiens 1200 gcgaggagtg tgtgtttgtg agtgt 25 1201 25 DNA Homo sapiens 1201 cgaggagtgt gtgtttgtga gtgtg 25 1202 25 DNA Homo sapiens 1202 gaggagtgtg tgtttgtgag tgtgt 25 1203 25 DNA Homo sapiens 1203 aggagtgtgt gtttgtgagt gtgta 25 1204 25 DNA Homo sapiens 1204 ggagtgtgtg tttgtgagtg tgtat 25 1205 25 DNA Homo sapiens 1205 gagtgtgtgt ttgtgagtgt gtatt 25 1206 25 DNA Homo sapiens 1206 agtgtgtgtt tgtgagtgtg tattt 25 1207 25 DNA Homo sapiens 1207 gtgtgtgttt gtgagtgtgt atttg 25 1208 25 DNA Homo sapiens 1208 tgtgtgtttg tgagtgtgta tttga 25 1209 25 DNA Homo sapiens 1209 gtgtgtttgt gagtgtgtat ttgag 25 1210 25 DNA Homo sapiens 1210 tgtgtttgtg agtgtgtatt tgaga 25 1211 25 DNA Homo sapiens 1211 gtgtttgtga gtgtgtattt gagag 25 1212 25 DNA Homo sapiens 1212 tgtttgtgag tgtgtatttg agaga 25 1213 25 DNA Homo sapiens 1213 gtttgtgagt gtgtatttga gagac 25 1214 25 DNA Homo sapiens 1214 tttgtgagtg tgtatttgag agact 25 1215 25 DNA Homo sapiens 1215 ttgtgagtgt gtatttgaga gactt 25 1216 25 DNA Homo sapiens 1216 tgtgagtgtg tatttgagag acttg 25 1217 25 DNA Homo sapiens 1217 gtgagtgtgt atttgagaga cttgg 25 1218 25 DNA Homo sapiens 1218 tgagtgtgta tttgagagac ttggc 25 1219 25 DNA Homo sapiens 1219 gagtgtgtat ttgagagact tggct 25 1220 25 DNA Homo sapiens 1220 agtgtgtatt tgagagactt ggctc 25 1221 25 DNA Homo sapiens 1221 gtgtgtattt gagagacttg gctca 25 1222 25 DNA Homo sapiens 1222 tgtgtatttg agagacttgg ctcat 25 1223 25 DNA Homo sapiens 1223 gtgtatttga gagacttggc tcatg 25 1224 25 DNA Homo sapiens 1224 tgtatttgag agacttggct catgc 25 1225 25 DNA Homo sapiens 1225 gtatttgaga gacttggctc atgcc 25 1226 25 DNA Homo sapiens 1226 tatttgagag acttggctca tgcct 25 1227 25 DNA Homo sapiens 1227 atttgagaga cttggctcat gcctg 25 1228 25 DNA Homo sapiens 1228 tttgagagac ttggctcatg cctgt 25 1229 25 DNA Homo sapiens 1229 ttgagagact tggctcatgc ctgtg 25 1230 25 DNA Homo sapiens 1230 tgagagactt ggctcatgcc tgtgg 25 1231 25 DNA Homo sapiens 1231 gagagacttg gctcatgcct gtggg 25 1232 25 DNA Homo sapiens 1232 agagacttgg ctcatgcctg tgggt 25 1233 25 DNA Homo sapiens 1233 gagacttggc tcatgcctgt gggtc 25 1234 25 DNA Homo sapiens 1234 agacttggct catgcctgtg ggtct 25 1235 25 DNA Homo sapiens 1235 gacttggctc atgcctgtgg gtctt 25 1236 25 DNA Homo sapiens 1236 acttggctca tgcctgtggg tcttc 25 1237 25 DNA Homo sapiens 1237 cttggctcat gcctgtgggt cttct 25 1238 25 DNA Homo sapiens 1238 ttggctcatg cctgtgggtc ttctc 25 1239 25 DNA Homo sapiens 1239 tggctcatgc ctgtgggtct tctct 25 1240 25 DNA Homo sapiens 1240 ggctcatgcc tgtgggtctt ctctt 25 1241 25 DNA Homo sapiens 1241 gctcatgcct gtgggtcttc tcttc 25 1242 25 DNA Homo sapiens 1242 ctcatgcctg tgggtcttct cttct 25 1243 25 DNA Homo sapiens 1243 tcatgcctgt gggtcttctc ttcta 25 1244 25 DNA Homo sapiens 1244 catgcctgtg ggtcttctct tctag 25 1245 25 DNA Homo sapiens 1245 atgcctgtgg gtcttctctt ctagt 25 1246 25 DNA Homo sapiens 1246 tgcctgtggg tcttctcttc tagta 25 1247 25 DNA Homo sapiens 1247 gcctgtgggt cttctcttct agtat 25 1248 25 DNA Homo sapiens 1248 cctgtgggtc ttctcttcta gtatc 25 1249 25 DNA Homo sapiens 1249 ctgtgggtct tctcttctag tatca 25 1250 25 DNA Homo sapiens 1250 tgtgggtctt ctcttctagt atcag 25 1251 25 DNA Homo sapiens 1251 gtgggtcttc tcttctagta tcagt 25 1252 25 DNA Homo sapiens 1252 tgggtcttct cttctagtat cagtg 25 1253 25 DNA Homo sapiens 1253 gggtcttctc ttctagtatc agtga 25 1254 25 DNA Homo sapiens 1254 ggtcttctct tctagtatca gtgag 25 1255 25 DNA Homo sapiens 1255 gtcttctctt ctagtatcag tgagg 25 1256 25 DNA Homo sapiens 1256 tcttctcttc tagtatcagt gaggg 25 1257 25 DNA Homo sapiens 1257 cttctcttct agtatcagtg agggg 25 1258 25 DNA Homo sapiens 1258 ttctcttcta gtatcagtga gggga 25 1259 25 DNA Homo sapiens 1259 tctcttctag tatcagtgag gggag 25 1260 25 DNA Homo sapiens 1260 ctcttctagt atcagtgagg ggagg 25 1261 25 DNA Homo sapiens 1261 tcttctagta tcagtgaggg gaggg 25 1262 25 DNA Homo sapiens 1262 cttctagtat cagtgagggg aggga 25 1263 25 DNA Homo sapiens 1263 ttctagtatc agtgagggga gggat 25 1264 25 DNA Homo sapiens 1264 tctagtatca gtgaggggag ggatt 25 1265 25 DNA Homo sapiens 1265 ctagtatcag tgaggggagg gatta 25 1266 25 DNA Homo sapiens 1266 tagtatcagt gaggggaggg attac 25 1267 25 DNA Homo sapiens 1267 agtatcagtg aggggaggga ttact 25 1268 25 DNA Homo sapiens 1268 gtatcagtga ggggagggat tactg 25 1269 25 DNA Homo sapiens 1269 tatcagtgag gggagggatt actga 25 1270 25 DNA Homo sapiens 1270 atcagtgagg ggagggatta ctgaa 25 1271 25 DNA Homo sapiens 1271 tcagtgaggg gagggattac tgaag 25 1272 25 DNA Homo sapiens 1272 cagtgagggg agggattact gaaga 25 1273 25 DNA Homo sapiens 1273 agtgagggga gggattactg aagaa 25 1274 25 DNA Homo sapiens 1274 gtgaggggag ggattactga agaag 25 1275 25 DNA Homo sapiens 1275 tgaggggagg gattactgaa gaaga 25 1276 25 DNA Homo sapiens 1276 gaggggaggg attactgaag aagaa 25 1277 25 DNA Homo sapiens 1277 aggggaggga ttactgaaga agaag 25 1278 25 DNA Homo sapiens 1278 ggggagggat tactgaagaa gaagg 25 1279 25 DNA Homo sapiens 1279 gggagggatt actgaagaag aaggg 25 1280 25 DNA Homo sapiens 1280 ggagggatta ctgaagaaga agggg 25 1281 25 DNA Homo sapiens 1281 gagggattac tgaagaagaa ggggg 25 1282 25 DNA Homo sapiens 1282 agggattact gaagaagaag ggggg 25 1283 25 DNA Homo sapiens 1283 gggattactg aagaagaagg gggga 25 1284 25 DNA Homo sapiens 1284 ggattactga agaagaaggg gggaa 25 1285 25 DNA Homo sapiens 1285 gattactgaa gaagaagggg ggaaa 25 1286 25 DNA Homo sapiens 1286 attactgaag aagaaggggg gaaaa 25 1287 25 DNA Homo sapiens 1287 ttactgaaga agaagggggg aaaaa 25 1288 25 DNA Homo sapiens 1288 tactgaagaa gaagggggga aaaaa 25 1289 25 DNA Homo sapiens 1289 actgaagaag aaggggggaa aaaaa 25 1290 25 DNA Homo sapiens 1290 ctgaagaaga aggggggaaa aaaaa 25 1291 25 DNA Homo sapiens 1291 tgaagaagaa ggggggaaaa aaaaa 25 1292 25 DNA Homo sapiens 1292 gaagaagaag gggggaaaaa aaaag 25 1293 25 DNA Homo sapiens 1293 aagaagaagg ggggaaaaaa aaaga 25 1294 25 DNA Homo sapiens 1294 agaagaaggg gggaaaaaaa aagaa 25 1295 25 DNA Homo sapiens 1295 gaagaagggg ggaaaaaaaa agaaa 25 1296 25 DNA Homo sapiens 1296 aagaaggggg gaaaaaaaaa gaaag 25 1297 25 DNA Homo sapiens 1297 agaagggggg aaaaaaaaag aaaga 25 1298 25 DNA Homo sapiens 1298 gaagggggga aaaaaaaaga aagaa 25 1299 25 DNA Homo sapiens 1299 aaggggggaa aaaaaaagaa agaaa 25 1300 25 DNA Homo sapiens 1300 aggggggaaa aaaaaagaaa gaaat 25 1301 25 DNA Homo sapiens 1301 ggggggaaaa aaaaagaaag aaatc 25 1302 25 DNA Homo sapiens 1302 gggggaaaaa aaaagaaaga aatct 25 1303 25 DNA Homo sapiens 1303 ggggaaaaaa aaagaaagaa atctg 25 1304 25 DNA Homo sapiens 1304 gggaaaaaaa aagaaagaaa tctga 25 1305 25 DNA Homo sapiens 1305 ggaaaaaaaa agaaagaaat ctgag 25 1306 25 DNA Homo sapiens 1306 gaaaaaaaaa gaaagaaatc tgagc 25 1307 25 DNA Homo sapiens 1307 aaaaaaaaag aaagaaatct gagct 25 1308 25 DNA Homo sapiens 1308 aaaaaaaaga aagaaatctg agctt 25 1309 25 DNA Homo sapiens 1309 aaaaaaagaa agaaatctga gcttt 25 1310 25 DNA Homo sapiens 1310 aaaaaagaaa gaaatctgag ctttc 25 1311 25 DNA Homo sapiens 1311 aaaaagaaag aaatctgagc tttct 25 1312 25 DNA Homo sapiens 1312 aaaagaaaga aatctgagct ttctg 25 1313 25 DNA Homo sapiens 1313 aaagaaagaa atctgagctt tctgg 25 1314 25 DNA Homo sapiens 1314 aagaaagaaa tctgagcttt ctggg 25 1315 25 DNA Homo sapiens 1315 agaaagaaat ctgagctttc tggga 25 1316 25 DNA Homo sapiens 1316 gaaagaaatc tgagctttct gggag 25 1317 25 DNA Homo sapiens 1317 aaagaaatct gagctttctg ggagg 25 1318 25 DNA Homo sapiens 1318 aagaaatctg agctttctgg gagga 25 1319 25 DNA Homo sapiens 1319 agaaatctga gctttctggg aggaa 25 1320 25 DNA Homo sapiens 1320 gaaatctgag ctttctggga ggaaa 25 1321 25 DNA Homo sapiens 1321 aaatctgagc tttctgggag gaaat 25 1322 25 DNA Homo sapiens 1322 aatctgagct ttctgggagg aaatt 25 1323 25 DNA Homo sapiens 1323 atctgagctt tctgggagga aattc 25 1324 25 DNA Homo sapiens 1324 tctgagcttt ctgggaggaa attca 25 1325 25 DNA Homo sapiens 1325 ctgagctttc tgggaggaaa ttcaa 25 1326 25 DNA Homo sapiens 1326 tgagctttct gggaggaaat tcaaa 25 1327 25 DNA Homo sapiens 1327 gagctttctg ggaggaaatt caaag 25 1328 25 DNA Homo sapiens 1328 agctttctgg gaggaaattc aaagg 25 1329 25 DNA Homo sapiens 1329 gctttctggg aggaaattca aagga 25 1330 25 DNA Homo sapiens 1330 ctttctggga ggaaattcaa aggaa 25 1331 25 DNA Homo sapiens 1331 tttctgggag gaaattcaaa ggaac 25 1332 25 DNA Homo sapiens 1332 ttctgggagg aaattcaaag gaacc 25 1333 25 DNA Homo sapiens 1333 tctgggagga aattcaaagg aacca 25 1334 25 DNA Homo sapiens 1334 ctgggaggaa attcaaagga accaa 25 1335 25 DNA Homo sapiens 1335 tgggaggaaa ttcaaaggaa ccaag 25 1336 25 DNA Homo sapiens 1336 gggaggaaat tcaaaggaac caaga 25 1337 25 DNA Homo sapiens 1337 ggaggaaatt caaaggaacc aagag 25 1338 25 DNA Homo sapiens 1338 gaggaaattc aaaggaacca agaga 25 1339 25 DNA Homo sapiens 1339 aggaaattca aaggaaccaa gagaa 25 1340 25 DNA Homo sapiens 1340 ggaaattcaa aggaaccaag agaaa 25 1341 25 DNA Homo sapiens 1341 gaaattcaaa ggaaccaaga gaaat 25 1342 25 DNA Homo sapiens 1342 aaattcaaag gaaccaagag aaatt 25 1343 25 DNA Homo sapiens 1343 aattcaaagg aaccaagaga aatta 25 1344 25 DNA Homo sapiens 1344 attcaaagga accaagagaa attaa 25 1345 25 DNA Homo sapiens 1345 ttcaaaggaa ccaagagaaa ttaac 25 1346 25 DNA Homo sapiens 1346 tcaaaggaac caagagaaat taact 25 1347 25 DNA Homo sapiens 1347 caaaggaacc aagagaaatt aactt 25 1348 25 DNA Homo sapiens 1348 aaaggaacca agagaaatta acttc 25 1349 25 DNA Homo sapiens 1349 aaggaaccaa gagaaattaa cttcg 25 1350 25 DNA Homo sapiens 1350 aggaaccaag agaaattaac ttcgt 25 1351 25 DNA Homo sapiens 1351 ggaaccaaga gaaattaact tcgtt 25 1352 25 DNA Homo sapiens 1352 gaaccaagag aaattaactt cgttc 25 1353 25 DNA Homo sapiens 1353 aaccaagaga aattaacttc gttct 25 1354 25 DNA Homo sapiens 1354 accaagagaa attaacttcg ttctg 25 1355 25 DNA Homo sapiens 1355 ccaagagaaa ttaacttcgt tctgc 25 1356 25 DNA Homo sapiens 1356 caagagaaat taacttcgtt ctgca 25 1357 25 DNA Homo sapiens 1357 aagagaaatt aacttcgttc tgcaa 25 1358 25 DNA Homo sapiens 1358 agagaaatta acttcgttct gcaag 25 1359 25 DNA Homo sapiens 1359 gagaaattaa cttcgttctg caagg 25 1360 25 DNA Homo sapiens 1360 agaaattaac ttcgttctgc aagga 25 1361 25 DNA Homo sapiens 1361 gaaattaact tcgttctgca aggac 25 1362 25 DNA Homo sapiens 1362 aaattaactt cgttctgcaa ggact 25 1363 25 DNA Homo sapiens 1363 aattaacttc gttctgcaag gacta 25 1364 25 DNA Homo sapiens 1364 attaacttcg ttctgcaagg actaa 25 1365 25 DNA Homo sapiens 1365 ttaacttcgt tctgcaagga ctaaa 25 1366 25 DNA Homo sapiens 1366 taacttcgtt ctgcaaggac taaag 25 1367 25 DNA Homo sapiens 1367 aacttcgttc tgcaaggact aaagt 25 1368 25 DNA Homo sapiens 1368 acttcgttct gcaaggacta aagta 25 1369 25 DNA Homo sapiens 1369 cttcgttctg caaggactaa agtac 25 1370 25 DNA Homo sapiens 1370 ttcgttctgc aaggactaaa gtaca 25 1371 25 DNA Homo sapiens 1371 tcgttctgca aggactaaag tacag 25 1372 25 DNA Homo sapiens 1372 cgttctgcaa ggactaaagt acagc 25 1373 25 DNA Homo sapiens 1373 gttctgcaag gactaaagta cagca 25 1374 25 DNA Homo sapiens 1374 ttctgcaagg actaaagtac agcaa 25 1375 25 DNA Homo sapiens 1375 tctgcaagga ctaaagtaca gcaag 25 1376 25 DNA Homo sapiens 1376 ctgcaaggac taaagtacag caaga 25 1377 25 DNA Homo sapiens 1377 tgcaaggact aaagtacagc aagag 25 1378 25 DNA Homo sapiens 1378 gcaaggacta aagtacagca agagg 25 1379 25 DNA Homo sapiens 1379 caaggactaa agtacagcaa gagga 25 1380 25 DNA Homo sapiens 1380 aaggactaaa gtacagcaag aggag 25 1381 25 DNA Homo sapiens 1381 aggactaaag tacagcaaga ggaga 25 1382 25 DNA Homo sapiens 1382 ggactaaagt acagcaagag gagag 25 1383 25 DNA Homo sapiens 1383 gactaaagta cagcaagagg agaga 25 1384 25 DNA Homo sapiens 1384 actaaagtac agcaagagga gagag 25 1385 25 DNA Homo sapiens 1385 ctaaagtaca gcaagaggag agagg 25 1386 25 DNA Homo sapiens 1386 taaagtacag caagaggaga gaggt 25 1387 25 DNA Homo sapiens 1387 aaagtacagc aagaggagag aggtc 25 1388 25 DNA Homo sapiens 1388 aagtacagca agaggagaga ggtca 25 1389 25 DNA Homo sapiens 1389 agtacagcaa gaggagagag gtcaa 25 1390 25 DNA Homo sapiens 1390 gtacagcaag aggagagagg tcaag 25 1391 25 DNA Homo sapiens 1391 tacagcaaga ggagagaggt caagc 25 1392 25 DNA Homo sapiens 1392 acagcaagag gagagaggtc aagcg 25 1393 25 DNA Homo sapiens 1393 cagcaagagg agagaggtca agcga 25 1394 25 DNA Homo sapiens 1394 agcaagagga gagaggtcaa gcgag 25 1395 25 DNA Homo sapiens 1395 gcaagaggag agaggtcaag cgaga 25 1396 25 DNA Homo sapiens 1396 caagaggaga gaggtcaagc gagaa 25 1397 25 DNA Homo sapiens 1397 aagaggagag aggtcaagcg agaag 25 1398 25 DNA Homo sapiens 1398 agaggagaga ggtcaagcga gaagc 25 1399 25 DNA Homo sapiens 1399 gaggagagag gtcaagcgag aagcg 25 1400 25 DNA Homo sapiens 1400 aggagagagg tcaagcgaga agcgt 25 1401 25 DNA Homo sapiens 1401 ggagagaggt caagcgagaa gcgtg 25 1402 25 DNA Homo sapiens 1402 gagagaggtc aagcgagaag cgtgc 25 1403 25 DNA Homo sapiens 1403 agagaggtca agcgagaagc gtgcg 25 1404 25 DNA Homo sapiens 1404 gagaggtcaa gcgagaagcg tgcgg 25 1405 25 DNA Homo sapiens 1405 agaggtcaag cgagaagcgt gcggg 25 1406 25 DNA Homo sapiens 1406 gaggtcaagc gagaagcgtg cggga 25 1407 25 DNA Homo sapiens 1407 aggtcaagcg agaagcgtgc gggaa 25 1408 25 DNA Homo sapiens 1408 ggtcaagcga gaagcgtgcg ggaag 25 1409 25 DNA Homo sapiens 1409 gtcaagcgag aagcgtgcgg gaagc 25 1410 25 DNA Homo sapiens 1410 tcaagcgaga agcgtgcggg aagca 25 1411 25 DNA Homo sapiens 1411 caagcgagaa gcgtgcggga agcac 25 1412 25 DNA Homo sapiens 1412 aagcgagaag cgtgcgggaa gcaca 25 1413 25 DNA Homo sapiens 1413 agcgagaagc gtgcgggaag cacat 25 1414 25 DNA Homo sapiens 1414 gcgagaagcg tgcgggaagc acatg 25 1415 25 DNA Homo sapiens 1415 cgagaagcgt gcgggaagca catgc 25 1416 25 DNA Homo sapiens 1416 gagaagcgtg cgggaagcac atgcc 25 1417 25 DNA Homo sapiens 1417 agaagcgtgc gggaagcaca tgccc 25 1418 25 DNA Homo sapiens 1418 gaagcgtgcg ggaagcacat gccct 25 1419 25 DNA Homo sapiens 1419 aagcgtgcgg gaagcacatg ccctg 25 1420 25 DNA Homo sapiens 1420 agcgtgcggg aagcacatgc cctgg 25 1421 25 DNA Homo sapiens 1421 gcgtgcggga agcacatgcc ctggg 25 1422 25 DNA Homo sapiens 1422 cgtgcgggaa gcacatgccc tgggg 25 1423 25 DNA Homo sapiens 1423 gtgcgggaag cacatgccct gggga 25 1424 25 DNA Homo sapiens 1424 tgcgggaagc acatgccctg gggag 25 1425 25 DNA Homo sapiens 1425 gcgggaagca catgccctgg ggagg 25 1426 25 DNA Homo sapiens 1426 cgggaagcac atgccctggg gaggc 25 1427 25 DNA Homo sapiens 1427 gggaagcaca tgccctgggg aggca 25 1428 25 DNA Homo sapiens 1428 ggaagcacat gccctgggga ggcat 25 1429 25 DNA Homo sapiens 1429 gaagcacatg ccctggggag gcata 25 1430 25 DNA Homo sapiens 1430 aagcacatgc cctggggagg catag 25 1431 25 DNA Homo sapiens 1431 agcacatgcc ctggggaggc ataga 25 1432 25 DNA Homo sapiens 1432 gcacatgccc tggggaggca tagaa 25 1433 25 DNA Homo sapiens 1433 cacatgccct ggggaggcat agaag 25 1434 25 DNA Homo sapiens 1434 acatgccctg gggaggcata gaagc 25 1435 25 DNA Homo sapiens 1435 catgccctgg ggaggcatag aagcc 25 1436 25 DNA Homo sapiens 1436 atgccctggg gaggcataga agcca 25 1437 25 DNA Homo sapiens 1437 tgccctgggg aggcatagaa gccac 25 1438 25 DNA Homo sapiens 1438 gccctgggga ggcatagaag ccaca 25 1439 25 DNA Homo sapiens 1439 ccctggggag gcatagaagc cacac 25 1440 25 DNA Homo sapiens 1440 cctggggagg catagaagcc acact 25 1441 25 DNA Homo sapiens 1441 ctggggaggc atagaagcca cactg 25 1442 25 DNA Homo sapiens 1442 tggggaggca tagaagccac actgg 25 1443 25 DNA Homo sapiens 1443 ggggaggcat agaagccaca ctggc 25 1444 25 DNA Homo sapiens 1444 gggaggcata gaagccacac tggca 25 1445 25 DNA Homo sapiens 1445 ggaggcatag aagccacact ggcag 25 1446 25 DNA Homo sapiens 1446 gaggcataga agccacactg gcaga 25 1447 25 DNA Homo sapiens 1447 aggcatagaa gccacactgg cagag 25 1448 25 DNA Homo sapiens 1448 ggcatagaag ccacactggc agagc 25 1449 25 DNA Homo sapiens 1449 gcatagaagc cacactggca gagcg 25 1450 25 DNA Homo sapiens 1450 catagaagcc acactggcag agcgg 25 1451 25 DNA Homo sapiens 1451 atagaagcca cactggcaga gcggc 25 1452 25 DNA Homo sapiens 1452 tagaagccac actggcagag cggcc 25 1453 25 DNA Homo sapiens 1453 agaagccaca ctggcagagc ggcca 25 1454 25 DNA Homo sapiens 1454 gaagccacac tggcagagcg gccag 25 1455 25 DNA Homo sapiens 1455 aagccacact ggcagagcgg ccagc 25 1456 25 DNA Homo sapiens 1456 agccacactg gcagagcggc cagca 25 1457 25 DNA Homo sapiens 1457 gccacactgg cagagcggcc agcac 25 1458 25 DNA Homo sapiens 1458 ccacactggc agagcggcca gcaca 25 1459 25 DNA Homo sapiens 1459 cacactggca gagcggccag cacag 25 1460 25 DNA Homo sapiens 1460 acactggcag agcggccagc acagg 25 1461 25 DNA Homo sapiens 1461 cactggcaga gcggccagca caggt 25 1462 25 DNA Homo sapiens 1462 actggcagag cggccagcac aggta 25 1463 25 DNA Homo sapiens 1463 ctggcagagc ggccagcaca ggtag 25 1464 25 DNA Homo sapiens 1464 tggcagagcg gccagcacag gtagc 25 1465 25 DNA Homo sapiens 1465 ggcagagcgg ccagcacagg tagcc 25 1466 25 DNA Homo sapiens 1466 gcagagcggc cagcacaggt agcca 25 1467 25 DNA Homo sapiens 1467 cagagcggcc agcacaggta gccag 25 1468 25 DNA Homo sapiens 1468 agagcggcca gcacaggtag ccagc 25 1469 25 DNA Homo sapiens 1469 gagcggccag cacaggtagc cagca 25 1470 25 DNA Homo sapiens 1470 agcggccagc acaggtagcc agcag 25 1471 25 DNA Homo sapiens 1471 gcggccagca caggtagcca gcaga 25 1472 25 DNA Homo sapiens 1472 cggccagcac aggtagccag cagag 25 1473 25 DNA Homo sapiens 1473 ggccagcaca ggtagccagc agagg 25 1474 25 DNA Homo sapiens 1474 gccagcacag gtagccagca gaggc 25 1475 25 DNA Homo sapiens 1475 ccagcacagg tagccagcag aggca 25 1476 25 DNA Homo sapiens 1476 cagcacaggt agccagcaga ggcat 25 1477 25 DNA Homo sapiens 1477 agcacaggta gccagcagag gcatt 25 1478 25 DNA Homo sapiens 1478 gcacaggtag ccagcagagg cattc 25 1479 25 DNA Homo sapiens 1479 cacaggtagc cagcagaggc attct 25 1480 25 DNA Homo sapiens 1480 acaggtagcc agcagaggca ttctt 25 1481 25 DNA Homo sapiens 1481 caggtagcca gcagaggcat tcttg 25 1482 25 DNA Homo sapiens 1482 aggtagccag cagaggcatt cttgg 25 1483 25 DNA Homo sapiens 1483 ggtagccagc agaggcattc ttggg 25 1484 25 DNA Homo sapiens 1484 gtagccagca gaggcattct tgggg 25 1485 25 DNA Homo sapiens 1485 tagccagcag aggcattctt ggggc 25 1486 25 DNA Homo sapiens 1486 agccagcaga ggcattcttg gggct 25 1487 25 DNA Homo sapiens 1487 gccagcagag gcattcttgg ggcta 25 1488 25 DNA Homo sapiens 1488 ccagcagagg cattcttggg gctat 25 1489 25 DNA Homo sapiens 1489 cagcagaggc attcttgggg ctatt 25 1490 25 DNA Homo sapiens 1490 agcagaggca ttcttggggc tattt 25 1491 25 DNA Homo sapiens 1491 gcagaggcat tcttggggct atttg 25 1492 25 DNA Homo sapiens 1492 cagaggcatt cttggggcta tttga 25 1493 25 DNA Homo sapiens 1493 agaggcattc ttggggctat ttgaa 25 1494 25 DNA Homo sapiens 1494 gaggcattct tggggctatt tgaaa 25 1495 25 DNA Homo sapiens 1495 aggcattctt ggggctattt gaaaa 25 1496 25 DNA Homo sapiens 1496 ggcattcttg gggctatttg aaaaa 25 1497 25 DNA Homo sapiens 1497 gcattcttgg ggctatttga aaaag 25 1498 25 DNA Homo sapiens 1498 cattcttggg gctatttgaa aaagt 25 1499 25 DNA Homo sapiens 1499 attcttgggg ctatttgaaa aagtt 25 1500 25 DNA Homo sapiens 1500 ttcttggggc tatttgaaaa agttt 25 1501 25 DNA Homo sapiens 1501 tcttggggct atttgaaaaa gtttg 25 1502 25 DNA Homo sapiens 1502 cttggggcta tttgaaaaag tttgg 25 1503 25 DNA Homo sapiens 1503 ttggggctat ttgaaaaagt ttggt 25 1504 25 DNA Homo sapiens 1504 tggggctatt tgaaaaagtt tggtc 25 1505 25 DNA Homo sapiens 1505 ggggctattt gaaaaagttt ggtct 25 1506 25 DNA Homo sapiens 1506 gggctatttg aaaaagtttg gtctg 25 1507 25 DNA Homo sapiens 1507 ggctatttga aaaagtttgg tctgt 25 1508 25 DNA Homo sapiens 1508 gctatttgaa aaagtttggt ctgtg 25 1509 25 DNA Homo sapiens 1509 ctatttgaaa aagtttggtc tgtga 25 1510 25 DNA Homo sapiens 1510 tatttgaaaa agtttggtct gtgaa 25 1511 25 DNA Homo sapiens 1511 atttgaaaaa gtttggtctg tgaac 25 1512 25 DNA Homo sapiens 1512 tttgaaaaag tttggtctgt gaaca 25 1513 25 DNA Homo sapiens 1513 ttgaaaaagt ttggtctgtg aacaa 25 1514 25 DNA Homo sapiens 1514 tgaaaaagtt tggtctgtga acaaa 25 1515 25 DNA Homo sapiens 1515 gaaaaagttt ggtctgtgaa caaaa 25 1516 25 DNA Homo sapiens 1516 aaaaagtttg gtctgtgaac aaaac 25 1517 25 DNA Homo sapiens 1517 aaaagtttgg tctgtgaaca aaaca 25 1518 25 DNA Homo sapiens 1518 aaagtttggt ctgtgaacaa aacag 25 1519 25 DNA Homo sapiens 1519 aagtttggtc tgtgaacaaa acagt 25 1520 25 DNA Homo sapiens 1520 agtttggtct gtgaacaaaa cagtt 25 1521 25 DNA Homo sapiens 1521 gtttggtctg tgaacaaaac agttt 25 1522 25 DNA Homo sapiens 1522 tttggtctgt gaacaaaaca gtttc 25 1523 25 DNA Homo sapiens 1523 ttggtctgtg aacaaaacag tttcc 25 1524 25 DNA Homo sapiens 1524 tggtctgtga acaaaacagt ttccc 25 1525 25 DNA Homo sapiens 1525 ggtctgtgaa caaaacagtt tccct 25 1526 25 DNA Homo sapiens 1526 gtctgtgaac aaaacagttt ccctg 25 1527 25 DNA Homo sapiens 1527 tctgtgaaca aaacagtttc cctgg 25 1528 25 DNA Homo sapiens 1528 ctgtgaacaa aacagtttcc ctggt 25 1529 25 DNA Homo sapiens 1529 tgtgaacaaa acagtttccc tggtg 25 1530 25 DNA Homo sapiens 1530 gtgaacaaaa cagtttccct ggtga 25 1531 25 DNA Homo sapiens 1531 tgaacaaaac agtttccctg gtgac 25 1532 25 DNA Homo sapiens 1532 gaacaaaaca gtttccctgg tgact 25 1533 25 DNA Homo sapiens 1533 aacaaaacag tttccctggt gactg 25 1534 25 DNA Homo sapiens 1534 acaaaacagt ttccctggtg actgc 25 1535 25 DNA Homo sapiens 1535 caaaacagtt tccctggtga ctgca 25 1536 25 DNA Homo sapiens 1536 aaaacagttt ccctggtgac tgcaa 25 1537 25 DNA Homo sapiens 1537 aaacagtttc cctggtgact gcaaa 25 1538 25 DNA Homo sapiens 1538 aacagtttcc ctggtgactg caaat 25 1539 25 DNA Homo sapiens 1539 acagtttccc tggtgactgc aaatc 25 1540 25 DNA Homo sapiens 1540 cagtttccct ggtgactgca aatcc 25 1541 25 DNA Homo sapiens 1541 agtttccctg gtgactgcaa atcca 25 1542 25 DNA Homo sapiens 1542 gtttccctgg tgactgcaaa tccat 25 1543 25 DNA Homo sapiens 1543 tttccctggt gactgcaaat ccatt 25 1544 25 DNA Homo sapiens 1544 ttccctggtg actgcaaatc cattg 25 1545 25 DNA Homo sapiens 1545 tccctggtga ctgcaaatcc attgc 25 1546 25 DNA Homo sapiens 1546 ccctggtgac tgcaaatcca ttgct 25 1547 25 DNA Homo sapiens 1547 cctggtgact gcaaatccat tgcta 25 1548 25 DNA Homo sapiens 1548 ctggtgactg caaatccatt gctag 25 1549 25 DNA Homo sapiens 1549 tggtgactgc aaatccattg ctagc 25 1550 25 DNA Homo sapiens 1550 ggtgactgca aatccattgc tagct 25 1551 25 DNA Homo sapiens 1551 gtgactgcaa atccattgct agctg 25 1552 25 DNA Homo sapiens 1552 tgactgcaaa tccattgcta gctgc 25 1553 25 DNA Homo sapiens 1553 gactgcaaat ccattgctag ctgcc 25 1554 25 DNA Homo sapiens 1554 actgcaaatc cattgctagc tgcct 25 1555 25 DNA Homo sapiens 1555 ctgcaaatcc attgctagct gcctc 25 1556 25 DNA Homo sapiens 1556 tgcaaatcca ttgctagctg cctct 25 1557 25 DNA Homo sapiens 1557 gcaaatccat tgctagctgc ctctt 25 1558 25 DNA Homo sapiens 1558 caaatccatt gctagctgcc tcttt 25 1559 25 DNA Homo sapiens 1559 aaatccattg ctagctgcct ctttc 25 1560 25 DNA Homo sapiens 1560 aatccattgc tagctgcctc tttct 25 1561 25 DNA Homo sapiens 1561 atccattgct agctgcctct ttctc 25 1562 25 DNA Homo sapiens 1562 tccattgcta gctgcctctt tctcg 25 1563 25 DNA Homo sapiens 1563 ccattgctag ctgcctcttt ctcgt 25 1564 25 DNA Homo sapiens 1564 cattgctagc tgcctctttc tcgtc 25 1565 25 DNA Homo sapiens 1565 attgctagct gcctctttct cgtct 25 1566 25 DNA Homo sapiens 1566 ttgctagctg cctctttctc gtctg 25 1567 25 DNA Homo sapiens 1567 tgctagctgc ctctttctcg tctgc 25 1568 25 DNA Homo sapiens 1568 gctagctgcc tctttctcgt ctgcc 25 1569 25 DNA Homo sapiens 1569 ctagctgcct ctttctcgtc tgccc 25 1570 25 DNA Homo sapiens 1570 tagctgcctc tttctcgtct gccca 25 1571 25 DNA Homo sapiens 1571 agctgcctct ttctcgtctg cccat 25 1572 25 DNA Homo sapiens 1572 gctgcctctt tctcgtctgc ccatc 25 1573 25 DNA Homo sapiens 1573 ctgcctcttt ctcgtctgcc catca 25 1574 25 DNA Homo sapiens 1574 tgcctctttc tcgtctgccc atcac 25 1575 25 DNA Homo sapiens 1575 gcctctttct cgtctgccca tcact 25 1576 25 DNA Homo sapiens 1576 cctctttctc gtctgcccat cactc 25 1577 25 DNA Homo sapiens 1577 ctctttctcg tctgcccatc actct 25 1578 25 DNA Homo sapiens 1578 tctttctcgt ctgcccatca ctctg 25 1579 25 DNA Homo sapiens 1579 ctttctcgtc tgcccatcac tctgg 25 1580 25 DNA Homo sapiens 1580 tttctcgtct gcccatcact ctggt 25 1581 25 DNA Homo sapiens 1581 ttctcgtctg cccatcactc tggtg 25 1582 25 DNA Homo sapiens 1582 tctcgtctgc ccatcactct ggtgt 25 1583 25 DNA Homo sapiens 1583 ctcgtctgcc catcactctg gtgtg 25 1584 25 DNA Homo sapiens 1584 tcgtctgccc atcactctgg tgtgg 25 1585 25 DNA Homo sapiens 1585 cgtctgccca tcactctggt gtggt 25 1586 25 DNA Homo sapiens 1586 gtctgcccat cactctggtg tggta 25 1587 25 DNA Homo sapiens 1587 tctgcccatc actctggtgt ggtac 25 1588 25 DNA Homo sapiens 1588 ctgcccatca ctctggtgtg gtacc 25 1589 25 DNA Homo sapiens 1589 tgcccatcac tctggtgtgg taccc 25 1590 25 DNA Homo sapiens 1590 gcccatcact ctggtgtggt accca 25 1591 25 DNA Homo sapiens 1591 cccatcactc tggtgtggta cccag 25 1592 25 DNA Homo sapiens 1592 ccatcactct ggtgtggtac ccaga 25 1593 25 DNA Homo sapiens 1593 catcactctg gtgtggtacc cagaa 25 1594 25 DNA Homo sapiens 1594 atcactctgg tgtggtaccc agaag 25 1595 25 DNA Homo sapiens 1595 tcactctggt gtggtaccca gaagt 25 1596 25 DNA Homo sapiens 1596 cactctggtg tggtacccag aagtt 25 1597 25 DNA Homo sapiens 1597 actctggtgt ggtacccaga agttg 25 1598 25 DNA Homo sapiens 1598 ctctggtgtg gtacccagaa gttga 25 1599 25 DNA Homo sapiens 1599 tctggtgtgg tacccagaag ttgac 25 1600 25 DNA Homo sapiens 1600 ctggtgtggt acccagaagt tgact 25 1601 25 DNA Homo sapiens 1601 tggtgtggta cccagaagtt gactt 25 1602 25 DNA Homo sapiens 1602 ggtgtggtac ccagaagttg acttc 25 1603 25 DNA Homo sapiens 1603 gtgtggtacc cagaagttga cttct 25 1604 25 DNA Homo sapiens 1604 tgtggtaccc agaagttgac ttctg 25 1605 25 DNA Homo sapiens 1605 gtggtaccca gaagttgact tctgg 25 1606 25 DNA Homo sapiens 1606 tggtacccag aagttgactt ctggt 25 1607 25 DNA Homo sapiens 1607 ggtacccaga agttgacttc tggtt 25 1608 25 DNA Homo sapiens 1608 gtacccagaa gttgacttct ggttc 25 1609 25 DNA Homo sapiens 1609 tacccagaag ttgacttctg gttct 25 1610 25 DNA Homo sapiens 1610 acccagaagt tgacttctgg ttctg 25 1611 25 DNA Homo sapiens 1611 cccagaagtt gacttctggt tctgt 25 1612 25 DNA Homo sapiens 1612 ccagaagttg acttctggtt ctgta 25 1613 25 DNA Homo sapiens 1613 cagaagttga cttctggttc tgtag 25 1614 25 DNA Homo sapiens 1614 agaagttgac ttctggttct gtaga 25 1615 25 DNA Homo sapiens 1615 gaagttgact tctggttctg tagaa 25 1616 25 DNA Homo sapiens 1616 aagttgactt ctggttctgt agaaa 25 1617 25 DNA Homo sapiens 1617 agttgacttc tggttctgta gaaag 25 1618 25 DNA Homo sapiens 1618 gttgacttct ggttctgtag aaaga 25 1619 25 DNA Homo sapiens 1619 ttgacttctg gttctgtaga aagag 25 1620 25 DNA Homo sapiens 1620 tgacttctgg ttctgtagaa agagc 25 1621 25 DNA Homo sapiens 1621 gacttctggt tctgtagaaa gagct 25 1622 25 DNA Homo sapiens 1622 acttctggtt ctgtagaaag agcta 25 1623 25 DNA Homo sapiens 1623 cttctggttc tgtagaaaga gctag 25 1624 25 DNA Homo sapiens 1624 ttctggttct gtagaaagag ctagg 25 1625 25 DNA Homo sapiens 1625 tctggttctg tagaaagagc taggg 25 1626 25 DNA Homo sapiens 1626 ctggttctgt agaaagagct agggg 25 1627 25 DNA Homo sapiens 1627 tggttctgta gaaagagcta gggga 25 1628 25 DNA Homo sapiens 1628 ggttctgtag aaagagctag gggag 25 1629 25 DNA Homo sapiens 1629 gttctgtaga aagagctagg ggagg 25 1630 25 DNA Homo sapiens 1630 ttctgtagaa agagctaggg gaggt 25 1631 25 DNA Homo sapiens 1631 tctgtagaaa gagctagggg aggta 25 1632 25 DNA Homo sapiens 1632 ctgtagaaag agctagggga ggtat 25 1633 25 DNA Homo sapiens 1633 tgtagaaaga gctaggggag gtatg 25 1634 25 DNA Homo sapiens 1634 gtagaaagag ctaggggagg tatga 25 1635 25 DNA Homo sapiens 1635 tagaaagagc taggggaggt atgat 25 1636 25 DNA Homo sapiens 1636 agaaagagct aggggaggta tgatg 25 1637 25 DNA Homo sapiens 1637 gaaagagcta ggggaggtat gatgt 25 1638 25 DNA Homo sapiens 1638 aaagagctag gggaggtatg atgtg 25 1639 25 DNA Homo sapiens 1639 aagagctagg ggaggtatga tgtgc 25 1640 25 DNA Homo sapiens 1640 agagctaggg gaggtatgat gtgct 25 1641 25 DNA Homo sapiens 1641 gagctagggg aggtatgatg tgctt 25 1642 25 DNA Homo sapiens 1642 agctagggga ggtatgatgt gctta 25 1643 25 DNA Homo sapiens 1643 gctaggggag gtatgatgtg cttaa 25 1644 25 DNA Homo sapiens 1644 ctaggggagg tatgatgtgc ttaaa 25 1645 25 DNA Homo sapiens 1645 taggggaggt atgatgtgct taaag 25 1646 25 DNA Homo sapiens 1646 aggggaggta tgatgtgctt aaaga 25 1647 25 DNA Homo sapiens 1647 ggggaggtat gatgtgctta aagat 25 1648 25 DNA Homo sapiens 1648 gggaggtatg atgtgcttaa agatc 25 1649 25 DNA Homo sapiens 1649 ggaggtatga tgtgcttaaa gatcc 25 1650 25 DNA Homo sapiens 1650 gaggtatgat gtgcttaaag atcct 25 1651 25 DNA Homo sapiens 1651 aggtatgatg tgcttaaaga tccta 25 1652 25 DNA Homo sapiens 1652 ggtatgatgt gcttaaagat cctaa 25 1653 25 DNA Homo sapiens 1653 gtatgatgtg cttaaagatc ctaag 25 1654 25 DNA Homo sapiens 1654 tatgatgtgc ttaaagatcc taaga 25 1655 25 DNA Homo sapiens 1655 atgatgtgct taaagatcct aagaa 25 1656 25 DNA Homo sapiens 1656 tgatgtgctt aaagatccta agaat 25 1657 25 DNA Homo sapiens 1657 gatgtgctta aagatcctaa gaata 25 1658 25 DNA Homo sapiens 1658 atgtgcttaa agatcctaag aataa 25 1659 25 DNA Homo sapiens 1659 tgtgcttaaa gatcctaaga ataag 25 1660 25 DNA Homo sapiens 1660 gtgcttaaag atcctaagaa taagc 25 1661 25 DNA Homo sapiens 1661 tgcttaaaga tcctaagaat aagcc 25 1662 25 DNA Homo sapiens 1662 gcttaaagat cctaagaata agcct 25 1663 25 DNA Homo sapiens 1663 cttaaagatc ctaagaataa gcctg 25 1664 25 DNA Homo sapiens 1664 ttaaagatcc taagaataag cctgg 25 1665 25 DNA Homo sapiens 1665 taaagatcct aagaataagc ctggc 25 1666 25 DNA Homo sapiens 1666 aaagatccta agaataagcc tggcg 25 1667 25 DNA Homo sapiens 1667 aagatcctaa gaataagcct ggcga 25 1668 25 DNA Homo sapiens 1668 agatcctaag aataagcctg gcgat 25 1669 25 DNA Homo sapiens 1669 gatcctaaga ataagcctgg cgatt 25 1670 25 DNA Homo sapiens 1670 atcctaagaa taagcctggc gattt 25 1671 25 DNA Homo sapiens 1671 tcctaagaat aagcctggcg atttt 25 1672 25 DNA Homo sapiens 1672 cctaagaata agcctggcga ttttg 25 1673 25 DNA Homo sapiens 1673 ctaagaataa gcctggcgat tttgg 25 1674 25 DNA Homo sapiens 1674 taagaataag cctggcgatt ttggc 25 1675 25 DNA Homo sapiens 1675 aagaataagc ctggcgattt tggct 25 1676 25 DNA Homo sapiens 1676 agaataagcc tggcgatttt ggctg 25 1677 25 DNA Homo sapiens 1677 gaataagcct ggcgattttg gctgg 25 1678 25 DNA Homo sapiens 1678 aataagcctg gcgattttgg ctggg 25 1679 25 DNA Homo sapiens 1679 ataagcctgg cgattttggc tgggt 25 1680 25 DNA Homo sapiens 1680 taagcctggc gattttggct gggtg 25 1681 25 DNA Homo sapiens 1681 aagcctggcg attttggctg ggtgg 25 1682 25 DNA Homo sapiens 1682 agcctggcga ttttggctgg gtggg 25 1683 25 DNA Homo sapiens 1683 gcctggcgat tttggctggg tgggc 25 1684 25 DNA Homo sapiens 1684 cctggcgatt ttggctgggt gggca 25 1685 25 DNA Homo sapiens 1685 ctggcgattt tggctgggtg ggcac 25 1686 25 DNA Homo sapiens 1686 tggcgatttt ggctgggtgg gcact 25 1687 25 DNA Homo sapiens 1687 ggcgattttg gctgggtggg cactc 25 1688 25 DNA Homo sapiens 1688 gcgattttgg ctgggtgggc actct 25 1689 25 DNA Homo sapiens 1689 cgattttggc tgggtgggca ctctg 25 1690 25 DNA Homo sapiens 1690 gattttggct gggtgggcac tctgt 25 1691 25 DNA Homo sapiens 1691 attttggctg ggtgggcact ctgtt 25 1692 25 DNA Homo sapiens 1692 ttttggctgg gtgggcactc tgttc 25 1693 25 DNA Homo sapiens 1693 tttggctggg tgggcactct gttct 25 1694 25 DNA Homo sapiens 1694 ttggctgggt gggcactctg ttctg 25 1695 25 DNA Homo sapiens 1695 tggctgggtg ggcactctgt tctgc 25 1696 25 DNA Homo sapiens 1696 ggctgggtgg gcactctgtt ctgcc 25 1697 25 DNA Homo sapiens 1697 gctgggtggg cactctgttc tgcca 25 1698 25 DNA Homo sapiens 1698 ctgggtgggc actctgttct gccaa 25 1699 25 DNA Homo sapiens 1699 tgggtgggca ctctgttctg ccaac 25 1700 25 DNA Homo sapiens 1700 gggtgggcac tctgttctgc caact 25 1701 25 DNA Homo sapiens 1701 ggtgggcact ctgttctgcc aactc 25 1702 25 DNA Homo sapiens 1702 gtgggcactc tgttctgcca actct 25 1703 25 DNA Homo sapiens 1703 tgggcactct gttctgccaa ctctg 25 1704 25 DNA Homo sapiens 1704 gggcactctg ttctgccaac tctga 25 1705 25 DNA Homo sapiens 1705 ggcactctgt tctgccaact ctgag 25 1706 25 DNA Homo sapiens 1706 gcactctgtt ctgccaactc tgagc 25 1707 25 DNA Homo sapiens 1707 cactctgttc tgccaactct gagct 25 1708 25 DNA Homo sapiens 1708 actctgttct gccaactctg agctg 25 1709 25 DNA Homo sapiens 1709 ctctgttctg ccaactctga gctgg 25 1710 25 DNA Homo sapiens 1710 tctgttctgc caactctgag ctggg 25 1711 25 DNA Homo sapiens 1711 ctgttctgcc aactctgagc tgggc 25 1712 17 DNA Homo sapiens 1712 gtcataagcc agttgtt 17 1713 17 DNA Homo sapiens 1713 tcataagcca gttgttg 17 1714 17 DNA Homo sapiens 1714 cataagccag ttgttgc 17 1715 17 DNA Homo sapiens 1715 ataagccagt tgttgct 17 1716 17 DNA Homo sapiens 1716 taagccagtt gttgctg 17 1717 17 DNA Homo sapiens 1717 aagccagttg ttgctgc 17 1718 17 DNA Homo sapiens 1718 agccagttgt tgctgct 17 1719 17 DNA Homo sapiens 1719 gccagttgtt gctgctt 17 1720 17 DNA Homo sapiens 1720 ccagttgttg ctgcttg 17 1721 17 DNA Homo sapiens 1721 cagttgttgc tgcttgt 17 1722 17 DNA Homo sapiens 1722 agttgttgct gcttgtg 17 1723 17 DNA Homo sapiens 1723 gttgttgctg cttgtgt 17 1724 17 DNA Homo sapiens 1724 ttgttgctgc ttgtgtt 17 1725 17 DNA Homo sapiens 1725 tgttgctgct tgtgttc 17 1726 17 DNA Homo sapiens 1726 gttgctgctt gtgttcc 17 1727 17 DNA Homo sapiens 1727 ttgctgcttg tgttccc 17 1728 17 DNA Homo sapiens 1728 tgctgcttgt gttccca 17 1729 17 DNA Homo sapiens 1729 gctgcttgtg ttcccat 17 1730 17 DNA Homo sapiens 1730 ctgcttgtgt tcccatt 17 1731 17 DNA Homo sapiens 1731 tgcttgtgtt cccattg 17 1732 17 DNA Homo sapiens 1732 gcttgtgttc ccattgt 17 1733 17 DNA Homo sapiens 1733 cttgtgttcc cattgtc 17 1734 17 DNA Homo sapiens 1734 ttgtgttccc attgtcc 17 1735 17 DNA Homo sapiens 1735 tgtgttccca ttgtccc 17 1736 17 DNA Homo sapiens 1736 gtgttcccat tgtccca 17 1737 17 DNA Homo sapiens 1737 tgttcccatt gtcccag 17 1738 17 DNA Homo sapiens 1738 gttcccattg tcccagc 17 1739 17 DNA Homo sapiens 1739 ttcccattgt cccagca 17 1740 17 DNA Homo sapiens 1740 tcccattgtc ccagcaa 17 1741 17 DNA Homo sapiens 1741 cccattgtcc cagcaag 17 1742 17 DNA Homo sapiens 1742 ccattgtccc agcaaga 17 1743 17 DNA Homo sapiens 1743 cattgtccca gcaagaa 17 1744 17 DNA Homo sapiens 1744 attgtcccag caagaac 17 1745