U.S. patent application number 17/412827 was filed with the patent office on 2022-07-21 for methods and compositions for producing vitamin k dependent proteins.
The applicant listed for this patent is The University of North Carolina at Chapel Hill. Invention is credited to Tao Li, Darrel W. Stafford.
Application Number | 20220228131 17/412827 |
Document ID | / |
Family ID | 1000006242556 |
Filed Date | 2022-07-21 |
United States Patent
Application |
20220228131 |
Kind Code |
A1 |
Stafford; Darrel W. ; et
al. |
July 21, 2022 |
METHODS AND COMPOSITIONS FOR PRODUCING VITAMIN K DEPENDENT
PROTEINS
Abstract
The present invention relates to methods and compositions for
improving the productivity of recombinant vitamin K dependent
protein expression in host cells.
Inventors: |
Stafford; Darrel W.;
(Carrboro, NC) ; Li; Tao; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of North Carolina at Chapel Hill |
Chapel Hill |
NC |
US |
|
|
Family ID: |
1000006242556 |
Appl. No.: |
17/412827 |
Filed: |
August 26, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16869701 |
May 8, 2020 |
|
|
|
17412827 |
|
|
|
|
16575083 |
Sep 18, 2019 |
|
|
|
16869701 |
|
|
|
|
16272290 |
Feb 11, 2019 |
|
|
|
16575083 |
|
|
|
|
16020731 |
Jun 27, 2018 |
|
|
|
16272290 |
|
|
|
|
15794843 |
Oct 26, 2017 |
|
|
|
16020731 |
|
|
|
|
15228740 |
Aug 4, 2016 |
|
|
|
15794843 |
|
|
|
|
14490244 |
Sep 18, 2014 |
9441208 |
|
|
15228740 |
|
|
|
|
14072108 |
Nov 5, 2013 |
|
|
|
14490244 |
|
|
|
|
12612154 |
Nov 4, 2009 |
8603823 |
|
|
14072108 |
|
|
|
|
11787072 |
Apr 13, 2007 |
7645602 |
|
|
12612154 |
|
|
|
|
10573131 |
Apr 18, 2006 |
7687233 |
|
|
11787072 |
|
|
|
|
PCT/US2005/008643 |
Mar 15, 2005 |
|
|
|
10573131 |
|
|
|
|
60505527 |
Sep 23, 2003 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6883 20130101;
C12Y 401/0109 20130101; C12Q 2600/106 20130101; C12N 9/0006
20130101; C12N 9/88 20130101; C12Q 2600/172 20130101; C12Q 2600/156
20130101; C12N 9/50 20130101; C12Y 304/21006 20130101; C12Q
2600/158 20130101; C12N 9/64 20130101; C12Y 101/04001 20130101 |
International
Class: |
C12N 9/04 20060101
C12N009/04; C12Q 1/6883 20060101 C12Q001/6883; C12N 9/64 20060101
C12N009/64; C12N 9/50 20060101 C12N009/50; C12N 9/88 20060101
C12N009/88 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant
Number HL006350 and HL048318 awarded by the National Institutes of
Health. The government has certain rights in the invention.
Claims
1. A host cell comprising a recombinant nucleic acid coding for a
vitamin K epoxide reductase (VKOR) or a functionally active
derivative thereof, and a recombinant nucleic acid coding for a
vitamin K dependent protein or a functionally active derivative
thereof, wherein both the recombinant VKOR and the recombinant
Vitamin K dependent protein are expressed in said host cell.
Description
STATEMENT OF PRIORITY
[0001] The present application is a continuation application of,
and claims priority to, U.S. application Ser. No. 16/869,701, filed
May 8, 2020, which is a continuation of U.S. application Ser. No.
16/575,083, filed Sep. 18, 2019 (abandoned), which is a
continuation application of U.S. application Ser. No. 16/272,290,
filed Feb. 11, 2019 (abandoned), which is a continuation
application of U.S. application Ser. No. 16/020,731, filed Jun. 27,
2018 (abandoned), which is a continuation application of U.S.
application Ser. No. 15/794,843, filed Oct. 26, 2017 (abandoned),
which is a continuation application of U.S. application Ser. No.
15/228,740, filed Aug. 4, 2016 (abandoned), which is a continuation
application of U.S. application Ser. No. 14/490,244, filed Sep. 18,
2014 and issued as U.S. Pat. No. 9,441,208 on Sep. 13, 2016, which
is a continuation application of U.S. application Ser. No.
14/072,108, filed Nov. 5, 2013 (abandoned), which is a continuation
application of U.S. application Ser. No. 12/612,154, filed Nov. 4,
2009 and issued as U.S. Pat. No. 8,603,823 on Dec. 12, 2013, which
is a continuation application of and claims priority to, U.S.
application Ser. No. 11/787,072, filed Apr. 13, 2007, and issued as
U.S. Pat. No. 7,645,602 on Jan. 12, 2010, which is a
continuation-in-part application of U.S. application Ser. No.
10/573,131, filed Apr. 18, 2006 and issued as U.S. Pat. No.
7,687,233 on Mar. 30, 2010, which claims the benefit, under 35
U.S.C. .sctn. 119(e), of U.S. Provisional Application Ser. No.
60/505,527, filed Sep. 23, 2003, and is a continuation-in-part of
PCT Application No. PCT/US2005/008643, filed Mar. 15, 2005, the
entire contents of each of which are incorporated by reference
herein.
FIELD OF THE INVENTION
[0003] The present invention relates to methods and compositions
for improving the productivity of recombinant vitamin K-dependent
protein expression in host cells.
BACKGROUND OF THE INVENTION
[0004] 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.
SUMMARY OF THE INVENTION
[0005] In one embodiment, the present invention relates to 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.
[0006] 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.
[0007] 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.
[0008] A further aspect of the present invention is an
oligonucleotide that hybridizes to an isolated nucleic acid
encoding VKOR as described herein.
[0009] 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.
[0010] 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, several
embodiments of the present invention further provide 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.
[0011] An isolated and/or purified host cell or organism is
disclosed in accordance with an embodiment of the present
invention. In one embodiment, the isolated host cell comprises a
recombinant nucleic acid coding for a vitamin K epoxide reductase
(VKOR) or a functionally active derivative thereof, and a
recombinant nucleic acid coding for a vitamin K dependent protein
or a functionally active derivative thereof, wherein both the
recombinant VKOR and the recombinant Vitamin K dependent protein
are expressed in the host cell. One of skill in the art will
understand that VKOR and vitamin K reductase complex subunit 1
(VKORC1) refer to the same enzyme, and that the terms can be used
interchangeably herein. Although in some embodiments, the host cell
can also include vitamin K dependent carboxylase, the carboxylase
is an optional compound and need not be included.
[0012] In a variation to the isolated host cell, the nucleic acid
coding for recombinant VKOR or the nucleic acid coding for the
recombinant Vitamin K dependent protein or both are expressed via
an expression mode selected from the group consisting of induced,
transient, and permanent expression, or combinations thereof.
[0013] In another variation, the isolated host cell is a mammalian
cell. The mammalian cell may be a cell derived from a mammalian
cell line selected from the group consisting of CHO cells and
HEK293 cells, or combinations thereof.
[0014] In another variation to the isolated host cell, the
recombinant Vitamin K dependent protein is a coagulation factor or
a functionally active derivative thereof. The coagulation factor
may be selected from the group consisting of factor II, factor VII,
factor IX, factor X, prothrombin, Protein C and Protein S. In one
preferred embodiment, the coagulation factor is human factor IX, or
combinations thereof.
[0015] A cell culture system is disclosed in accordance with
another embodiment of the present invention. The cell culture
system comprises isolated cells that contain a recombinant nucleic
acid coding for a vitamin K epoxide reductase (VKOR) or a
functionally active derivative thereof and a recombinant nucleic
acid coding for a vitamin K dependent protein or a functionally
active derivative thereof, wherein both the recombinant VKOR and
the recombinant Vitamin K dependent protein are expressed in the
cells.
[0016] In a variation to the cell culture system, the cultured
cells are mammalian cells. In another variation, the mammalian
cells may be selected from the group consisting of CHO cells and
HEK293 cells, or combinations thereof.
[0017] In another variation to cell culture system, the recombinant
Vitamin K dependent protein is a coagulation factor or a
functionally active derivative thereof. Preferably, the coagulation
factor is selected from the group consisting of factor II, factor
VII, factor IX, factor X, prothrombin, Protein C and Protein S. In
one preferred embodiment, the coagulation factor is human factor
IX.
[0018] A method for improving the productivity of recombinant
vitamin K dependent protein expression in an isolated and/or
purified host cell or organism is disclosed in accordance with
another embodiment of the present invention. In one embodiment, the
method comprises the steps of: providing an isolated host cell;
inserting a recombinant nucleic acid coding for a Vitamin K
dependent protein or a functionally active derivative thereof into
the host cell; inserting a recombinant nucleic acid coding for a
vitamin K epoxide reductase complex (VKOR) or a functionally active
derivative thereof into the host cell; and expressing the
recombinant nucleic acids.
[0019] Another method for improving the productivity of recombinant
vitamin K dependent protein expression in a host cell is disclosed
in accordance with another embodiment of the invention. The method
comprises the steps of: providing an isolated host cell having a
recombinant nucleic acid coding for a Vitamin K dependent protein
or a functionally active derivative thereof integrated into its
genome; inserting a recombinant nucleic acid coding for a vitamin K
epoxide reductase (VKOR) or a functionally active derivative
thereof into the host cell; and expressing the nucleic acids. In a
variation to the method, the recombinant nucleic acid coding for
the Vitamin K dependent protein or a functionally active derivative
thereof is stably expressed.
[0020] A method is disclosed in accordance with another embodiment
of the invention for improving the productivity of recombinant
vitamin K dependent protein expression or a functionally active
derivative thereof in a host cell. The method comprises the steps
of: providing an isolated host cell having a recombinant nucleic
acid coding for a vitamin K epoxide reductase (VKOR) or a
functionally active derivative thereof integrated into its genome;
inserting a recombinant nucleic acid coding for a Vitamin K
dependent protein or a functionally active derivative thereof into
the host cell; and expressing the nucleic acids. The recombinant
nucleic acid coding for VKOR or a functionally active derivative
thereof is preferably stably expressed.
[0021] A recombinant Vitamin K dependent protein is disclosed in
accordance with another embodiment of the invention. The protein is
obtainable by inserting a recombinant nucleic acid coding for a
Vitamin K epoxide reductase (VKOR) or a functionally active
derivative thereof and a recombinant nucleic acid coding for the
recombinant Vitamin K dependent protein or a functionally active
derivative thereof into a host cell, expressing the nucleic acids,
and recovering the recombinant Vitamin K dependent protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIGS. 1A-D Comparisons of warfarin dosages in wild type,
heterozygous and homozygous subjects for SNPs vk 2581, vk3294 and
vk4769, as well as a comparison of warfarin dosages in wild type
and heterozygous subjects for P450 2Y9.
[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. Panel 6A: Control HEK293 cells producing factor X without
exogenous VKOR or VKGC. Panel 6B: HEK 293 cells producing factor X
and exogenous VKGC alone. Panel 6C: HEK293 cells producing factor X
and exogenous VKOR alone. Panel 6D: 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] The phrase "functionally active derivative" shall be given
its ordinary meaning and shall include naturally-occurring or
synthetic fragments, variants, and analogs that exhibit at least
one function that is substantially similar to that of the compound
from which it is derived. Functionally active derivatives may be,
but need not be, structurally or chemically similar to the compound
from which they are derived.
[0037] 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 60-70.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.).
[0038] 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.
[0039] 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).
[0040] 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.
[0041] 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).
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] In other embodiments, 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.
[0051] Several embodiments of the invention comprise 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.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] In one embodiment, the invention also comprises 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.
[0058] In another aspect, the invention comprises 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.
[0059] In another embodiment, 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.
[0060] Also encompassed by one embodiment of 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.
[0061] 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.
[0062] Thus, in some embodiments, the present invention also
provides a method of producing a vitamin K dependent protein,
comprising:
[0063] 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
[0064] b) optionally collecting the vitamin K dependent protein
from the cell.
[0065] In one embodiment, 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.
[0066] 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.
[0067] In addition, in another embodiment, 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.
[0068] Furthermore, in another embodiment, 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).
[0069] 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.
[0070] The present invention further comprises embodiments wherein
nucleic acid encoding vitamin K epoxide reductase can be introduced
into a cell to improve the growth characteristics (e.g., enhance
growth rate; increase survival time, etc.) of the cell.
[0071] In some embodiments of this invention, the nucleic acid
encoding vitamin K epoxide reductase can be a nucleic acid that is
naturally present in the cell (i.e., endogenous to the cell). In
other embodiments, the nucleic acid encoding vitamin K epoxide
reductase can be an exogenous nucleic acid that is introduced into
the cell. In further embodiments, the cell of this invention can
comprise an endogenous nucleic acid encoding vitamin K epoxide
reductase and an exogenous nucleic acid encoding vitamin K epoxide
reductase.
[0072] 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.
[0073] According to several embodiments, 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.
[0074] 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).
[0075] 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, activated 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. The nucleotide sequences and amino acid
sequences of the vitamin K dependent proteins of this invention are
known in the art. Nonlimiting examples of sequences of some of the
vitamin K dependent proteins of this invention include human factor
VII, which has GenBank Accession No. BC130468, human factor Vila,
which has SwissProt Accession No. P08709 and Protein C, which has
GenBank Accession No. NM_000312. These examples demonstrate the
availability of these sequences in the art and are not intended to
be limiting in any way, as the present invention includes any
vitamin K dependent protein, in any combination.
[0076] 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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] 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.
[0082] In additional embodiments, 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.
[0083] Also provided herein as another embodiment 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).
[0084] 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.
[0085] 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).
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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.
[0090] In additional embodiments, 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.
[0091] 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).
[0092] 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.
[0093] 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
[0094] 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
[0095] 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
[0096] 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/.mu.L.
3. Sequencing of the Genomic DNA Samples
[0097] 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
[0098] 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.
[0099] 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
[0100] 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
[0101] 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).
[0102] 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.
[0103] 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
[0104] 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.
[0105] 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
[0106] 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
[0107] 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
[0108] 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)
[0109] 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
[0110] 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
[0111] 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
[0112] 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 (Nara 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
(Nolen 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.
[0113] 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
[0114] 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).
[0115] 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
[0116] 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.
[0117] 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.
[0118] 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).
[0119] 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
[0120] 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 Vila 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 undercarboxylated. 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.
[0121] 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.TM.
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.
[0122] Construction of mammalian cell expression vector containing
VKOR. Two primers were designed to amplify the VKOR cDNA.
TABLE-US-00002 Primer1: (SEQ ID NO: 35) 5'-CCGGAATTC ATGGGCA
GCACCTGGGGGAGCCCTGGCTGGGTGCGG
introduced a Kozak sequence (underlined) and a 5' Eco R I site.
Primer2: 5'-CGGGCGGCCGCTCAGTGCCTCTTAGCCTTGCC (SEQ ID NO:36)
introduced a NotI 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.
[0123] Construction of mammalian cell expression vector containing
HGC. Two primers were designed to amplify HGC cDNA.
TABLE-US-00003 Primer3: (SEQ ID NO: 37) 5'-CGCGGATCC GCCGCCACCAT
GGCGGTGTCTGCCGGGTCCGCGCGGACCTCGCCC
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.
[0124] 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.
[0125] 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.
[0126] 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.
[0127] 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 was 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.
[0128] 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.
[0129] 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.
[0130] 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.
[0131] 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.
[0132] 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.
[0133] 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.
[0134] 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
[0135] 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.
[0136] 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.
[0137] 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 can not be reused in the
carboxylation system in vivo.
[0138] 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.
[0139] 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.
[0140] 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.
[0141] 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.
[0142] 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.
[0143] 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.
[0144] 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 Heterozygous
SNPs position AA change ratio vk563 5'- N/A 1/58 G > A UTR (SEQ
ID NO: 15) vk2581 G > C Intro N/A 17/58 (SEQ ID n2 NO: 12)
vk3294 T > C Intro N/A 25/58 (SEQ ID n2 NO: 13) vk4501 C > T
Exo Leu120Leu 1/58 (SEQ ID n3 NO: 16 vk4769 G > A 3'- N/A 19/58
(SEQ ID UTR NO: 14
TABLE-US-00005 TABLE 2 VIC FAM Probe Probe Forward Reverse SNPs
Sequence Sequence Primer Primer vk2581 TCAT TCAT GGTG CCTG G > C
CACG CACC ATCC TTAG GAGC GAGC ACAC TTAC GTC GTC AGCT CTCC (SEQ (SEQ
GACA CCAC ID ID (SEQ ATC NO: NO: ID (SEQ 17) 18) NO: ID 19) NO: 20)
vk3294 CCAG CCAG GCTC GCCA T > C GACC GACC CAGA AGTC ATGG GTGG
GAAG TGAA TGC TGC GCAT CCAT (SEQ (SEQ CACT GTGT ID ID (SEQ CA NO:
NO: ID (SEQ 21) 22) NO: ID 23) NO: 24) vk4769 ATAC CATA GTCC GTGT G
> A CCGC CCCA CTAG GGCA ACAT CACA AAGG CATT GAC TGAC CCCT TGGT
(SEQ (SEQ AGAT CCAT ID ID GT T NO: NO: (SEQ (SEQ 25) 26) ID ID NO:
NO: 27) 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
* * * * *