METHODS AND COMPOSITIONS FOR PRODUCING ACTIVE VITAMIN K-DEPENDENT PROTEINS

Abstract

The present invention provides a stable cell line overexpressing a recombinant vitamin K-dependent (VKD) protein and both a recombinant vitamin K epoxide reductase (VKOR) and a recombinant vitamin K dependent gamma carboxylase (VKGC), wherein the amount of fully carboxylated VKD protein produced in the cell is increased 10% as compared to the amount of fully carboxylated VKD protein produced in the cell in the absence of the recombinant VKOR and the recombinant VKGC.

Description

STATEMENT OF PRIORITY

[0001] This application is a continuation application of, and claims priority to, U.S. application Ser. No. 16/951,320, filed Nov. 18, 2020, which is a continuation application of U.S. application Ser. No. 16/552,757, filed Aug. 27, 2019 (abandoned), which is a continuation application of U.S. application Ser. No. 16/252,074, filed Jan. 18, 2019 (abandoned), which is a continuation application of U.S. application Ser. No. 16/001,476, filed Jun. 6, 2018 (abandoned), which is a continuation application of U.S. application Ser. No. 15/794,788, filed Oct. 26, 2017 (abandoned), which is a continuation application of U.S. application Ser. No. 14/496,801 filed Sep. 25, 2014, now U.S. Pat. No. 9,828,588, issued Nov. 28, 2017, which is a continuation application of U.S. application Ser. No. 11/885,067 filed Jan. 9, 2008 (abandoned), which is a 35 U.S.C. .sctn. 371 national phase application of International Application No. PCT/US2005/008643, filed Mar. 15, 2005; the entire contents of each of which are incorporated herein by reference.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

[0003] A Sequence Listing in ASCII text format, submitted under 37 C.F.R. .sctn. 1.821, entitled 5470-472TSCT7_ST25.txt, 62,713 bytes in size, generated on Jul. 15, 2021 and filed via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is incorporated by reference into the specification for its disclosures.

FIELD OF THE INVENTION

[0004] The present invention concerns isolated nucleic acids, host cells containing the same, and methods of use thereof, as well as methods and compositions directed to identification of single nucleotide polymorphisms (SNPs) in the Vitamin K epoxide reductase (VKOR) gene and their correlation with sensitivity to warfarin.

BACKGROUND OF THE INVENTION

[0005] The function of numerous proteins requires the modification of multiple glutamic acid residues to .gamma.-carboxyglutamate. Among these vitamin K-dependent (VKD) coagulation proteins, FIX (Christmas factor), FVII, and prothrombin are the best known. The observation that a knock-out of the gene for matrix Gla protein results in calcification of the mouse's arteries (Luo et al. (1997) "Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein" Nature 386:78-81) emphasizes the importance of the vitamin K cycle for proteins with functions other than coagulation. Moreover, Gas6 and other Gla proteins of unknown function are expressed in neural tissue and warfarin exposure in utero results in mental retardation and facial abnormalities. This is consistent with the observation that the expression of VKD carboxylase, the enzyme that accomplishes the Gla modification, is temporally regulated in a tissue-specific manner with high expression in the nervous system during early embryonic stages. Concomitant with carboxylation, reduced vitamin K, a co-substrate of the reaction, is converted to vitamin K epoxide. Because the amount of vitamin K in the human diet is limited, vitamin K epoxide must be converted back to vitamin K by vitamin K epoxide reductase (VKOR) to prevent its depletion. Warfarin, the most widely used anticoagulation drug, targets VKOR and prevents the regeneration of vitamin K. The consequence is a decrease in the concentration of reduced vitamin K, which results in a reduced rate of carboxylation by the .gamma.-glutamyl carboxylase and in the production of undercarboxylated vitamin K-dependent proteins.

[0006] In the United States alone, warfarin is prescribed to more than one million patients per year and in Holland, it has been reported that approximately 2% of the population is on long term warfarin therapy. Because the dose of warfarin required for a therapeutic level of anticoagulation varies greatly between patients, the utilization of warfarin is accompanied by a significant risk of side effects. For example, it has been reported that following initiation of warfarin therapy, major bleeding episodes occurred in 1-2% of patients and death occurred in 0.1-0.7% of patients. In spite of the dangers, it has been estimated that warfarin use can prevent 20 strokes per induced bleeding episode and is probably underutilized because of the fear of induced bleeding.

[0007] The present invention overcomes previous shortcomings in the art by providing methods and compositions for correlating single nucleotide polymorphisms in a subject with an increased or decreased sensitivity to warfarin, thereby allowing for more accurate and rapid determination of therapeutic and maintenance doses of warfarin at reduced risk to the subject.

SUMMARY OF THE INVENTION

[0008] The present invention provides a method of identifying a human subject having increased or decreased sensitivity to warfarin, comprising detecting in the subject the presence of a single nucleotide polymorphism in the VKOR gene, wherein the single nucleotide polymorphism is correlated with increased or decreased sensitivity to warfarin, thereby identifying the subject having increased or decreased sensitivity to warfarin.

[0009] Additionally provided is a method of identifying a human subject having increased or decreased sensitivity to warfarin, comprising: a) correlating the presence of a single nucleotide polymorphism in the VKOR gene with increased or decreased sensitivity to warfarin; and b) detecting the single nucleotide polymorphism of step (a) in the subject, thereby identifying a subject having increased or decreased sensitivity to warfarin.

[0010] In a further embodiment, the present invention provides a method of identifying a single nucleotide polymorphism in the VKOR gene correlated with increased or decreased sensitivity to warfarin, comprising:

[0011] a) identifying a subject having increased or decreased sensitivity to warfarin;

[0012] b) detecting in the subject the presence of a single nucleotide polymorphism in the VKOR gene; and

[0013] c) correlating the presence of the single nucleotide polymorphism of step (b) with the increased or decreased sensitivity to warfarin in the subject, thereby identifying a single nucleotide polymorphism in the VKOR gene correlated with increased or decreased sensitivity to warfarin.

[0014] In addition, the present invention provides a method of correlating a single nucleotide polymorphism in the VKOR gene of a subject with increased or decreased sensitivity to warfarin, comprising: a) identifying a subject having increased or decreased sensitivity to warfarin; b) determining the nucleotide sequence of the VKOR gene of the subject of (a); c) comparing the nucleotide sequence of step (b) with the wild type nucleotide sequence of the VKOR gene; d) detecting a single nucleotide polymorphism in the nucleotide sequence of (b); and e) correlating the single nucleotide polymorphism of (d) with increased or decreased sensitivity to warfarin in the subject of (a).

[0015] A further aspect of the present invention is an isolated nucleic acid encoding vitamin K epoxide reductase (VKOR), particularly mammalian (e.g., human, ovine, bovine, monkey, etc.) VKOR. Examples include (a) nucleic acids as disclosed herein, such as isolated nucleic acids having the nucleotide sequence as set forth in SEQ ID NO: 8 or SEQ ID NO: 9; (b) nucleic acids that hybridize to isolated nucleic acids of (a) above or the complement thereof (e.g., under stringent conditions), and/or have substantial sequence identity to nucleic acids of (a) above (e.g., are 80, 85, 90 95 or 99% identical to nucleic acids of (a) above), and encode a VKOR; and (c) nucleic acids that differ from the nucleic acids of (a) or (b) above due to the degeneracy of the genetic code, but code for a VKOR encoded by a nucleic acid of (a) or (b) above.

[0016] The term "stringent" as used here refers to hybridization conditions that are commonly understood in the art to define the commodities of the hybridization procedure. Stringency conditions can be low, high or medium, as those terms are commonly know in the art and well recognized by one of ordinary skill. High stringency hybridization conditions that will permit homologous nucleotide sequences to hybridize to a nucleotide sequence as given herein are well known in the art. As one example, hybridization of such sequences to the nucleic acid molecules disclosed herein can be carried out in 25% formamide, 5.times.SSC, 5.times.Denhardt's solution and 5% dextran sulfate at 42.degree. C., with wash conditions of 25% formamide, 5.times.SSC and 0.1% SDS at 42.degree. C., to allow hybridization of sequences of about 60% homology. Another example includes hybridization conditions of 6.times.SSC, 0.1% SDS at about 45.degree. C., followed by wash conditions of 0.2.times.SSC, 0.1% SDS at 50-65.degree. C. Another example of stringent conditions is represented by a wash stringency of 0.3 M NaCl, 0.03M sodium citrate, 0.1% SDS at 60.sup.-70.degree. C. using a standard hybridization assay (see SAMBROOK et al., EDS., MOLECULAR CLONING: A LABORATORY MANUAL 2d ed. (Cold Spring Harbor, N.Y. 1989, the entire contents of which are incorporated by reference herein). In various embodiments, stringent conditions can include, for example, highly stringent (i.e., high stringency) conditions (e.g., hybridization to filter-bound DNA in 0.5 M NaHPO.sub.4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65.degree. C., and washing in 0.1.times.SSC/0.1% SDS at 68.degree. C.), and/or moderately stringent (i.e., medium stringency) conditions (e.g., washing in 0.2.times.SSC/0.1% SDS at 42.degree. C.).

[0017] An additional aspect of the present invention is a recombinant nucleic acid comprising a nucleic acid encoding vitamin K epoxide reductase as described herein operatively associated with a heterologous promoter.

[0018] A further aspect of the present invention is a cell that contains and expresses a recombinant nucleic acid as described above. Suitable cells include plant, animal, mammal, insect, yeast and bacterial cells.

[0019] A further aspect of the present invention is an oligonucleotide that hybridizes to an isolated nucleic acid encoding VKOR as described herein.

[0020] A further aspect of the present invention is isolated and purified VKOR (e.g., VKOR purified to homogeneity) encoded by a nucleic acid as described herein. For example, the VKOR of this invention can comprise the amino acid sequence as set forth in SEQ ID NO:10.

[0021] A further aspect of the present invention is a method of making a vitamin K dependent protein which comprises culturing a host cell that expresses a nucleic acid encoding a vitamin K dependent protein in the presence of vitamin K and produces a vitamin K dependent protein, and then harvesting the vitamin K dependent protein from the culture, the host cell containing and expressing a heterologous nucleic acid encoding vitamin K dependent carboxylase, and the host cell further containing and expressing a heterologous nucleic acid encoding vitamin K epoxide reductase (VKOR) and producing VKOR as described herein. Thus, the present invention further provides a cell comprising a heterologous nucleic acid encoding vitamin K dependent carboxylase and a heterologous nucleic acid encoding vitamin K epoxide reductase. The cell can further comprise nucleic acid encoding a vitamin K dependent protein, which nucleic acid can be heterologous to the cell or endogenous to the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIGS. 1A-1D Comparisons of warfarin dosages in wild type, heterozygous and homozygous subjects for SNPs vk2581 and vk3294 (FIGS. 1A-1B) and vk4769, as well as a comparison of warfarin dosages in wild type and heterozygous subjects for P450 2Y9 (FIGS. 1C-1D).

[0023] FIG. 2. For each of the 13 siRNA pools, three T7 flasks containing A549 cells were transfected and VKOR activity determined after 72 h. The VKOR assay used 25 .mu.M vitamin K epoxide. One siRNA pool specific for gene gi:13124769 reduced VKOR activity by 64%-70% in eight repetitions.

[0024] FIG. 3. Time course of inhibition of VKOR activity by the siRNA pool specific for gi:13124769 in A549 cells. VKOR activity decreased continuously during this time period while the level of its mRNA decreased rapidly to about 20% of normal. 25 .mu.M vitamin K epoxide was used for this assay. The siRNA did not affect the activity of VKD carboxylase or the level of lamin A/C mRNA.

[0025] FIG. 4. VKOR activity was detected when mGC_11276 was expressed in Sf9 insect cells. .about.1.times.10.sup.6 cells were used in this assay. Reactions were performed using 32 .mu.M KO at 30.degree. C. for 30 minutes in Buffer D. Blank Sf9 cells served as a negative control and A549 cells as a reference.

[0026] FIG. 5. Inhibition of VKOR by warfarin. Reactions were performed using 1.6 mg microsomal proteins made from VKOR_Sf9 cells, 60 .mu.M KO, and various concentration of warfarin at 30.degree. C. for 15 minutes in Buffer D.

[0027] FIGS. 6A-D. Carboxylation of a vitamin K dependent protein, factor X. A: Control HEK293 cells producing factor X without exogenous VKOR or VKGC. B: HEK 293 cells producing factor X and exogenous VKGC alone. C: HEK293 cells producing factor X and exogenous VKOR alone. D: HEK293 cells producing factor X and both exogenous VKOR and CKGC.

DETAILED DESCRIPTION OF THE INVENTION

[0028] As used herein, "a," "an" or "the" can mean one or more than one. For example, "a" cell can mean a single cell or a multiplicity of cells.

[0029] The present invention is explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure which do not depart from the instant invention. Hence, the following specification is intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

[0030] The "Sequence Listing" attached hereto forms a part of the instant specification as if fully set forth herein.

[0031] The present invention may be carried out based on the instant disclosure and further utilizing methods, components and features known in the art, including but not limited to those described in U.S. Pat. No. 5,268,275 to Stafford and Wu and U.S. Pat. No. 6,531,298 to Stafford and Chang, the disclosures of which are incorporated by reference herein in their entirety as if fully set forth herein.

[0032] As used herein, "nucleic acids" encompass both RNA and DNA, including cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA and chimeras of RNA and DNA. The nucleic acid may be double-stranded or single-stranded. Where single-stranded, the nucleic acid may be a sense strand or an antisense strand. The nucleic acid may be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.

[0033] An "isolated nucleic acid" is a DNA or RNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promoter) sequences that are immediately contiguous to the coding sequence. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment), independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.

[0034] The term "isolated" can refer to a nucleic acid or polypeptide that is substantially free of cellular material, viral material, or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Moreover, an "isolated nucleic acid fragment" is a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state.

[0035] The term "oligonucleotide" refers to a nucleic acid sequence of at least about six nucleotides to about 100 nucleotides, for example, about 15 to 30 nucleotides, or about 20 to 25 nucleotides, which can be used, for example, as a primer in a PCR amplification or as a probe in a hybridization assay or in a microarray. Oligonucleotides may be natural or synthetic, e.g., DNA, RNA, modified backbones, etc.

[0036] Where a particular nucleotide sequence is said to have a specific percent identity to a reference nucleotide sequence, the percent identity is relative to the reference nucleotide sequence. For example, a nucleotide sequence that is 50%, 75%, 85%, 90%, 95% or 99% identical to a reference nucleotide sequence that is 100 bases long can have 50, 75, 85, 90, 95 or 99 bases that are completely identical to a 50, 75, 85, 90, 95 or 99 nucleotide sequence of the reference nucleotide sequence. The nucleotide sequence can also be a 100 base long nucleotide sequence that is 50%, 75%, 85%, 90%, 95% or 99% identical to the reference nucleotide sequence over its entire length. Of course, there are other nucleotide sequences that will also meet the same criteria.

[0037] A nucleic acid sequence that is "substantially identical" to a VKOR nucleotide sequence is at least 80%, 85% 90%, 95% or 99% identical to the nucleotide sequence of SEQ ID NO:8 or 9. For purposes of comparison of nucleic acids, the length of the reference nucleic acid sequence will generally be at least 40 nucleotides, e.g., at least 60 nucleotides or more nucleotides. Sequence identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705).

[0038] As is known in the art, a number of different programs can be used to identify whether a nucleic acid or amino acid has sequence identity or similarity to a known sequence. Sequence identity or similarity may be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2, 482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48,443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al., Nucl. Acid Res. 12, 387-395 (1984), preferably using the default settings, or by inspection.

[0039] An example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35, 351-360 (1987); the method is similar to that described by Higgins & Sharp, CABIOS 5:151-153 (1989).

[0040] Another example of a useful algorithm is the BLAST algorithm, described in Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993). A particularly useful BLAST program is the WU-BLAST-2 program that was obtained from Altschul et al., Methods in Enzymology, 266, 460-480 (1996). WU-BLAST-2 uses several search parameters, which are preferably set to the default values. The parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity. An additional useful algorithm is gapped BLAST as reported by Altschul et al. Nucleic Acids Res. 25, 3389-3402.

[0041] The CLUSTAL program can also be used to determine sequence similarity. This algorithm is described by Higgins et al. (1988) Gene 73:237; Higgins et al. (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Res. 16: 10881-90; Huang et al. (1992) CABIOS 8: 155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24: 307-331.

[0042] In addition, for sequences that contain either more or fewer nucleotides than the nucleic acids disclosed herein, it is understood that in one embodiment, the percentage of sequence identity will be determined based on the number of identical nucleotides in relation to the total number of nucleotide bases. Thus, for example, sequence identity of sequences shorter than a sequence specifically disclosed herein will be determined using the number of nucleotide bases in the shorter sequence, in one embodiment. In percent identity calculations, relative weight is not assigned to various manifestations of sequence variation, such as, insertions, deletions, substitutions, etc.

[0043] The VKOR polypeptides of the invention include, but are not limited to, recombinant polypeptides, synthetic peptides and natural polypeptides. The invention also encompasses nucleic acid sequences that encode forms of VKOR polypeptides in which naturally occurring amino acid sequences are altered or deleted. Preferred nucleic acids encode polypeptides that are soluble under normal physiological conditions. Also within the invention are nucleic acids encoding fusion proteins in which all or a portion of VKOR is fused to an unrelated polypeptide (e.g., a marker polypeptide or a fusion partner) to create a fusion protein. For example, the polypeptide can be fused to a hexa-histidine tag to facilitate purification of bacterially expressed polypeptides, or to a hemagglutinin tag to facilitate purification of polypeptides expressed in eukaryotic cells, or to an HPC4 tag to facilitate purification of polypeptides by affinity chromatography or immunoprecipitation. The invention also includes isolated polypeptides (and the nucleic acids that encode these polypeptides) that include a first portion and a second portion; the first portion includes, e.g., all or a portion of a VKOR polypeptide, and the second portion includes, e.g., a detectable marker.

[0044] The fusion partner can be, for example, a polypeptide that facilitates secretion, e.g., a secretory sequence. Such a fused polypeptide is typically referred to as a preprotein. The secretory sequence can be cleaved by the cell to form the mature protein. Also within the invention are nucleic acids that encode VKOR fused to a polypeptide sequence to produce an inactive preprotein. Preproteins can be converted into the active form of the protein by removal of the inactivating sequence.

[0045] The invention also includes nucleic acids that hybridize, e.g., under stringent hybridization conditions (as defined herein) to all or a portion of the nucleotide sequence of SEQ ID NOS: 1-6, 8 or 9 or their complements. In particular embodiments, the hybridizing portion of the hybridizing nucleic acid is typically at least 15 (e.g., 20, 30, or 50) nucleotides in length. The hybridizing portion of the hybridizing nucleic acid is at least 80%, e.g., at least 95%, at least 98% or 100%, identical to the sequence of a portion or all of a nucleic acid encoding a VKOR polypeptide. Hybridizing nucleic acids of the type described herein can be used, for example, as a cloning probe, a primer (e.g., a PCR primer), or a diagnostic probe. Also included within the invention are small inhibitory RNAs (siRNAs) and/or antisense RNAs that inhibit the function of VKOR, as determined, for example, in an activity assay, as described herein and as is known in the art.

[0046] In another embodiment, the invention features cells, e.g., transformed cells, which contain a nucleic acid of this invention. A "transformed cell" is a cell into which (or into an ancestor of which) has been introduced, by means of recombinant nucleic acid techniques, a nucleic acid encoding all or a part of a VKOR polypeptide, and/or an antisense nucleic acid or siRNA. Both prokaryotic and eukaryotic cells are included, e.g., bacteria, yeast, insect, mouse, rat, human, plant and the like.

[0047] The invention also features nucleic acid constructs (e.g., vectors and plasmids) that include a nucleic acid of the invention that is operably linked to a transcription and/or translation control elements to enable expression, e.g., expression vectors. By "operably linked" is meant that a selected nucleic acid, e.g., a DNA molecule encoding a VKOR polypeptide, is positioned adjacent to one or more regulatory elements, e.g., a promoter, which directs transcription and/or translation of the sequence such that the regulatory elements can control transcription and/or translation of the selected nucleic acid.

[0048] The present invention further provides fragments or oligonucleotides of the nucleic acids of this invention, which can be used as primers or probes. Thus, in some embodiments, a fragment or oligonucleotide of this invention is a nucleotide sequence that is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1500, 2000, 2500 or 3000 contiguous nucleotides of the nucleotide sequence set forth in SEQ ID NO:8 or SEQ ID NO:9. Examples of oligonucleotides of this invention are provided in the Sequence Listing included herewith. Such fragments or oligonucleotides can be detectably labeled or modified, for example, to include and/or incorporate a restriction enzyme cleavage site when employed as a primer in an amplification (e.g., PCR) assay.

[0049] The invention also features purified or isolated VKOR polypeptides, such as, for example, a polypeptide comprising, consisting essentially of and/or consisting of the amino acid sequence of SEQ ID NO:10 or a biologically active fragment or peptide thereof. Such fragments or peptides are typically at least about ten amino acids of the amino acid sequence of SEQ ID NO:10 (e.g., 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 85, 95, 100, 125, or 150 amino acids of the amino acid sequence of SEQ ID NO:10) and can be peptides or fragment of contiguous amino acids of the amino acid sequence of the VKOR protein (e.g., as set forth in SEQ ID NO:10). The biological activity of a fragment or peptide of this invention can be determined according to the methods provided herein and as are known in the art for identifying VKOR activity. The fragments and peptides of the VKOR protein of this invention can also be active as antigens for the production of antibodies. The identification of epitopes on a fragment or peptide of this invention is carried out by well known protocols and would be within the ordinary skill of one in the art.

[0050] As used herein, both "protein" and "polypeptide" mean any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation or N-myristylation). Thus, the term "VKOR polypeptide" includes full-length, naturally occurring VKOR proteins, respectively, as well as recombinantly or synthetically produced polypeptides that correspond to a full-length, naturally occurring VKOR protein, or to a portion of a naturally occurring or synthetic VKOR polypeptide.

[0051] A "purified" or "isolated" compound or polypeptide is a composition that is at least 60% by weight the compound of interest, e.g., a VKOR polypeptide or antibody that is separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide. As used herein, the "isolated" polypeptide is at least about 25%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more pure (w/w). Preferably the preparation is at least 75% (e.g., at least 90% or 99%) by weight the compound of interest. Purity can be measured by any appropriate standard method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0052] Preferred VKOR polypeptides include a sequence substantially identical to all or a portion of a naturally occurring VKOR polypeptide. Polypeptides "substantially identical" to the VKOR polypeptide sequences described herein have an amino acid sequence that is at least 80% or 85% (e.g., 90%, 95% or 99%) identical to the amino acid sequence of the VKOR polypeptides of SEQ ID NO: 10. For purposes of comparison, the length of the reference VKOR polypeptide sequence will generally be at least 16 amino acids, e.g., at least 20, 25, 30, 35, 40, 45, 50, 75, or 100 amino acids.

[0053] In the case of polypeptide sequences that are less than 100% identical to a reference sequence, the non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence. Conservative substitutions typically include, but are not limited to, substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.

[0054] Where a particular polypeptide is said to have a specific percent identity to a reference polypeptide of a defined length, the percent identity is relative to the reference polypeptide. Thus, for example, a polypeptide that is 50%, 75%, 85%, 90%, 95% or 99% identical to a reference polypeptide that is 100 amino acids long can be a 50, 75, 85, 90, 95 or 99 amino acid polypeptide that is completely identical to a 50, 75, 85, 90, 95 or 99 amino acid long portion of the reference polypeptide. It can also be a 100 amino acid long polypeptide that is 50%, 75%, 85%, 90%, 95% or 99% identical to the reference polypeptide over its entire length. Of course, other polypeptides also will meet the same criteria.

[0055] The invention also features purified or isolated antibodies that specifically bind to a VKOR polypeptide of this invention or to a fragment thereof. By "specifically binds" is meant that an antibody recognizes and binds a particular antigen, e.g., a VKOR polypeptide, or an epitope on a fragment or peptide of a VKOR polypeptide, but does not substantially recognize and bind other molecules in a sample. In one embodiment the antibody is a monoclonal antibody and in other embodiments, the antibody is a polyclonal antibody. The production of both monoclonal and polyclonal antibodies, including chimeric antibodies, humanized antibodies, single chain antibodies, bi-specific antibodies, antibody fragments, etc., is well known in the art.

[0056] In another aspect, the invention features a method for detecting a VKOR polypeptide in a sample. This method comprises contacting the sample with an antibody that specifically binds a VKOR polypeptide or a fragment thereof under conditions that allow the formation of a complex between an antibody and VKOR; and detecting the formation of a complex, if any, as detection of a VKOR polypeptide or fragment thereof in the sample. Such immunoassays are well known in the art and include immunoprecipitation assays, immunoblotting assays, immunolabeling assays, ELISA, etc.

[0057] The present invention further provides a method of detecting a nucleic acid encoding a VKOR polypeptide in a sample, comprising contacting the sample with a nucleic acid of this invention that encodes VKOR or a fragment thereof, or a complement of a nucleic acid that encodes VKOR or a fragment thereof, under conditions whereby a hybridization complex can form, and detecting formation of a hybridization complex, thereby detecting a nucleic acid encoding a VKOR polypeptide in a sample. Such hybridization assays are well known in the art and include probe detection assays and nucleic acid amplification assays.

[0058] Also encompassed by the invention is a method of obtaining a gene related to (i.e., a functional homologue of) the VKOR gene. Such a method entails obtaining or producing a detectably-labeled probe comprising an isolated nucleic acid which encodes all or a portion of VKOR, or a homolog thereof; screening a nucleic acid fragment library with the labeled probe under conditions that allow hybridization of the probe to nucleic acid fragments in the library, thereby forming nucleic acid duplexes; isolating labeled duplexes, if any; and preparing a full-length gene sequence from the nucleic acid fragments in any labeled duplex to obtain a gene related to the VKOR gene.

[0059] A further aspect of the present invention is a method of making a vitamin K dependent protein, comprising culturing a cell that expresses a nucleic acid encoding a vitamin K dependent protein that, in the presence of vitamin K, produces a vitamin K dependent protein; and then harvesting the vitamin K dependent protein from the culture medium, wherein the cell comprises and expresses an exogenous nucleic acid encoding vitamin K epoxide reductase (VKOR), thereby producing VKOR and in some embodiments the cell further comprises and expresses an exogenous nucleic acid encoding vitamin K dependent carboxylase, thereby producing vitamin K dependent carboxylase as described herein. In some embodiments, the expression of the VKOR-encoding nucleic acid and the production of the VKOR causes the cell to produce greater levels of the vitamin K dependent protein and/or greater levels of active (e.g., fully carboxylated) vitamin K dependent protein than would be produced in the absence of the VKOR or in the absence of the VKOR and carboxylase.

[0060] Thus, in some embodiments, the present invention also provides a method of producing a vitamin K dependent protein, comprising:

[0061] a) introducing into a cell a nucleic acid that encodes a vitamin K dependent protein under conditions whereby the nucleic acid is expressed and the vitamin K dependent protein is produced in the presence of vitamin K, wherein the cell comprises a heterologous nucleic acid encoding vitamin K dependent carboxylase and further comprises a heterologous nucleic acid encoding vitamin K epoxide reductase; and

[0062] b) optionally collecting the vitamin K dependent protein from the cell.

[0063] The present invention also provides a method of increasing the amount of carboxylated vitamin K dependent protein in a cell, comprising introducing into a cell that expresses a first nucleic acid encoding a vitamin K dependent protein a second heterologous nucleic acid encoding vitamin K epoxide reductase (VKOR) under conditions whereby said first and second nucleic acids are expressed to produce a vitamin K dependent protein and VKOR, respectively.

[0064] Further provided herein is a method of increasing the carboxylation of a vitamin K dependent protein, comprising introducing into a cell that expresses a first nucleic acid encoding a vitamin K dependent protein a second heterologous nucleic acid encoding vitamin K epoxide reductase (VKOR) under conditions whereby said first and second nucleic acids are expressed to produce a vitamin K dependent protein and VKOR, respectively.

[0065] In addition, the present invention provides a method of producing a carboxylated (e.g., fully carboxylated) vitamin K dependent protein in a cell, comprising introducing into a cell that expresses a first nucleic acid encoding a vitamin K dependent protein a second heterologous nucleic acid encoding vitamin K epoxide reductase (VKOR) under conditions whereby said first and second nucleic acids are expressed to produce a vitamin K dependent protein and VKOR, respectively, wherein the amount of carboxylated vitamin K dependent protein produced in the cell in the presence of VKOR is increased as compared to the amount of carboxylated vitamin K dependent protein produced in the cell in the absence of VKOR.

[0066] Furthermore, the present invention provides a method of producing a vitamin K dependent protein in a cell, comprising introducing into a cell that expresses a first nucleic acid encoding a vitamin K dependent protein a second exogenous nucleic acid encoding vitamin K epoxide reductase (VKOR) under conditions whereby said first and second nucleic acids are expressed to produce a vitamin K dependent protein and VKOR, respectively, wherein 100%, 90%, 80%, 70% or 60% of the vitamin K dependent protein produced in the cell in the presence of VKOR is carboxylated (e.g., fully carboxylated).

[0067] Also included herein is a method of producing a vitamin K dependent protein in a cell, comprising introducing into a cell that expresses a first nucleic acid encoding a vitamin K dependent protein a second heterologous nucleic acid encoding vitamin K epoxide reductase (VKOR) under conditions whereby said first and second nucleic acids are expressed to produce a vitamin K dependent protein and VKOR, respectively.

[0068] In some embodiments of the methods described above, the cell can further comprise a third nucleic acid encoding a vitamin K dependent carboxylase, which can be, but is not limited to, a bovine vitamin K dependent carboxylase. In particular embodiments, the vitamin K-dependent carboxylase is vitamin K gamma glutamyl carboxylase (VKGC). The VKGC used in the methods of this invention can be VKGC from any vertebrate or invertebrate species that produces VKGC, as are known in the art.

[0069] In methods of this invention where the amount of carboxylated vitamin K-dependent protein is increased in a cell in the presence of VKOR and/or VKGC, the amount of carboxylated or fully carboxylated vitamin K dependent protein produced in the cell in the presence of VKOR and/or VKGC can be increased 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 100% 125% 150%, 200% or 300%, as compared to the amount of carboxylated or fully carboxylated vitamin K dependent protein produced in the cell in the absence of VKOR and/or VKGC.

[0070] By "fully carboxylated" in some embodiments is meant that all sites (or in some embodiments, the majority of sites) on a vitamin K dependent protein that can undergo carboxylation are carboxylated. In some embodiments, fully carboxylated can mean that all vitamin K dependent proteins are carboxylated to some extent and/or that all vitamin K dependent proteins are carboxylated at all or at the majority of carboxylation sites. A carboxylated vitamin K dependent protein or fully carboxylated vitamin K dependent protein is an active protein. By "active protein" is meant that the vitamin K dependent protein has or is capable of activity in carrying out its biological function (e.g., an enzymatic activity for factor IX or factor X).

[0071] The vitamin K dependent protein that can be produced according to the methods of this invention can be any vitamin K dependent protein now known or later identified as such, including but not limited to, Factor VII, Factor VIIA, Factor IX, Factor X, Protein C, activated Protein C, Protein S, bone Gla protein (osteocalcin), matrix Gla protein and prothrombin, including modified versions of such proteins as described herein, in any combination.

[0072] Any cell that can be transformed with the nucleic acids described herein can be used as described herein, although in some embodiments non-human or even non-mammalian cells can be used. Thus, a cell or cell line of this invention can be, for example, a human cell, an animal cell, a plant cell and/or an insect cell. Nucleic acids encoding vitamin K dependent carboxylase and nucleic acids encoding vitamin K dependent proteins as described herein are well known in the art and their introduction into cells for expression would be carried out according to routine protocols. Thus, in some embodiments, the present invention provides a cell that comprises a nucleic acid (either endogenous or exogenous to the cell) that encodes a vitamin K dependent protein. The vitamin K dependent protein is produced in the cell in the presence of vitamin K. The cell further comprises a heterologous (i.e., exogenous) nucleic acid encoding vitamin K epoxide reductase (VKOR) and/or a vitamin K dependent carboxylase. The cell can be maintained under conditions known in the art whereby the nucleic acid encoding VKOR and/or the vitamin K dependent carboxylase are expressed and VKOR and/or the carboxylase are produced in the cell.

[0073] Certain embodiments of this invention are based on the inventors' discovery that a subject's therapeutic dose of warfarin for anticoagulation therapy can be correlated with the presence of one or more single nucleotide polymorphisms in the VKOR gene of the subject. Thus, the present invention also provides a method of identifying a human subject having increased or decreased sensitivity to warfarin, comprising detecting in the subject the presence of a single nucleotide polymorphism (SNP) in the VKOR gene, wherein the single nucleotide polymorphism is correlated with increased or decreased sensitivity to warfarin, thereby identifying the subject as having increased or decreased sensitivity to warfarin.

[0074] An example of a SNP correlated with an increased sensitivity to warfarin is a G.fwdarw.C alteration at nucleotide 2581 (SEQ ID NO:12) (in intron 2 of the VKOR gene; GenBank accession no. refSNP ID: rs8050894, incorporated by reference herein) of the nucleotide sequence of SEQ ID NO:11, which is a reference sequence encompassing the genomic sequence of SEQ ID NO:8 and approximately 1000 nucleotides preceding and following this sequence. This sequence can be located as having the genome position "human chromosome 16p11.2" or in the physical map in the NCBI database as human chromosome 16: 31009700-31013800.

[0075] Examples of SNPs correlated with a decreased sensitivity to warfarin are a T.fwdarw.C alteration at nucleotide 3294 (SEQ ID NO:13) (in intron 2 of the VKOR gene; GenBank accession no. refSNP ID: rs2359612, incorporated by reference herein) of the nucleotide sequence of SEQ ID NO:11 and a G.fwdarw.A alteration at nucleotide 4769 (SEQ ID NO:14) (in the 3' UTR of the VKOR gene; GenBank accession no. refSNP ID: rs7294, incorporated by reference herein) of the nucleotide sequence of SEQ ID NO:11.

[0076] As used herein, a subject having an "increased sensitivity to warfarin" is a subject for whom a suitable therapeutic or maintenance dose of warfarin is lower than the therapeutic or maintenance dose of warfarin that would suitable for a normal subject, i.e., a subject who did not carry a SNP in the VKOR gene that imparts a phenotype of increased sensitivity to warfarin. Conversely, as used herein, a subject having a "decreased sensitivity to warfarin" is a subject for whom a suitable therapeutic or maintenance dose of warfarin is higher than the therapeutic or maintenance dose of warfarin that would suitable for a normal subject, i.e., a subject who did not carry a SNP in the VKOR gene that imparts a phenotype of decreased sensitivity to warfarin. An example of a typical therapeutic dose of warfarin for a normal subject is 35 mg per week, although this amount can vary (e.g., a dose range of 3.5 to 420 mg per week is described in Aithal et al. (1999) Lancet 353:717-719). A typical therapeutic dose of warfarin can be determined for a given study group according to the methods described herein, which can be used to identify subjects with therapeutic warfarin doses above or below this dose, thereby identifying subjects having decreased or increased sensitivity to warfarin.

[0077] Further provided herein is a method of identifying a human subject having increased or decreased sensitivity to warfarin, comprising: a) correlating the presence of a single nucleotide polymorphism in the VKOR gene with increased or decreased sensitivity to warfarin; and b) detecting the single nucleotide polymorphism of step (a) in the subject, thereby identifying a subject having increased or decreased sensitivity to warfarin.

[0078] In addition, the present invention provides a method of identifying a single nucleotide polymorphism in the VKOR gene correlated with increased or decreased sensitivity to warfarin, comprising: a) identifying a subject having increased or decreased sensitivity to warfarin; b) detecting in the subject the presence of a single nucleotide polymorphism in the VKOR gene; and c) correlating the presence of the single nucleotide polymorphism of step (b) with the increased or decreased sensitivity to warfarin in the subject, thereby identifying a single nucleotide polymorphism in the VKOR gene correlated with increased or decreased sensitivity to warfarin.

[0079] Also provided herein is a method of correlating a single nucleotide polymorphism in the VKOR gene of a subject with increased or decreased sensitivity to warfarin, comprising: a) identifying a subject having increased or decreased sensitivity to warfarin; b) determining the nucleotide sequence of the VKOR gene of the subject of (a); c) comparing the nucleotide sequence of step (b) with the wild type nucleotide sequence of the VKOR gene; d) detecting a single nucleotide polymorphism in the nucleotide sequence of (b); and e) correlating the single nucleotide polymorphism of (d) with increased or decreased sensitivity to warfarin in the subject of (a).

[0080] A subject is identified as having an increased or decreased sensitivity to warfarin by establishing a therapeutic or maintenance dose of warfarin for anticoagulation therapy according to well known protocols and comparing the therapeutic or maintenance dose for that subject with the therapeutic or maintenance dose of warfarin for anticoagulation therapy of a population of normal subjects (e.g., subjects lacking any SNPs in the VKOR gene correlated with increased or decreased sensitivity to warfarin) from which an average or mean therapeutic or maintenance dose of warfarin is calculated. A subject having a therapeutic or maintenance dose of warfarin that is below the average therapeutic or maintenance dose of warfarin (e.g., the dose of warfarin that is therapeutic or provides a maintenance level for a subject that has a wild type VKOR gene, i.e., lacking any single nucleotide polymorphisms associated with warfarin sensitivity) is a subject identified as having an increased sensitivity to warfarin. A subject having a therapeutic or maintenance dose of warfarin that is above the average therapeutic or maintenance of warfarin is a subject identified as having a decreased sensitivity to warfarin. An average therapeutic or maintenance dose of warfarin for a subject with a wild type VKOR gene would be readily determined by one skilled in the art.

[0081] The nucleotide sequence of the VKOR gene of a subject is determined according to methods standard in the art, and as described in the Examples provided herein. For example, genomic DNA is extracted from cells of a subject and the VKOR gene is located and sequenced according to known protocols. Single nucleotide polymorphisms in the VKOR gene are identified by a comparison of a subject's sequence with the wild type sequence as known in the art (e.g., the reference sequence as shown herein as SEQ ID NO:11).

[0082] A SNP in the VKOR gene is correlated with an increased or decreased sensitivity to warfarin by identifying the presence of a SNP or multiple SNPs in the VKOR gene of a subject also identified as having increased or decreased sensitivity to warfarin, i.e., having a maintenance or therapeutic dose of warfarin that is above or below the average dose and performing a statistical analysis of the association of the SNP or SNPs with the increased or decreased sensitivity to warfarin, according to well known methods of statistical analysis. An analysis that identifies a statistical association (e.g., a significant association) between the SNP(s) (genotype) and increased or decreased warfarin sensitivity (phenotype) establishes a correlation between the presence of the SNP(s) in a subject and an increased or decreased sensitivity to warfarin in that subject.

[0083] It is contemplated that a combination of factors, including the presence of one or more SNPs in the VKOR gene of a subject, can be correlated with an increased or decreased sensitivity to warfarin in that subject. Such factors can include, but are not limited to cytochrome p450 2C9 polymorphisms, race, age, gender, smoking history and hepatic disease.

[0084] Thus, in a further embodiment, the present invention provides a method of identifying a human subject having increased or decreased sensitivity to warfarin, comprising identifying in the subject the presence of a combination of factors correlated with an increased or decreased sensitivity to warfarin selected from the group consisting of one or more single nucleotide polymorphisms of the VKOR gene, one or more cytochrome p450 2C9 polymorphisms, race, age, gender, smoking history, hepatic disease and any combination of two or more of these factors, wherein the combination of factors is correlated with increased or decreased sensitivity to warfarin, thereby identifying the subject having increased or decreased sensitivity to warfarin.

[0085] Further provided herein is a method of identifying a human subject having increased or decreased sensitivity to warfarin, comprising: a) correlating the presence of a combination of factors with an increased or decreased sensitivity to warfarin, wherein the factors are selected from the group consisting of one or more single nucleotide polymorphisms of the VKOR gene, one or more cytochrome p450 2C9 polymorphisms, race, age, gender, smoking history, hepatic disease and any combination of two or more of these factors; and b) detecting the combination of factors of step (a) in the subject, thereby identifying a subject having increased or decreased sensitivity to warfarin.

[0086] In addition, the present invention provides a method of identifying a combination of factors correlated with an increased or decreased sensitivity to warfarin, wherein the factors are selected from the group consisting of one or more single nucleotide polymorphisms of the VKOR gene, one or more cytochrome p450 2C9 polymorphisms, race, age, gender, smoking history, hepatic disease and any combination of two or more of these factors, comprising: a) identifying a subject having increased or decreased sensitivity to warfarin; b) detecting in the subject the presence of a combination of the factors; and c) correlating the presence of the combination of factors of step (b) with the increased or decreased sensitivity to warfarin in the subject, thereby identifying a combination of factors correlated with increased or decreased sensitivity to warfarin.

[0087] Also provided herein is a method of correlating a combination of factors, wherein the factors are selected from the group consisting of one or more single nucleotide polymorphisms of the VKOR gene, one or more cytochrome p450 2C9 polymorphisms, race, age, gender, smoking history, hepatic disease and any combination of two or more of these factors, with increased or decreased sensitivity to warfarin, comprising: a) identifying a subject having increased or decreased sensitivity to warfarin; b) identifying the presence of a combination of the factors in the subject; and c) correlating the combination of the factors of (b) with increased or decreased sensitivity to warfarin in the subject of (a).

[0088] A combination of factors as described herein is correlated with an increased or decreased sensitivity to warfarin by identifying the presence of the combination of factors in a subject also identified as having increased or decreased sensitivity to warfarin and performing a statistical analysis of the association of the combination of factors with the increased or decreased sensitivity to warfarin, according to well known methods of statistical analysis. An analysis that identifies a statistical association (e.g., a significant association) between the combination of factors and the warfarin sensitivity phenotype (increased or decreased) establishes a correlation between the presence of the combination of factors in a subject and an increased or decreased sensitivity to warfarin in that subject.

[0089] Further provided herein are nucleic acids encoding VKOR and comprising one or more SNPs as described herein. Thus, the present invention further provides nucleic acids comprising, consisting essentially of and/or consisting of the nucleotide sequence as set forth in SEQ ID NOs:12, 13, 14, 15 and 16. The nucleic acids can be present in a vector and the vector can be present in a cell. Further included are proteins encoded by a nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NOs:12, 13, 14, 15 and 16, as well as antibodies that specifically bind a protein encoded by a nucleic acid comprising a nucleotide sequence as set forth in SEQ ID NOs:12, 13, 14, 15 and 16. The present invention is more particularly described in the following examples that are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.

EXAMPLES

Example I Correlation Between SNPS in VKOR Gene and Increased or Decreased Sensitivity to Warfarin

[0090] The most prevalent isoform of the VKOR gene is about 4 kb long, has three exons and encodes an enzyme of 163 amino acids with a mass of 18.4 kDa. In the present study, three mutations vk2581(G>C), vk3294(T>C) and vk4769(G>A), identified as SNPs (heterozygosity ratios of 46.9%, 46.8% and 46.3%, respectively) were examined for a correlation between their presence in a subject and the maintenance dose of warfarin required to achieve a therapeutically effective response.

1. Selection of Subjects

[0091] Subjects were obtained from the UNC Coagulation Clinic in the Ambulatory Care Center. Informed consent was obtained by a trained genetic counselor. Subjects not fluent in English were excluded because of the lack of translators and the requirement for consent. To qualify for the study, subjects had warfarin for at least six months, were older than 18 and were followed by the UNC Coagulation clinic at the Ambulatory Care Clinic.

2. Extraction of Genomic DNA from Whole Blood

[0092] Genomic DNAs were extracted from the whole blood of subjects using QIAamp DNA Blood Mini Kit (QIAGEN cat #51104). The DNA concentration was adjusted to 10 ng/4.

3. Sequencing of the Genomic DNA Samples

[0093] Approximately 10 ng of DNA was used for polymerase chain reaction (PCR) assays. The primers used to amplify the VKOR gene were: Exon 1-5' CCAATCGCCGAGTCAGAGG (SEQ ID NO:29) and Exon 1-3' CCCAGTCCCCAGCACTGTCT (SEQ ID NO:30) for the 5'-UTR and Exon 1 region; Exon 2-5' AGGGGAGGATAGGGTCAGTG (SEQ ID NO:31) and Exon 2-3' CCTGTTAGTTACCTCCCCACA (SEQ ID NO:32) for the Exon 2 region; and Exon 3-5' ATACGTGCGTAAGCCACCAC (SEQ ID NO:33) and Exon 3-3' ACCCAGATATGCCCCCTTAG (SEQ ID NO:34) for the Exon3 and 3'-UTR region. Automated high throughput capillary electrophoresis DNA sequencing was used for detecting SNPs in the VKOR gene.

4. Detection of Known SNPs Using Real-Time PCR

[0094] The assay reagents for SNP genotyping were from the Assay-by-Design.TM. service (Applied Biosystems, cat #4332072). The primers and probes (FAM.TM. and VIC.TM. dye-labeled) were designed using Primer Express software and were synthesized in an Applied Biosystems synthesizer. The primer pairs for each SNP are located at the upstream/downstream position of the SNP site and can generate less than 100 bp length of a DNA fragment in the PCR reaction. The FAM.TM. and VIC.TM. dye-labeled probes were designed to cover the SNP sites with a length of 15-16 nt. The primer and probe sequences for each VKOR SNP are shown in Table 2.

[0095] The 2.times. TaqMan.TM. Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems, cat #4324018) was used in the PCR reactions. Forty cycles of real-time PCR were performed in an Opticon II (MJ Research) machine. There was a 10 minute 95.degree. C. preheat followed by 92.degree. C. for 15 sec, 60.degree. C. for 1 min. and then a plate reading. The results were read according to the signal value of FAM and VIC dye.

5. Statistical Analysis

[0096] The difference of average dose between different genotypes was compared by analysis of variance (ANOVA) using SAS version 8.0 (SAS, Inc., Cary, N.C.). A two-sided p value less than 0.05 was considered significant. Examination of the distribution and residuals for the average dose of treatment among the SNP groups indicated that a log transformation was necessary to satisfy the assumption of homogeneity of variance.

6. Correlation of SNPs with Warfarin Dosage

[0097] By direct genomic DNA sequencing and SNP real-time PCR detection, five SNPs were identified in the VKOR gene: one in the 5'-UTR, two in intron II, one in the coding region and one in the 3'-UTR (Table 1).

[0098] Among these SNPs, the vk563 and vk4501 SNPs allele were carried by only one of the 58 subjects of the study (a triple heterozygous, also carrying the 3'-UTR SNP allele), while the other SNPs were identified in 17-25 heterozygous patients.

[0099] Each marker was first analyzed independently. FIG. 1A shows that the average warfarin dose for patients with the vk2581 wild type allele was 50.19.+-.3.20 mg per week (n=26), while those heterozygous and homozygous for this polymorphism were 35.19.+-.3.73 (n=17) and 31.14.+-.6.2 mg per week (n=15), respectively. FIG. 1B shows that the average warfarin dose for patients with the wild-type vk3294 allele was 25.29.+-.3.05 mg per week (n=11), while patients bearing the heterozygous and homozygous alleles were 41.68.+-.4.92 (n=25) and 47.73.+-.2.75 mg per week (n=22), respectively. FIG. 1C shows the average warfarin dose for patients with vk4769 SNP wild type was 35.35.+-.4.01 mg per week (n=27), while patients with the heterozygous and homozygous alleles required 44.48.+-.4.80 (n=19) and 47.56.+-.3.86 mg per week (n=12), respectively. It was also observed that P450 2C9*3 has a significant effect on warfarin dose (FIG. 1D), as previously reported (Joffe et al. (2004) "Warfarin dosing and cytochrome P450 2C9 polymorphisms" Thromb Haemost 91:1123-1128). The average warfarin dose for patients with P450 2C9*1 (wild type) was 43.82.+-.2.75 mg per week (n=50), while patients heterozygous for this allele required 22.4.+-.4.34 mg per week (n=8).

7. Statistical Analysis

[0100] The association of the Log.sub.e (warfarin average dosage)(LnDose) with the SNPs in the VKOR gene was examined by analysis of variance (ANOVA). SAS was used first to do a repeated procedure in which a series of factors (race, gender, smoking history, hepatic diseases, SNPs at cytochrome P450 2Y9 gene, etc.) were examined to identify factors, excluding VKOR SNPs, which might affect dosage. P450 2C9*3 was significantly associated with the average dose of warfarin; thus, it was included as a covariant for further analysis. The analysis indicated that the three VKOR SNPs were still significantly associated with weekly warfarin dose (vk2581, P<0.0001; vk3294, P<0.0001; and vk4769, P=0.0044), when the covariance is included.

[0101] To specifically test if the three SNPs of VKOR were independently associated with warfarin dosage, the analysis was repeated in which two SNPs in the VKOR gene were included as covariates for the other SNP. The three VKOR SNPs are located within 2 kb distance of one another and are expected to be closely linked. It was clear from inspection that, at least for Caucasians, one haplotype (where A=vk2581 guanine and a=vk2581 cytosine; B=vk3294 thymine and b=vk3924 cytosine; C=vk4769 guanine and c=vk4769 adenine) was AAbbcc and another aaBBCC. The distribution of individual SNPs in patients was found to be significantly correlated with the others (R=0.63-0.87, p<0.001). Indeed, subjects with the haplotype AAbbcc (n=7) required a significantly higher dosage of warfarin (warfarin dosage=48.98.+-.3.93) compared to those patients with haplotype aaBBCC (25.29.+-.3.05; p<0.001).

Example 2 siRNA Design and Synthesis

[0102] siRNAs were selected using an advanced version of a rational design algorithm (Reynolds et al. (2004) "Rational siRNA design for RNA interference" Nature Biotechnology 22:326-330). For each of the 13 genes, four siRNAs duplexes with the highest scores were selected and a BLAST search was conducted using the Human EST database. To minimize the potential for off-target silencing effects, only those sequence targets with more than three mismatches against un-related sequences were selected (Jackson et al. (2003) "Expression profiling reveals off-target gene regulation by RNAi" Nat Biotechnol 21:635-7). All duplexes were synthesized in Dharmacon (Lafayette, Colo.) as 21-mers with UU overhangs using a modified method of 2'-ACE chemistry (Scaringe (2000) "Advanced 5'-silyl-2'-orthoester approach to RNA oligonucleotide synthesis" Methods Enzymol 317:3-18) and the AS strand was chemically phosphorylated to ensure maximum activity (Martinez et al. (2002) "Single-stranded antisense siRNAs guide target RNA cleavage in RNAi" Cell 110:563-74).

Example 3 siRNA Transfection

[0103] Transfection was essentially as previously described (Harborth et al. (2001) "Identification of essential genes in cultured mammalian cells using small interfering RNAs" J Cell Sci 114:4557-65) with minor modifications.

Example 4 VKOR Activity Assay

[0104] siRNA transfected A549 cells were trypsinized and washed twice with cold PBS. 1.5.times.10.sup.7 cells were taken for each VKOR assay. 200 .mu.L buffer D (250 mM Na.sub.2HPO.sub.4--NaH.sub.2PO.sub.4, 500 mM KCl, 20% glycerol and 0.75% CHAPS, pH 7.4) was added to the cell pellet, followed by sonication of the cell lysate. For assays of solubilized microsomes, microsomes were prepared from 2.times.10.sup.9 cells as described (Lin et al. (2002) "The putative vitamin K-dependent gamma-glutamyl carboxylase internal propeptide appears to be the propeptide binding site" J Biol Chem 277:28584-91); 10 to 50 .mu.L of solubilized microsomes were used for each assay. Vitamin K epoxide was added to the concentration indicated in the figure legends and DTT was added to 4 mM to initiate the reaction. The reaction mixture was incubated in yellow light at 30.degree. C. for 30 minutes and stopped by adding 500 .mu.L 0.05 M AgNO.sub.3: isopropanol (5:9). 500 .mu.L hexane was added and the mixture was vortexed vigorously for 1 minute to extract the vitamin K and KO. After 5 minutes centrifugation, the upper organic layer was transferred to a 5-mL brown vial and dried with N.sub.2. 150 .mu.L HPLC buffer, acetonitrile:isopropanol:water (100:7:2), was added to dissolve the vitamin K and KO and the sample was analyzed by HPLC on an A C-18 column (Vydac, cat #218TP54).

Example 5. RT-qPCR (Reverse Transcriptase Quantitative PCR)

[0105] 1.times.10.sup.6 cells were washed with PBS twice and total RNA was isolated with Trizol reagent according to the manufacturer's protocol (Invitrogen). 1 .mu.g of RNA was digested by RQ1 DNaseI (Promega) and heat-inactivated. First strand cDNA was made with M-MLV reverse transcriptase (Invitrogen). cDNAs were mixed with DyNAmo SYBR Green qPCR pre-mix (Finnzymes) and real-time PCR was performed with an Opticon II PCR thermal cycler (MJ Research). The following primers were used:

TABLE-US-00001 13124769-5' (F): (TCCAACAGCATATTCGGTTGC, SEQ ID NO: 1); 13124769-3 (R)': (TTCTTGGACCTTCCGGAAACT, SEQ ID NO: 2); GAPDH-F: (GAAGGTGAAGGTCGGAGTC, SEQ ID NO: 3); GAPDH-R: (GAAGATGGTGATGGGATTTC, SEQ ID NO: 4); Lamin-RT-F: (CTAGGTGAGGCCAAGAAGCAA, SEQ ID NO: 5) and Lamin-RT-R: (CTGTTCCTCTCAGCAGACTGC, SEQ ID NO: 6).

Example 6. Over-Expression of VKOR in Sf9 Insect Cell Line

[0106] The cDNA for the mGC11276 coding region was cloned into pVL1392 (Pharmingen), with the HPC4 tag (EDQVDPRLIDGK, SEQ ID NO: 7) at its amino terminus and expressed in Sf9 cells as described (Li et al. (2000) "Identification of a Drosophila vitamin K-dependent gamma-glutamyl carboxylase" J Biol Chem 275:18291-6).

Example 7. Gene Selection

[0107] The search for the VKOR gene was focused on human chromosome sixteen between markers D16S3131 and D16S419. This region corresponds to chromosome 16 at 50cM-65cM on the genetic map and 26-46.3 Mb on the physical map. 190 predicted coding sequences in this region were analyzed by a BLASTX search of the NCBI non-redundant protein database. Those human genes and orthologs from related species with known function were eliminated. Because VKOR appears to be a transmembrane protein (Carlisle & Suttie (1980) "Vitamin K dependent carboxylase: subcellular location of the carboxylase and enzymes involved in vitamin K metabolism in rat liver" Biochemistry 19:1161-7), the remaining genes were translated according to the cDNA sequences in the NCBI database and analyzed with the programs TMHMM and TMAP (Biology WorkBench, San Diego Supercomputer System) to predict those with transmembrane domains. Thirteen genes predicted to code for integral membrane proteins were chosen for further analysis.

Example 8. Cell Line Screening for VKOR Activity

[0108] The strategy was to identify a cell line expressing relatively high amounts of VKOR activity and use siRNA to systematically knock down all thirteen candidate genes. siRNA, double stranded RNA of 21-23 nucleotides, has been shown to cause specific RNA degradation in cell culture (Hara et al. (2002) "Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action" Cell 110:177-89; Krichevsky & Kosik (2002) "RNAi functions in cultured mammalian neurons" Proc Natl Acad Sci USA 99:11926-9; Burns et al. (2003) "Silencing of the Novel p53 Target Gene Snk/Plk2 Leads to Mitotic Catastrophe in Paclitaxel (Taxol)-Exposed Cells" Mol Cell Biol 23:5556-71). However, application of siRNA for large scale screening in mammalian cells has not previously been reported because of the difficulty in identifying a functional target for a specific mammalian cell mRNA (Holen et al. (2003) "Similar behaviour of single-strand and double-strand siRNAs suggests they act through a common RNAi pathway" Nucleic Acids Res 31:2401-7). The development of a rational selection algorithm (Reynolds et al.) for siRNA design increases the probability that a specific siRNA can be developed; furthermore, the probability of success can be increased by pooling four rationally selected siRNAs. Using siRNA to search for previously unidentified genes has the advantage that, even if VKOR activity requires the product of more than one gene for activity, the screen should still be effective because the assay determines the loss of enzymatic activity.

[0109] Fifteen cell lines were screened and a human lung carcinoma line, A549, was identified to exhibit sufficient warfarin-sensitive VKOR activity for facile measurement. A second human colorectal adenocarcinoma cell line, HT29, which expressed very little VKOR activity, was used as a reference.

Example 9 siRNA Inhibition of VKOR Activity in A549 Cells

[0110] Each of the thirteen pools of siRNA were transfected in triplicate into A549 cells and assayed for VKOR activity after 72 hours. One siRNA pool specific for gene gi:13124769 reduced VKOR activity by 64%-70% in eight separate assays (FIG. 2).

[0111] One possible reason that VKOR activity was inhibited to only .about.35% of its initial activity after 72 hours is that the half-life of mammalian proteins varies greatly (from minutes to days) (Zhang et al. (1996) "The major calpain isozymes are long-lived proteins. Design of an antisense strategy for calpain depletion in cultured cells" J Biol Chem 271:18825-30; Bohley (1996) "Surface hydrophobicity and intracellular degradation of proteins" Biol Chem 377:425-35; Dice & Goldberg (1975) "Relationship between in vivo degradative rates and isoelectric points of proteins" Proc Natl Acad Sci USA 72:3893-7), and mRNA translation is being inhibited, not enzyme activity. Therefore, the cells were carried through eleven days and their VKOR activity followed. FIG. 3 shows that the level of mRNA for gi:13124769 mRNA decreased rapidly to about 20% of normal while VKOR activity decreased continuously during this time period. This reduction in activity is not a general effect of the siRNA or the result of cell death because the level of VKD carboxylase activity and lamin A/C mRNA remained constant. Furthermore, the level of gi:132124769 mRNA is four fold lower in HT-29 cells, which have low VKOR activity, than in A549 cells that exhibit high VKOR activity. These data indicate that gi:13124769 corresponds to the VKOR gene.

Example 10 Identification of Gene Encoding VKOR

[0112] The gene, IMAGE 3455200 (gi:13124769, SEQ ID NO: 8), identified herein to encode VKOR, maps to human chromosome 16p11.2, mouse chromosome 7F3, and rat chromosome 1:180.8 Mb. There are 338 cDNA clones in the NCBI database representing seven different splicing patterns (NCBI AceView program). These are composed of all or part of two to four exons. Among these, the most prevalent isoform, mGC11276, has three exons and is expressed at high levels in lung and liver cells. This three exon transcript (SEQ ID NO: 9) encodes a predicted protein of 163 amino acids with a mass of 18.2 kDa (SEQ ID NO: 10). It is a putative N-myristylated endoplasmic reticulum protein with one to three transmembrane domains, depending upon the program used for prediction. It has seven cysteine residues, which is consistent with observations that the enzymatic activity is dependent upon thiol reagents (Thijssen et al. (1994) "Microsomal lipoamide reductase provides vitamin K epoxide reductase with reducing equivalents" Biochem J 297:277-80). Five of the seven cysteines are conserved among human, mice, rat, zebrafish, Xenopus and Anopheles.

[0113] To confirm that the VKOR gene had been identified, the most prevalent form of the enzyme (three exons) was expressed in Spodoptera frugiperda, Sf9 cells. Sf9 cells have no measurable VKOR activity but exhibit warfarin sensitive activity when transfected with mGC11276 cDNA (FIG. 4). VKOR activity is observed from constructs with an epitope tag at either their amino or carboxyl terminus. This tag should assist in the purification of VKOR.

[0114] VKOR should exhibit warfarin sensitivity, therefore microsomes were made from Sf9 cells expressing VKOR and tested for warfarin sensitivity. The VKOR activity is warfarin-sensitive (FIG. 5).

[0115] In summary, the present invention provides the first example of using siRNA in mammalian cells to identify an unknown gene. The identity of the VKOR gene was confirmed by its expression in insect cells. The VKOR gene encodes several isoforms. It will be important to characterize the activity and expression pattern of each isoform. Millions of people world-wide utilize warfarin to inhibit coagulation; therefore it is important to further characterize VKOR as it can lead to more accurate dosing or design of safer, more effective, anti-coagulants.

Example 11 Studies on Carboxylation of Factor X

[0116] Post translational modification of glutamic acid to gamma carboxy glutamic acid is required for the activity of a number of proteins, most of them related to coagulation. Of these, several have become useful tools for treating various bleeding disorders. For example, recombinant human factor IX now accounts for most of the factor IX used for treating hemophilia B patients. In addition factor VIIa is widely used for treating patients with auto-antibodies (inhibitors) to either factor IX or factor VIII and for bleeding that results from general trauma. Another Gla protein, activated protein C, is used for the treatment of sepsis. These vitamin K dependent proteins can be produced in cell culture utilizing cells such as Chinese hamster ovary (CHO), baby hamster kidney cells (BHK) and human embryo kidney cells (HEK 293). A common problem for all of these cell lines is that, if significant overproduction is achieved, then a significant fraction of the recombinant protein produced is under-carboxylated. Originally it was thought that the limiting factor in carboxylation was the vitamin K dependent gamma glutamyl carboxylase. However, after its purification and cloning, it was reported that co-expression of factor IX and carboxylase failed to improve the degree of carboxylation of factor IX in a CHO cell line over-expressing human factor IX. The percentage of carboxylated factor X in the HEK 293 cell line can be increased by reducing the affinity of the factor X's propeptide. However, if the level of expression of factor X bearing the prothrombin propeptide is sufficiently high, the level of expression still exceeds the ability of the cell to achieve complete post-translational modification. The present study demonstrates that co-expressing vitamin K epoxide reductase in a cell line over-expressing factor X (with prothrombin propeptide) to the extent that only about 50% of the factor X is carboxylated, results in its near complete carboxylation.

[0117] Materials. All restriction enzymes were from New England Biolabs. Pfu DNA polymerase was obtained from Stratagene. Lipofectamine, hygromycin B and pcDNA3.1/Hygro vector were from Invitrogen. Trypsin-EDTA, fetal bovine serum and Dulbecco's phosphate buffered saline were from Sigma. Antibiotic-antimycotic, G418 (Geneticin) and DMEM F-12 were from GIBCO. Puromycin and the pIRESpuro3 vector were from BD Biosciences. Human factor X was from Enzyme Research Laboratories. Goat anti-human factor X (affinity-purified IgG) and rabbit anti-human factor X (IgG-peroxidase conjugate) were from Affinity Biologicals Corporation. Peroxidase-conjugated AffiniPure rabbit anti-goat IgG was from Jackson ImmunoResearch Laboratories INC. Q-sepharose Fast Flow was obtained from Amersham Pharmacia Biotech. The calcium-dependent monoclonal human FX antibody [MoAb, 4G3] was obtained from Dr. Harold James, University of Texas, Tyler, Tex. Bio-Scale CHT5-I Hydroxyapatite was from Bio-Rad Laboratories.

[0118] Construction of mammalian cell expression vector containing VKOR. Two primers were designed to amplify the VKOR cDNA.

TABLE-US-00002 Primer 1: (SEQ ID NO: 35) 5'-CCGGAATTCGCCGCCACCATGGGCAGCACCTGGGGGAGCCCTGGC TGGGTGCGG

introduced a Kozak sequence (underlined) and a 5' Eco R I site. Primer2: 5'-CGGGCGGCCGCTCAGTGCCTCTTAGCCTTGCC (SEQ ID NO:36) introduced a Nod site at the 3' terminus of the cDNA. After PCR amplification and digestion with EcoRI and NotI, the PCR product was inserted into pIRESpuro3, which has a CMV virus major immediate early promoter/enhancer and confers puromycin resistance upon the transformed cells.

[0119] Construction of mammalian cell expression vector containing HGC. Two primers were designed to amplify HGC cDNA.

TABLE-US-00003 Primer 3: (SEQ ID NO: 37) 5'-CGCGGATCCGCCGCCACCATGGCGGTGTCTGCCGGGTCCGCGCGGA CCTC GCCC

introduced a Bam H1 site and a Kozak sequence (underlined) at the 5' terminus and Primer4: 5'-CGGGCGGCCGCTCAGAACTCTGAGTGGACAGGATCAGGATTTGACTC (SEQ ID NO:38) introduced a NotI site at the 3' terminus. After digestion with BamHI and NotI, the PCR product was inserted into pcDNA3.1/Hygro, which has a CMV promoter and confers hygromycin resistance upon the transformed cell.

[0120] Stable cell lines expressing Human VKOR. A cell line expressing mutated factor X (HEK293-FXI16L) that produces factor X (half of which is fully carboxylated) at about 10-12 mg per liter was used. HEK293-FXI16L was prepared as described (Camire, 2000) and was selected with the neomycin analogue, G418. HEK293-FXI16L was transfected with the plasmid pIRESpuro3-VKOR using lipofectin (Invitrogen) according to the manufacturer's protocol. Selection was done with 450 .mu.l/ml G418 and 1.75 .mu.l/ml puromycin. Resistant colonies were picked and screened for VKOR activity. The colony with the highest VKOR activity was selected for further analysis.

[0121] Stable cell lines expressing Human GGCX. HEK293-FXI16L was transfected, using lipofectin, with the Plasmid pcDNA3.1/Hygro-HGGCX. Transformed colonies were selected with 300 .mu.g/ml of hygromycin and 450 .mu.g/ml of G418 and 18 clones were selected for assay of GGCX activity with the small peptide substrate FLEEL (SEQ ID NO:39). The colony with the highest GGCX activity was selected for further studies.

[0122] Stable cell lines co-expressing Human VKOR and HGC. To obtain a HEK293-FXI16L cell line over-expressing both VKOR and GGCX, HEK293-FXI16L-VKOR was transfected with the plasmid pcDNA3.1/Hygro-HGGCX and 18 resistant colonies were selected for analysis. HEK293-FXI16L-HGGCX was also transfected with HEK293-FXI16L-VKOR and from this selection, only one resistant colony was obtained. HEK293-FXI16L was transfected with both pIRESpuro3-VKOR and pcDNA3.1/Hygro-HGC, yielding 10 resistant colonies. The 29 isolated colonies were then assayed for both VKOR and GGCX activity. The clone with the highest levels of both activities was selected for further analysis.

[0123] Level of FXI16L production by each cell line. For the sandwich ELISA antibody assay, goat anti-human Factor X (Affinity-Purified IgG) IgG-Peroxidase Conjugate was used as the capture antibody and rabbit anti-human Factor X was used as the detecting antibody. P-OD was used as the substrate for color development. Human factor X was used to make a standard curve. HEK293-FXI16L, HEK293-FXI16L-VKOR, HEK293-FXI16L-HGGCX, and HEK293-FXI16L-VKOR-HGGCX were grown in T25 flasks until they were confluent, then the medium were replaced with serum-free medium containing vitamin K1. The serum-free medium was changed at 12 hours and after 24 hours the conditioned medium was collected and analyzed for FXI16L expression.

[0124] Expression of FXI16L from each cell line in roller bottles. The 4 stable cell lines, HEK293-FXI16L, HEK293-FXI16L-VKOR, HEK293-FXI16L-HGGCX, and HEK293-FXI16L-VKOR-HGGCX, were grown in T-225 flasks to confluency and transferred into roller bottles. At 24 and 36 hours the medium was replaced with serum-free medium containing Vitamin K1. The medium was collected from each cell line every 24 hours until a total of three liters was obtained.

[0125] Purification of FXI16L from each cell line. The conditioned medium was thawed and passed over a 0.45 .mu.m HVLP filter. EDTA was then added to 5 mM and 0.25 ml of a 100.times. stock protease inhibitor cocktail was added per liter of conditioned medium. The conditioned media was loaded on a Q-Sepharose.TM. Fast Flow column equilibrated with 20 mM Tris (pH 7.2)/60 mM NaCl/5 mM EDTA and the column was washed with the same buffer until the baseline was steady. 20 mM Tris (pH 7.2)/700 mM NaCl was used to elute FXI16L from the column. The protein containing fractions were pooled and dialyzed into 8 mM Tris(pH 7.4)/60 mM NaCl. Each sample was made 2 mM CaCl.sub.2 and applied to an immunoaffinity (4G3) column that had been equilibrated with 8 mM Tris(pH 7.4)/60 mM NaCl/2 mM CaCl.sub.2. After washing with the same buffer, eluted factor X was eluted with a linear gradient of 0-8 mM EDTA in the same buffer. The fractions containing protein were pooled and dialyzed overnight into 1 mM Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4(pH 6.8). The dialyzed samples were applied to a Bio-Scale CHT5-I hydroxyapatite column pre-equilibrated with the starting buffer. A linear gradient of 1 to 400 mM Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4(pH 6.8) was used to separate carboxylated and non-carboxylated factor X.

[0126] Western blotting of sample post Q-sepharose and SDS-PAGE of sample post 4G3. After purification by using Q-Sepharose.TM. Fast Flow, fractions from four cell lines (HEK293-FXI16L, HEK293-FXI16L-VKOR, HEK293-FXI16L-HGC, and HEK293-VKOR-HGC) were identified by Western blotting. Goat anti-human factor X (Affinity-Purified IgG) was used as first antibody, peroxidase-conjugated affinipure rabbit anti-goat IgG was used as second antibody and ECL substrates were used for developing. After purification by affinity antibody chromatography, some samples were checked for purity.

[0127] Analysis of mRNA expression levels for VKOR, HGC and FXI16L among each cell line using real-time Q-PCR. A total of 1.times.10.sup.6 cells for each cell line (HEK293-FXI16L, HEK293-FXI16L-VKOR, HEK293-FXI16L-HGC and HEK293-FXI16L-VKOR-HGC) were seeded in a 12 well plate. Total RNA was extracted from each cell line.

[0128] VKOR & HGC activity for each cell line (HEK293-FXI16L, HEK293-FXI16L-VKOR, HEK293-FXI16L-HGC and HEK293-FXI16L-VKOR-HGC). pIRESpuro3-VKOR was transfected into HEK293-FXI16L and selected with 1.75 .mu.g/ml puromycin and 450 .mu.g/ml G418. Eighteen single clones were screened for VKOR activity. A single clone that contained a very high level of VKOR activity was kept as a stable cell line, HEK293-FXI16L-VKOR. After pcDNA3.1/Hygro-HGC was transfected into HEK293-FXI16L, transfectants can be selected at 300 .mu.g/ml hygromycin and 450 .mu.g/ml G418. A total of 18 single clones were screened for HGC activity. A single clone that contained a very high level of HGC activity was kept as a stable cell line, HEK293-FXI16L-HGC.

[0129] Three methods were used to make the stable cell line that contains a high level of both VKOR and HGC activity. A total of 29 single clones were screened for VKOR and HGC activity. A single clone that contained a high level of both VKOR and HGC activity was kept as a stable cell line HEK293-FXI16L-VKOR-HGC.

[0130] FXI16L production in each of the cell line. HEK293-FXI16L-VKOR, HEK293-FXI16L-HGC and HEK293-FXI16L-VKOR-HGC all expressed FXI16L at levels at least as high as the host cell. This experiment was done for comparative purposes in 25 ml T-flasks and the levels of expression were lower than when the protein was prepared in roller bottles. These experiments show that selecting cells over-producing carboxylase or VKOR did not result in loss of factor X expression

[0131] Three liters of medium were collected from cells grown in roller bottles and the factor X from each cell line was purified by Q-sepharose and factor X antibody affinity chromatography as described.

[0132] Analyzing carboxylation ratio alteration of rFXI16L among each cell line by using hydroxyapatite chromatography. After being dialyzed to 1 mM Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 (pH 6.8), fractions post 4G3 were applied to a Bio-Scale CHT5-I Hydroxyapatite column. A linear gradient of 0-100% of 400 mM Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 (pH 6.8) was used to elute column. A total of two pools can be obtained from each sample. The first pool is composed of uncarboxylated human FXI16L, the second pool is composed of fully .gamma.-carboxylated human FXI16L. For each cell line, the amount of fully .gamma.-carboxylated human FXI16L is divided by total amount of human FXI16L, carboxylated to obtain a ratio. The carboxylated ratio for host cell line HEK293-FXI16L is 52% [4.5 mg/(4.13 mg+4.5 mg)=52%]. The carboxylated ratio for the other three cell lines (HEK293-FXI16L-VKOR, HEK293-FXI16L-HGC and HEK293-FXI16L-VKOR-HGC) is 92% [10.5 mg/(0.9 mg+10.5 mg)=92%], 57% [6.4 mg/(4.78 mg+6.4 mg)=57%] and .about.100% [2.4 mg/2.4 mg=100%], respectively.

[0133] The big difference in carboxylation ratios between cell lines HEK293-FXI16L and HEK293-FXI16L-VKOR indicates that VKOR improves the .gamma.-carboxylation reaction in vivo dramatically. The smaller difference in carboxylation ratios between cell lines HEK293-FXI16L and HEK293-FXI16L-HGC indicates that although HGC catalyzes the carboxylation reaction, HGC is not the limiting factor of the carboxylation reaction in vivo, and it can only improves the carboxylation reaction in vivo a little. A carboxylation ratio of almost 100% in the cell line HEK293-FXI16L-VKOR-HGC indicates that VKOR can be the limiting factor of the carboxylation reaction in vivo. VKOR not only reduces vitamin K epoxide (KO) to vitamin K, but it also reduces vitamin K to reduced vitamin K (KH.sub.2). Without the second function, which can reduce K to KH.sub.2, vitamin K cannot be reused in the carboxylation system in vivo.

[0134] In summary, this study demonstrates that a nucleic acid encoding vitamin K epoxide reductase (VKOR), when transfected into cells that have been transfected with and are producing a vitamin K dependent protein, such as factor X, results in the production of a vitamin K dependent protein with increased carboxylation, thereby increasing the amount of active vitamin K dependent protein in the cell.

[0135] To do these experiments, a human embryo kidney (HEK) cell line expressing about 12-14 mg per liter of a mutant factor X (with a prothrombin propeptide) was used. This factor X had been modified by replacing its propeptide with the propeptide of prothrombin (Camire et al. "Enhanced gamma-carboxylation of recombinant factor X using a chimeric construct containing the prothrombin propeptide" Biochemistry 39(46):14322-9 (2000)) and was over-producing coagulation factor X to such a great extent that only .about.50% of the factor X was carboxylated.

[0136] This cell line making about 12-14 mg per liter of factor X was used for the starting and control cells. At this level of expression, the HEK cells could not completely carboxylate the factor X, even with the prothrombin propeptide instead of the normal factor X propeptide. The HEK 293 cells expressing factor X at about 12-14 mg per liter were transfected with 1) vitamin K epoxide reductase (VKOR), 2) vitamin K gamma glutamyl carboxylase, or 3) both vitamin K epoxide reductase and vitamin K gamma glutamyl carboxylase (VKGC). Several cell lines were selected that were shown to produce a large amount of carboxylase, VKOR or both VKOR and carboxylase. In each of these selected cell lines, the level of expression of factor X was at least as high as the starting cell line (within experimental limits). The results of these experiments are shown in FIGS. 6A-D. The comparison in all cases is with the original factor X expressing cell line, which is expressing factor X that is about 50% carboxylated.

[0137] Three liters of media were collected from each of the experimental cell lines and the factor X was purified over QAE sephadex, a factor X antibody column and finally a hydroxylapatite column. The figures shown are for the final hydroxylapatite columns. It has previously been shown that the first peak is uncarboxylated factor X and the second peak is fully carboxylated factor X (Camire et al.). FIG. 6A shows results of carboxylation of factor X in the original cell line without exogenous VKOR or VKGC. The second peak (centered around fraction 26) is the fully carboxylated peak. By area, 52% of factor X is fully carboxylated. FIG. 6B shows that adding carboxylase alone to the cell line expressing factor X did not significantly increase the percentage of carboxylated factor X. The extent of full carboxylation increases marginally to 57% fully carboxylated. In this case the fully carboxylated peak is centered around fraction 25. FIG. 6C shows that cells transfected with VKOR alone exhibited dramatically increased levels of fully carboxylated factor X. In this case the fully carboxylated peak (centered around fraction 26) and the extent of full carboxylation is increased to 92% of the total factor X made. FIG. 6D shows that when cells are transfected with both VKOR and VKGC, 100% of the factor X is fully carboxylated. In this situation, expression of the VKOR gene is the main determinant of complete carboxylation of a vitamin K dependent protein. In other situations where the turnover of the substrate is slower, i.e., when the propeptide binds much tighter than the factor X with the prothrombin propeptide and overexpression of the factor X is very high, it is likely that expression of the carboxylase gene will also be limiting. These results can be extended to all vitamin K dependent proteins, in addition to factor X.

[0138] These results demonstrate that VKOR (and probably VKGC) facilitates the production of fully carboxylated vitamin K dependent proteins. This provides a mechanism to increase the efficiency of producing fully active, modified proteins.

[0139] The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

[0140] All publications, patent applications, patents, patent publications and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented.

TABLE-US-00004 TABLE 1 Five SNPs examined in VKOR gene SNPs position AA change Heterozygous ratio vk563 G > A 5'- N/A 1/58 (SEQ ID NO: 15) UTR vk2581 G > C Intro N/A 17/58 (SEQ ID NO: 12) n2 vk3294 T > C Intro N/A 25/58 (SEQ ID NO: 13) n2 vk4501 C > T Exon Leu120Leu 1/58 (SEQ ID NO: 16 3 vk4769 G > A 3'- N/A 19/58 (SEQ ID NO: 14 UTR

TABLE-US-00005 TABLE 2 SNPs VIC Probe Sequence FAM Probe Sequence Forward Primer Reverse Primer vk2581 TCATCACGGAGCGTC TCATCACCGAGCGTC GGTGATCCACACAGCTGACA CCTGTTAGTTACCTCCCCACATC G > C (SEQ ID NO: 17) (SEQ ID NO: 18) (SEQ ID NO: 19) (SEQ ID NO:20) vk3294 CCAGGACCATGGTGC CCAGGACCGTGGTGC GCTCCAGAGAAGGCATCACT GCCAAGTCTGAACCATGTGTCA T > C (SEQ ID NO: 21) (SEQ ID NO: 22) (SEQ ID NO: 23) (SEQ ID NO:24) vk4769 ATACCCGCACATGAC CATACCCACACATGAC GTCCCTAGAAGGCCCTAGATGT GTGTGGCACATTTGGTCCATT G > A (SEQ ID NO: 25) (SEQ ID NO: 26) (SEQ ID NO: 27) (SEQ ID NO:28)

Sequence CWU 1

1

39121DNAArtificial sequenceSynthetic oligonucleotide primer 1tccaacagca tattcggttg c 21221DNAArtificial sequenceSynthetic oligonucleotide primer 2ttcttggacc ttccggaaac t 21319DNAArtificial sequenceSynthetic oligonucleotide primer 3gaaggtgaag gtcggagtc 19420DNAArtificial sequenceSynthetic oligonucleotide primer 4gaagatggtg atgggatttc 20521DNAArtificial sequenceSynthetic oligonucleotide primer 5ctaggtgagg ccaagaagca a 21621DNAArtificial sequenceSynthetic oligonucleotide primer 6ctgttcctct cagcagactg c 21712PRTArtificial sequenceHPC4 tag sequence 7Glu Asp Gln Val Asp Pro Arg Leu Ile Asp Gly Lys1 5 1083915DNAHomo sapiens 8ggttttctcc gcgggcgcct cgggcggaac ctggagataa tgggcagcac ctgggggagc 60cctggctggg tgcggctcgc tctttgcctg acgggcttag tgctctcgct ctacgcgctg 120cacgtgaagg cggcgcgcgc ccgggaccgg gattaccgcg cgctctgcga cgtgggcacc 180gccatcagct gttcgcgcgt cttctcctcc aggtgtgcac gggagtggga ggcgtggggc 240ctcggagcag ggcggccagg atgccagatg attattctgg agtctgggat cggtgtgccc 300ggggaacgga cacggggctg gactgctcgc ggggtcgttg cacaggggct gagctaccca 360gcgatactgg tgttcgaaat aagagtgcga ggcaagggac cagacagtgc tggggactgg 420gattattccg gggactcgca cgtgaattgg atgccaagga ataacggtga ccaggaaagg 480cggggaggca ggatggcggt agagattgac gatggtctca aggacggcgc gcaggtgaag 540gggggtgttg gcgatggctg cgcccaggaa caaggtggcc cggtctggct gtgcgtgatg 600gccaggcgtt agcataatga cggaatacag aggaggcgag tgagtggcca gggagctgga 660gattctgggg tccagggcaa agataatctg cccccgactc ccagtctctg atgcaaaacc 720gagtgaaccg ttataccagc cttgccattt taagaattac ttaagggccg ggcgcggtgg 780cccactcctg taatcccagc actttgggag gccgaggcgg atggatcact tgaagtcagg 840agttgaccag cctggccaac atggtgaaag cctgtctcta ccaaaaatag aaaaattaat 900cgggcgctat ggcgggtgcc ttaatcccag ctactcgggg gggctaaggc aggagaatcg 960cttgaacccg ggaggcggag gtttcagtga gccgagatcg cgccactgca ctccagcctg 1020ggccagagtg agactccgtc tcaaaaaaaa aaaaaaaaaa aaaaaaaaag agacttactt 1080aaggtctaag atgaaaagca gggcctacgg agtagccacg tccgggcctg gtctggggag 1140aggggaggat agggtcagtg acatggaatc ctgacgtggc caaaggtgcc cggtgccagg 1200agatcatcga cccttggact aggatgggag gtcggggaac agaggatagc ccaggtggct 1260tcttggaaat cacctttctc gggcagggtc caaggcactg ggttgacagt cctaacctgg 1320ttccacccca ccccacccct ctgccaggtg gggcaggggt ttcgggctgg tggagcatgt 1380gctgggacag gacagcatcc tcaatcaatc caacagcata ttcggttgca tcttctacac 1440actacagcta ttgttaggtg agtggctccg ccccctccct gcccgccccg ccccgcccct 1500catccccctt ggtcagctca gccccactcc atgcaatctt ggtgatccac acagctgaca 1560gccagctagc tgctcatcac ggagcgtcct gcgggtgggg atgtggggag gtaactaaca 1620ggagtctttt aattggttta agtactgtta gaggctgaag ggcccttaaa gacatcctag 1680gtccccaggt tttttgtttg ttgttgtttt gagacagggt ctggctctgt tgcccaaagt 1740gaggtctagg atgcccttag tgtgcactgg cgtgatctca gttcatggca acctctgcct 1800ccctgcccaa gggatcctcc caccttagcc tcccaagcag ctggaatcac aggcgtgcac 1860cactatgccc agctaatttt tgtttttgtt tttttttggt agagatggtg tctcgccatg 1920ttgcccaggc tggtctcaag caatctgtct gcctcagcct cccaaagtgc tggggggatt 1980acaggcgtga gctaccatgc cccaccaaca ccccagtttt gtggaaaaga tgccgaaatt 2040cctttttaag gagaagctga gcatgagcta tcttttgtct catttagtgc tcagcaggaa 2100aatttgtatc tagtcccata agaacagaga gaggaaccaa gggagtggaa gacgatggcg 2160ccccaggcct tgctgatgcc atatgccgga gatgagacta tccattacca cccttcccag 2220caggctccca cgctcccttt gagtcaccct tcccagctcc agagaaggca tcactgaggg 2280aggcccagca ccatggtcct ggctgacaca tggttcagac ttggccgatt tatttaagaa 2340attttattgc tcagaacttt ccctccctgg gcaatggcaa gagcttcaga gaccagtccc 2400ttggagggga cctgttgaag ccttcttttt tttttttttt aagaaataat cttgctctgt 2460tgcccaggct ggagtgcagt ggcacaatca tagctcactg taacctggct caagcgatcc 2520tcctgagtag ctaggactat aggcatgtca ctgcacccag ctaatttttt tttttttttt 2580tttttttttt ttgcgacata gtctcgctct gtcaccaggc tggagtgcag tggcacgatc 2640ttggctcact gcaacctctg cctcccgggt tcaagcaatt ttcctgcctc agcctcctga 2700gtagctggga ctacaggcgc gtgtcaccac gcccagctaa tttttgtatt tttagtggag 2760acagggtttc accatgttgg ctaggatggt ctcaatctct tgacctggtg atccatccgc 2820cttggcctcc caaagtgcta ggattacagg cgtgagtcaa cctcaccggg catttttttt 2880ttgagacgaa gtcttgctct tgctgcccaa gctggaatgt ggtggcatga tctcggctca 2940ctgcaacctc cacctcctag gttcaagcga ttctccacct tagcctcccc agcagctggg 3000attacaggtg cccatcaaca cacccggcta atttttgtat ttttattaga gatggggttt 3060tgccatgttg gccaggctgc tctcgaactc ctaacctcag gtgatccacc cccattggcc 3120tcccaaaata ctgggattac aggcatgagc caccgtgccc agctgaattt ctaaattttt 3180gatagagatc gggtctttct atgttgccca agctggtctt gaactcctag cctaaagcag 3240tcttcccacc tcggcctccc agagtgtttg gaatacgtgc gtaagccacc acatctgccc 3300tggagcctct tgttttagag acccttccca gcagctcctg gcatctaggt agtgcagtga 3360catcatggag tgttcgggag gtggccagtg cctgaagccc acaccggacc ctcttctgcc 3420ttgcaggttg cctgcggaca cgctgggcct ctgtcctgat gctgctgagc tccctggtgt 3480ctctcgctgg ttctgtctac ctggcctgga tcctgttctt cgtgctctat gatttctgca 3540ttgtttgtat caccacctat gctatcaacg tgagcctgat gtggctcagt ttccggaagg 3600tccaagaacc ccagggcaag gctaagaggc actgagccct caacccaagc caggctgacc 3660tcatctgctt tgctttggca tgtgagcctt gcctaagggg gcatatctgg gtccctagaa 3720ggccctagat gtggggcttc tagattaccc cctcctcctg ccatacccgc acatgacaat 3780ggaccaaatg tgccacacgc tcgctctttt ttacacccag tgcctctgac tctgtcccca 3840tgggctggtc tccaaagctc tttccattgc ccagggaggg aaggttctga gcaataaagt 3900ttcttagatc aatca 39159806DNAHomo sapiensCDS(48)..(536) 9ggcacgaggg ttttctccgc gggcgcctcg ggcggaacct ggagata atg ggc agc 56 Met Gly Ser 1acc tgg ggg agc cct ggc tgg gtg cgg ctc gct ctt tgc ctg acg ggc 104Thr Trp Gly Ser Pro Gly Trp Val Arg Leu Ala Leu Cys Leu Thr Gly 5 10 15tta gtg ctc tcg ctc tac gcg ctg cac gtg aag gcg gcg cgc gcc cgg 152Leu Val Leu Ser Leu Tyr Ala Leu His Val Lys Ala Ala Arg Ala Arg20 25 30 35gac cgg gat tac cgc gcg ctc tgc gac gtg ggc acc gcc atc agc tgt 200Asp Arg Asp Tyr Arg Ala Leu Cys Asp Val Gly Thr Ala Ile Ser Cys 40 45 50tcg cgc gtc ttc tcc tcc agg tgg ggc agg ggt ttc ggg ctg gtg gag 248Ser Arg Val Phe Ser Ser Arg Trp Gly Arg Gly Phe Gly Leu Val Glu 55 60 65cat gtg ctg gga cag gac agc atc ctc aat caa tcc aac agc ata ttc 296His Val Leu Gly Gln Asp Ser Ile Leu Asn Gln Ser Asn Ser Ile Phe 70 75 80ggt tgc atc ttc tac aca cta cag cta ttg tta ggt tgc ctg cgg aca 344Gly Cys Ile Phe Tyr Thr Leu Gln Leu Leu Leu Gly Cys Leu Arg Thr 85 90 95cgc tgg gcc tct gtc ctg atg ctg ctg agc tcc ctg gtg tct ctc gct 392Arg Trp Ala Ser Val Leu Met Leu Leu Ser Ser Leu Val Ser Leu Ala100 105 110 115ggt tct gtc tac ctg gcc tgg atc ctg ttc ttc gtg ctc tat gat ttc 440Gly Ser Val Tyr Leu Ala Trp Ile Leu Phe Phe Val Leu Tyr Asp Phe 120 125 130tgc att gtt tgt atc acc acc tat gct atc aac gtg agc ctg atg tgg 488Cys Ile Val Cys Ile Thr Thr Tyr Ala Ile Asn Val Ser Leu Met Trp 135 140 145ctc agt ttc cgg aag gtc caa gaa ccc cag ggc aag gct aag agg cac 536Leu Ser Phe Arg Lys Val Gln Glu Pro Gln Gly Lys Ala Lys Arg His 150 155 160tgagccctca acccaagcca ggctgacctc atctgctttg ctttggcatg tgagccttgc 596ctaagggggc atatctgggt ccctagaagg ccctagatgt ggggcttcta gattaccccc 656tcctcctgcc atacccgcac atgacaatgg accaaatgtg ccacacgctc gctctttttt 716acacccagtg cctctgactc tgtccccatg ggctggtctc caaagctctt tccattgccc 776agggagggaa ggttctgagc aataaagttt 80610163PRTHomo sapiens 10Met Gly Ser Thr Trp Gly Ser Pro Gly Trp Val Arg Leu Ala Leu Cys1 5 10 15Leu Thr Gly Leu Val Leu Ser Leu Tyr Ala Leu His Val Lys Ala Ala 20 25 30Arg Ala Arg Asp Arg Asp Tyr Arg Ala Leu Cys Asp Val Gly Thr Ala 35 40 45Ile Ser Cys Ser Arg Val Phe Ser Ser Arg Trp Gly Arg Gly Phe Gly 50 55 60Leu Val Glu His Val Leu Gly Gln Asp Ser Ile Leu Asn Gln Ser Asn65 70 75 80Ser Ile Phe Gly Cys Ile Phe Tyr Thr Leu Gln Leu Leu Leu Gly Cys 85 90 95Leu Arg Thr Arg Trp Ala Ser Val Leu Met Leu Leu Ser Ser Leu Val 100 105 110Ser Leu Ala Gly Ser Val Tyr Leu Ala Trp Ile Leu Phe Phe Val Leu 115 120 125Tyr Asp Phe Cys Ile Val Cys Ile Thr Thr Tyr Ala Ile Asn Val Ser 130 135 140Leu Met Trp Leu Ser Phe Arg Lys Val Gln Glu Pro Gln Gly Lys Ala145 150 155 160Lys Arg His115915DNAHomo sapiens 11caccatcaga tgggacgtct gtgaaggaga gacctcatct ggcccacagc ttggaaagga 60gagactgact gttgagttga tgcaagctca ggtgttgcca ggcgggcgcc atgatagtag 120agaggttagg atactgtcaa gggtgtgtgt ggccaaagga gtggttctgt gaatgtatgg 180gagaaaggga gaccgaccac caggaagcac tggtgaggca ggacccggga ggatgggagg 240ctgcagcccg aatggtgcct gaaatagttt caggggaaat gcttggttcc cgaatcggat 300cgccgtattc gctggatccc ctgatccgct ggtctctagg tcccggatgc tgcaattctt 360acaacaggac ttggcatagg gtaagcgcaa atgctgttaa ccacactaac acactttttt 420ttttcttttt tttttttgag acagagtctc actctgtcgg cctggctgga gtgcagtggc 480acgatctcgg ctcactgcaa cctccggctc cccggctcaa gcaattctcc tgcctcagcc 540tcccgagtag ctgggattac aggcatgtgc caccacgccc ggctaatttt tgtattttta 600gttgagatgg ggtttcacca tgttggcgag gctggtcttg aactcctgac ctcaggtaat 660ccgccagcct cggcctccca aagtgctggg attacaagcg tgagccaccg tgcccggcca 720acagttttta aatctgtgga gacttcattt cccttgatgc cttgcagccg cgccgactac 780aactcccatc atgcctggca gccgctgggg ccgcgattcc gcacgtccct tacccgcttc 840actagtcccg gcattcttcg ctgttttcct aactcgcccg cttgactagc gccctggaac 900agccatttgg gtcgtggagt gcgagcacgg ccggccaatc gccgagtcag agggccagga 960ggggcgcggc cattcgccgc ccggcccctg ctccgtggct ggttttctcc gcgggcgcct 1020cgggcggaac ctggagataa tgggcagcac ctgggggagc cctggctggg tgcggctcgc 1080tctttgcctg acgggcttag tgctctcgct ctacgcgctg cacgtgaagg cggcgcgcgc 1140ccgggaccgg gattaccgcg cgctctgcga cgtgggcacc gccatcagct gttcgcgcgt 1200cttctcctcc aggtgtgcac gggagtggga ggcgtggggc ctcggagcag ggcggccagg 1260atgccagatg attattctgg agtctgggat cggtgtgccc ggggaacgga cacggggctg 1320gactgctcgc ggggtcgttg cacaggggct gagctaccca gcgatactgg tgttcgaaat 1380aagagtgcga ggcaagggac cagacagtgc tggggactgg gattattccg gggactcgca 1440cgtgaattgg atgccaagga ataacggtga ccaggaaagg cggggaggca ggatggcggt 1500agagattgac gatggtctca aggacggcgc gcaggtgaag gggggtgttg gcgatggctg 1560cgcccaggaa caaggtggcc cggtctggct gtgcgtgatg gccaggcgtt agcataatga 1620cggaatacag aggaggcgag tgagtggcca gggagctgga gattctgggg tccagggcaa 1680agataatctg cccccgactc ccagtctctg atgcaaaacc gagtgaaccg ttataccagc 1740cttgccattt taagaattac ttaagggccg ggcgcggtgg cccactcctg taatcccagc 1800actttgggag gccgaggcgg atggatcact tgaagtcagg agttgaccag cctggccaac 1860atggtgaaag cctgtctcta ccaaaaatag aaaaattaat cgggcgctat ggcgggtgcc 1920ttaatcccag ctactcgggg gggctaaggc aggagaatcg cttgaacccg ggaggcggag 1980gtttcagtga gccgagatcg cgccactgca ctccagcctg ggccagagtg agactccgtc 2040tcaaaaaaaa aaaaaaaaaa aaaaaaaaag agacttactt aaggtctaag atgaaaagca 2100gggcctacgg agtagccacg tccgggcctg gtctggggag aggggaggat agggtcagtg 2160acatggaatc ctgacgtggc caaaggtgcc cggtgccagg agatcatcga cccttggact 2220aggatgggag gtcggggaac agaggatagc ccaggtggct tcttggaaat cacctttctc 2280gggcagggtc caaggcactg ggttgacagt cctaacctgg ttccacccca ccccacccct 2340ctgccaggtg gggcaggggt ttcgggctgg tggagcatgt gctgggacag gacagcatcc 2400tcaatcaatc caacagcata ttcggttgca tcttctacac actacagcta ttgttaggtg 2460agtggctccg ccccctccct gcccgccccg ccccgcccct catccccctt ggtcagctca 2520gccccactcc atgcaatctt ggtgatccac acagctgaca gccagctagc tgctcatcac 2580ggagcgtcct gcgggtgggg atgtggggag gtaactaaca ggagtctttt aattggttta 2640agtactgtta gaggctgaag ggcccttaaa gacatcctag gtccccaggt tttttgtttg 2700ttgttgtttt gagacagggt ctggctctgt tgcccaaagt gaggtctagg atgcccttag 2760tgtgcactgg cgtgatctca gttcatggca acctctgcct ccctgcccaa gggatcctcc 2820caccttagcc tcccaagcag ctggaatcac aggcgtgcac cactatgccc agctaatttt 2880tgtttttgtt tttttttggt agagatggtg tctcgccatg ttgcccaggc tggtctcaag 2940caatctgtct gcctcagcct cccaaagtgc tggggggatt acaggcgtga gctaccatgc 3000cccaccaaca ccccagtttt gtggaaaaga tgccgaaatt cctttttaag gagaagctga 3060gcatgagcta tcttttgtct catttagtgc tcagcaggaa aatttgtatc tagtcccata 3120agaacagaga gaggaaccaa gggagtggaa gacgatggcg ccccaggcct tgctgatgcc 3180atatgccgga gatgagacta tccattacca cccttcccag caggctccca cgctcccttt 3240gagtcaccct tcccagctcc agagaaggca tcactgaggg aggcccagca ccatggtcct 3300ggctgacaca tggttcagac ttggccgatt tatttaagaa attttattgc tcagaacttt 3360ccctccctgg gcaatggcaa gagcttcaga gaccagtccc ttggagggga cctgttgaag 3420ccttcttttt tttttttttt aagaaataat cttgctctgt tgcccaggct ggagtgcagt 3480ggcacaatca tagctcactg taacctggct caagcgatcc tcctgagtag ctaggactat 3540aggcatgtca ctgcacccag ctaatttttt tttttttttt tttttttttt ttgcgacata 3600gtctcgctct gtcaccaggc tggagtgcag tggcacgatc ttggctcact gcaacctctg 3660cctcccgggt tcaagcaatt ttcctgcctc agcctcctga gtagctggga ctacaggcgc 3720gtgtcaccac gcccagctaa tttttgtatt tttagtggag acagggtttc accatgttgg 3780ctaggatggt ctcaatctct tgacctggtg atccatccgc cttggcctcc caaagtgcta 3840ggattacagg cgtgagtcaa cctcaccggg catttttttt ttgagacgaa gtcttgctct 3900tgctgcccaa gctggaatgt ggtggcatga tctcggctca ctgcaacctc cacctcctag 3960gttcaagcga ttctccacct tagcctcccc agcagctggg attacaggtg cccatcaaca 4020cacccggcta atttttgtat ttttattaga gatggggttt tgccatgttg gccaggctgc 4080tctcgaactc ctaacctcag gtgatccacc cccattggcc tcccaaaata ctgggattac 4140aggcatgagc caccgtgccc agctgaattt ctaaattttt gatagagatc gggtctttct 4200atgttgccca agctggtctt gaactcctag cctaaagcag tcttcccacc tcggcctccc 4260agagtgtttg gaatacgtgc gtaagccacc acatctgccc tggagcctct tgttttagag 4320acccttccca gcagctcctg gcatctaggt agtgcagtga catcatggag tgttcgggag 4380gtggccagtg cctgaagccc acaccggacc ctcttctgcc ttgcaggttg cctgcggaca 4440cgctgggcct ctgtcctgat gctgctgagc tccctggtgt ctctcgctgg ttctgtctac 4500ctggcctgga tcctgttctt cgtgctctat gatttctgca ttgtttgtat caccacctat 4560gctatcaacg tgagcctgat gtggctcagt ttccggaagg tccaagaacc ccagggcaag 4620gctaagaggc actgagccct caacccaagc caggctgacc tcatctgctt tgctttggca 4680tgtgagcctt gcctaagggg gcatatctgg gtccctagaa ggccctagat gtggggcttc 4740tagattaccc cctcctcctg ccatacccgc acatgacaat ggaccaaatg tgccacacgc 4800tcgctctttt ttacacccag tgcctctgac tctgtcccca tgggctggtc tccaaagctc 4860tttccattgc ccagggaggg aaggttctga gcaataaagt ttcttagatc aatcagccaa 4920gtctgaacca tgtgtctgcc atggactgtg gtgctgggcc tccctcggtg ttgccttctc 4980tggagctggg aagggtgagt cagagggaga gtggagggcc tgctgggaag ggtggttatg 5040ggtagtctca tctccagtgt gtggagtcag caaggcctgg ggcaccattg gcccccaccc 5100ccaggaaaca ggctggcagc tcgctcctgc tgcccacagg agccaggcct cctctcctgg 5160gaaggctgag cacacacctg gaagggcagg ctgcccttct ggttctgtaa atgcttgctg 5220ggaagttctt ccttgagttt aactttaacc cctccagttg ccttatcgac cattccaagc 5280cagtattggt agccttggag ggtcagggcc aggttgtgaa ggtttttgtt ttgcctatta 5340tgccctgacc acttacctac atgccaagca ctgtttaaga acttgtgttg gcagggtgca 5400gtggctcaca cctgtaatcc ctgtactttg ggaggccaag gcaggaggat cacttgaggc 5460caggagttcc agaccagcct gggcaaaata gtgagacccc tgtctctaca aaaaaaaaaa 5520aaaaaaaaaa ttagccaggc atggtggtgt atgtacctat agtcccaact aatcgggaag 5580ctggcgggaa gactgcttga gcccagaagg ttgaggctgc agtgagccat gatcactgca 5640ctccagcctg agcaacagag caagaccgtc tccaaaaaaa aacaaaaaac aaaaaaaaac 5700ttgtgttaac gtgttaaact cgtttaatct ttacagtgat ttatgaggtg ggtactatta 5760ttatccctat cttgatgata gggacagagt ggctaattag tatgcctgag atcacacagc 5820tactgcagga ggctctcagg atttgaatcc acctggtcca tctggctcca gcatctatat 5880gctttttttt ttgttggttt gtttttgaga cggac 5915125915DNAHomo sapiens 12caccatcaga tgggacgtct gtgaaggaga gacctcatct ggcccacagc ttggaaagga 60gagactgact gttgagttga tgcaagctca ggtgttgcca ggcgggcgcc atgatagtag 120agaggttagg atactgtcaa gggtgtgtgt ggccaaagga gtggttctgt gaatgtatgg 180gagaaaggga gaccgaccac caggaagcac tggtgaggca ggacccggga ggatgggagg 240ctgcagcccg aatggtgcct gaaatagttt caggggaaat gcttggttcc cgaatcggat 300cgccgtattc gctggatccc ctgatccgct ggtctctagg tcccggatgc tgcaattctt 360acaacaggac ttggcatagg gtaagcgcaa atgctgttaa ccacactaac acactttttt 420ttttcttttt tttttttgag acagagtctc actctgtcgg cctggctgga gtgcagtggc 480acgatctcgg ctcactgcaa cctccggctc cccggctcaa gcaattctcc tgcctcagcc 540tcccgagtag ctgggattac aggcatgtgc caccacgccc ggctaatttt tgtattttta 600gttgagatgg ggtttcacca tgttggcgag gctggtcttg aactcctgac ctcaggtaat 660ccgccagcct cggcctccca aagtgctggg attacaagcg tgagccaccg tgcccggcca 720acagttttta aatctgtgga gacttcattt cccttgatgc cttgcagccg cgccgactac 780aactcccatc atgcctggca gccgctgggg ccgcgattcc gcacgtccct tacccgcttc 840actagtcccg gcattcttcg ctgttttcct aactcgcccg cttgactagc gccctggaac 900agccatttgg gtcgtggagt gcgagcacgg ccggccaatc gccgagtcag agggccagga 960ggggcgcggc cattcgccgc ccggcccctg ctccgtggct ggttttctcc gcgggcgcct 1020cgggcggaac ctggagataa tgggcagcac ctgggggagc cctggctggg tgcggctcgc 1080tctttgcctg acgggcttag tgctctcgct ctacgcgctg cacgtgaagg cggcgcgcgc 1140ccgggaccgg gattaccgcg cgctctgcga cgtgggcacc gccatcagct gttcgcgcgt 1200cttctcctcc aggtgtgcac gggagtggga

ggcgtggggc ctcggagcag ggcggccagg 1260atgccagatg attattctgg agtctgggat cggtgtgccc ggggaacgga cacggggctg 1320gactgctcgc ggggtcgttg cacaggggct gagctaccca gcgatactgg tgttcgaaat 1380aagagtgcga ggcaagggac cagacagtgc tggggactgg gattattccg gggactcgca 1440cgtgaattgg atgccaagga ataacggtga ccaggaaagg cggggaggca ggatggcggt 1500agagattgac gatggtctca aggacggcgc gcaggtgaag gggggtgttg gcgatggctg 1560cgcccaggaa caaggtggcc cggtctggct gtgcgtgatg gccaggcgtt agcataatga 1620cggaatacag aggaggcgag tgagtggcca gggagctgga gattctgggg tccagggcaa 1680agataatctg cccccgactc ccagtctctg atgcaaaacc gagtgaaccg ttataccagc 1740cttgccattt taagaattac ttaagggccg ggcgcggtgg cccactcctg taatcccagc 1800actttgggag gccgaggcgg atggatcact tgaagtcagg agttgaccag cctggccaac 1860atggtgaaag cctgtctcta ccaaaaatag aaaaattaat cgggcgctat ggcgggtgcc 1920ttaatcccag ctactcgggg gggctaaggc aggagaatcg cttgaacccg ggaggcggag 1980gtttcagtga gccgagatcg cgccactgca ctccagcctg ggccagagtg agactccgtc 2040tcaaaaaaaa aaaaaaaaaa aaaaaaaaag agacttactt aaggtctaag atgaaaagca 2100gggcctacgg agtagccacg tccgggcctg gtctggggag aggggaggat agggtcagtg 2160acatggaatc ctgacgtggc caaaggtgcc cggtgccagg agatcatcga cccttggact 2220aggatgggag gtcggggaac agaggatagc ccaggtggct tcttggaaat cacctttctc 2280gggcagggtc caaggcactg ggttgacagt cctaacctgg ttccacccca ccccacccct 2340ctgccaggtg gggcaggggt ttcgggctgg tggagcatgt gctgggacag gacagcatcc 2400tcaatcaatc caacagcata ttcggttgca tcttctacac actacagcta ttgttaggtg 2460agtggctccg ccccctccct gcccgccccg ccccgcccct catccccctt ggtcagctca 2520gccccactcc atgcaatctt ggtgatccac acagctgaca gccagctagc tgctcatcac 2580cgagcgtcct gcgggtgggg atgtggggag gtaactaaca ggagtctttt aattggttta 2640agtactgtta gaggctgaag ggcccttaaa gacatcctag gtccccaggt tttttgtttg 2700ttgttgtttt gagacagggt ctggctctgt tgcccaaagt gaggtctagg atgcccttag 2760tgtgcactgg cgtgatctca gttcatggca acctctgcct ccctgcccaa gggatcctcc 2820caccttagcc tcccaagcag ctggaatcac aggcgtgcac cactatgccc agctaatttt 2880tgtttttgtt tttttttggt agagatggtg tctcgccatg ttgcccaggc tggtctcaag 2940caatctgtct gcctcagcct cccaaagtgc tggggggatt acaggcgtga gctaccatgc 3000cccaccaaca ccccagtttt gtggaaaaga tgccgaaatt cctttttaag gagaagctga 3060gcatgagcta tcttttgtct catttagtgc tcagcaggaa aatttgtatc tagtcccata 3120agaacagaga gaggaaccaa gggagtggaa gacgatggcg ccccaggcct tgctgatgcc 3180atatgccgga gatgagacta tccattacca cccttcccag caggctccca cgctcccttt 3240gagtcaccct tcccagctcc agagaaggca tcactgaggg aggcccagca ccatggtcct 3300ggctgacaca tggttcagac ttggccgatt tatttaagaa attttattgc tcagaacttt 3360ccctccctgg gcaatggcaa gagcttcaga gaccagtccc ttggagggga cctgttgaag 3420ccttcttttt tttttttttt aagaaataat cttgctctgt tgcccaggct ggagtgcagt 3480ggcacaatca tagctcactg taacctggct caagcgatcc tcctgagtag ctaggactat 3540aggcatgtca ctgcacccag ctaatttttt tttttttttt tttttttttt ttgcgacata 3600gtctcgctct gtcaccaggc tggagtgcag tggcacgatc ttggctcact gcaacctctg 3660cctcccgggt tcaagcaatt ttcctgcctc agcctcctga gtagctggga ctacaggcgc 3720gtgtcaccac gcccagctaa tttttgtatt tttagtggag acagggtttc accatgttgg 3780ctaggatggt ctcaatctct tgacctggtg atccatccgc cttggcctcc caaagtgcta 3840ggattacagg cgtgagtcaa cctcaccggg catttttttt ttgagacgaa gtcttgctct 3900tgctgcccaa gctggaatgt ggtggcatga tctcggctca ctgcaacctc cacctcctag 3960gttcaagcga ttctccacct tagcctcccc agcagctggg attacaggtg cccatcaaca 4020cacccggcta atttttgtat ttttattaga gatggggttt tgccatgttg gccaggctgc 4080tctcgaactc ctaacctcag gtgatccacc cccattggcc tcccaaaata ctgggattac 4140aggcatgagc caccgtgccc agctgaattt ctaaattttt gatagagatc gggtctttct 4200atgttgccca agctggtctt gaactcctag cctaaagcag tcttcccacc tcggcctccc 4260agagtgtttg gaatacgtgc gtaagccacc acatctgccc tggagcctct tgttttagag 4320acccttccca gcagctcctg gcatctaggt agtgcagtga catcatggag tgttcgggag 4380gtggccagtg cctgaagccc acaccggacc ctcttctgcc ttgcaggttg cctgcggaca 4440cgctgggcct ctgtcctgat gctgctgagc tccctggtgt ctctcgctgg ttctgtctac 4500ctggcctgga tcctgttctt cgtgctctat gatttctgca ttgtttgtat caccacctat 4560gctatcaacg tgagcctgat gtggctcagt ttccggaagg tccaagaacc ccagggcaag 4620gctaagaggc actgagccct caacccaagc caggctgacc tcatctgctt tgctttggca 4680tgtgagcctt gcctaagggg gcatatctgg gtccctagaa ggccctagat gtggggcttc 4740tagattaccc cctcctcctg ccatacccgc acatgacaat ggaccaaatg tgccacacgc 4800tcgctctttt ttacacccag tgcctctgac tctgtcccca tgggctggtc tccaaagctc 4860tttccattgc ccagggaggg aaggttctga gcaataaagt ttcttagatc aatcagccaa 4920gtctgaacca tgtgtctgcc atggactgtg gtgctgggcc tccctcggtg ttgccttctc 4980tggagctggg aagggtgagt cagagggaga gtggagggcc tgctgggaag ggtggttatg 5040ggtagtctca tctccagtgt gtggagtcag caaggcctgg ggcaccattg gcccccaccc 5100ccaggaaaca ggctggcagc tcgctcctgc tgcccacagg agccaggcct cctctcctgg 5160gaaggctgag cacacacctg gaagggcagg ctgcccttct ggttctgtaa atgcttgctg 5220ggaagttctt ccttgagttt aactttaacc cctccagttg ccttatcgac cattccaagc 5280cagtattggt agccttggag ggtcagggcc aggttgtgaa ggtttttgtt ttgcctatta 5340tgccctgacc acttacctac atgccaagca ctgtttaaga acttgtgttg gcagggtgca 5400gtggctcaca cctgtaatcc ctgtactttg ggaggccaag gcaggaggat cacttgaggc 5460caggagttcc agaccagcct gggcaaaata gtgagacccc tgtctctaca aaaaaaaaaa 5520aaaaaaaaaa ttagccaggc atggtggtgt atgtacctat agtcccaact aatcgggaag 5580ctggcgggaa gactgcttga gcccagaagg ttgaggctgc agtgagccat gatcactgca 5640ctccagcctg agcaacagag caagaccgtc tccaaaaaaa aacaaaaaac aaaaaaaaac 5700ttgtgttaac gtgttaaact cgtttaatct ttacagtgat ttatgaggtg ggtactatta 5760ttatccctat cttgatgata gggacagagt ggctaattag tatgcctgag atcacacagc 5820tactgcagga ggctctcagg atttgaatcc acctggtcca tctggctcca gcatctatat 5880gctttttttt ttgttggttt gtttttgaga cggac 5915135915DNAHomo sapiens 13caccatcaga tgggacgtct gtgaaggaga gacctcatct ggcccacagc ttggaaagga 60gagactgact gttgagttga tgcaagctca ggtgttgcca ggcgggcgcc atgatagtag 120agaggttagg atactgtcaa gggtgtgtgt ggccaaagga gtggttctgt gaatgtatgg 180gagaaaggga gaccgaccac caggaagcac tggtgaggca ggacccggga ggatgggagg 240ctgcagcccg aatggtgcct gaaatagttt caggggaaat gcttggttcc cgaatcggat 300cgccgtattc gctggatccc ctgatccgct ggtctctagg tcccggatgc tgcaattctt 360acaacaggac ttggcatagg gtaagcgcaa atgctgttaa ccacactaac acactttttt 420ttttcttttt tttttttgag acagagtctc actctgtcgg cctggctgga gtgcagtggc 480acgatctcgg ctcactgcaa cctccggctc cccggctcaa gcaattctcc tgcctcagcc 540tcccgagtag ctgggattac aggcatgtgc caccacgccc ggctaatttt tgtattttta 600gttgagatgg ggtttcacca tgttggcgag gctggtcttg aactcctgac ctcaggtaat 660ccgccagcct cggcctccca aagtgctggg attacaagcg tgagccaccg tgcccggcca 720acagttttta aatctgtgga gacttcattt cccttgatgc cttgcagccg cgccgactac 780aactcccatc atgcctggca gccgctgggg ccgcgattcc gcacgtccct tacccgcttc 840actagtcccg gcattcttcg ctgttttcct aactcgcccg cttgactagc gccctggaac 900agccatttgg gtcgtggagt gcgagcacgg ccggccaatc gccgagtcag agggccagga 960ggggcgcggc cattcgccgc ccggcccctg ctccgtggct ggttttctcc gcgggcgcct 1020cgggcggaac ctggagataa tgggcagcac ctgggggagc cctggctggg tgcggctcgc 1080tctttgcctg acgggcttag tgctctcgct ctacgcgctg cacgtgaagg cggcgcgcgc 1140ccgggaccgg gattaccgcg cgctctgcga cgtgggcacc gccatcagct gttcgcgcgt 1200cttctcctcc aggtgtgcac gggagtggga ggcgtggggc ctcggagcag ggcggccagg 1260atgccagatg attattctgg agtctgggat cggtgtgccc ggggaacgga cacggggctg 1320gactgctcgc ggggtcgttg cacaggggct gagctaccca gcgatactgg tgttcgaaat 1380aagagtgcga ggcaagggac cagacagtgc tggggactgg gattattccg gggactcgca 1440cgtgaattgg atgccaagga ataacggtga ccaggaaagg cggggaggca ggatggcggt 1500agagattgac gatggtctca aggacggcgc gcaggtgaag gggggtgttg gcgatggctg 1560cgcccaggaa caaggtggcc cggtctggct gtgcgtgatg gccaggcgtt agcataatga 1620cggaatacag aggaggcgag tgagtggcca gggagctgga gattctgggg tccagggcaa 1680agataatctg cccccgactc ccagtctctg atgcaaaacc gagtgaaccg ttataccagc 1740cttgccattt taagaattac ttaagggccg ggcgcggtgg cccactcctg taatcccagc 1800actttgggag gccgaggcgg atggatcact tgaagtcagg agttgaccag cctggccaac 1860atggtgaaag cctgtctcta ccaaaaatag aaaaattaat cgggcgctat ggcgggtgcc 1920ttaatcccag ctactcgggg gggctaaggc aggagaatcg cttgaacccg ggaggcggag 1980gtttcagtga gccgagatcg cgccactgca ctccagcctg ggccagagtg agactccgtc 2040tcaaaaaaaa aaaaaaaaaa aaaaaaaaag agacttactt aaggtctaag atgaaaagca 2100gggcctacgg agtagccacg tccgggcctg gtctggggag aggggaggat agggtcagtg 2160acatggaatc ctgacgtggc caaaggtgcc cggtgccagg agatcatcga cccttggact 2220aggatgggag gtcggggaac agaggatagc ccaggtggct tcttggaaat cacctttctc 2280gggcagggtc caaggcactg ggttgacagt cctaacctgg ttccacccca ccccacccct 2340ctgccaggtg gggcaggggt ttcgggctgg tggagcatgt gctgggacag gacagcatcc 2400tcaatcaatc caacagcata ttcggttgca tcttctacac actacagcta ttgttaggtg 2460agtggctccg ccccctccct gcccgccccg ccccgcccct catccccctt ggtcagctca 2520gccccactcc atgcaatctt ggtgatccac acagctgaca gccagctagc tgctcatcac 2580ggagcgtcct gcgggtgggg atgtggggag gtaactaaca ggagtctttt aattggttta 2640agtactgtta gaggctgaag ggcccttaaa gacatcctag gtccccaggt tttttgtttg 2700ttgttgtttt gagacagggt ctggctctgt tgcccaaagt gaggtctagg atgcccttag 2760tgtgcactgg cgtgatctca gttcatggca acctctgcct ccctgcccaa gggatcctcc 2820caccttagcc tcccaagcag ctggaatcac aggcgtgcac cactatgccc agctaatttt 2880tgtttttgtt tttttttggt agagatggtg tctcgccatg ttgcccaggc tggtctcaag 2940caatctgtct gcctcagcct cccaaagtgc tggggggatt acaggcgtga gctaccatgc 3000cccaccaaca ccccagtttt gtggaaaaga tgccgaaatt cctttttaag gagaagctga 3060gcatgagcta tcttttgtct catttagtgc tcagcaggaa aatttgtatc tagtcccata 3120agaacagaga gaggaaccaa gggagtggaa gacgatggcg ccccaggcct tgctgatgcc 3180atatgccgga gatgagacta tccattacca cccttcccag caggctccca cgctcccttt 3240gagtcaccct tcccagctcc agagaaggca tcactgaggg aggcccagca ccacggtcct 3300ggctgacaca tggttcagac ttggccgatt tatttaagaa attttattgc tcagaacttt 3360ccctccctgg gcaatggcaa gagcttcaga gaccagtccc ttggagggga cctgttgaag 3420ccttcttttt tttttttttt aagaaataat cttgctctgt tgcccaggct ggagtgcagt 3480ggcacaatca tagctcactg taacctggct caagcgatcc tcctgagtag ctaggactat 3540aggcatgtca ctgcacccag ctaatttttt tttttttttt tttttttttt ttgcgacata 3600gtctcgctct gtcaccaggc tggagtgcag tggcacgatc ttggctcact gcaacctctg 3660cctcccgggt tcaagcaatt ttcctgcctc agcctcctga gtagctggga ctacaggcgc 3720gtgtcaccac gcccagctaa tttttgtatt tttagtggag acagggtttc accatgttgg 3780ctaggatggt ctcaatctct tgacctggtg atccatccgc cttggcctcc caaagtgcta 3840ggattacagg cgtgagtcaa cctcaccggg catttttttt ttgagacgaa gtcttgctct 3900tgctgcccaa gctggaatgt ggtggcatga tctcggctca ctgcaacctc cacctcctag 3960gttcaagcga ttctccacct tagcctcccc agcagctggg attacaggtg cccatcaaca 4020cacccggcta atttttgtat ttttattaga gatggggttt tgccatgttg gccaggctgc 4080tctcgaactc ctaacctcag gtgatccacc cccattggcc tcccaaaata ctgggattac 4140aggcatgagc caccgtgccc agctgaattt ctaaattttt gatagagatc gggtctttct 4200atgttgccca agctggtctt gaactcctag cctaaagcag tcttcccacc tcggcctccc 4260agagtgtttg gaatacgtgc gtaagccacc acatctgccc tggagcctct tgttttagag 4320acccttccca gcagctcctg gcatctaggt agtgcagtga catcatggag tgttcgggag 4380gtggccagtg cctgaagccc acaccggacc ctcttctgcc ttgcaggttg cctgcggaca 4440cgctgggcct ctgtcctgat gctgctgagc tccctggtgt ctctcgctgg ttctgtctac 4500ctggcctgga tcctgttctt cgtgctctat gatttctgca ttgtttgtat caccacctat 4560gctatcaacg tgagcctgat gtggctcagt ttccggaagg tccaagaacc ccagggcaag 4620gctaagaggc actgagccct caacccaagc caggctgacc tcatctgctt tgctttggca 4680tgtgagcctt gcctaagggg gcatatctgg gtccctagaa ggccctagat gtggggcttc 4740tagattaccc cctcctcctg ccatacccgc acatgacaat ggaccaaatg tgccacacgc 4800tcgctctttt ttacacccag tgcctctgac tctgtcccca tgggctggtc tccaaagctc 4860tttccattgc ccagggaggg aaggttctga gcaataaagt ttcttagatc aatcagccaa 4920gtctgaacca tgtgtctgcc atggactgtg gtgctgggcc tccctcggtg ttgccttctc 4980tggagctggg aagggtgagt cagagggaga gtggagggcc tgctgggaag ggtggttatg 5040ggtagtctca tctccagtgt gtggagtcag caaggcctgg ggcaccattg gcccccaccc 5100ccaggaaaca ggctggcagc tcgctcctgc tgcccacagg agccaggcct cctctcctgg 5160gaaggctgag cacacacctg gaagggcagg ctgcccttct ggttctgtaa atgcttgctg 5220ggaagttctt ccttgagttt aactttaacc cctccagttg ccttatcgac cattccaagc 5280cagtattggt agccttggag ggtcagggcc aggttgtgaa ggtttttgtt ttgcctatta 5340tgccctgacc acttacctac atgccaagca ctgtttaaga acttgtgttg gcagggtgca 5400gtggctcaca cctgtaatcc ctgtactttg ggaggccaag gcaggaggat cacttgaggc 5460caggagttcc agaccagcct gggcaaaata gtgagacccc tgtctctaca aaaaaaaaaa 5520aaaaaaaaaa ttagccaggc atggtggtgt atgtacctat agtcccaact aatcgggaag 5580ctggcgggaa gactgcttga gcccagaagg ttgaggctgc agtgagccat gatcactgca 5640ctccagcctg agcaacagag caagaccgtc tccaaaaaaa aacaaaaaac aaaaaaaaac 5700ttgtgttaac gtgttaaact cgtttaatct ttacagtgat ttatgaggtg ggtactatta 5760ttatccctat cttgatgata gggacagagt ggctaattag tatgcctgag atcacacagc 5820tactgcagga ggctctcagg atttgaatcc acctggtcca tctggctcca gcatctatat 5880gctttttttt ttgttggttt gtttttgaga cggac 5915145915DNAHomo sapiens 14caccatcaga tgggacgtct gtgaaggaga gacctcatct ggcccacagc ttggaaagga 60gagactgact gttgagttga tgcaagctca ggtgttgcca ggcgggcgcc atgatagtag 120agaggttagg atactgtcaa gggtgtgtgt ggccaaagga gtggttctgt gaatgtatgg 180gagaaaggga gaccgaccac caggaagcac tggtgaggca ggacccggga ggatgggagg 240ctgcagcccg aatggtgcct gaaatagttt caggggaaat gcttggttcc cgaatcggat 300cgccgtattc gctggatccc ctgatccgct ggtctctagg tcccggatgc tgcaattctt 360acaacaggac ttggcatagg gtaagcgcaa atgctgttaa ccacactaac acactttttt 420ttttcttttt tttttttgag acagagtctc actctgtcgg cctggctgga gtgcagtggc 480acgatctcgg ctcactgcaa cctccggctc cccggctcaa gcaattctcc tgcctcagcc 540tcccgagtag ctgggattac aggcatgtgc caccacgccc ggctaatttt tgtattttta 600gttgagatgg ggtttcacca tgttggcgag gctggtcttg aactcctgac ctcaggtaat 660ccgccagcct cggcctccca aagtgctggg attacaagcg tgagccaccg tgcccggcca 720acagttttta aatctgtgga gacttcattt cccttgatgc cttgcagccg cgccgactac 780aactcccatc atgcctggca gccgctgggg ccgcgattcc gcacgtccct tacccgcttc 840actagtcccg gcattcttcg ctgttttcct aactcgcccg cttgactagc gccctggaac 900agccatttgg gtcgtggagt gcgagcacgg ccggccaatc gccgagtcag agggccagga 960ggggcgcggc cattcgccgc ccggcccctg ctccgtggct ggttttctcc gcgggcgcct 1020cgggcggaac ctggagataa tgggcagcac ctgggggagc cctggctggg tgcggctcgc 1080tctttgcctg acgggcttag tgctctcgct ctacgcgctg cacgtgaagg cggcgcgcgc 1140ccgggaccgg gattaccgcg cgctctgcga cgtgggcacc gccatcagct gttcgcgcgt 1200cttctcctcc aggtgtgcac gggagtggga ggcgtggggc ctcggagcag ggcggccagg 1260atgccagatg attattctgg agtctgggat cggtgtgccc ggggaacgga cacggggctg 1320gactgctcgc ggggtcgttg cacaggggct gagctaccca gcgatactgg tgttcgaaat 1380aagagtgcga ggcaagggac cagacagtgc tggggactgg gattattccg gggactcgca 1440cgtgaattgg atgccaagga ataacggtga ccaggaaagg cggggaggca ggatggcggt 1500agagattgac gatggtctca aggacggcgc gcaggtgaag gggggtgttg gcgatggctg 1560cgcccaggaa caaggtggcc cggtctggct gtgcgtgatg gccaggcgtt agcataatga 1620cggaatacag aggaggcgag tgagtggcca gggagctgga gattctgggg tccagggcaa 1680agataatctg cccccgactc ccagtctctg atgcaaaacc gagtgaaccg ttataccagc 1740cttgccattt taagaattac ttaagggccg ggcgcggtgg cccactcctg taatcccagc 1800actttgggag gccgaggcgg atggatcact tgaagtcagg agttgaccag cctggccaac 1860atggtgaaag cctgtctcta ccaaaaatag aaaaattaat cgggcgctat ggcgggtgcc 1920ttaatcccag ctactcgggg gggctaaggc aggagaatcg cttgaacccg ggaggcggag 1980gtttcagtga gccgagatcg cgccactgca ctccagcctg ggccagagtg agactccgtc 2040tcaaaaaaaa aaaaaaaaaa aaaaaaaaag agacttactt aaggtctaag atgaaaagca 2100gggcctacgg agtagccacg tccgggcctg gtctggggag aggggaggat agggtcagtg 2160acatggaatc ctgacgtggc caaaggtgcc cggtgccagg agatcatcga cccttggact 2220aggatgggag gtcggggaac agaggatagc ccaggtggct tcttggaaat cacctttctc 2280gggcagggtc caaggcactg ggttgacagt cctaacctgg ttccacccca ccccacccct 2340ctgccaggtg gggcaggggt ttcgggctgg tggagcatgt gctgggacag gacagcatcc 2400tcaatcaatc caacagcata ttcggttgca tcttctacac actacagcta ttgttaggtg 2460agtggctccg ccccctccct gcccgccccg ccccgcccct catccccctt ggtcagctca 2520gccccactcc atgcaatctt ggtgatccac acagctgaca gccagctagc tgctcatcac 2580ggagcgtcct gcgggtgggg atgtggggag gtaactaaca ggagtctttt aattggttta 2640agtactgtta gaggctgaag ggcccttaaa gacatcctag gtccccaggt tttttgtttg 2700ttgttgtttt gagacagggt ctggctctgt tgcccaaagt gaggtctagg atgcccttag 2760tgtgcactgg cgtgatctca gttcatggca acctctgcct ccctgcccaa gggatcctcc 2820caccttagcc tcccaagcag ctggaatcac aggcgtgcac cactatgccc agctaatttt 2880tgtttttgtt tttttttggt agagatggtg tctcgccatg ttgcccaggc tggtctcaag 2940caatctgtct gcctcagcct cccaaagtgc tggggggatt acaggcgtga gctaccatgc 3000cccaccaaca ccccagtttt gtggaaaaga tgccgaaatt cctttttaag gagaagctga 3060gcatgagcta tcttttgtct catttagtgc tcagcaggaa aatttgtatc tagtcccata 3120agaacagaga gaggaaccaa gggagtggaa gacgatggcg ccccaggcct tgctgatgcc 3180atatgccgga gatgagacta tccattacca cccttcccag caggctccca cgctcccttt 3240gagtcaccct tcccagctcc agagaaggca tcactgaggg aggcccagca ccatggtcct 3300ggctgacaca tggttcagac ttggccgatt tatttaagaa attttattgc tcagaacttt 3360ccctccctgg gcaatggcaa gagcttcaga gaccagtccc ttggagggga cctgttgaag 3420ccttcttttt tttttttttt aagaaataat cttgctctgt tgcccaggct ggagtgcagt 3480ggcacaatca tagctcactg taacctggct caagcgatcc tcctgagtag ctaggactat 3540aggcatgtca ctgcacccag ctaatttttt tttttttttt tttttttttt ttgcgacata 3600gtctcgctct gtcaccaggc tggagtgcag tggcacgatc ttggctcact gcaacctctg 3660cctcccgggt tcaagcaatt ttcctgcctc agcctcctga gtagctggga ctacaggcgc 3720gtgtcaccac gcccagctaa tttttgtatt tttagtggag acagggtttc accatgttgg 3780ctaggatggt ctcaatctct tgacctggtg atccatccgc cttggcctcc caaagtgcta 3840ggattacagg cgtgagtcaa cctcaccggg catttttttt ttgagacgaa gtcttgctct 3900tgctgcccaa gctggaatgt ggtggcatga tctcggctca ctgcaacctc cacctcctag 3960gttcaagcga ttctccacct tagcctcccc agcagctggg attacaggtg cccatcaaca 4020cacccggcta atttttgtat ttttattaga gatggggttt tgccatgttg gccaggctgc 4080tctcgaactc ctaacctcag gtgatccacc cccattggcc tcccaaaata ctgggattac 4140aggcatgagc caccgtgccc agctgaattt ctaaattttt gatagagatc gggtctttct 4200atgttgccca agctggtctt gaactcctag cctaaagcag tcttcccacc tcggcctccc 4260agagtgtttg gaatacgtgc gtaagccacc acatctgccc tggagcctct tgttttagag 4320acccttccca gcagctcctg gcatctaggt agtgcagtga

catcatggag tgttcgggag 4380gtggccagtg cctgaagccc acaccggacc ctcttctgcc ttgcaggttg cctgcggaca 4440cgctgggcct ctgtcctgat gctgctgagc tccctggtgt ctctcgctgg ttctgtctac 4500ctggcctgga tcctgttctt cgtgctctat gatttctgca ttgtttgtat caccacctat 4560gctatcaacg tgagcctgat gtggctcagt ttccggaagg tccaagaacc ccagggcaag 4620gctaagaggc actgagccct caacccaagc caggctgacc tcatctgctt tgctttggca 4680tgtgagcctt gcctaagggg gcatatctgg gtccctagaa ggccctagat gtggggcttc 4740tagattaccc cctcctcctg ccatacccac acatgacaat ggaccaaatg tgccacacgc 4800tcgctctttt ttacacccag tgcctctgac tctgtcccca tgggctggtc tccaaagctc 4860tttccattgc ccagggaggg aaggttctga gcaataaagt ttcttagatc aatcagccaa 4920gtctgaacca tgtgtctgcc atggactgtg gtgctgggcc tccctcggtg ttgccttctc 4980tggagctggg aagggtgagt cagagggaga gtggagggcc tgctgggaag ggtggttatg 5040ggtagtctca tctccagtgt gtggagtcag caaggcctgg ggcaccattg gcccccaccc 5100ccaggaaaca ggctggcagc tcgctcctgc tgcccacagg agccaggcct cctctcctgg 5160gaaggctgag cacacacctg gaagggcagg ctgcccttct ggttctgtaa atgcttgctg 5220ggaagttctt ccttgagttt aactttaacc cctccagttg ccttatcgac cattccaagc 5280cagtattggt agccttggag ggtcagggcc aggttgtgaa ggtttttgtt ttgcctatta 5340tgccctgacc acttacctac atgccaagca ctgtttaaga acttgtgttg gcagggtgca 5400gtggctcaca cctgtaatcc ctgtactttg ggaggccaag gcaggaggat cacttgaggc 5460caggagttcc agaccagcct gggcaaaata gtgagacccc tgtctctaca aaaaaaaaaa 5520aaaaaaaaaa ttagccaggc atggtggtgt atgtacctat agtcccaact aatcgggaag 5580ctggcgggaa gactgcttga gcccagaagg ttgaggctgc agtgagccat gatcactgca 5640ctccagcctg agcaacagag caagaccgtc tccaaaaaaa aacaaaaaac aaaaaaaaac 5700ttgtgttaac gtgttaaact cgtttaatct ttacagtgat ttatgaggtg ggtactatta 5760ttatccctat cttgatgata gggacagagt ggctaattag tatgcctgag atcacacagc 5820tactgcagga ggctctcagg atttgaatcc acctggtcca tctggctcca gcatctatat 5880gctttttttt ttgttggttt gtttttgaga cggac 5915155915DNAHomo sapiens 15caccatcaga tgggacgtct gtgaaggaga gacctcatct ggcccacagc ttggaaagga 60gagactgact gttgagttga tgcaagctca ggtgttgcca ggcgggcgcc atgatagtag 120agaggttagg atactgtcaa gggtgtgtgt ggccaaagga gtggttctgt gaatgtatgg 180gagaaaggga gaccgaccac caggaagcac tggtgaggca ggacccggga ggatgggagg 240ctgcagcccg aatggtgcct gaaatagttt caggggaaat gcttggttcc cgaatcggat 300cgccgtattc gctggatccc ctgatccgct ggtctctagg tcccggatgc tgcaattctt 360acaacaggac ttggcatagg gtaagcgcaa atgctgttaa ccacactaac acactttttt 420ttttcttttt tttttttgag acagagtctc actctgtcgg cctggctgga gtgcagtggc 480acgatctcgg ctcactgcaa cctccggctc cccggctcaa gcaattctcc tgcctcagcc 540tcccgagtag ctgggattac agacatgtgc caccacgccc ggctaatttt tgtattttta 600gttgagatgg ggtttcacca tgttggcgag gctggtcttg aactcctgac ctcaggtaat 660ccgccagcct cggcctccca aagtgctggg attacaagcg tgagccaccg tgcccggcca 720acagttttta aatctgtgga gacttcattt cccttgatgc cttgcagccg cgccgactac 780aactcccatc atgcctggca gccgctgggg ccgcgattcc gcacgtccct tacccgcttc 840actagtcccg gcattcttcg ctgttttcct aactcgcccg cttgactagc gccctggaac 900agccatttgg gtcgtggagt gcgagcacgg ccggccaatc gccgagtcag agggccagga 960ggggcgcggc cattcgccgc ccggcccctg ctccgtggct ggttttctcc gcgggcgcct 1020cgggcggaac ctggagataa tgggcagcac ctgggggagc cctggctggg tgcggctcgc 1080tctttgcctg acgggcttag tgctctcgct ctacgcgctg cacgtgaagg cggcgcgcgc 1140ccgggaccgg gattaccgcg cgctctgcga cgtgggcacc gccatcagct gttcgcgcgt 1200cttctcctcc aggtgtgcac gggagtggga ggcgtggggc ctcggagcag ggcggccagg 1260atgccagatg attattctgg agtctgggat cggtgtgccc ggggaacgga cacggggctg 1320gactgctcgc ggggtcgttg cacaggggct gagctaccca gcgatactgg tgttcgaaat 1380aagagtgcga ggcaagggac cagacagtgc tggggactgg gattattccg gggactcgca 1440cgtgaattgg atgccaagga ataacggtga ccaggaaagg cggggaggca ggatggcggt 1500agagattgac gatggtctca aggacggcgc gcaggtgaag gggggtgttg gcgatggctg 1560cgcccaggaa caaggtggcc cggtctggct gtgcgtgatg gccaggcgtt agcataatga 1620cggaatacag aggaggcgag tgagtggcca gggagctgga gattctgggg tccagggcaa 1680agataatctg cccccgactc ccagtctctg atgcaaaacc gagtgaaccg ttataccagc 1740cttgccattt taagaattac ttaagggccg ggcgcggtgg cccactcctg taatcccagc 1800actttgggag gccgaggcgg atggatcact tgaagtcagg agttgaccag cctggccaac 1860atggtgaaag cctgtctcta ccaaaaatag aaaaattaat cgggcgctat ggcgggtgcc 1920ttaatcccag ctactcgggg gggctaaggc aggagaatcg cttgaacccg ggaggcggag 1980gtttcagtga gccgagatcg cgccactgca ctccagcctg ggccagagtg agactccgtc 2040tcaaaaaaaa aaaaaaaaaa aaaaaaaaag agacttactt aaggtctaag atgaaaagca 2100gggcctacgg agtagccacg tccgggcctg gtctggggag aggggaggat agggtcagtg 2160acatggaatc ctgacgtggc caaaggtgcc cggtgccagg agatcatcga cccttggact 2220aggatgggag gtcggggaac agaggatagc ccaggtggct tcttggaaat cacctttctc 2280gggcagggtc caaggcactg ggttgacagt cctaacctgg ttccacccca ccccacccct 2340ctgccaggtg gggcaggggt ttcgggctgg tggagcatgt gctgggacag gacagcatcc 2400tcaatcaatc caacagcata ttcggttgca tcttctacac actacagcta ttgttaggtg 2460agtggctccg ccccctccct gcccgccccg ccccgcccct catccccctt ggtcagctca 2520gccccactcc atgcaatctt ggtgatccac acagctgaca gccagctagc tgctcatcac 2580ggagcgtcct gcgggtgggg atgtggggag gtaactaaca ggagtctttt aattggttta 2640agtactgtta gaggctgaag ggcccttaaa gacatcctag gtccccaggt tttttgtttg 2700ttgttgtttt gagacagggt ctggctctgt tgcccaaagt gaggtctagg atgcccttag 2760tgtgcactgg cgtgatctca gttcatggca acctctgcct ccctgcccaa gggatcctcc 2820caccttagcc tcccaagcag ctggaatcac aggcgtgcac cactatgccc agctaatttt 2880tgtttttgtt tttttttggt agagatggtg tctcgccatg ttgcccaggc tggtctcaag 2940caatctgtct gcctcagcct cccaaagtgc tggggggatt acaggcgtga gctaccatgc 3000cccaccaaca ccccagtttt gtggaaaaga tgccgaaatt cctttttaag gagaagctga 3060gcatgagcta tcttttgtct catttagtgc tcagcaggaa aatttgtatc tagtcccata 3120agaacagaga gaggaaccaa gggagtggaa gacgatggcg ccccaggcct tgctgatgcc 3180atatgccgga gatgagacta tccattacca cccttcccag caggctccca cgctcccttt 3240gagtcaccct tcccagctcc agagaaggca tcactgaggg aggcccagca ccatggtcct 3300ggctgacaca tggttcagac ttggccgatt tatttaagaa attttattgc tcagaacttt 3360ccctccctgg gcaatggcaa gagcttcaga gaccagtccc ttggagggga cctgttgaag 3420ccttcttttt tttttttttt aagaaataat cttgctctgt tgcccaggct ggagtgcagt 3480ggcacaatca tagctcactg taacctggct caagcgatcc tcctgagtag ctaggactat 3540aggcatgtca ctgcacccag ctaatttttt tttttttttt tttttttttt ttgcgacata 3600gtctcgctct gtcaccaggc tggagtgcag tggcacgatc ttggctcact gcaacctctg 3660cctcccgggt tcaagcaatt ttcctgcctc agcctcctga gtagctggga ctacaggcgc 3720gtgtcaccac gcccagctaa tttttgtatt tttagtggag acagggtttc accatgttgg 3780ctaggatggt ctcaatctct tgacctggtg atccatccgc cttggcctcc caaagtgcta 3840ggattacagg cgtgagtcaa cctcaccggg catttttttt ttgagacgaa gtcttgctct 3900tgctgcccaa gctggaatgt ggtggcatga tctcggctca ctgcaacctc cacctcctag 3960gttcaagcga ttctccacct tagcctcccc agcagctggg attacaggtg cccatcaaca 4020cacccggcta atttttgtat ttttattaga gatggggttt tgccatgttg gccaggctgc 4080tctcgaactc ctaacctcag gtgatccacc cccattggcc tcccaaaata ctgggattac 4140aggcatgagc caccgtgccc agctgaattt ctaaattttt gatagagatc gggtctttct 4200atgttgccca agctggtctt gaactcctag cctaaagcag tcttcccacc tcggcctccc 4260agagtgtttg gaatacgtgc gtaagccacc acatctgccc tggagcctct tgttttagag 4320acccttccca gcagctcctg gcatctaggt agtgcagtga catcatggag tgttcgggag 4380gtggccagtg cctgaagccc acaccggacc ctcttctgcc ttgcaggttg cctgcggaca 4440cgctgggcct ctgtcctgat gctgctgagc tccctggtgt ctctcgctgg ttctgtctac 4500ctggcctgga tcctgttctt cgtgctctat gatttctgca ttgtttgtat caccacctat 4560gctatcaacg tgagcctgat gtggctcagt ttccggaagg tccaagaacc ccagggcaag 4620gctaagaggc actgagccct caacccaagc caggctgacc tcatctgctt tgctttggca 4680tgtgagcctt gcctaagggg gcatatctgg gtccctagaa ggccctagat gtggggcttc 4740tagattaccc cctcctcctg ccatacccgc acatgacaat ggaccaaatg tgccacacgc 4800tcgctctttt ttacacccag tgcctctgac tctgtcccca tgggctggtc tccaaagctc 4860tttccattgc ccagggaggg aaggttctga gcaataaagt ttcttagatc aatcagccaa 4920gtctgaacca tgtgtctgcc atggactgtg gtgctgggcc tccctcggtg ttgccttctc 4980tggagctggg aagggtgagt cagagggaga gtggagggcc tgctgggaag ggtggttatg 5040ggtagtctca tctccagtgt gtggagtcag caaggcctgg ggcaccattg gcccccaccc 5100ccaggaaaca ggctggcagc tcgctcctgc tgcccacagg agccaggcct cctctcctgg 5160gaaggctgag cacacacctg gaagggcagg ctgcccttct ggttctgtaa atgcttgctg 5220ggaagttctt ccttgagttt aactttaacc cctccagttg ccttatcgac cattccaagc 5280cagtattggt agccttggag ggtcagggcc aggttgtgaa ggtttttgtt ttgcctatta 5340tgccctgacc acttacctac atgccaagca ctgtttaaga acttgtgttg gcagggtgca 5400gtggctcaca cctgtaatcc ctgtactttg ggaggccaag gcaggaggat cacttgaggc 5460caggagttcc agaccagcct gggcaaaata gtgagacccc tgtctctaca aaaaaaaaaa 5520aaaaaaaaaa ttagccaggc atggtggtgt atgtacctat agtcccaact aatcgggaag 5580ctggcgggaa gactgcttga gcccagaagg ttgaggctgc agtgagccat gatcactgca 5640ctccagcctg agcaacagag caagaccgtc tccaaaaaaa aacaaaaaac aaaaaaaaac 5700ttgtgttaac gtgttaaact cgtttaatct ttacagtgat ttatgaggtg ggtactatta 5760ttatccctat cttgatgata gggacagagt ggctaattag tatgcctgag atcacacagc 5820tactgcagga ggctctcagg atttgaatcc acctggtcca tctggctcca gcatctatat 5880gctttttttt ttgttggttt gtttttgaga cggac 5915165915DNAHomo sapiens 16caccatcaga tgggacgtct gtgaaggaga gacctcatct ggcccacagc ttggaaagga 60gagactgact gttgagttga tgcaagctca ggtgttgcca ggcgggcgcc atgatagtag 120agaggttagg atactgtcaa gggtgtgtgt ggccaaagga gtggttctgt gaatgtatgg 180gagaaaggga gaccgaccac caggaagcac tggtgaggca ggacccggga ggatgggagg 240ctgcagcccg aatggtgcct gaaatagttt caggggaaat gcttggttcc cgaatcggat 300cgccgtattc gctggatccc ctgatccgct ggtctctagg tcccggatgc tgcaattctt 360acaacaggac ttggcatagg gtaagcgcaa atgctgttaa ccacactaac acactttttt 420ttttcttttt tttttttgag acagagtctc actctgtcgg cctggctgga gtgcagtggc 480acgatctcgg ctcactgcaa cctccggctc cccggctcaa gcaattctcc tgcctcagcc 540tcccgagtag ctgggattac aggcatgtgc caccacgccc ggctaatttt tgtattttta 600gttgagatgg ggtttcacca tgttggcgag gctggtcttg aactcctgac ctcaggtaat 660ccgccagcct cggcctccca aagtgctggg attacaagcg tgagccaccg tgcccggcca 720acagttttta aatctgtgga gacttcattt cccttgatgc cttgcagccg cgccgactac 780aactcccatc atgcctggca gccgctgggg ccgcgattcc gcacgtccct tacccgcttc 840actagtcccg gcattcttcg ctgttttcct aactcgcccg cttgactagc gccctggaac 900agccatttgg gtcgtggagt gcgagcacgg ccggccaatc gccgagtcag agggccagga 960ggggcgcggc cattcgccgc ccggcccctg ctccgtggct ggttttctcc gcgggcgcct 1020cgggcggaac ctggagataa tgggcagcac ctgggggagc cctggctggg tgcggctcgc 1080tctttgcctg acgggcttag tgctctcgct ctacgcgctg cacgtgaagg cggcgcgcgc 1140ccgggaccgg gattaccgcg cgctctgcga cgtgggcacc gccatcagct gttcgcgcgt 1200cttctcctcc aggtgtgcac gggagtggga ggcgtggggc ctcggagcag ggcggccagg 1260atgccagatg attattctgg agtctgggat cggtgtgccc ggggaacgga cacggggctg 1320gactgctcgc ggggtcgttg cacaggggct gagctaccca gcgatactgg tgttcgaaat 1380aagagtgcga ggcaagggac cagacagtgc tggggactgg gattattccg gggactcgca 1440cgtgaattgg atgccaagga ataacggtga ccaggaaagg cggggaggca ggatggcggt 1500agagattgac gatggtctca aggacggcgc gcaggtgaag gggggtgttg gcgatggctg 1560cgcccaggaa caaggtggcc cggtctggct gtgcgtgatg gccaggcgtt agcataatga 1620cggaatacag aggaggcgag tgagtggcca gggagctgga gattctgggg tccagggcaa 1680agataatctg cccccgactc ccagtctctg atgcaaaacc gagtgaaccg ttataccagc 1740cttgccattt taagaattac ttaagggccg ggcgcggtgg cccactcctg taatcccagc 1800actttgggag gccgaggcgg atggatcact tgaagtcagg agttgaccag cctggccaac 1860atggtgaaag cctgtctcta ccaaaaatag aaaaattaat cgggcgctat ggcgggtgcc 1920ttaatcccag ctactcgggg gggctaaggc aggagaatcg cttgaacccg ggaggcggag 1980gtttcagtga gccgagatcg cgccactgca ctccagcctg ggccagagtg agactccgtc 2040tcaaaaaaaa aaaaaaaaaa aaaaaaaaag agacttactt aaggtctaag atgaaaagca 2100gggcctacgg agtagccacg tccgggcctg gtctggggag aggggaggat agggtcagtg 2160acatggaatc ctgacgtggc caaaggtgcc cggtgccagg agatcatcga cccttggact 2220aggatgggag gtcggggaac agaggatagc ccaggtggct tcttggaaat cacctttctc 2280gggcagggtc caaggcactg ggttgacagt cctaacctgg ttccacccca ccccacccct 2340ctgccaggtg gggcaggggt ttcgggctgg tggagcatgt gctgggacag gacagcatcc 2400tcaatcaatc caacagcata ttcggttgca tcttctacac actacagcta ttgttaggtg 2460agtggctccg ccccctccct gcccgccccg ccccgcccct catccccctt ggtcagctca 2520gccccactcc atgcaatctt ggtgatccac acagctgaca gccagctagc tgctcatcac 2580ggagcgtcct gcgggtgggg atgtggggag gtaactaaca ggagtctttt aattggttta 2640agtactgtta gaggctgaag ggcccttaaa gacatcctag gtccccaggt tttttgtttg 2700ttgttgtttt gagacagggt ctggctctgt tgcccaaagt gaggtctagg atgcccttag 2760tgtgcactgg cgtgatctca gttcatggca acctctgcct ccctgcccaa gggatcctcc 2820caccttagcc tcccaagcag ctggaatcac aggcgtgcac cactatgccc agctaatttt 2880tgtttttgtt tttttttggt agagatggtg tctcgccatg ttgcccaggc tggtctcaag 2940caatctgtct gcctcagcct cccaaagtgc tggggggatt acaggcgtga gctaccatgc 3000cccaccaaca ccccagtttt gtggaaaaga tgccgaaatt cctttttaag gagaagctga 3060gcatgagcta tcttttgtct catttagtgc tcagcaggaa aatttgtatc tagtcccata 3120agaacagaga gaggaaccaa gggagtggaa gacgatggcg ccccaggcct tgctgatgcc 3180atatgccgga gatgagacta tccattacca cccttcccag caggctccca cgctcccttt 3240gagtcaccct tcccagctcc agagaaggca tcactgaggg aggcccagca ccatggtcct 3300ggctgacaca tggttcagac ttggccgatt tatttaagaa attttattgc tcagaacttt 3360ccctccctgg gcaatggcaa gagcttcaga gaccagtccc ttggagggga cctgttgaag 3420ccttcttttt tttttttttt aagaaataat cttgctctgt tgcccaggct ggagtgcagt 3480ggcacaatca tagctcactg taacctggct caagcgatcc tcctgagtag ctaggactat 3540aggcatgtca ctgcacccag ctaatttttt tttttttttt tttttttttt ttgcgacata 3600gtctcgctct gtcaccaggc tggagtgcag tggcacgatc ttggctcact gcaacctctg 3660cctcccgggt tcaagcaatt ttcctgcctc agcctcctga gtagctggga ctacaggcgc 3720gtgtcaccac gcccagctaa tttttgtatt tttagtggag acagggtttc accatgttgg 3780ctaggatggt ctcaatctct tgacctggtg atccatccgc cttggcctcc caaagtgcta 3840ggattacagg cgtgagtcaa cctcaccggg catttttttt ttgagacgaa gtcttgctct 3900tgctgcccaa gctggaatgt ggtggcatga tctcggctca ctgcaacctc cacctcctag 3960gttcaagcga ttctccacct tagcctcccc agcagctggg attacaggtg cccatcaaca 4020cacccggcta atttttgtat ttttattaga gatggggttt tgccatgttg gccaggctgc 4080tctcgaactc ctaacctcag gtgatccacc cccattggcc tcccaaaata ctgggattac 4140aggcatgagc caccgtgccc agctgaattt ctaaattttt gatagagatc gggtctttct 4200atgttgccca agctggtctt gaactcctag cctaaagcag tcttcccacc tcggcctccc 4260agagtgtttg gaatacgtgc gtaagccacc acatctgccc tggagcctct tgttttagag 4320acccttccca gcagctcctg gcatctaggt agtgcagtga catcatggag tgttcgggag 4380gtggccagtg cctgaagccc acaccggacc ctcttctgcc ttgcaggttg cctgcggaca 4440cgctgggcct ctgtcctgat gctgctgagc tccctggtgt ctctcgctgg ttctgtctac 4500ttggcctgga tcctgttctt cgtgctctat gatttctgca ttgtttgtat caccacctat 4560gctatcaacg tgagcctgat gtggctcagt ttccggaagg tccaagaacc ccagggcaag 4620gctaagaggc actgagccct caacccaagc caggctgacc tcatctgctt tgctttggca 4680tgtgagcctt gcctaagggg gcatatctgg gtccctagaa ggccctagat gtggggcttc 4740tagattaccc cctcctcctg ccatacccgc acatgacaat ggaccaaatg tgccacacgc 4800tcgctctttt ttacacccag tgcctctgac tctgtcccca tgggctggtc tccaaagctc 4860tttccattgc ccagggaggg aaggttctga gcaataaagt ttcttagatc aatcagccaa 4920gtctgaacca tgtgtctgcc atggactgtg gtgctgggcc tccctcggtg ttgccttctc 4980tggagctggg aagggtgagt cagagggaga gtggagggcc tgctgggaag ggtggttatg 5040ggtagtctca tctccagtgt gtggagtcag caaggcctgg ggcaccattg gcccccaccc 5100ccaggaaaca ggctggcagc tcgctcctgc tgcccacagg agccaggcct cctctcctgg 5160gaaggctgag cacacacctg gaagggcagg ctgcccttct ggttctgtaa atgcttgctg 5220ggaagttctt ccttgagttt aactttaacc cctccagttg ccttatcgac cattccaagc 5280cagtattggt agccttggag ggtcagggcc aggttgtgaa ggtttttgtt ttgcctatta 5340tgccctgacc acttacctac atgccaagca ctgtttaaga acttgtgttg gcagggtgca 5400gtggctcaca cctgtaatcc ctgtactttg ggaggccaag gcaggaggat cacttgaggc 5460caggagttcc agaccagcct gggcaaaata gtgagacccc tgtctctaca aaaaaaaaaa 5520aaaaaaaaaa ttagccaggc atggtggtgt atgtacctat agtcccaact aatcgggaag 5580ctggcgggaa gactgcttga gcccagaagg ttgaggctgc agtgagccat gatcactgca 5640ctccagcctg agcaacagag caagaccgtc tccaaaaaaa aacaaaaaac aaaaaaaaac 5700ttgtgttaac gtgttaaact cgtttaatct ttacagtgat ttatgaggtg ggtactatta 5760ttatccctat cttgatgata gggacagagt ggctaattag tatgcctgag atcacacagc 5820tactgcagga ggctctcagg atttgaatcc acctggtcca tctggctcca gcatctatat 5880gctttttttt ttgttggttt gtttttgaga cggac 59151715DNAArtificialvk2581 G>C VIC probe sequence 17tcatcacgga gcgtc 151815DNAArtificialvk2581 G>C FAM probe sequence 18tcatcaccga gcgtc 151920DNAArtificialPCR primer 19ggtgatccac acagctgaca 202023DNAArtificialPCR primer 20cctgttagtt acctccccac atc 232115DNAArtificialvk3294 T>C VIC probe sequence 21ccaggaccat ggtgc 152215DNAArtificialvk3294 T>C FAM probe sequence 22ccaggaccgt ggtgc 152320DNAArtificialPCR primer 23gctccagaga aggcatcact 202422DNAArtificialPCR primer 24gccaagtctg aaccatgtgt ca 222515DNAArtificialvk4769 G>A VIC probe sequence 25atacccgcac atgac 152616DNAArtificialvk4769 G>A FAM probe sequence 26catacccaca catgac 162722DNAArtificialPCR primer 27gtccctagaa ggccctagat gt 222821DNAArtificialPCR primer 28gtgtggcaca tttggtccat t 212919DNAArtificialPCR primer 29ccaatcgccg agtcagagg 193020DNAArtificialPCR primer 30cccagtcccc agcactgtct 203120DNAArtificialPCR primer 31aggggaggat agggtcagtg 203221DNAArtificialPCR primer 32cctgttagtt acctccccac a 213320DNAArtificialPCR primer 33atacgtgcgt

aagccaccac 203420DNAArtificialPCR primer 34acccagatat gcccccttag 203552DNAArtificial sequencePCR primer 35ccggaattcg ccgccaccat gggcagcacc tgggggagcc ctggctgggt gc 523632DNAArtificial sequencePCR primer 36cgggcggccg ctcagtgcct cttagccttg cc 323752DNAArtificial sequencePCR primer 37cgcggatccg ccgccaccat ggcggtgtct gccgggtccg cgcggacctc gc 523847DNAArtificial sequencePCR primer 38cgggcggccg ctcagaactc tgagtggaca ggatcaggat ttgactc 47395PRTArtificial sequenceGGCX peptide substrate 39Phe Leu Glu Glu Leu1 5

Claims

1. A method of increasing the amount of carboxylated vitamin K dependent protein in a cell, comprising introducing into a cell that expresses a first nucleic acid encoding a vitamin K dependent protein a second heterologous nucleic acid encoding vitamin K epoxide reductase (VKOR) under conditions whereby said first and second nucleic acids are expressed to produce a vitamin K dependent protein and VKOR, respectively.