U.S. patent application number 15/343998 was filed with the patent office on 2017-05-04 for therapeutic methods employing nitric oxide precursors.
The applicant listed for this patent is Vanderbilt University. Invention is credited to Frederick E. Barr, Brian W. Christman, Marshall L. SUMMAR.
Application Number | 20170119715 15/343998 |
Document ID | / |
Family ID | 34911448 |
Filed Date | 2017-05-04 |
United States Patent
Application |
20170119715 |
Kind Code |
A1 |
SUMMAR; Marshall L. ; et
al. |
May 4, 2017 |
THERAPEUTIC METHODS EMPLOYING NITRIC OXIDE PRECURSORS
Abstract
Isolated polynucleotide molecules and peptides encoded by these
molecules are used in the analysis of human carbamyl phosphate
synthetase I phenotypes, as well as in diagnostic and therapeutic
applications, relating to a human carbamyl phosphate synthetase I
polymorphism. By analyzing genomic DNA or amplified genomic DNA, or
amplified cDNA derived from mRNA, it is possible to type a human
carbamyl phosphate synthetase I with regard to the human carbamyl
phosphate synthetase I polymorphism, for example, in the context of
diagnosing and treating hepatic veno-occlusive disease (HVOD)
associated with bone marrow transplants.
Inventors: |
SUMMAR; Marshall L.;
(Brentwood, TN) ; Christman; Brian W.; (Nashville,
TN) ; Barr; Frederick E.; (Nashville, TN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Vanderbilt University |
Nashville |
TN |
US |
|
|
Family ID: |
34911448 |
Appl. No.: |
15/343998 |
Filed: |
November 4, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10785374 |
Feb 24, 2004 |
9486429 |
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15343998 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 37/00 20180101;
A61P 1/04 20180101; A61K 31/195 20130101; A61P 11/06 20180101; A61P
9/00 20180101; A61P 29/00 20180101; A61P 9/12 20180101; A61P 31/04
20180101; A61K 38/00 20130101; A61P 43/00 20180101; A61P 37/06
20180101; A01K 2217/05 20130101; C12N 9/93 20130101; A61P 1/16
20180101; A61P 15/10 20180101; A61P 11/00 20180101; A61K 31/198
20130101 |
International
Class: |
A61K 31/198 20060101
A61K031/198 |
Goverment Interests
GRANT STATEMENT
[0002] This work was supported by NIH grants R29-DK46965, NIH HL
55198, NIH ES 09915 and NIH 1 P30 CA 68485. Thus, the U.S.
Government has certain rights in the invention.
Claims
1-10. (canceled)
11. A method of treating or preventing bone marrow transplant
toxicity in a subject undergoing bone marrow transplant comprising
intravenously or orally administering to the subject a
therapeutically effective amount of a nitric oxide precursor,
whereby bone marrow transplant toxicity is treated or prevented in
the subject.
12-17. (canceled)
18. A method of treating a disorder selected from the group
consisting hepatitis, cirrhosis, necrotizing enterocolitis (NEC),
Acute Respiratory Distress Syndrome, ethnic specific endothelial
dysfunction, erectile dysfunction, asthma, chemotherapy, increased
oxidative stress, septic shock, hypoxia, hepatotoxin exposure, or a
combination thereof in a subject comprising administering to a
subject in need thereof an effective amount of a nitric oxide
precursor.
19. The method of claim 18, wherein the administering is
intravenously or orally.
20. The method of claim 18, wherein the nitric oxide precursor is
selected from the group consisting of citrulline, arginine and
combinations thereof.
21. The method of claim 18, wherein the nitric oxide precursor is
administered in a dose ranging from about 100 mg to about 30,000
mg.
22. The method of claim 21, wherein the nitric oxide precursor is
administered in a dose ranging from about 250 mg to about 1,000
mg.
23. The method of claim 18, wherein the subject is a human.
24. The method of claim 18, wherein the disorder is necrotizing
enterocolitis (NEC).
25-33. (canceled)
34. The method of claim 18, wherein the asthma is acute asthma
attack.
35. The method of claim 18, wherein the nitric oxide precursor is
administered in a dose ranging from about 200 mg/kg, 400 mg/kg, 600
mg/kg, or 800 mg/kg.
36. A method for treating or preventing pulmonary hypertension
comprising administering to a subject in need thereof an effective
amount of a nitric oxide precursor.
Description
RELATED APPLICATION INFORMATION
[0001] This is a continuation-in-part of U.S. patent application
Ser. No. 09/585,077, filed Jun. 1, 2000, which is a
continuation-in-part of U.S. patent application Ser. No.
09/323,472, filed Jun. 1, 1999, now U.S. Pat. No. 6,346,382, the
entire contents of which are herein incorporated by reference.
TECHNICAL FIELD
[0003] The present invention relates to isolated polynucleotide
molecules useful for analyzing carbamyl phosphate synthetase I
phenotypes, to peptides encoded by these molecules, and to the
diagnostic and therapeutic uses thereof relating to a newly
identified carbamyl phosphate synthetase I polymorphism. Among such
uses are methods for determining the susceptibility of a subject to
hyperammonemia, decreased production of arginine and to bone marrow
transplant toxicity based on an analysis of a nucleic acid sample
isolated from tissue biopsies from the subject.
TABLE-US-00001 Table of Abbreviations ABG arterial blood gas(es)
ALI acute lung injury ASO allele-specific oligonucleotide ATP
adenosine triphosphate BCAA branched chain amino acid(s) BMT bone
marrow transplant BSA bovine serum albumin BuCy busulfan,
cyclophosphamide BUN blood urea nitrogen CBVP16 cyclophosphamide,
bis- chloroethylnitrosourea, etoposide cc cubic centimeters CPSI
carbamyl phosphate synthetase I CTC cyclophosphamide, thiotepa,
carboplatin CVP16TBI cyclophosphamide, etoposide, total body
irradiation ECMO extracorpreal membrane oxygenation fl full length
GSHosc glutathione synthetase HAT hypoxanthine, aminopterin,
thymidine HVOD hepatic veno-occlusive disease iNO inhaled nitric
oxide KDa kilodalton KLH keyhole limpet hemocyanin I liter LAT
ligation activated translation LCR ligase chain reaction MAS
meconium aspiration syndrome NAG n-acetyl glutamate NASDA .TM.
nucleic acid sequence-based amplification NO or NO.sub.x nitric
oxide NOS nitric oxide synthetase O/C ornithine/citrulline PBSCT
peripheral blood stem-cell transplantation PPHN persistant
pulmonary hypertension in newborns PCR polymerase chain reaction
RCR repair chain reaction RDS respiratory distress syndrome REF
restriction endonuclease finger-printing RT reverse transcriptase
SSCP single strand conformation polymorphism SDA strand
displacement activation SNP single nucleotide polymorphism TC
thiotepa, cyclophosphamide TEAA total essential amino acids UC urea
cycle UCF urea cycle function VPA valproic acid
BACKGROUND ART
[0004] The in vivo synthetic pathway for arginine commences with
ornithine. Ornithine is combined with carbamyl phosphate to produce
citrulline, which in turn is combined with aspartate, in the
presence of adenosine triphosphate (ATP), to produce
argininosuccinate. In the final step, fumarate is split from
argininosuccinate, to produce arginine. The degradative pathway for
arginine is by the hydrolytic action of arginase, to produce
ornithine and urea. These reactions form the urea cycle. The urea
cycle serves as the primary pathway for removing waste nitrogen
produced by the metabolism of endogenous and exogenous proteins,
and is shown schematically in FIG. 1.
[0005] Disruption of metabolic processes is a frequent side effect
of chemotherapy. Indeed, the agents used in high-dose chemotherapy
affect a number of cellular processes. Metabolic processes
localized in chemo-sensitive tissues, such as the liver and
gastrointestinal tract, face a particularly great risk to
disruption.
[0006] The constant turn-over and processing of nitrogen involves
all the tissues in the body, but the first critical steps of the
urea cycle are limited to the liver and gut. The high-dose
chemotherapy associated with bone marrow transplant (BMT)
interferes with liver function and is toxic to the intestine.
Idiopathic hyperammonemia, which is suggestive of urea cycle
dysfunction, has been reported to be associated with high mortality
in patients undergoing bone marrow transplant. Davies et al., Bone
Marrow Transplantation, 17:1119-1125 (1996); Tse et al., American
Journal of Hematology, 38:140-141 (1991); and Mitchell et al.,
American Journal of Medicine, 85:662-667 (1988).
[0007] A common complication of BMT is hepatic veno-occlusive
disease (HVOD). HVOD is associated with jaundice, increased liver
size and disruption of normal hepatic blood flow. HVOD occurs in
approximately 20 to 40% of patients and is associated with severe
morbidity and mortality.
[0008] Nitric oxide (NO) plays a role in regulating vascular tone
and in maintaining patency of hepatic and pulmonary venules
following high-dose chemotherapy. Intact urea cycle function is
important not only for excretion of ammonia but in maintaining
adequate tissue levels of arginine, the precursor of NO.
[0009] Carbamyl phosphate synthetase I (CPSI) is the rate limiting
enzyme catalyzing the first committed step of ureagenesis via the
urea cycle. CPSI is highly tissue specific, with function and
production substantially limited to liver and intestines.
Genomically encoded, CPSI is produced in the cytoplasm and
transported into the mitochondria where it is cleaved into its
mature 160 kDA monomeric form. The enzyme combines ammonia and
bicarbonate to form carbamyl with the expenditure of two ATP
molecules and using the co-factor N-acetyl-glutamate (NAG).
[0010] Any genetic predisposition to decreased urea cycle function
would lead to hyperammonemia and would likely contribute to the
severity of disorders associated with sub-optimal urea cycle
function, including BMT-related toxicity. Thus, there is a need in
the art for characterization of alleles present in populations
suffering from disorders associated with suboptimal urea cycle
funtion, undergoing BMT or otherwise facing exposure to
environmental or pharmacological hepatotoxins. In view of the role
of CPSI in the urea cycle, there is a particular need for
characterization of CPSI alleles present in such populations.
SUMMARY OF THE INVENTION
[0011] A method of screening for susceptibility to sub-optimal urea
cycle function in a subject is disclosed. The method comprising the
steps of: (a) obtaining a nucleic acid sample from the subject; and
(b) detecting a polymorphism of a carbamyl phosphate synthase I
(CPSI) gene in the nucleic acid sample from the subject, the
presence of the polymorphism indicating that the susceptibility of
the subject to sub-optimal urea cycle function. In accordance with
the present invention, detection of the polymorphism is
particularly provided with respect to determining the
susceptibility of a subject to bone marrow transplant toxicity.
[0012] Preferably, the polymorphism of the carbamyl phosphate
synthetase polypeptide comprises a C to A transversion in exon 36
of the CPSI gene, more preferably at nucleotide 4340 of a cDNA that
corresponds to the CPSI gene. More preferably, the C to A
transversion at nucleotide 4340 of the cDNA that corresponds to the
CPSI gene further comprises a change in the triplet code from AAC
to ACC, which encodes a CPSI polypeptide having an threonine moiety
at amino acid 1405.
[0013] The present invention also provides an isolated and purified
biologically active CPSI polypeptide. Preferably, a polypeptide of
the invention is a recombinant polypeptide. More preferably, a
polypeptide of the present invention comprises human CPSI having an
asparagine moiety at amino acid 1405.
[0014] The present invention also provides an isolated and purified
polynucleotide that encodes a biologically active CPSI polypeptide.
In a preferred embodiment, a polynucleotide of the present
invention comprises a DNA molecule from a human. More preferably, a
polynucleotide of the present invention comprises a cDNA that
corresponds to the CPSI gene and which includes a C to A
transversion at nucleotide 4340. Even more preferably, a
polynucleotide of the present invention further comprises a cDNA
that corresponds to the CPSI gene that includes a change in the
triplet code from ACC to AAC at nucleotide 4340, and encodes a CPSI
polypeptide having an asparagine moiety at amino acid 1405.
[0015] Kits and reagents, including oligonucleotides, nucleic acid
probes and antibodies suitable for use in carrying out the methods
of the present invention and for use in detecting the polypeptides
and polynucleotides of the present invention are also disclosed
herein. Methods for preparing the polynucleotides and polypeptides
of the present invention are also disclosed herein.
[0016] In a further embodiment, this invention pertains to
therapeutic methods based upon a polymorphism of a carbamyl
phosphate synthase I (CPSI) gene as described herein. Such
therapeutic methods include administration of nitric oxide
precursors in the treatment and prophylaxis of disorders mediated
or modulated by sub-optimal urea cycle function (e.g. bone marrow
transplant toxicity) and gene therapy approaches using an isolated
and purified polynucleotide of the present invention.
[0017] It is therefore an object of the present invention to
provide polynucleotide molecules that can be used in analyzing
carbamyl phosphate synthetase I (CPSI) in vertebrate subjects.
[0018] It is also an object of the present invention to provide for
the determination of CPSI phenotype in vertebrate subjects and
particularly human subjects, based on information obtained through
the analysis of nucleic acids, including genomic DNA and cDNA,
derived from tissues from the subject.
[0019] It is yet another object of the present invention to provide
a ready technique for determining CPSI phenotype.
[0020] It is still a further object of the present invention to
provide polypeptide and polynucleotide molecules for use in
generating antibodies that distinguish between the different forms
of CPSI which constitute the CPSI polymorphism.
[0021] It is yet a further object of the present invention is to
provide methods for diagnosing and treating clinical syndromes
related to and associated with the CPSI polymorphism.
[0022] Some of the objects of the invention having been stated
hereinabove, other objects will become evident as the description
proceeds, when taken in connection with the accompanying drawings
and examples as best described hereinbelow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 is a schematic of the urea cycle;
[0024] FIG. 2 is a schematic of the consensus CPSI protein which
does not reflect recognized mutations;
[0025] FIG. 3 is a schematic of the consensus CPSI protein
depicting several known mutations in the protein and depicting the
T1405N polymorphism of the present invention;
[0026] FIG. 4 is a schematic of recognized post-transcriptional
modification of CPSI;
[0027] FIG. 5 is a schematic of the human genomic locus for
CPSI;
[0028] FIG. 6 is a schematic of a cloning strategy for a full
length CPSI cDNA;
[0029] FIG. 7 is a schematic of an alternative cloning strategy for
a full length CPSI cDNA;
[0030] FIG. 8 is a graphical depiction of the metabolic activity of
the CPSI protein expressed in COS-7 cells;
[0031] FIG. 9 is a graphical presentation of the size and position
of introns in CPSI cDNA;
[0032] FIG. 10 is a diagram of exon 36 (SEQ ID NO:5) showing the
locations of preferred oligonucleotide primers of the present
invention;
[0033] FIG. 11 presents the amino acid sequence of T1405 CPSI (SEQ
ID NO:4) (stop codon translated as "X", 165049 MW, 1.163602e+07
CN), with the initial amino acid methionine considered to be at a
-1 position;
[0034] FIG. 12 presents the amino acid sequence of N1405 CPSI (SEQ
ID NO:2) (stop codon translated as "X", 165062 MW, 1.161634E+07
CN), with the initial amino acid methionine considered to be at a
-1 position;
[0035] FIG. 13 is a graph of a concentration curve of plasma
arginine levels.
DETAILED DESCRIPTION OF THE INVENTION
[0036] Disclosed herein is the surprising discovery of a
polymorphism of carbamyl phosphate synthetase I (CPSI), the enzyme
that catalyzes the rate limiting first step of the urea cycle.
Particularly, the polymorphism is characterized by an amino acid
substitution, threonine/asparagine at amino acid 1405
(heterozygosity=0.44) in CPSI.
[0037] Also disclosed herein is the surprising observation that a
single nucleotide change in the CPSI gene is responsible for the
polymorphism of CPSI. Particularly, a C to A transversion with exon
36 of the CPSI gene changes the triplet code from ACC to AAC and
leads to the T1405N change in the encoded CPSI polypeptide.
[0038] In light of these discoveries, manipulation of nucleic acid
molecules derived from the tissues of vertebrate subjects can be
effected to provide for the analysis of CPSI phenotypes, for the
generation of peptides encoded by such nucleic acid molecules, and
for diagnostic and therapeutic methods relating to the CPSI
polymorphism. Nucleic acid molecules utilized in these contexts may
be amplified, as described below, and generally include RNA,
genomic DNA and cDNA derived from RNA.
A. General Considerations
[0039] Most of the currently available structural information on
CPSI is derived from studies of the rat CPSI enzyme. The rat CPSI
enzyme and the human CPSI enzyme each comprise a single polypeptide
of 1,500 residues and exhibit about 95% sequence identity. Rat CPSI
polypeptide and nucleic acid sequence information is disclosed by
Nyunoya, H., et al., Journal of Biological Chemistry 260:9346-9356
(1985) and at GenBank accession numbers AH005315, M12335, M12328,
M12327, M12326, M12325, M12324, M12323, M12322, M12321, M12320,
M12319, M12318 and M11710, herein incorporated by reference. The
structural information about rat CPSI is derived from sequence
homology and substrate and co-factor binding studies; however, no
crystallographic data is available.
[0040] Mature CPSI is modular in nature, containing 2 main regions.
The first region, residues 39-406, is homologous to the small
subunit of the heterodimeric CPS of Escherichia coli. Bacterial and
yeast CPSI polypeptide and nucleic acid sequence information is
disclosed at GenBank accession numbers AB005063, X67573, M27174,
P07258, P03965, BAA21088, SYBYCP, SYBYCS, and SYECCS, herein
incorporated by reference.
[0041] The other region, residues 417-1500 (referred to hereinafter
as the "CPS domain"), is homologous to the large subunit of E. coli
CPS. Meister, A., Adv. Enzymol. Relat. Areas Mol. Biol. 62:315-374
(1989). This subunit is responsible for carbamyl phosphate
synthesis from ammonia and for the binding of the substrates and
cofactors. Meister, A., Adv. Enzymol. Relat. Areas Mol. Biol.
62:315-374 (1989). The CPS domain arose by gene duplication and
tandem fusion in the pro-genome, and, as depicted schematically in
FIG. 2, is itself composed of two phosphorylation domains and a
C-terminal regulatory domain involved in the binding of
n-acetyl-glutamate (NAG). Nyunoya, H., et al., Journal of
Biological Chemistry 260:9346-9356 (1985).
[0042] As depicted schematically in FIG. 2, residues 407-416 act as
a bridge between the two major subunits, and residues 1-38
constitute the leader peptide that directs immature CPSI to the
mitochondria prior to being removed. Continuing with FIG. 2, the
small subunit-like region is composed of two approximately equal
subdomains. The interaction subdomain, residues 39-212, corresponds
to the region which, in the small subunit of the CPS from E. coli,
is necessary for association with the large subunit. The
glutaminase subdomain, residues 213-406, is homologous to several
glutamine amidotransferases and to the region of CPSI that when
generated free from other components exhibited considerable
glutaminase activity, as described by Guillou, F., et al. Proc Natl
Acad Sci 86:8304-8308 (1989); Nyunoya, H., et al., Journal of
Biological Chemistry 260:9346-9356 (1985); and Guy, H. I. et al.,
Journal of Biological Chemistry 270:2190-2197 (1995). Since CPSI
has lost the cysteine residue necessary to split glutamine, the
function of the glutaminase subdomain is uncertain in this
enzyme.
[0043] The CPS domain (corresponding to the large subunit in E.
coli) is believed to catalyze the synthesis of carbamyl phosphate
from ammonia, according to the reaction:
2 ATP+bicarbonate+.fwdarw.2 ADP+phosphate+ammonia carbamyl
phosphate
As shown schematically in FIGS. 1 and 2, this reaction comprises
three steps: bicarbonate phosphorylation by an ATP molecule that is
designated herein as ATP.sub.A, giving carboxyphosphate; carbamate
synthesis from carboxyphosphate and ammonia; and carbamate
phosphorylation by another ATP molecule (ATP.sub.B), giving
carbamyl phosphate, as described by Rubio, V. and Grisolia, S.,
Enzyme 26:233-239 (1981).
[0044] As shown schematically in FIG. 4, the CPS domain appears to
have arisen by duplication and tandem fusion of the duplicated
component; therefore, its amino and COOH-terminal halves are
homologous, as described by Nyunoya, H., et al., Journal of
Biological Chemistry 260:9346-9356 (1985)). Each homologous half
comprises an amino- and a COOH-terminal domain of about 40 and 20
kDa, respectively, of which the domain of 40 kDa of the amino-half
is believed to be involved in bicarbonate phosphorylation
(bicarbonate phosphorylation domain, residues 417-788) (FIG. 2).
The corresponding domain in the COOH-half is involved in carbamate
phosphorylation via the carbamate phosphorylation domain, residues
969-1329 (FIG. 2), as described by Alonso, E. and Rubio, V.,
European Journal of Biochemistry 229:377-384 (1995)).
[0045] These phosphorylation domains are homologous to biotin
carboxylase (Toh, H. et al., European Journal of Biochemistry
215:687-696 (1993)), an enzyme of known tri-dimensional structure
that phosphorylates bicarbonate as well as DD-ligase and
glutathione synthetase (GSHase), two enzymes that catalyze
analogous reactions (Artymiuk, P. J. et al., Nature Struct. Biol.
3:128-132 (1996)). Thus, information on these enzymes is helpful in
interpreting the mutations found in homologous domains in the
patients with CPSI deficiency.
[0046] Referring again to FIG. 2, of the 20-kDa domains of the
large subunit-like region, the function of the domain of the
amino-terminal half, residues 789-968, remains to be established.
In contrast, the corresponding COOH-terminal domain, residues
1330-1500, is called the allosteric domain, because the activator,
n-acetyl-glutamate (NAG) of CPSI and the nucleotide effectors of
the E. coli enzyme, UMP and IMP, bind in this domain, as described
by Rodriguez-Aparicio, L. B. et al., Biochemistry 28:3070-3074
(1989) and Cervera, J. et al., Biochemistry 35:7247-7255
(1996).
[0047] A.1. Enzyme Processing.
[0048] Human CPSI mRNA encodes a 165 kDA, 1500 amino acid
pre-protein. The amino terminus of this precursor contains 38
residues, including 8 basic residues, and 1 acidic residue with a
Pro-Gly sequence 4 residues before the start of the mature enzyme
(Nyunoya, H. et al., Journal of Biological Chemistry 260:9346-9356
(1985); Lagace, M. et al., Journal of Biological Chemistry
262:10415-10418 (1987). This highly conserved signal sequence
promotes enzyme entry into the mitochondrial matrix, where it is
then removed to produce the 160 kDA mature enzyme.
[0049] A.2. Normal Expression of CPSI.
[0050] CPSI enzymatic activity is first detected in human fetal
liver by 5-10 weeks gestation (Moorman, A. F. et al. Histochemical
Journal 22:457-468 (1990)). By 20 weeks gestation, the level of
CPSI reaches approximately 50% of the normal adult level, where it
remains until birth, after which it gradually increases to adult
levels by 20 years of age (Raiha, N. C. R. and Suihkonen, J. Acta
Paediatrica Scand 57:121-127 (1968)). Tissue expression of CPSI is
essentially limited to the liver, with trace amounts of activity in
the intestine and kidney. When the liver develops its mature acinar
structure in adulthood, CPSI is compartmentalized in parenchymal
cells around the terminal portal venules (Moorman, A. F. et al.
Histochemical Journal 22:457-468 (1990)).
[0051] In addition to its compartmentalization, several factors are
known to be important in the regulation of CPSI activity and
expression. For example, low or absent levels of ornithine decrease
CPSI activity, presumably due to an inhibitory effect from
accumulated carbamyl phosphate (CP) as described by Jackson, M. J.
et al., Annual Review of Genetics 20:431-464 (1986); and Rubio, V.,
Biochemical Society Transactions 21:198-202 (1993)). Levels of both
CPSI mRNA and enzyme increase with a high protein diet, and in
response to glucagon and glucocorticoids (Jackson, M. J. et al.,
Annual Review of Genetics 20:431-464 (1986); de Groot, C. J., et
al., Biochemical & Biophysical Research Communications
124:882-888 (1984)). In normal unstimulated hepatic tissue that has
been examined, an abundance of CPSI mRNA has been observed.
B. Screening Techniques
[0052] In accordance with the present invention, a method of
screening for susceptibility to sub-optimal urea cycle function
resulting in decreased ammonia clearance and decreased arginine
production in a subject is provided. The method comprises: (a)
obtaining a nucleic acid sample from the subject; and (b) detecting
a polymorphism of a carbamyl phosphate synthase I (CPSI) gene in
the nucleic acid sample from the subject, the presence of the
polymorphism indicating that the susceptibility of the subject to
sub-optimal urea cycle function resulting in decreased ammonia
clearance and decreased arginine production. In accordance with the
present invention, detection of the polymorphism is particularly
provided with respect to determining the susceptibility of a
subject to bone marrow transplant toxicity.
[0053] It is further noted that the polymorphism of the present
invention may be used to predict toxicity in a number of conditions
beyond BMT or valproic acid administration as disclosed herein and
in the Examples. The polymorphism is also implicated in the
mediation or modulation of disrupted ammonia clearance and arginine
production in situations such as adult hepatic cirrhosis, other
medication toxicities, newborns with impaired hepatic function, and
the like.
[0054] As used herein and in the claims, the term "polymorphism"
refers to the occurrence of two or more genetically determined
alternative sequences or alleles in a population. A polymorphic
marker is the locus at which divergence occurs. Preferred markers
have at least two alleles, each occurring at frequency of greater
than 1%. A polymorphic locus may be as small as one base pair.
[0055] Useful nucleic acid molecules according to the present
invention include those which will specifically hybridize to CPSI
sequences in the region of the C to A transversion at base 4340 and
within exon 36 changing the triplet code from ACC to AAC. This
transversion leads to the T1405N change in the encoded CPSI
polypeptide. Typically these are at least about 20 nucleotides in
length and have the nucleotide sequence corresponding to the region
of the C to A transversion at base 4340 of the consensus CPSI cDNA
sequence (EC6.3.4.16), which changes the triplet code from ACC to
AAC. The term "consensus sequence", as used herein, is meant to
refer to a nucleic acid or protein sequence for CSPI, the nucleic
or amino acids of which are known to occur with high frequency in a
population of individuals who carry the gene which codes for a
normally functioning protein, or which nucleic acid itself has
normal function.
[0056] Provided nucleic acid molecules can be labeled according to
any technique known in the art, such as with radiolabels,
fluorescent labels, enzymatic labels, sequence tags, etc. According
to another aspect of the invention, the nucleic acid molecules
contain the C to A transversion at base 4340. Such molecules can be
used as allele-specific oligonucleotide probes to track a
particular mutation, for example, through a family of subjects.
[0057] Body samples can be tested to determine whether the CPSI
gene contains the C to A transversion at base 4340. Suitable body
samples for testing include those comprising DNA, RNA or protein
obtained from biopsies, including liver and intestinal tissue
biopsies; or from blood, prenatal; or embryonic tissues, for
example.
[0058] In one embodiment of the invention a pair of isolated
oligonucleotide primers are provided:
5'-AGCTGTTTGCCACGGAAGCC-3'(SEQ ID NO:6) and
5'-CCCAGCCTCTCTTCCATCAGAAAGTAAG-3'(SEQ ID NO:7). These primers are
derived from CPSI exon 36 (the location of the polymorphism of the
present invention) and related intronic sequences (SEQ ID NO:5) and
produce a 119 base pair fragment. Other primers derived from CPSI
exon 36 (the location of the polymorphism of the present invention)
and related intronic sequences (SEQ ID NO:5) are provided in SEQ ID
NOs:8-10, in FIG. 10, and in Example 2 (SEQ ID NOs:15 and 16).
[0059] The oligonucleotide primers are useful in diagnosis of a
subject at risk for hyperammonemia such as can result as a BMT
complication or toxicity. The primers direct amplification of a
target polynucleotide prior to sequencing. These unique CPSI exon
36 oligonucleotide primers were designed and produced based upon
identification of the C to A transversion in exon 36.
[0060] In another embodiment of the invention isolated allele
specific oligonucleotides are provided. Sequences substantially
similar thereto are also provided in accordance with the present
invention. The allele specific oligonucleotides are useful in
diagnosis of a subject at risk for hyperammonemia, such as can
result as a BMT complication or toxicity. These unique CPSI exon
oligonucleotide primers were designed and produced based upon
identification of the C to A transversion in exon 36.
[0061] The terms "substantially complementary to" or "substantially
the sequence of" refer to sequences which hybridize to the
sequences provided (e.g. SEQ ID NOs: 5-10) under stringent
conditions and/or sequences having sufficient homology with any of
SEQ ID NOs: 5-10, such that the allele specific oligonucleotides of
the invention hybridize to the sequence. The term "isolated" as
used herein includes oligonucleotides substantially free of other
nucleic acids, proteins, lipids, carbohydrates or other materials
with which they may be associated, such association being either in
cellular material or in a synthesis medium. A "target
polynucleotide" or "target nucleic acid" refers to the nucleic acid
sequence of interest e.g., a CPSI-encoding polynucleotide. Other
primers which can be used for primer hybridization are readily
ascertainable to those of skill in the art based upon the
disclosure herein of the CPSI polymorphism.
[0062] The primers of the invention embrace oligonucleotides of
sufficient length and appropriate sequence so as to provide
initiation of polymerization on a significant number of nucleic
acids in the polymorphic locus. The CPSI locus is depicted
schematically in FIG. 5. Specifically, the term "primer" as used
herein refers to a sequence comprising two or more
deoxyribonucleotides or ribonucleotides, preferably more than
three, and more preferably more than eight and most preferably at
least about 20 nucleotides of the CPSI gene wherein the DNA
sequence contains the C to A transversion at base 4340 relative to
CPSI contained in SEQ ID NO's:1 and 3. The allele including
cytosine (C) at base 4340 relative to CPSI is referred to herein as
the "CPSIa allele", the "T1405 allele", or the "threonine-encoding
allele". The allele including adenosine (A) at base 4340 relative
to CPSI is referred to herein as the "CPSIb allele", the "N1405
allele", or the "arginine-encoding allele".
[0063] An oligonucleotide that distinguishes between the CPSIa and
the CPSIb alleles of the CPSI gene, wherein said oligonucleotide
hybridizes to a portion of said CPSI gene that includes nucleotide
4340 of the cDNA that corresponds to said CPSI gene when said
nucleotide 4340 is adenosine, but does not hybridize with said
portion of said CPSI gene when said nucleotide 4340 is cytosine is
also provided in accordance with the present invention. An
oligonucleotide that distinguishes between the CPSIa and the CPSIb
alleles of the CPSI gene, wherein said oligonucleotide hybridizes
to a portion of said CPSI gene that includes nucleotide 4340 of the
cDNA that corresponds to said CPSI gene when said nucleotide 4340
is cytosine, but does not hybridize with said portion of said CPSI
gene when said nucleotide 4340 is adenosine is also provided in
accordance with the present invention. Such oligonucleotides are
preferably between ten and thirty bases in length. Such
oligonucleotides may optionally further comprises a detectable
label.
[0064] Environmental conditions conducive to synthesis include the
presence of nucleoside triphosphates and an agent for
polymerization, such as DNA polymerase, and a suitable temperature
and pH. The primer is preferably single stranded for maximum
efficiency in amplification, but may be double stranded. If double
stranded, the primer is first treated to separate its strands
before being used to prepare extension products. The primer must be
sufficiently long to prime the synthesis of extension products in
the presence of the inducing agent for polymerization. The exact
length of primer will depend on many factors, including
temperature, buffer, and nucleotide composition. The
oligonucleotide primer typically contains 12-20 or more
nucleotides, although it may contain fewer nucleotides.
[0065] Primers of the invention are designed to be "substantially"
complementary to each strand of the genomic locus to be amplified.
This means that the primers must be sufficiently complementary to
hybridize with their respective strands under conditions which
allow the agent for polymerization to perform. In other words, the
primers should have sufficient complementarity with the 5' and 3'
sequences flanking the transversion to hybridize therewith and
permit amplification of the genomic locus.
[0066] Oligonucleotide primers of the invention are employed in the
amplification method which is an enzymatic chain reaction that
produces exponential quantities of polymorphic locus relative to
the number of reaction steps involved. Typically, one primer is
complementary to the negative (-) strand of the polymorphic locus
and the other is complementary to the positive (+) strand.
Annealing the primers to denatured nucleic acid followed by
extension with an enzyme, such as the large fragment of DNA
polymerase I (Klenow) and nucleotides, results in newly synthesized
+ and - strands containing the target polymorphic locus sequence.
Because these newly synthesized sequences are also templates,
repeated cycles of denaturing, primer annealing, and extension
results in exponential production of the region (i.e., the target
polymorphic locus sequence) defined by the primers. The product of
the chain reaction is a discreet nucleic acid duplex with termini
corresponding to the ends of the specific primers employed.
[0067] The oligonucleotide primers of the invention may be prepared
using any suitable method, such as conventional phosphotriester and
phosphodiester methods or automated embodiments thereof. In one
such automated embodiment, diethylphosphoramidites are used as
starting materials and may be synthesized as described by Beaucage
et al., Tetrahedron Letters 22:1859-1862 (1981). One method for
synthesizing oligonucleotides on a modified solid support is
described in U.S. Pat. No. 4,458,066.
[0068] Any nucleic acid specimen, in purified or non-purified form,
can be utilized as the starting nucleic acid or acids, providing it
contains, or is suspected of containing, a nucleic acid sequence
containing the polymorphic locus. Thus, the method may amplify, for
example, DNA or RNA, including messenger RNA, wherein DNA or RNA
may be single stranded or double stranded. In the event that RNA is
to be used as a template, enzymes, and/or conditions optimal for
reverse transcribing the template to DNA would be utilized. In
addition, a DNA-RNA hybrid which contains one strand of each may be
utilized. A mixture of nucleic acids may also be employed, or the
nucleic acids produced in a previous amplification reaction herein,
using the same or different primers may be so utilized. The
specific nucleic acid sequence to be amplified, i.e., the
polymorphic locus, may be a fraction of a larger molecule or can be
present initially as a discrete molecule, so that the specific
sequence constitutes the entire nucleic acid. It is not necessary
that the sequence to be amplified be present initially in a pure
form; it may be a minor fraction of a complex mixture, such as
contained in whole human DNA.
[0069] DNA utilized herein may be extracted from a body sample,
such as blood, tissue material, preferably liver tissue, and the
like by a variety of techniques such as that described by Maniatis
et. al. in Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor, N.Y., p 280-281 (1982). If the extracted sample is impure,
it may be treated before amplification with an amount of a reagent
effective to open the cells, or animal cell membranes of the
sample, and to expose and/or separate the strand(s) of the nucleic
acid(s). This lysing and nucleic acid denaturing step to expose and
separate the strands will allow amplification to occur much more
readily.
[0070] The deoxyribonucleotide triphosphates dATP, dCTP, dGTP, and
dTTP are added to the synthesis mixture, either separately or
together with the primers, in adequate amounts and the resulting
solution is heated to about 90-100.degree. C. from about 1 to 10
minutes, preferably from 1 to 4 minutes. After this heating period,
the solution is allowed to cool, which is preferable for the primer
hybridization. To the cooled mixture is added an appropriate agent
for effecting the primer extension reaction (called herein "agent
for polymerization"), and the reaction is allowed to occur under
conditions known in the art. The agent for polymerization may also
be added together with the other reagents if it is heat stable.
This synthesis (or amplification) reaction may occur at room
temperature up to a temperature above which the agent for
polymerization no longer functions. Thus, for example, if DNA
polymerase is used as the agent, the temperature is generally no
greater than about 40.degree. C. Most conveniently the reaction
occurs at room temperature.
[0071] The agent for polymerization may be any compound or system
which will function to accomplish the synthesis of primer extension
products, including enzymes. Suitable enzymes for this purpose
include, for example, E. coli DNA polymerase I, Klenow fragment of
E. coli DNA polymerase, polymerase muteins, reverse transcriptase,
other enzymes, including heat-stable enzymes (i.e., those enzymes
which perform primer extension after being subjected to
temperatures sufficiently elevated to cause denaturation), such as
Taq polymerase. Suitable enzyme will facilitate combination of the
nucleotides in the proper manner to form the primer extension
products which are complementary to each polymorphic locus nucleic
acid strand. Generally, the synthesis will be initiated at the 3'
end of each primer and proceed in the 5' direction along the
template strand, until synthesis terminates, producing molecules of
different lengths.
[0072] The newly synthesized strand and its complementary nucleic
acid strand will form a double-stranded molecule under hybridizing
conditions described above and this hybrid is used in subsequent
steps of the method. In the next step, the newly synthesized
double-stranded molecule is subjected to denaturing conditions
using any of the procedures described above to provide
single-stranded molecules.
[0073] The steps of denaturing, annealing, and extension product
synthesis can be repeated as often as needed to amplify the target
polymorphic locus nucleic acid sequence to the extent necessary for
detection. The amount of the specific nucleic acid sequence
produced will accumulate in an exponential fashion. PCR. A
Practical Approach, ILR Press, Eds. McPherson et al. (1992).
[0074] The amplification products may be detected by Southern blot
analysis with or without using radioactive probes. In one such
method, for example, a small sample of DNA containing a very low
level of the nucleic acid sequence of the polymorphic locus is
amplified, and analyzed via a Southern blotting technique or
similarly, using dot blot analysis. The use of non-radioactive
probes or labels is facilitated by the high level of the amplified
signal. Alternatively, probes used to detect the amplified products
can be directly or indirectly detectably labeled, for example, with
a radioisotope, a fluorescent compound, a bioluminescent compound,
a chemiluminescent compound, a metal chelator or an enzyme. Those
of ordinary skill in the art will know of other suitable labels for
binding to the probe, or will be able to ascertain such, using
routine experimentation.
[0075] Sequences amplified by the methods of the invention can be
further evaluated, detected, cloned, sequenced, and the like,
either in solution or after binding to a solid support, by any
method usually applied to the detection of a specific DNA sequence
such as dideoxy sequencing, PCR, oligomer restriction (Saiki et
al., Bio/Technology 3:1008-1012 (1985), allele-specific
oligonucleotide (ASO) probe analysis (Conner et al., Proc. Natl.
Acad. Sci. U.S.A. 80:278 (1983), oligonucleotide ligation assays
(OLAs) (Landgren et. al., Science 241:1007, 1988), and the like.
Molecular techniques for DNA analysis have been reviewed (Landgren
et. al., Science 242:229-237, 1988).
[0076] Preferably, the method of amplifying is by PCR, as described
herein and in U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188
each of which is hereby incorporated by reference; and as is
commonly used by those of ordinary skill in the art. Alternative
methods of amplification have been described and can also be
employed as long as the CPSI locus amplified by PCR using primers
of the invention is similarly amplified by the alternative means.
Such alternative amplification systems include but are not limited
to self-sustained sequence replication, which begins with a short
sequence of RNA of interest and a T7 promoter. Reverse
transcriptase copies the RNA into cDNA and degrades the RNA,
followed by reverse transcriptase polymerizing a second strand of
DNA.
[0077] Another nucleic acid amplification technique is nucleic acid
sequence-based amplification (NASBA.TM.) which uses reverse
transcription and T7 RNA polymerase and incorporates two primers to
target its cycling scheme. NASBA.TM. amplification can begin with
either DNA or RNA and finish with either, and amplifies to about
10.sup.8 copies within 60 to 90 minutes.
[0078] Alternatively, nucleic acid can be amplified by ligation
activated transcription (LAT). LAT works from a single-stranded
template with a single primer that is partially single-stranded and
partially double-stranded. Amplification is initiated by ligating a
cDNA to the promoter olignucleotide and within a few hours,
amplification is about 10.sup.8 to about 10.sup.9 fold. The QB
replicase system can be utilized by attaching an RNA sequence
called MDV-1 to RNA complementary to a DNA sequence of interest.
Upon mixing with a sample, the hybrid RNA finds its complement
among the specimen's mRNAs and binds, activating the replicase to
copy the tag-along sequence of interest. Another nucleic acid
amplification technique, lignase chain reaction (LCR), works by
using two differently labeled halves of a sequence of interest
which are covalently bonded by ligase in the presence of the
contiguous sequence in a sample, forming a new target. The repair
chain reaction (RCR) nucleic acid amplification technique uses two
complementary and target-specific oligonucleotide probe pairs,
thermostable polymerase and ligase, and DNA nucleotides to
geometrically amplify targeted sequences. A 2-base gap separates
the oligo probe pairs, and the RCR fills and joins the gap,
mimicking normal DNA repair.
[0079] Nucleic acid amplification by strand displacement activation
(SDA) utilizes a short primer containing a recognition site for
HindII with short overhang on the 5' end which binds to target DNA.
A DNA polymerase fills in the part of the primer opposite the
overhang with sulfur-containing adenine analogs. HincII is added
but only cuts the unmodified DNA strand. A DNA polymerase that
lacks 5' exonuclease activity enters at the cite of the nick and
begins to polymerize, displacing the initial primer strand
downstream and building a new one which serves as more primer.
[0080] SDA produces greater than about a 10.sup.7-fold
amplification in 2 hours at 37.degree. C. Unlike PCR and LCR, SDA
does not require instrumented temperature cycling. Another
amplification system useful in the method of the invention is the
QB Replicase System. Although PCR is the preferred method of
amplification if the invention, these other methods can also be
used to amplify the CPSI locus as described in the method of the
invention. Thus, the term "amplification technique" as used herein
and in the claims is meant to encompass all the foregoing
methods.
[0081] In another embodiment of the invention a method is provided
for diagnosing or identifying a subject having a predisposition or
higher susceptibility to (at risk of) hyperammonemia, comprising
sequencing a target nucleic acid of a sample from a subject by
dideoxy sequencing, preferably following amplification of the
target nucleic acid.
[0082] In another embodiment of the invention a method is provided
for diagnosing a subject having a predisposition or higher
susceptibility to (at risk of) hyperammonemia, comprising
contacting a target nucleic acid of a sample from a subject with a
reagent that detects the presence of the CPSI polymorphism and
detecting the reagent.
[0083] Another method comprises contacting a target nucleic acid of
a sample from a subject with a reagent that detects the presence of
the C to A transversion at base 4340, i.e. within exon 36, and
detecting the transversion. A number of hybridization methods are
well known to those skilled in the art. Many of them are useful in
carrying out the invention.
[0084] Hepatic veno-occlusive disease (HVOD) is a common toxicity
in bone marrow transplant (BMT). It occurs in approximately 20 to
40% of patients and is associated with severe morbidity and
mortality. In accordance with the present invention, the frequency
of both CPSI alleles was tested in an HVOD and a non-HVOD group
undergoing BMT in an effort to identify evidence of disequilibrium.
The results indicated the CPSI polymorphism disclosed herein
effects susceptibility to a BMT toxicity. Thus, a method of
screening subjects for susceptibility to BMT toxicity, and
particularly to HVOD, via detection of the CPSI polymorphism is
provided in accordance with the present invention.
[0085] The materials for use in the method of the invention are
ideally suited for the preparation of a diagnostic kit. Such a kit
may comprise a carrier means being compartmentalized to receive in
close confinement one or more container means such as vials, tubes,
and the like, each of the container means comprising one of the
separate elements to be used in the method. For example, one of the
container means may comprise means for amplifying CPSI DNA, the
means comprising the necessary enzyme(s) and oligonucleotide
primers for amplifying said target DNA from the subject.
[0086] The oligonucleotide primers include primers having a
sequence selected from the group including, but not limited to: SEQ
ID NOs:6-10, or primer sequences substantially complementary or
substantially homologous thereto. The target flanking 5' and 3'
polynucleotide sequence has substantially the sequence set forth in
SEQ ID NO:5, and sequences substantially complementary or
homologous thereto. Other oligonucleotide primers for amplifying
CPSI will be known or readily ascertainable to those of skill in
the art given the disclosure of the present invention presented
herein.
[0087] A kit in accordance with the present invention can further
comprise a reagent or reagents for extracting a nucleic acid sample
from a biological sample obtained from a subject. Any such reagents
as would be readily apparent to one of ordinary skill in the art
are contemplated to fall within the scope of the present invention.
By way of particular example, a suitable lysis buffer for the
tissue along with a suspension of glass beads for capturing the
nucleic acid sample and an elution buffer for eluting the nucleic
acid sample off of the glass beads comprise reagents for extracting
a nucleic acid sample from a biological sample obtained from a
subject.
[0088] Other examples include commercially available, such as the
GENOMIC ISOLATION KIT A.S.A.P..TM. (Boehringer Mannheim,
Indianapolis, Ind.), Genomic DNA Isolation System (GIBCO BRL,
Gaithersburg, Md.), ELU-QUIK.TM. DNA Purification Kit (Schleicher
& Schuell, Keene, N.H.), DNA Extraction Kit (Stratagene, La
Jolla, Calif.), TURBOGEN.TM. Isolation Kit (Invitrogen, San Diego,
Calif.), and the like. Use of these kits according to the
manufacturer's instructions is generally acceptable for
purification of DNA prior to practicing the methods of the present
invention.
C. Definitions Affecting CPSI-Encoding Polynucleotide and CPSI
Polypeptides Encoded by Same
[0089] In accordance with the present invention, purified and
isolated CPSI-encoding polynucleotides and CPSI polypeptides
encoded by same are provided. A particularly provided CPSI-encoding
polynucleotide comprises a CPSI encoding polynucleotide which
includes a C to A transversion at base 4340, i.e. within exon 36,
of the CPSI gene which changes the triplet code from ACC to AAC and
leads to the T1405N change in the encoded CPSI polypeptide. The
encoded CPSI polypeptide comprising the T1405N change is also
particularly provided. Thus, allelic variant polynucleotides and
polypeptides encoded by same are provided in accordance with the
present invention. Further, a biologically active CPSI polypeptide
is also provided in accordance with the present invention, as is a
CPSI-encoding polynucleotide encoding such a CPSI polypeptide.
Exemplary biological activities include the biological activity of
mediating the first step of the urea cycle and the biological
activity of cross-reacting with an anti-CPSI antibody.
[0090] The provided CPSI-encoding polynucleotides and polypeptides
have broad utility given the biological significance of the urea
cycle, as is known in the art. By way of example, the CPSI-encoding
polynucleotides and polypeptides are useful in the preparation of
screening assays and assay kits that are used to detect the
presence of the proteins and nucleic acids of this invention in
biological samples. Additionally, it is well known that isolated
and purified polypeptides have utility as feed additives for
livestock and polynucleotides encoding the polypeptides are thus
useful in producing the polypeptides.
[0091] Preferably, the provided CPSI polynucleotides and
polypeptides are isolated from vertebrate and invertebrate sources.
Thus, homologs of CPSI, including, but not limited to, mammalian,
yeast and bacterial homologs are provided in accordance with the
present invention. Preferred mammalian homologs of CPSI members
include, but are not limited to, rat and human homologs.
[0092] The terms "CPSI gene product", "CPSI protein" and "CPSI
polypeptide" refer to proteins having amino acid sequences which
are substantially identical to the native amino acid sequences in
CPSI and which are biologically active in that they are capable of
mediating the synthesis of carbamyl phosphate in the urea cycle, or
cross-reacting with anti-CPSI antibodies raised against a CPSI
polypeptide.
[0093] The terms "CPSI gene product", "CPSI protein" and "CPSI
polypeptide" also include analogs of CPSI molecules which exhibit
at least some biological activity in common with native CPSI gene
products. Furthermore, those skilled in the art of mutagenesis will
appreciate that other analogs, as yet undisclosed or undiscovered,
may be used to construct CPSI analogs. There is no need for an
"CPSI gene product", "CPSI protein" or "CPSI polypeptide" to
comprise all, or substantially all of the amino acid sequence of a
native CPSI gene product. Shorter or longer sequences are
anticipated to be of use in the invention. Thus, the term "CPSI
gene product" also includes fusion or recombinant CPSI polypeptides
and proteins. Methods of preparing such proteins are described
herein.
[0094] The terms "CPSI-encoding polynucleotide", "CPSI gene", "CPSI
gene sequence" and "CPSI gene segment" refer to any DNA sequence
that is substantially identical to a polynucleotide sequence
encoding a CPSI gene product, CPSI protein or CPSI polypeptide as
defined above. The terms also refer to RNA, or antisense sequences,
compatible with such DNA sequences. A "CPSI-encoding
polynucleotide", "CPSI gene", "CPSI gene sequence" and "CPSI gene
segment" may also comprise any combination of associated control
sequences.
[0095] The term "substantially identical", when used to define
either a CPSI gene product or CPSI amino acid sequence, or a CPSI
gene or CPSI nucleic acid sequence, means that a particular
sequence, for example, a mutant sequence, varies from the sequence
of a natural CPSI by one or more deletions, substitutions, or
additions, the net effect of which is to retain at least some of
biological activity of CPSI. Alternatively, DNA analog sequences
are "substantially identical" to specific DNA sequences disclosed
herein if: (a) the DNA analog sequence is derived from coding
regions of the natural CPSI gene; or (b) the DNA analog sequence is
capable of hybridization of DNA sequences of (a) under moderately
stringent conditions and which encode biologically active CPSI gene
product; or (c) the DNA sequences are degenerative as a result of
the genetic code to the DNA analog sequences defined in (a) and/or
(b). Substantially identical analog proteins will be greater than
about 60% identical to the corresponding sequence of the native
protein. Sequences having lesser degrees of similarity but
comparable biological activity are considered to be equivalents. In
determining nucleic acid sequences, all subject nucleic acid
sequences capable of encoding substantially similar amino acid
sequences are considered to be substantially similar to a reference
nucleic acid sequence, regardless of differences in codon
sequences.
[0096] C.1. Percent Similarity
[0097] Percent similarity may be determined, for example, by
comparing sequence information using the GAP computer program,
available from the University of Wisconsin Geneticist Computer
Group. The GAP program utilizes the alignment method of Needleman
et al., J. Mol. Biol. 48:443 (1970), as revised by Smith et al.,
Adv. Appl. Math. 2:482 (1981). Briefly, the GAP program defines
similarity as the number of aligned symbols (i.e. nucleotides or
amino acids) which are similar, divided by the total number of
symbols in the shorter of the two sequences. The preferred default
parameters for the GAP program include: (1) a unitary comparison
matrix (containing a value of 1 for identities and 0 for
non-identities) of nucleotides and the weighted comparison matrix
of Gribskov et al., Nuci. Acids. Res. 14:6745 (1986), as described
by Schwartz et al., eds., Atlas of Protein Sequence and Structure,
National Biomedical Research Foundation, pp. 357-358 (1979); (2) a
penalty of 3.0 for each gap and an additional 0.01 penalty for each
symbol and each gap; and (3) no penalty for end gaps. Other
comparison techniques are described in the Examples.
[0098] The term "homology" describes a mathematically based
comparison of sequence similarities which is used to identify genes
or proteins with similar functions or motifs. Accordingly, the term
"homology" is synonymous with the term "similarity" and "percent
similarity" as defined above. Thus, the phrases "substantial
homology" or "substantial similarity" have similar meanings.
[0099] C.2. Nucleic Acid Sequences
[0100] In certain embodiments, the invention concerns the use of
CPSI genes and gene products that include within their respective
sequences a sequence which is essentially that of a CPSI gene, or
the corresponding protein. The term "a sequence essentially as that
of a CPSI gene", means that the sequence substantially corresponds
to a portion of a CPSI polypeptide or CPSI encoding polynucleotide
and has relatively few bases or amino acids (whether DNA or
protein) which are not identical to those of a CPSI protein or CPSI
gene, (or a biologically functional equivalent of, when referring
to proteins). The term "biologically functional equivalent" is well
understood in the art and is further defined in detail herein.
Accordingly, sequences which have between about 70% and about 80%;
or more preferably, between about 81% and about 90%; or even more
preferably, between about 91% and about 99%; of amino acids which
are identical or functionally equivalent to the amino acids of a
CPSI protein or CPSI gene, will be sequences which are "essentially
the same".
[0101] CPSI gene products and CPSI genes which have functionally
equivalent codons are also covered by the invention. The term
"functionally equivalent codon" is used herein to refer to codons
that encode the same amino acid, such as the six codons for
arginine or serine, and also to refer to codons that encode
biologically equivalent amino acids (see Table 1).
TABLE-US-00002 TABLE 1 Table of the Genetic Code Amino Acids Codons
Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic Acid
Asp D GAC GAU Glumatic acid Glu E GAA GAG Phenylalanine Phe F UUC
UUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU
Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA
UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAU
Proline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg
R AGA AGG CGA CGC CGG CGU Serine Ser S ACG AGU UCA UCC UCG UCU
Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU
Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU
[0102] It will also be understood that amino acid and nucleic acid
sequences may include additional residues, such as additional N- or
C-terminal amino acids or 5' or 3' sequences, and yet still be
essentially as set forth in one of the sequences disclosed herein,
so long as the sequence meets the criteria set forth above,
including the maintenance of biological protein activity where
protein expression is concerned. The addition of terminal sequences
particularly applies to nucleic acid sequences which may, for
example, include various non-coding sequences flanking either of
the 5' or 3' portions of the coding region or may include various
internal sequences, i.e., introns, which are known to occur within
genes.
[0103] The present invention also encompasses the use of DNA
segments which are complementary, or essentially complementary, to
the sequences set forth in the specification. Nucleic acid
sequences which are "complementary" are those which are
base-pairing according to the standard Watson-Crick complementarity
rules. As used herein, the term "complementary sequences" means
nucleic acid sequences which are substantially complementary, as
may be assessed by the same nucleotide comparison set forth above,
or as defined as being capable of hybridizing to the nucleic acid
segment in question under relatively stringent conditions such as
those described herein. A particular example of a contemplated
complementary nucleic acid segment is an antisense
oligonucleotide.
[0104] Nucleic acid hybridization will be affected by such
conditions as salt concentration, temperature, or organic solvents,
in addition to the base composition, length of the complementary
strands, and the number of nucleotide base mismatches between the
hybridizing nucleic acids, as will be readily appreciated by those
skilled in the art. Stringent temperature conditions will generally
include temperatures in excess of 30 C, typically in excess of 37
C, and preferably in excess of 45 C. Stringent salt conditions will
ordinarily be less than 1,000 mM, typically less than 500 mM, and
preferably less than 200 mM. However, the combination of parameters
is much more important than the measure of any single parameter.
(See e.g., Wetmur & Davidson, J. Mol. Biol. 31:349-370
(1968)).
[0105] Probe sequences may also hybridize specifically to duplex
DNA under certain conditions to form triplex or other higher order
DNA complexes. The preparation of such probes and suitable
hybridization conditions are well known in the art.
[0106] As used herein, the term "DNA segment" refers to a DNA
molecule which has been isolated free of total genomic DNA of a
particular species. Furthermore, a DNA segment encoding a CPSI
polypeptide refers to a DNA segment which contains CPSI coding
sequences, yet is isolated away from, or purified free from, total
genomic DNA of a source species, such as Homo sapiens. Included
within the term "DNA segment" are DNA segments and smaller
fragments of such segments, and also recombinant vectors,
including, for example, plasmids, cosmids, phages, viruses, and the
like.
[0107] Similarly, a DNA segment comprising an isolated or purified
CPSI gene refers to a DNA segment including CPSI coding sequences
isolated substantially away from other naturally occurring genes or
protein encoding sequences. In this respect, the term "gene" is
used for simplicity to refer to a functional protein, polypeptide
or peptide encoding unit. As will be understood by those in the
art, this functional term includes both genomic sequences and cDNA
sequences. "Isolated substantially away from other coding
sequences" means that the gene of interest, in this case, the CPSI
gene, forms the significant part of the coding region of the DNA
segment, and that the DNA segment does not contain large portions
of naturally-occurring coding DNA, such as large chromosomal
fragments or other functional genes or cDNA coding regions. Of
course, this refers to the DNA segment as originally isolated, and
does not exclude genes or coding regions later added to the segment
by the hand of man.
[0108] In particular embodiments, the invention concerns isolated
DNA segments and recombinant vectors incorporating DNA sequences
which encode a CPSI polypeptide that includes within its amino acid
sequence an amino acid sequence of any of SEQ ID NOs:2, 4, 12 and
14. In other particular embodiments, the invention concerns
isolated DNA segments and recombinant vectors incorporating DNA
sequences which encode a protein that includes within its amino
acid sequence the amino acid sequence of a CPSI polypeptide
corresponding to human tissues.
[0109] It will also be understood that this invention is not
limited to the particular nucleic acid and amino acid sequences of
SEQ ID NOs:1-4 and 11-14. Recombinant vectors and isolated DNA
segments may therefore variously include the CPSI
polypeptide-encoding region itself, include coding regions bearing
selected alterations or modifications in the basic coding region,
or include encoded larger polypeptides which nevertheless include
CPSI polypeptide-encoding regions or may encode biologically
functional equivalent proteins or peptides which have variant amino
acid sequences.
[0110] In certain embodiments, the invention concerns isolated DNA
segments and recombinant vectors which encode a protein or peptide
that includes within its amino acid sequence an amino acid sequence
essentially as set forth in any of SEQ ID NOs:2, 4, 12 and 14.
Naturally, where the DNA segment or vector encodes a full length
CPSI gene product, the most preferred nucleic acid sequence is that
which is essentially as set forth in any of SEQ ID NOs: 1, 3, 11
and 13 and which encode a protein that exhibits activity in the
urea cycle, as may be determined by, for example, colorimetric
assays to detect production of carbonyl phosphate from ammonia, as
disclosed herein in Example 3.
[0111] The term "a sequence essentially as set forth in any of SEQ
ID NO:2, 4, 12 and 14" means that the sequence substantially
corresponds to a portion an amino acid sequence either of SEQ ID
NOs:2, 4, 12 and 14 and has relatively few amino acids which are
not identical to, or a biologically functional equivalent of, the
amino acids of an amino acid sequence of any of SEQ ID NOs:2, 4, 12
and 14. The term "biologically functional equivalent" is well
understood in the art and is further defined in detail herein.
Accordingly, sequences, which have between about 70% and about 80%;
or more preferably, between about 81% and about 90%; or even more
preferably, between about 91% and about 99%; of amino acids which
are identical or functionally equivalent to the amino acids in any
of SEQ ID NOs: 2, 4, 12 and 14, will be sequences which "a sequence
essentially as set forth in SEQ ID NOs:2, 4, 12 and 14".
[0112] In particular embodiments, the invention concerns gene
therapy methods that use isolated DNA segments and recombinant
vectors incorporating DNA sequences which encode a protein that
includes within its amino acid sequence an amino acid sequence of
any of SEQ ID NOs:2, 4, 12 and 14, SEQ ID NOs:2, 4, 12 and 14
including sequences which are derived from human tissue. In other
particular embodiments, the invention concerns isolated DNA
sequences and recombinant DNA vectors incorporating DNA sequences
which encode a protein that includes within its amino acid sequence
the amino acid sequence of the CPSI protein from human hepatic
tissue.
[0113] In certain other embodiments, the invention concerns
isolated DNA segments and recombinant vectors that include within
their sequence a nucleic acid sequence essentially as set forth in
any of SEQ ID NO:1, 3, 11 and 13. The term "a sequence essentially
as set forth in any of SEQ ID NO:1, 3, 11 and 13" is used in the
same sense as described above and means that the nucleic acid
sequence substantially corresponds to a portion of any of SEQ ID
NOs:1, 3, 11 and 13, respectively, and has relatively few codons
which are not identical, or functionally equivalent, to the codons
of any of SEQ ID NOs:1, 3, 11 and 13, respectively. Again, DNA
segments which encode gene products exhibiting activity in the urea
cycle, cross-reactivity with an anti-CPSI antibody, or other
biological activity of the CPSI gene product will be most
preferred. The term "functionally equivalent codon" is used herein
to refer to codons that encode the same amino acid, such as the six
codons for arginine or serine, and also to refer to codons that
encode biologically equivalent amino acids (see Table 1).
[0114] The nucleic acid segments of the present invention,
regardless of the length of the coding sequence itself, may be
combined with other DNA sequences, such as promoters, enhancers,
polyadenylation signals, additional restriction enzyme sites,
multiple cloning sites, other coding segments, and the like, such
that their overall length may vary considerably. It is therefore
contemplated that a nucleic acid fragment of almost any length may
be employed, with the total length preferably being limited by the
ease of preparation and use in the intended recombinant DNA
protocol. For example, nucleic acid fragments may be prepared which
include a short stretch complementary to a nucleic acid sequence
set for in any of SEQ ID NOs:1, 3, 11 and 13 respectively, such as
about 10 nucleotides, and which are up to 10,000 or 5,000 base
pairs in length, with segments of 3,000 being preferred in certain
cases. DNA segments with total lengths of about 1,000, 500, 200,
100 and about 50 base pairs in length are also contemplated to be
useful.
[0115] The DNA segments of the present invention encompass
biologically functional equivalent CPSI proteins and peptides. Such
sequences may rise as a consequence of codon redundancy and
functional equivalency which are known to occur naturally within
nucleic acid sequences and the proteins thus encoded.
Alternatively, functionally equivalent proteins or peptides may be
created via the application of recombinant DNA technology, in which
changes in the protein structure may be engineered, based on
considerations of the properties of the amino acids being
exchanged, e.g. substitution of Ile and Leu at amino acids 4 and 5
is SEQ ID NOs:11-14. Changes designed by man may be introduced
through the application of site-directed mutagenesis techniques,
e.g., to introduce improvements to the antigenicity of the protein
or to test CPSI mutants in order to examine activity in the urea
cycle, or other activity at the molecular level.
[0116] If desired, one may also prepare fusion proteins and
peptides, e.g., where the CPSI coding region is aligned within the
same expression unit with other proteins or peptides having desired
functions, such as for purification or immunodetection purposes
(e.g., proteins which may be purified by affinity chromatography
and enzyme label coding regions, respectively).
[0117] Recombinant vectors form important further aspects of the
present invention. Particularly useful vectors are contemplated to
be those vectors in which the coding portion of the DNA segment is
positioned under the control of a promoter. The promoter may be in
the form of the promoter which is naturally associated with the
CPSI gene, e.g., in mammalian tissues, as may be obtained by
isolating the 5' non-coding sequences located upstream of the
coding segment or exon, for example, using recombinant cloning
and/or PCR technology, in connection with the compositions
disclosed herein.
[0118] In other embodiments, it is contemplated that certain
advantages will be gained by positioning the coding DNA segment
under the control of a recombinant, or heterologous, promoter. As
used herein, a recombinant or heterologous promoter is intended to
refer to a promoter that is not normally associated with a CPSI
gene in its natural environment. Such promoters may include
promoters isolated from bacterial, viral, eukaryotic, or mammalian
cells. Naturally, it will be important to employ a promoter that
effectively directs the expression of the DNA segment in the cell
type chosen for expression. The use of promoter and cell type
combinations for protein expression is generally known to those of
skill in the art of molecular biology, for example, see Sambrook et
al., 1989, incorporated herein by reference. The promoters employed
may be constitutive, or inducible, and can be used under the
appropriate conditions to direct high level expression of the
introduced DNA segment, such as is advantageous in the large-scale
production of recombinant proteins or peptides. Appropriate
promoter systems provided for use in high-level expression include,
but are not limited to, the vaccina virus promoter and the
baculovirus promoter.
[0119] In an alternative embodiment, the present invention provides
an expression vector comprising a polynucleotide that encodes a
CPSI polypeptide having activity in the urea cycle, cross-reacting
with an anti-CPSI antibody, or other biological activity in
accordance with the present invention. Also preferably, an
expression vector of the present invention comprises a
polynucleotide that encodes a human CPSI gene product. More
preferably, an expression vector of the present invention comprises
a polynucleotide that encodes a polypeptide comprising an amino
acid residue sequence of any of SEQ ID NOs:2, 4, 12 and 14. More
preferably, an expression vector of the present invention comprises
a polynucleotide comprising the nucleotide base sequence of any of
SEQ ID NO:1, 3, 11 and 13.
[0120] Even more preferably, an expression vector of the invention
comprises a polynucleotide operatively linked to an
enhancer-promoter. More preferably still, an expression vector of
the invention comprises a polynucleotide operatively linked to a
prokaryotic promoter. Alternatively, an expression vector of the
present invention comprises a polynucleotide operatively linked to
an enhancer-promoter that is a eukaryotic promoter, and the
expression vector further comprises a polyadenylation signal that
is positioned 3' of the carboxy-terminal amino acid and within a
transcriptional unit of the encoded polypeptide.
[0121] In yet another embodiment, the present invention provides a
recombinant host cell transfected with a polynucleotide that
encodes a CPSI polypeptide having activity in the modulation of the
urea cycle, cross-reactivity with an anti-CPSI antibody, or other
biological activity in accordance with the present invention. SEQ
ID NO's: 1-4 and 11-14 set forth nucleotide and amino acid
sequences from an exemplary vertebrate, human. Also provided by the
present invention are homologous or biologically equivalent
polynucleotides and CPSI polypeptides found in other vertebrates,
including rat. Also provided by the present invention are
homologous or biologically equivalent polynucleotides and CPSI
polypeptides found in invertebrates, including bacteria and
yeast.
[0122] Preferably, a recombinant host cell of the present invention
is transfected with the polynucleotide that encodes human CPSI
polypeptide. More preferably, a recombinant host cell of the
present invention is transfected with the polynucleotide sequence
of any of SEQ ID NOs:1, 3, 11 and 13. Even more preferably, a host
cell of the invention is a eukaryotic host cell. Still more
preferably, a recombinant host cell of the present invention is a
vertebrate cell. Preferably, a recombinant host cell of the
invention is a mammalian cell.
[0123] In another aspect, a recombinant host cell of the present
invention is a prokaryotic host cell. Preferably, a recombinant
host cell of the invention is a bacterial cell, preferably a strain
of Escherichia coli. More preferably, a recombinant host cell
comprises a polynucleotide under the transcriptional control of
regulatory signals functional in the recombinant host cell, wherein
the regulatory signals appropriately control expression of the CPSI
polypeptide in a manner to enable all necessary transcriptional and
post-transcriptional modification.
[0124] In yet another embodiment, the present invention provides a
method of preparing a CPSI polypeptide comprising transfecting a
cell with polynucleotide that encodes a CPSI polypeptide having
activity in the urea cycle, cross-reacting with an anti-CPSI
antibody, or other biological activity in accordance with the
present invention, to produce a transformed host cell; and
maintaining the transformed host cell under biological conditions
sufficient for expression of the polypeptide. More preferably, the
transformed host cell is a eukaryotic cell. More preferably still,
the eukaryotic cell is a vertebrate cell. Alternatively, the host
cell is a prokaryotic cell. More preferably, the prokaryotic cell
is a bacterial cell of Escherichia coli. Even more preferably, a
polynucleotide transfected into the transformed cell comprises a
nucleotide base sequence of any of SEQ ID NOs:1, 3, 11 and 13. SEQ
ID NO's:1-4 and 11-14 set forth nucleotide and amino acid sequences
for an exemplary vertebrate, human. Also provided by the present
invention are homologues or biologically equivalent CPSI
polynucleotides and polypeptides found in other vertebrates,
particularly warm blooded vertebrates, and more particularly rat.
Also provided by the present invention are homologous or
biologically equivalent polynucleotides and CPSI polypeptides found
in invertebrates, including bacteria and yeast.
[0125] As mentioned above, in connection with expression
embodiments to prepare recombinant CPSI proteins and peptides, it
is contemplated that longer DNA segments will most often be used,
with DNA segments encoding the entire CPSI protein, functional
domains or cleavage products thereof, being most preferred.
However, it will be appreciated that the use of shorter DNA
segments to direct the expression of CPSI peptides or epitopic core
regions, such as may be used to generate anti-CPSI antibodies, also
falls within the scope of the invention.
[0126] DNA segments which encode peptide antigens from about 15 to
about 50 amino acids in length, or more preferably, from about 15
to about 30 amino acids in length are contemplated to be
particularly useful. DNA segments encoding peptides will generally
have a minimum coding length in the order of about 45 to about 150,
or to about 90 nucleotides. DNA segments encoding full length
proteins may have a minimum coding length on the order of about
4,500 to about 4,600 nucleotides for a protein in accordance with
any of SEQ ID NOs: 2, 4, 12 and 14.
[0127] Naturally, the present invention also encompasses DNA
segments which are complementary, or essentially complementary, to
the sequences set forth in any of SEQ ID NO's: 1, 3, 11 and 13. The
terms "complementary" and "essentially complementary" are defined
above. Excepting intronic or flanking regions, details of which are
disclosed graphically in FIG. 9, and allowing for the degeneracy of
the genetic code, sequences which have between about 70% and about
80%; or more preferably, between about 81% and about 90%; or even
more preferably, between about 91% and about 99%; of nucleotides
which are identical or functionally equivalent (i.e. encoding the
same amino acid) of nucleotides in any of SEQ ID NOs:1, 3, 11 and
13 will be sequences which are "a sequence essentially as set forth
in any of SEQ ID NOs:1, 3, 11 and 13". Sequences which are
essentially the same as those set forth in any of SEQ ID NOs:1, 3,
11 and 13 may also be functionally defined as sequences which are
capable of hybridizing to a nucleic acid segment containing the
complement in any of SEQ ID NOs:1, 3, 11 and 13 under relatively
stringent conditions. Suitable relatively stringent hybridization
conditions are described herein and will be well known to those of
skill in the art.
[0128] C.2. Biologically Functional Equivalents
[0129] As mentioned above, modification and changes may be made in
the structure of the CPSI proteins and peptides described herein
and still obtain a molecule having like or otherwise desirable
characteristics. For example, certain amino acids may be
substituted for other amino acids in a protein structure without
appreciable loss of interactive capacity with structures such as,
for example, in the nucleus of a cell. Since it is the interactive
capacity and nature of a protein that defines that protein's
biological functional activity, certain amino acid sequence
substitutions can be made in a protein sequence (or, of course, its
underlying DNA coding sequence) and nevertheless obtain a protein
with like or even countervailing properties (e.g., antagonistic v.
agonistic). It is thus contemplated by applicants that various
changes may be made in the sequence of the CPSI proteins and
peptides (or underlying DNA) without appreciable loss of their
biological utility or activity.
[0130] It is also well understood by the skilled artisan that,
inherent in the definition of a biologically functional equivalent
protein or peptide, is the concept that there is a limit to the
number of changes that may be made within a defined portion of the
molecule and still result in a molecule with an acceptable level of
equivalent biological activity. Biologically functional equivalent
peptides are thus defined herein as those peptides in which
certain, not most or all, of the amino acids may be substituted. Of
course, a plurality of distinct proteins/peptides with different
substitutions may easily be made and used in accordance with the
invention.
[0131] It is also well understood that where certain residues are
shown to be particularly important to the biological or structural
properties of a protein or peptide, e.g., residues in active sites,
such residues may not generally be exchanged. This is the case in
the present invention, where if any changes, for example, in the
phosphorylation domains of a CPSI polypeptide, could result in a
loss of an aspect of the utility of the resulting peptide for the
present invention.
[0132] Amino acid substitutions, such as those which might be
employed in modifying the CPSI proteins and peptides described
herein, are generally based on the relative similarity of the amino
acid side-chain substituents, for example, their hydrophobicity,
hydrophilicity, charge, size, and the like. An analysis of the
size, shape and type of the amino acid side-chain substituents
reveals that arginine, lysine and histidine are all positively
charged residues; that alanine, glycine and serine are all a
similar size; and that phenylalanine, tryptophan and tyrosine all
have a generally similar shape. Therefore, based upon these
considerations, arginine, lysine and histidine; alanine, glycine
and serine; and phenylalanine, tryptophan and tyrosine; are defined
herein as biologically functional equivalents.
[0133] In making such changes, the hydropathic index of amino acids
may be considered. Each amino acid has been assigned a hydropathic
index on the basis of their hydrophobicity and charge
characteristics, these are: isoleucine (+4.5); valine (+4.2);
leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine
(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline
(-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine
(-4.5).
[0134] The importance of the hydropathic amino acid index in
conferring interactive biological function on a protein is
generally understood in the art (Kyte & Doolittle, J. Mol.
Biol. 157:105-132 (1982), incorporated herein by reference). It is
known that certain amino acids may be substituted for other amino
acids having a similar hydropathic index or score and still retain
a similar biological activity. In making changes based upon the
hydropathic index, the substitution of amino acids whose
hydropathic indices are within .+-.2 of the original value is
preferred, those which are within .+-.1 of the original value are
particularly preferred, and those within .+-.0.5 of the original
value are even more particularly preferred.
[0135] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by
reference, states that the greatest local average hydrophilicity of
a protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with its immunogenicity and antigenicity, i.e.
with a biological property of the protein. It is understood that an
amino acid can be substituted for another having a similar
hydrophilicity value and still obtain a biologically equivalent
protein.
[0136] As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4).
[0137] In making changes based upon similar hydrophilicity values,
the substitution of amino acids whose hydrophilicity values are
within .+-.2 of the original value is preferred, those which are
within .+-.1 of the original value are particularly preferred, and
those within .+-.0.5 of the original value are even more
particularly preferred.
[0138] While discussion has focused on functionally equivalent
polypeptides arising from amino acid changes, it will be
appreciated that these changes may be effected by alteration of the
encoding DNA, taking into consideration also that the genetic code
is degenerate and that two or more codons may code for the same
amino acid.
[0139] C.3. Sequence Modification Techniques
[0140] Modifications to the CPSI proteins and peptides described
herein may be carried out using techniques such as site directed
mutagenesis. Site-specific mutagenesis is a technique useful in the
preparation of individual peptides, or biologically functional
equivalent proteins or peptides, through specific mutagenesis of
the underlying DNA. The technique further provides a ready ability
to prepare and test sequence variants, for example, incorporating
one or more of the foregoing considerations, by introducing one or
more nucleotide sequence changes into the DNA. Site-specific
mutagenesis allows the production of mutants through the use of
specific oligonucleotide sequences which encode the DNA sequence of
the desired mutation, as well as a sufficient number of adjacent
nucleotides, to provide a primer sequence of sufficient size and
sequence complexity to form a stable duplex on both sides of the
deletion junction being traversed. Typically, a primer of about 17
to 30 nucleotides in length is preferred, with about 5 to 10
residues on both sides of the junction of the sequence being
altered.
[0141] In general, the technique of site-specific mutagenesis is
well known in the art as exemplified by publications (e.g., Adelman
et al., 1983). As will be appreciated, the technique typically
employs a phage vector which exists in both a single stranded and
double stranded form. Typical vectors useful in site-directed
mutagenesis include vectors such as the M13 phage (Messing et al.,
1981). These phage are readily commercially available and their use
is generally well known to those skilled in the art. Double
stranded plasmids are also routinely employed in site directed
mutagenesis which eliminates the step of transferring the gene of
interest from a plasmid to a phage.
[0142] In general, site-directed mutagenesis in accordance herewith
is performed by first obtaining a single-stranded vector or melting
apart the two strands of a double stranded vector which includes
within its sequence a DNA sequence which encodes, for example, a
human CPSI polypeptide. An oligonucleotide primer bearing the
desired mutated sequence is prepared, generally synthetically, for
example by the method of Crea et al. (1978). This primer is then
annealed with the single-stranded vector, and subjected to DNA
polymerizing enzymes such as E. coli polymerase I Klenow fragment,
in order to complete the synthesis of the mutation-bearing strand.
Thus, a heteroduplex is formed wherein one strand encodes the
original non-mutated sequence and the second strand bears the
desired mutation. This heteroduplex vector is then used to
transform appropriate cells, such as E. coli cells, and clones are
selected which include recombinant vectors bearing the mutated
sequence arrangement.
[0143] The preparation of sequence variants of the selected gene
using site-directed mutagenesis is provided as a means of producing
potentially useful CPSI polypeptide or other species having
activity in the urea cycle and is not meant to be limiting as there
are other ways in which sequence variants of these peptides may be
obtained. For example, recombinant vectors encoding the desired
genes may be treated with mutagenic agents to obtain sequence
variants (see, e.g., a method described by Eichenlaub, 1979) for
the mutagenesis of plasmid DNA using hydroxylamine.
[0144] C.4. Other Structural Equivalents
[0145] In addition to the CPSI peptidyl compounds described herein,
the inventors also contemplate that other sterically similar
compounds may be formulated to mimic the key portions of the
peptide structure. Such compounds may be used in the same manner as
the peptides of the invention and hence are also functional
equivalents. The generation of a structural functional equivalent
may be achieved by the techniques of modeling and chemical design
known to those of skill in the art. It will be understood that all
such sterically similar constructs fall within the scope of the
present invention.
D. Introduction of Gene Products
[0146] Where the gene itself is employed to introduce the gene
products, a convenient method of introduction will be through the
use of a recombinant vector which incorporates the desired gene,
together with its associated control sequences. The preparation of
recombinant vectors is well known to those of skill in the art and
described in many references, such as, for example, Sambrook et al.
(1989), specifically incorporated herein by reference.
[0147] In vectors, it is understood that the DNA coding sequences
to be expressed, in this case those encoding the CPSI gene
products, are positioned adjacent to and under the control of a
promoter. It is understood in the art that to bring a coding
sequence under the control of such a promoter, one generally
positions the 5' end of the transcription initiation site of the
transcriptional reading frame of the gene product to be expressed
between about 1 and about 50 nucleotides "downstream" of (i.e., 3'
of) the chosen promoter. One may also desire to incorporate into
the transcriptional unit of the vector an appropriate
polyadenylation site (e.g., 5''-AATAAA-3'), if one was not
contained within the original inserted DNA. Typically, these poly A
addition sites are placed about 30 to 2000 nucleotides "downstream"
of the coding sequence at a position prior to transcription
termination.
[0148] While use of the control sequences of the specific gene
(i.e., a CPSI promoter for a CPSI gene) will be preferred, there is
no reason why other control sequences could not be employed, so
long as they are compatible with the genotype of the cell being
treated. Thus, one may mention other useful promoters by way of
example, including, e.g., an SV40 early promoter, a long terminal
repeat promoter from retrovirus, an actin promoter, a heat shock
promoter, a metallothionein promoter, and the like.
[0149] As is known in the art, a promoter is a region of a DNA
molecule typically within about 100 nucleotide pairs in front of
(upstream of) the point at which transcription begins (i.e., a
transcription start site). That region typically contains several
types of DNA sequence elements that are located in similar relative
positions in different genes. As used herein, the term "promoter"
includes what is referred to in the art as an upstream promoter
region, a promoter region or a promoter of a generalized eukaryotic
RNA Polymerase II transcription unit.
[0150] Another type of discrete transcription regulatory sequence
element is an enhancer. An enhancer provides specificity of time,
location and expression level for a particular encoding region
(e.g., gene). A major function of an enhancer is to increase the
level of transcription of a coding sequence in a cell that contains
one or more transcription factors that bind to that enhancer.
Unlike a promoter, an enhancer can function when located at
variable distances from transcription start sites so long as a
promoter is present.
[0151] As used herein, the phrase "enhancer-promoter" means a
composite unit that contains both enhancer and promoter elements.
An enhancer-promoter is operatively linked to a coding sequence
that encodes at least one gene product. As used herein, the phrase
"operatively linked" means that an enhancer-promoter is connected
to a coding sequence in such a way that the transcription of that
coding sequence is controlled and regulated by that
enhancer-promoter. Means for operatively linking an
enhancer-promoter to a coding sequence are well known in the art.
As is also well known in the art, the precise orientation and
location relative to a coding sequence whose transcription is
controlled, is dependent inter alia upon the specific nature of the
enhancer-promoter. Thus, a TATA box minimal promoter is typically
located from about 25 to about 30 base pairs upstream of a
transcription initiation site and an upstream promoter element is
typically located from about 100 to about 200 base pairs upstream
of a transcription initiation site. In contrast, an enhancer can be
located downstream from the initiation site and can be at a
considerable distance from that site.
[0152] An enhancer-promoter used in a vector construct of the
present invention can be any enhancer-promoter that drives
expression in a cell to be transfected. By employing an
enhancer-promoter with well-known properties, the level and pattern
of gene product expression can be optimized.
[0153] For introduction of, for example, the human CPSI gene
including allelic variations thereof, it is proposed that one will
desire to preferably employ a vector construct that will deliver
the desired gene to the affected cells. This will, of course,
generally require that the construct be delivered to the targeted
cells, for example, mammalian hepatic cells. It is proposed that
this may be achieved most preferably by introduction of the desired
gene through the use of a viral vector to carry the CPSI sequence
to efficiently infect the cells. These vectors will preferably be
an adenoviral, a retroviral, a vaccinia viral vector or
adeno-associated virus. These vectors are preferred because they
have been successfully used to deliver desired sequences to cells
and tend to have a high infection efficiency. Suitable vector-CPSI
gene constructs are adapted for administration as pharmaceutical
compositions, as described herein below.
[0154] Commonly used viral promoters for expression vectors are
derived from polyoma, cytomegalovirus, Adenovirus 2, and Simian
Virus 40 (SV40). The early and late promoters of SV40 virus are
particularly useful because both are obtained easily from the virus
as a fragment which also contains the SV40 viral origin of
replication. Smaller or larger SV40 fragments may also be used,
provided there is included the approximately 250 bp sequence
extending from the Hind III site toward the Bgl I site located in
the viral origin of replication. Further, it is also possible, and
often desirable, to utilize promoter or control sequences normally
associated with the desired gene sequence, provided such control
sequences are compatible with the host cell systems.
[0155] The origin of replication may be provided either by
construction of the vector to include an exogenous origin, such as
may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV,
BPV) source, or may be provided by the host cell chromosomal
replication mechanism. If the vector is integrated into the host
cell chromosome, the latter is often sufficient.
[0156] Where a CPSI gene itself is employed it will be most
convenient to simply use a wild type CPSI gene directly. The CPSI
gene can thus comprise the threonine encoding allele such that
amino acid 1405 of the encoded polypeptide comprises threonine.
Alternatively, the CPSI gene comprises the arginine encoding allele
such that amino acid 1405 of the encoded polypeptide comprises
arginine. Additionally, it is envisioned that certain regions of a
CPSI gene can be employed exclusively without employing an entire
wild type CPSI gene or an entire allelic variant thereof. It is
proposed that it will ultimately be preferable to employ the
smallest region needed to modulate the urea cycle so that one is
not introducing unnecessary DNA into cells which receive a CPSI
gene construct. Techniques well known to those of skill in the art,
such as the use of restriction enzymes, will allow for the
generation of small regions of an exemplary CPSI gene. The ability
of these regions to modulate the urea cycle can easily be
determined by the assays reported in the Examples. In general,
techniques for assessing the modulation of the urea cycle are known
in the art.
[0157] D.1. Transgenic Animals
[0158] It is also provided within the scope of the present
invention to prepare a transgenic non-human animal which expresses
a CPSI gene of the present invention or in which expression of a
CPSI gene is "knocked-out". Provided transgenic non-human animals
express either the T1405 form of CPSI or the N1405 form of CPSI. A
preferred transgenic animal is a mouse.
[0159] Techniques for the preparation of transgenic animals are
known in the art. Exemplary techniques are described in U.S. Pat.
No. 5,489,742 (transgenic rats); U.S. Pat. Nos. 4,736,866,
5,550,316, 5,614,396, 5,625,125 and 5,648,061 (transgenic mice);
U.S. Pat. No. 5,573,933 (transgenic pigs); U.S. Pat. No. 5,162,215
(transgenic avian species) and U.S. Pat. No. 5,741,957 (transgenic
bovine species), the entire contents of each of which are herein
incorporated by reference.
[0160] With respect to an exemplary method for the preparation of a
transgenic mouse, cloned recombinant or synthetic DNA sequences or
DNA segments encoding a CPSI gene product are injected into
fertilized mouse eggs. The injected eggs are implanted in pseudo
pregnant females and are grown to term to provide transgenic mice
whose cells express a CPSI gene product. Preferably, the injected
sequences are constructed having promoter sequences connected so as
to express the desired protein in hepatic cells of the transgenic
mouse.
[0161] D.2. Gene Therapy
[0162] CPSI genes can be used for gene therapy in accordance with
the present invention. Exemplary gene therapy methods, including
liposomal transfection of nucleic acids into host cells, are
described in U.S. Pat. Nos. 5,279,833; 5,286,634; 5,399,346;
5,646,008; 5,651,964; 5,641,484; and 5,643,567, the contents of
each of which are herein incorporated by reference.
[0163] Briefly, CPSI gene therapy directed toward modulation of the
urea cycle in a target cell is described. Target cells include but
are not limited to hepatic cells and intestinal cells. In one
embodiment, a therapeutic method of the present invention provides
a method for modulating of the urea cycle in a cell comprising the
steps of: (a) delivering to the cell an effective amount of a DNA
molecule comprising a polynucleotide that encodes a CPSI
polypeptide that modulates the urea cycle; and (b) maintaining the
cell under conditions sufficient for expression of said
polypeptide.
[0164] Delivery is preferably accomplished by injecting the DNA
molecule into the cell. Where the cell is in a subject delivering
is preferably administering the DNA molecule into the circulatory
system of the subject. In a preferred embodiment, administering
comprises the steps of: (a) providing a vehicle that contains the
DNA molecule; and (b) administering the vehicle to the subject.
[0165] A vehicle is preferably a cell transformed or transfected
with the DNA molecule or a transfected cell derived from such a
transformed or transfected cell. An exemplary and preferred
transformed or transfected cell is a hepatic cell. Means for
transforming or transfecting a cell with a DNA molecule of the
present invention are set forth above.
[0166] Alternatively, the vehicle is a virus or an antibody that
specifically infects or immunoreacts with an antigen of the tumor.
Retroviruses used to deliver the constructs to the host target
tissues generally are viruses in which the 3'-LTR (linear transfer
region) has been inactivated. That is, these are enhancerless
3'-LTR's, often referred to as SIN (self-inactivating viruses)
because after productive infection into the host cell, the 3'-LTR
is transferred to the 5'-end and both viral LTR's are inactive with
respect to transcriptional activity. A use of these viruses well
known to those skilled in the art is to clone genes for which the
regulatory elements of the cloned gene are inserted in the space
between the two LTR's. An advantage of a viral infection system is
that it allows for a very high level of infection into the
appropriate recipient cell.
[0167] Antibodies have been used to target and deliver DNA
molecules. An N-terminal modified poly-L-lysine (NPLL)-antibody
conjugate readily forms a complex with plasmid DNA. A complex of
monoclonal antibodies against a cell surface thrombomodulin
conjugated with NPLL was used to target a foreign plasmid DNA to an
antigen-expressing mouse lung endothelial cell line and mouse lung.
Those targeted endothelial cells expressed the product encoded by
that foreign DNA.
[0168] It is also envisioned that this embodiment of the present
invention can be practiced using alternative viral or phage
vectors, including retroviral vectors and vaccinia viruses whose
genome has been manipulated in alternative ways so as to render the
virus non-pathogenic. Methods for creating such a viral mutation
are set forth in detail in U.S. Pat. No. 4,769,331, incorporated
herein by reference.
[0169] By way of specific example, a human CPSI-encoding
polynucleotide or a CPSI-encoding polynucleotide homolog from
another warm-blooded vertebrate or a CPSI-encoding homolog from an
invertebrate source, such as bacteria or yeast is introduced into
isolated hepatic cells or other relevant cells. The re-injection of
the transgene-carrying cells into the liver or other relevant
tissues provides a treatment for susceptibility to hyperammonemia
or other relevant diseases in human and animals.
E. Supplementation Therapy
[0170] In addition to its role in nitrogen clearance, the urea
cycle is the body's intrinsic source of arginine which acts as a
precursor of nitric oxide (NO), a potent vasodilator. Methods of
treating suboptimal urea cycle function are provided in accordance
with the present invention, including treatment by administration
of nitric oxide precursors such as citrulline. Typically, the
suboptimal urea cycle function is associated with the polymorphism
disclosed herein. The sub-optimal urea cycle function can further
comprise hyperammonemia or decreased citrulline and/or arginine
production.
[0171] The subject to be treated can be suffering from a disorder
associated with sub-optimal urea cycle function, such as but not
limited to a disorder associated with impaired production of nitric
oxide precursors. Such disorders include but are not limited to
disorders that involve impaired or damaged liver and/or gut tissue.
Representative disorders include but are not limited to hepatitis
(including hepatitis A, B and C), sclerosis, asthma, pulmonary
hypertension (including primary and secondary), bone marrow
transplant toxicity in a subject undergoing bone marrow transplant,
and combinations thereof.
[0172] The subject to be treated can also exposed or about to be
exposed to an environmental stimulus associated with sub-optimal
urea cycle function. Such environmental stimuli include but are not
limited to stimuli that involve impairment or damage to liver
and/or gut tissue. Representative environmental stimuli include but
are not limited to chemotherapy or other pharmaceutical therapy,
cardiac surgery (represented in some situations as increased
postoperative pulmonary vascular tone), increased oxidative stress,
bone marrow transplant, sepsis, acute asthma attack, hypoxia,
hepatotoxin exposure, and combinations thereof. Representative
cardiac surgeries include repair of congential heart defects, and
further includes cardiopulmonary bypass used for correction of
congenital heart defects. Cardiac defects associated with excess
pulmonary blood flow, such as an atrioventricular septal defect
(AVSD) or large unrestrictive ventricular septal defect (VSD) are
representative cardiac defects. Sustained pulmonary overcirculation
can cause hypertrophy and hyperreactivity of pulmonary vascular
smooth muscle. Preoperatively, these patients often have congestive
heart failure and poor weight gain. Surgical repair is scheduled as
early as possible in order to reduce this postoperative
complication.
[0173] Additional cardic defect correct procedures are
bidirectional Glenn and modified Fontan procedures. In such
procedures patients with single ventricle lesions require surgical
procedures where success depends on maintenance of low
postoperative pulmonary vascular tone. Staged correction of a
single ventricle lesion requires a series of 3 surgical procedures
aimed at separating the pulmonary and systemic circulations. The
first of these procedures, often performed in the neonatal period,
is a Blalock-Taussig shunt for those patients with a hypoplastic
right ventricle or a Norwood I procedure for those patients with
hypoplastic left heart syndrome. The second surgery is a
bidirectional Glenn shunt where superior vena cava flow is diverted
directly into the pulmonary artery. The third and final stage is a
modified Fontan procedure where inferior vena cava flow is diverted
into the pulmonary artery, thereby completing separation of the
pulmonary and systemic circulations. With the Glenn and Fontan
procedures, pulmonary blood flow is entirely passive and relies on
an adequate pressure gradient between the venous system (SVC and
IVC pressure) and the PA pressure. Any elevation in the pulmonary
vascular tone in the immediate postoperative period can lead to
decreased pulmonary blood flow and a subsequent fall in cardiac
output. On a longer term, elevated pulmonary vascular tone after
these procedures can lead to persistent pleural effusions,
prolonged requirement for pleural or mediastinal drainage tubes,
prolonged ventilation, and prolonged ICU stays.
[0174] Additional cardic defect correct procedures are Norwood I
procedures. Postoperative care of infants with hypoplastic left
heart syndrome (HLHS) undergoing a Norwood I procedure relies
heavily on balancing pulmonary and systemic flow. Abrupt elevations
in pulmonary vascular resistance can cause significant hypoxemia
and desaturation. Rarely, low pulmonary vascular resistance can be
detrimental if blood flow is shunted to the lungs at the expense of
systemic and coronary circulation. With refined surgical techniques
and optimal sizing of the central shunt, this complication is much
less common than problems with inadequate pulmonary blood flow.
[0175] Additional cardic defect correct procedures are arterial
Switch Procedures. Transposition of the great arteries (TGA) is a
complex cardiac lesion that requires surgical correction in the
immediate neonatal period. Timing of the arterial switch procedure
for correction of TGA specifically takes into account pulmonary
vascular tone issues. Frequently, surgery is not performed until
5-7 days of age when perinatal pulmonary vascular tone has
partially decreased. Because the right ventricle is the systemic
ventricle before surgical correction, postoperative elevations in
pulmonary vascular resistance are usually well tolerated and
pulmonary artery pressure is usually not measured. However, if
postoperative pulmonary vascular tone is increased; it may
partially explain why some infants with favorable anatomy and short
bypass times still have a complicated postoperative course.
[0176] A method of treating or preventing a disorder related to
sub-optimal urea cycle function in a subject is provided in
accordance with the present invention. The method comprises
administering to the subject a therapeutically effective amount of
a nitric oxide precursor, whereby treatment or prevention of the
disorder is accomplished. The nitric oxide precursor can include
but is not limited to citrulline, arginine and combinations
thereof. In some embodiments, sub-optimal nitric oxide formation
resulting from sub-optimal urea cycle function can be treated.
[0177] A method of treating or preventing a disorder selected from
the group consisting hepatitis, cirrhosis, pulmonary hypertension
(both primary and secondary), necrotizing enterocolitis (NEC),
Acute Respiratory Distress Syndrome, ethnic specific endothelial
dysfunction, erectile dysfunction, asthma, and combinations
thereof, in a subject is also disclosed. In some embodiments the
method comprises administering to a subject in need thereof a
therapeutically effective amount of a nitric oxide precursor. The
administering can be intravenous or oral administration. The nitric
oxide precursor can be selected from the group consisting of
citrulline, arginine and combinations thereof. In some embodiments
the disorder is necrotizing enterocolitis (NEC) and the subject is
a premature infant.
[0178] A method of raising a level of a nitric acid precursor in a
subject in need thereof is also disclosed. In some embodiments the
method comprises administering to the subject a therapeutically
effective amount of a nitric oxide precursor, whereby a level of a
nitric oxide precursor in the subject is raised. The administering
can be intravenous or oral administration. The nitric oxide
precursor can be selected from the group consisting of citrulline,
arginine and combinations thereof.
[0179] Optionally, a supplementation therapy method of the present
invention further comprises the step of initially detecting a
polymorphism of a carbamyl phosphate synthase I (CPSI) gene in the
subject. The polymorphism of the carbamyl phosphate synthetase
polypeptide preferably comprises a C to A transversion within CPSI
exon 36, more preferably comprises a C to A transversion at
nucleotide 4340 of a cDNA that corresponds to the CPSI gene, and
ever more preferably, the C to A transversion at nucleotide 4340 of
the cDNA that corresponds to the CPSI gene further comprises a
change in the triplet code from AAC to ACC, which encodes a CPSI
polypeptide having an threonine moiety at amino acid 1405.
[0180] A significant decrease in urea cycle intermediates
(citrulline, arginine) was observed in subjects undergoing BMT
associated with the T1405N CPSI polymorphism disclosed herein. In
accordance with the present invention, a method for the treatment
or prophylaxis of BMT toxicity, such as HVOD and/or acute lung
injury, comprising administering a therapeutically effective amount
of a NO precursor, such as citrulline and/or arginine, to a subject
in need thereof is also provided in accordance with the present
invention. Preferably, the T1405N CPSI polymorphism disclosed
herein is present in the subject. More preferably, a
therapeutically effective amount of citrulline is administered to
the subject.
[0181] In accordance with the present invention, a method of
reducing toxicity and/or the occurrence of HVOD in a subject
undergoing BMT is thus provided. This method comprises
administering the BMT subject an effective amount of arginine
and/or citrulline, with citrulline being preferred, to bolster
arginine and NO synthesis in the subject. The bolstering of
arginine and NO synthesis in the subject will reduce and/or
substantially prevent the occurrence of HVOD associated with BMT.
Citrulline is a preferred supplementation agent given that it is
more readily converted to NO. Additionally and preferably, subjects
having the CPSI polymorphism of the present invention are
contemplated to be preferred candidates for supplementation in
accordance with this method.
[0182] The subject treated in the present invention in its many
embodiments is desirably a human subject, although it is to be
understood that the principles of the invention indicate that the
invention is effective with respect to all vertebrate species,
including warm-blooded vertebrates such as mammals and birds, which
are intended to be included in the term "subject". In this context,
a mammal is understood to include any mammalian species in which
treatment of hyperammonemia, BMT toxicity and other diseases
associated with impaired urea cycle tunction is desirable,
particularly agricultural and domestic mammalian species.
[0183] Thus, contemplated is the treatment of mammals such as
humans, as well as those mammals of importance due to being
endangered (such as Siberian tigers), of economical importance
(animals raised on farms for consumption by humans) and/or social
importance (animals kept as pets or in zoos) to humans, for
instance, carnivores other than humans (such as cats and dogs),
swine (pigs, hogs, and wild boars), ruminants (such as cattle,
oxen, sheep, giraffes, deer, goats, bison, and camels), and horses.
Also contemplated is the treatment of birds, including the
treatment of those kinds of birds that are endangered, kept in
zoos, as well as fowl, and more particularly domesticated fowl,
i.e., poultry, such as turkeys, chickens, ducks, geese, guinea
fowl, and the like, as they are also of economical importance to
humans. Thus, contemplated is the treatment of livestock,
including, but not limited to, domesticated swine (pigs and hogs),
ruminants, horses, poultry, and the like.
[0184] The amount of active ingredient that may be combined with
the carrier materials to produce a single dosage form will vary
depending upon the host treated and the particular mode of
administration. For example, a formulation intended for
administration to humans may contain from 0.5 mg to 5 g of active
agent compounded with an appropriate and convenient amount of
carrier material which may vary from about 5 to about 95 percent of
the total composition. For example, in a human adult, the doses per
person per administration are generally between 1 mg and 500 mg up
to several times per day. Thus, dosage unit forms will generally
contain between from about 1 mg to about 500 mg of an active
ingredient, typically 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg,
500 mg, 600 mg, 800 mg, or 1000 mg.
[0185] The nitric oxide precursor is administered in a dose ranging
from about 0.01 mg to about 1,000 mg, preferably in a dose ranging
from about 0.5 mg to about 500 mg, and more preferably in a dose
ranging from about 1.0 mg to about 250 mg. The nitric oxide
precursor can also be administered in a dose ranging from about 100
mg to about 30,000 mg, in some embodiments in a dose ranging from
about 250 mg to about 1,000 mg. A representative dose is 3.8
g/m2/day of arginine or citrulline (molar equivalents, MW
L-citrulline 175.2, MW L-arginine 174.2).
[0186] Representative intravenous citrulline solutions can comprise
a 100 mg/ml (10%) solution. Representative intravenous citrulline
dosages can comprise 200 mg/kg, 400 mg/kg, 600 mg/kg, and 800
mg/kg. In some embodiments, for example but not limited to a 600 or
800 mg/kg dosage, the dose can be decreased by an amount ranging
from 50 mg/kg and 100 mg/kg to mitigate observed undesired effects
on systemic blood pressure.
[0187] In a representative embodiment doses can be administered to
a subject, prior to exposure to an environmental stimulus (e.g. one
dose 30 minutes before initiation of a cardiac surgery such as
cardiopulmonary bypass and/or up to 1, 2, 3, 4, 5, 6 or more
dosages over a perioperative period, such as every 12 hours over a
period of time prior to surgery) after exposure to an environmental
stimulus (e.g. upon arrival to a postoperative care setting, and/or
up to 1, 2, 3, 4, 5, 6 or more dosages over a postoperative period,
such as every 12 hours over a period of time after surgery).
[0188] It will be understood, however, that the specific dose level
for any particular subject will depend upon a variety of factors
including the age, body weight, general health, sex, diet, time of
administration, route of administration, rate of excretion, drug
combination and the severity of the particular disease undergoing
therapy.
F. Pharmaceutical Compositions
[0189] In a preferred embodiment, the present invention provides
pharmaceutical compositions comprising a polypeptide or
polynucleotide of the present invention and a physiologically
acceptable carrier. More preferably, a pharmaceutical composition
comprises a polynucleotide that encodes a biologically active CPSI
polypeptide. Alternatively, provided pharmaceutical compositions
comprise citrulline or arginine in dosages as described above.
[0190] A composition of the present invention is typically
administered orally or parenterally in dosage unit formulations
containing standard, well-known nontoxic physiologically acceptable
carriers, adjuvants, and vehicles as desired. The term "parenteral"
as used herein includes intravenous, intra-muscular, intra-arterial
injection, or infusion techniques.
[0191] Injectable preparations, for example sterile injectable
aqueous or oleaginous suspensions, are formulated according to the
known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation can also be a
sterile injectable solution or suspension in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol.
[0192] Among the acceptable vehicles and solvents that may be
employed are water, Ringer's solution, and isotonic sodium chloride
solution. In addition, sterile, fixed oils are conventionally
employed as a solvent or suspending medium. For this purpose any
bland fixed oil can be employed including synthetic mono- or
di-glycerides. In addition, fatty acids such as oleic acid find use
in the preparation of injectables.
[0193] Preferred carriers include neutral saline solutions buffered
with phosphate, lactate, Tris, and the like. Of course, in the case
of a pharmaceutical composition provided for use in gene therapy,
one purifies the vector sufficiently to render it essentially free
of undesirable contaminants, such as defective interfering
adenovirus particles or endotoxins and other pyrogens such that it
does not cause any untoward reactions in the individual receiving
the vector construct. A preferred means of purifying the vector
involves the use of buoyant density gradients, such as cesium
chloride gradient centrifugation.
[0194] A transfected cell can also serve as a carrier. By way of
example, a liver cell can be removed from an organism, transfected
with a polynucleotide of the present invention using methods set
forth above and then the transfected cell returned to the organism
(e.g. injected intra-vascularly).
G. Generation of Antibodies
[0195] In still another embodiment, the present invention provides
an antibody immunoreactive with a polypeptide or polynucleotide of
the present invention. Preferably, an antibody of the invention is
a monoclonal antibody. Means for preparing and characterizing
antibodies are well known in the art (See, e.g., Antibodies A
Laboratory Manual, E. Howell and D. Lane, Cold Spring Harbor
Laboratory, 1988). More preferred antibodies distinguish between
the different forms of CPSI which comprise the CPSI
polymorphism.
[0196] Briefly, a polyclonal antibody is prepared by immunizing an
animal with an immunogen comprising a polypeptide or polynucleotide
of the present invention, and collecting antisera from that
immunized animal. A wide range of animal species can be used for
the production of antisera. Typically an animal used for production
of anti-antisera is a rabbit, a mouse, a rat, a hamster or a guinea
pig. Because of the relatively large blood volume of rabbits, a
rabbit is a preferred choice for production of polyclonal
antibodies.
[0197] As is well known in the art, a given polypeptide or
polynucleotide may vary in its immunogenicity. It is often
necessary therefore to couple the immunogen (e.g., a polypeptide or
polynucleotide) of the present invention) with a carrier. Exemplary
and preferred carriers are keyhole limpet hemocyanin (KLH) and
bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse
serum albumin or rabbit serum albumin can also be used as
carriers.
[0198] Means for conjugating a polypeptide or a polynucleotide to a
carrier protein are well known in the art and include
glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester,
carbodiimide and bis-biazotized benzidine.
[0199] As is also well known in the art, immunogencity to a
particular immunogen can be enhanced by the use of non-specific
stimulators of the immune response known as adjuvants. Exemplary
and preferred adjuvants include complete Freund's adjuvant,
incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
[0200] The amount of immunogen used of the production of polyclonal
antibodies varies, inter alia, upon the nature of the immunogen as
well as the animal used for immunization. A variety of routes can
be used to administer the immunogen, e.g. subcutaneous,
intramuscular, intradermal, intravenous and intraperitoneal. The
production of polyclonal antibodies is monitored by sampling blood
of the immunized animal at various points following immunization.
When a desired level of immunogenicity is obtained, the immunized
animal can be bled and the serum isolated and stored.
[0201] In another aspect, the present invention provides a method
of producing an antibody immunoreactive with a CPSI polypeptide,
the method comprising the steps of (a) transfecting recombinant
host cells with a polynucleotide that encodes that polypeptide; (b)
culturing the host cells under conditions sufficient for expression
of the polypeptide; (c) recovering the polypeptide; and (d)
preparing antibodies to the polypeptide. Preferably, the CPSI
polypeptide is capable of mediating the first step of the urea
cycle, cross-reacting with anti-CPSI antibody, or other biological
activity in accordance with the present invention. Even more
preferably, the present invention provides antibodies prepared
according to the method described above.
[0202] A monoclonal antibody of the present invention can be
readily prepared through use of well-known techniques such as those
exemplified in U.S. Pat. No. 4,196,265, herein incorporated by
reference. Typically, a technique involves first immunizing a
suitable animal with a selected antigen (e.g., a polypeptide or
polynucleotide of the present invention) in a manner sufficient to
provide an immune response. Rodents such as mice and rats are
preferred animals. Spleen cells from the immunized animal are then
fused with cells of an immortal myeloma cell. Where the immunized
animal is a mouse, a preferred myeloma cell is a murine NS-1
myeloma cell.
[0203] The fused spleen/myeloma cells are cultured in a selective
medium to select fused spleen/myeloma cells from the parental
cells. Fused cells are separated from the mixture of non-fused
parental cells, for example, by the addition of agents that block
the de novo synthesis of nucleotides in the tissue culture media.
Exemplary and preferred agents are aminopterin, methotrexate, and
azaserine. Aminopterin and methotrexate block de novo synthesis of
both purines and pyrimidines, whereas azaserine blocks only purine
synthesis. Where aminopterin or methotrexate is used, the media is
supplemented with hypoxanthine and thymidine as a source of
nucleotides. Where azaserine is used, the media is supplemented
with hypoxanthine.
[0204] This culturing provides a population of hybridomas from
which specific hybridomas are selected. Typically, selection of
hybridomas is performed by culturing the cells by single-clone
dilution in microtiter plates, followed by testing the individual
clonal supernatants for reactivity with an antigen-polypeptides.
The selected clones can then be propagated indefinitely to provide
the monoclonal antibody.
[0205] By way of specific example, to produce an antibody of the
present invention, mice are injected intraperitoneally with between
about 1-200 .mu.g of an antigen comprising a polypeptide of the
present invention. B lymphocyte cells are stimulated to grow by
injecting the antigen in association with an adjuvant such as
complete Freund's adjuvant (a non-specific stimulator of the immune
response containing killed Mycobacterium tuberculosis). At some
time (e.g., at least two weeks) after the first injection, mice are
boosted by injection with a second dose of the antigen mixed with
incomplete Freund's adjuvant.
[0206] A few weeks after the second injection, mice are tail bled
and the sera titered by immunoprecipitation against radiolabeled
antigen. Preferably, the process of boosting and titering is
repeated until a suitable titer is achieved. The spleen of the
mouse with the highest titer is removed and the spleen lymphocytes
are obtained by homogenizing the spleen with a syringe. Typically,
a spleen from an immunized mouse contains approximately
5.times.10.sup.7 to 2.times.10.sup.8 lymphocytes.
[0207] Mutant lymphocyte cells known as myeloma cells are obtained
from laboratory animals in which such cells have been induced to
grow by a variety of well-known methods. Myeloma cells lack the
salvage pathway of nucleotide biosynthesis. Because myeloma cells
are tumor cells, they can be propagated indefinitely in tissue
culture, and are thus denominated immortal. Numerous cultured cell
lines of myeloma cells from mice and rats, such as murine NS-1
myeloma cells, have been established.
[0208] Myeloma cells are combined under conditions appropriate to
foster fusion with the normal antibody-producing cells from the
spleen of the mouse or rat injected with the antigen/polypeptide of
the present invention. Fusion conditions include, for example, the
presence of polyethylene glycol. The resulting fused cells are
hybridoma cells. Like myeloma cells, hybridoma cells grow
indefinitely in culture.
[0209] Hybridoma cells are separated from unfused myeloma cells by
culturing in a selection medium such as HAT media (hypoxanthine,
aminopterin, thymidine). Unfused myeloma cells lack the enzymes
necessary to synthesize nucleotides from the salvage pathway
because they are killed in the presence of aminopterin,
methotrexate, or azaserine. Unfused lymphocytes also do not
continue to grow in tissue culture. Thus, only cells that have
successfully fused (hybridoma cells) can grow in the selection
media.
[0210] Each of the surviving hybridoma cells produces a single
antibody. These cells are then screened for the production of the
specific antibody immunoreactive with an antigen/polypeptide of the
present invention. Single cell hybridomas are isolated by limiting
dilutions of the hybridomas. The hybridomas are serially diluted
many times and, after the dilutions are allowed to grow, the
supernatant is tested for the presence of the monoclonal antibody.
The clones producing that antibody are then cultured in large
amounts to produce an antibody of the present invention in
convenient quantity.
[0211] By use of a monoclonal antibody of the present invention,
specific polypeptides and polynucleotide of the invention can be
recognized as antigens, and thus identified. Once identified, those
polypeptides and polynucleotide can be isolated and purified by
techniques such as antibody-affinity chromatography. In
antibody-affinity chromatography, a monoclonal antibody is bound to
a solid substrate and exposed to a solution containing the desired
antigen. The antigen is removed from the solution through an
immunospecific reaction with the bound antibody. The polypeptide or
polynucleotide is then easily removed from the substrate and
purified.
H. Detecting a Polynucleotide or a Polypeptide of the Present
Invention
[0212] Alternatively, the present invention provides a method of
detecting a polypeptide of the present invention, wherein the
method comprises immunoreacting the polypeptides with antibodies
prepared according to the methods described above to form
antibody-polypeptide conjugates, and detecting the conjugates.
[0213] In yet another embodiment, the present invention provides a
method of detecting messenger RNA transcripts that encode a
polypeptide of the present invention, wherein the method comprises
hybridizing the messenger RNA transcripts with polynucleotide
sequences that encode the polypeptide to form duplexes; and
detecting the duplex. Alternatively, the present invention provides
a method of detecting DNA molecules that encode a polypeptide of
the present invention, wherein the method comprises hybridizing DNA
molecules with a polynucleotide that encodes that polypeptide to
form duplexes; and detecting the duplexes.
[0214] The detection and screening assays disclosed herein can be
used as a prognosis tool. Human CPSI-encoding polynucleotides as
well as their protein products can be readily used in clinical
setting as a prognostic indicator for screening for susceptibility
to hyperammonemia and to other heritable CPSI-related diseases in
humans.
[0215] The detection and screening assays disclosed herein can be
also used as a part of a diagnostic method. Human CPSI-encoding
polynucleotides as well as their protein products can be readily
used in clinical setting to diagnose susceptibility to
hyperammonemia and to other heritable CPSI-related diseases in
humans.
[0216] H.1. Screening Assays for a Polypeptide of the Present
Invention
[0217] The present invention provides a method of screening a
biological sample for the presence of a CPSI polypeptide.
Preferably, the CPSI polypeptide possesses activity in the urea
cycle, cross-reactivity with an anti-CPSI antibody, or other
biological activity in accordance with the present invention. A
biological sample to be screened can be a biological fluid such as
extracellular or intracellular fluid or a cell or tissue extract or
homogenate. A biological sample can also be an isolated cell (e.g.,
in culture) or a collection of cells such as in a tissue sample or
histology sample. A tissue sample can be suspended in a liquid
medium or fixed onto a solid support such as a microscope slide.
Hepatic tissues comprise particularly contemplated tissues.
[0218] Preferably, antibodies which distinguish between the N1405
CPSI polypeptide and the T1405 CPSI polypeptide are provided. Such
antibodies may compare polyclonal antibodies but are preferably
monoclonal antibodies prepared as described hereinabove.
[0219] In accordance with a screening assay method, a biological
sample is exposed to an antibody immunoreactive with the
polypeptide whose presence is being assayed. Typically, exposure is
accomplished by forming an admixture in a liquid medium that
contains both the antibody and the candidate polypeptide. Either
the antibody or the sample with the polypeptide can be affixed to a
solid support (e.g., a column or a microtiter plate).
[0220] The biological sample is exposed to the antibody under
biological reaction conditions and for a period of time sufficient
for antibody-polypeptide conjugate formation. Biological reaction
conditions include ionic composition and concentration,
temperature, pH and the like.
[0221] Ionic composition and concentration can range from that of
distilled water to a 2 molal solution of NaCl. Preferably,
osmolality is from about 100 mosmols/l to about 400 mosmols/l and,
more preferably from about 200 mosmols/l to about 300 mosmols/l.
Temperature preferably is from about 4.degree. C. to about
100.degree. C., more preferably from about 15.degree. C. to about
50.degree. C. and, even more preferably from about 25.degree. C. to
about 40.degree. C. pH is preferably from about a value of 4.0 to a
value of about 9.0, more preferably from about a value of 6.5 to a
value of about 8.5 and, even more preferably from about a value of
7.0 to a value of about 7.5. The only limit on biological reaction
conditions is that the conditions selected allow for
antibody-polypeptide conjugate formation and that the conditions do
not adversely affect either the antibody or the polypeptide.
[0222] Exposure time will vary inter alia with the biological
conditions used, the concentration of antibody and polypeptide and
the nature of the sample (e.g., fluid or tissue sample). Means for
determining exposure time are well known to one of ordinary skill
in the art. Typically, where the sample is fluid and the
concentration of polypeptide in that sample is about 10.sup.10M,
exposure time is from about 10 minutes to about 200 minutes.
[0223] The presence of polypeptide in the sample is detected by
detecting the formation and presence of antibody-polypeptide
conjugates. Means for detecting such antibody-antigen (e.g.,
receptor polypeptide) conjugates or complexes are well known in the
art and include such procedures as centrifugation, affinity
chromatography and the like, binding of a secondary antibody to the
antibody-candidate receptor complex.
[0224] In one embodiment, detection is accomplished by detecting an
indicator affixed to the antibody. Exemplary and well known such
indicators include radioactive labels (e.g., .sup.32P, .sup.125I,
.sup.14C), a second antibody or an enzyme such as horse radish
peroxidase. Means for affixing indicators to antibodies are well
known in the art. Commercial kits are available.
[0225] H.2. Screening Assay for Anti-Polypeptide Antibody
[0226] In another aspect, the present invention provides a method
of screening a biological sample for the presence of antibodies
immunoreactive with a CPSI polypeptide. Preferably the CPSI
polypeptide has activity in the urea cycle, cross-reactivity with
an anti-CPSI antibody, or other biological activity in accordance
with the present invention. In accordance with such a method, a
biological sample is exposed to a CPSI polypeptide under biological
conditions and for a period of time sufficient for
antibody-polypeptide conjugate formation and the formed conjugates
are detected.
[0227] H.3. Screening Assay for Polynucleotide That Encodes a CPSI
Polypeptide of the Present Invention
[0228] A nucleic acid molecule and, particularly a probe molecule,
can be used for hybridizing as an oligonucleotide probe to a
nucleic acid source suspected of encoding a CPSI polypeptide of the
present invention. Optimally, the CPSI polypeptide has activity in
the urea cycle, cross-reactivity with an anti-CPSI antibody, or
other biological activity in accordance with the present invention.
The probing is usually accomplished by hybridizing the
oligonucleotide to a DNA source suspected of possessing a CPSI
gene. In some cases, the probes constitute only a single probe, and
in others, the probes constitute a collection of probes based on a
certain amino acid sequence or sequences of the polypeptide and
account in their diversity for the redundancy inherent in the
genetic code.
[0229] A suitable source of DNA for probing in this manner is
capable of expressing a polypeptide of the present invention and
can be a genomic library of a cell line of interest. Alternatively,
a source of DNA can include total DNA from the cell line of
interest. Once the hybridization method of the invention has
identified a candidate DNA segment, one confirms that a positive
clone has been obtained by further hybridization, restriction
enzyme mapping, sequencing and/or expression and testing.
[0230] Alternatively, such DNA molecules can be used in a number of
techniques including their use as: (1) diagnostic tools to detect
normal and abnormal DNA sequences in DNA derived from subject's
cells, such as a CPSI polymorphism described herein; (2) means for
detecting and isolating other members of the polypeptide family and
related polypeptides from a DNA library potentially containing such
sequences; (3) primers for hybridizing to related sequences for the
purpose of amplifying those sequences; (4) primers for altering
native CPSI DNA sequences; as well as other techniques which rely
on the similarity of the DNA sequences to those of the DNA segments
herein disclosed.
[0231] As set forth above, in certain aspects, DNA sequence
information provided by the invention allows for the preparation of
relatively short DNA (or RNA) sequences (e.g., probes) that
specifically hybridize to encoding sequences of a selected CPSI
gene. In these aspects, nucleic acid probes of an appropriate
length are prepared based on a consideration of the encoding
sequence for a polypeptide of this invention. The ability of such
nucleic acid probes to specifically hybridize to other encoding
sequences lend them particular utility in a variety of embodiments.
Most importantly, the probes can be used in a variety of assays for
detecting the presence of complementary sequences in a given
sample. However, other uses are envisioned, including the use of
the sequence information for the preparation of mutant species
primers, or primers for use in preparing other genetic
constructions.
[0232] To provide certain of the advantages in accordance with the
invention, a preferred nucleic acid sequence employed for
hybridization studies or assays includes probe sequences that are
complementary to at least a 14 to 40 or so long nucleotide stretch
of a nucleic acid sequence of the present invention, such as a
sequence shown in any of SEQ ID NOs:1, 3, 11 and 13. A size of at
least 14 nucleotides in length helps to ensure that the fragment is
of sufficient length to form a duplex molecule that is both stable
and selective. Molecules having complementary sequences over
stretches greater than 14 bases in length are generally preferred,
though, to increase stability and selectivity of the hybrid, and
thereby improve the quality and degree of specific hybrid molecules
obtained. One will generally prefer to design nucleic acid
molecules having gene-complementary stretches of 14 to 20
nucleotides, or even longer where desired. Such fragments can be
readily prepared by, for example, directly synthesizing the
fragment by chemical means, by application of nucleic acid
reproduction technology, such as the PCR technology of U.S. Pat.
No. 4,683,202, herein incorporated by reference, or by introducing
selected sequences into recombinant vectors for recombinant
production.
[0233] Accordingly, a nucleotide sequence of the present invention
can be used for its ability to selectively form duplex molecules
with complementary stretches of the gene. Depending on the
application envisioned, one employs varying conditions of
hybridization to achieve varying degrees of selectivity of the
probe toward the target sequence. For applications requiring a high
degree of selectivity, one typically employs relatively stringent
conditions to form the hybrids. For example, one selects relatively
low salt and/or high temperature conditions, such as provided by
0.02M-0.15M salt at temperatures of about 50.degree. C. to about
70.degree. C. including particularly temperatures of about
55.degree. C., about 60.degree. C. and about 65.degree. C. Such
conditions are particularly selective, and tolerate little, if any,
mismatch between the probe and the template or target strand.
[0234] Of course, for some applications, for example, where one
desires to prepare mutants employing a mutant primer strand
hybridized to an underlying template or where one seeks to isolate
polypeptide coding sequences from related species, functional
equivalents, or the like, less stringent hybridization conditions
are typically needed to allow formation of the heteroduplex. Under
such circumstances, one employs conditions such as 0.15M-0.9M salt,
at temperatures ranging from about 20.degree. C. to about
55.degree. C., including particularly temperatures of about
25.degree. C., about 37.degree. C., about 45.degree. C., and about
50.degree. C. Cross-hybridizing species can thereby be readily
identified as positively hybridizing signals with respect to
control hybridizations. In any case, it is generally appreciated
that conditions can be rendered more stringent by the addition of
increasing amounts of formamide, which serves to destabilize the
hybrid duplex in the same manner as increased temperature. Thus,
hybridization conditions can be readily manipulated, and thus will
generally be a method of choice depending on the desired
results.
[0235] In certain embodiments, it is advantageous to employ a
nucleic acid sequence of the present invention in combination with
an appropriate means, such as a label, for determining
hybridization. A wide variety of appropriate indicator means are
known in the art, including radioactive, enzymatic or other
ligands, such as avidin/biotin, which are capable of giving a
detectable signal. In preferred embodiments, one likely employs an
enzyme tag such a urease, alkaline phosphatase or peroxidase,
instead of radioactive or other environmentally undesirable
reagents. In the case of enzyme tags, calorimetric indicator
substrates are known which can be employed to provide a means
visible to the human eye or spectrophotometrically, to identify
specific hybridization with complementary nucleic acid-containing
samples.
[0236] In general, it is envisioned that the hybridization probes
described herein are useful both as reagents in solution
hybridization as well as in embodiments employing a solid phase. In
embodiments involving a solid phase, the sample containing test DNA
(or RNA) is adsorbed or otherwise affixed to a selected matrix or
surface. This fixed, single-stranded nucleic acid is then subjected
to specific hybridization with selected probes under desired
conditions. The selected conditions depend inter alia on the
particular circumstances based on the particular criteria required
(depending, for example, on the G+C contents, type of target
nucleic acid, source of nucleic acid, size of hybridization probe,
etc.). Following washing of the hybridized surface so as to remove
nonspecifically bound probe molecules, specific hybridization is
detected, or even quantified, by means of the label.
[0237] H.4. Assay Kits
[0238] In another aspect, the present invention provides a
diagnostic assay kit for detecting the presence of a polypeptide of
the present invention in biological samples, where the kit
comprises a first container containing a first antibody capable of
immunoreacting with the polypeptide, with the first antibody
present in an amount sufficient to perform at least one assay.
Preferably, the assay kits of the invention further comprise a
second container containing a second antibody that immunoreacts
with the first antibody. More preferably, the antibodies used in
the assay kits of the present invention are monoclonal antibodies.
Even more preferably, the first antibody is affixed to a solid
support. More preferably still, the first and second antibodies
comprise an indicator, and, preferably, the indicator is a
radioactive label or an enzyme.
[0239] The present invention also provides a diagnostic kit for
screening agents. Such a kit can contain a polypeptide of the
present invention. The kit can contain reagents for detecting an
interaction between an agent and a receptor of the present
invention. The provided reagent can be radiolabeled. The kit can
contain a known radiolabelled agent capable of binding or
interacting with a receptor of the present invention.
[0240] In an alternative aspect, the present invention provides
diagnostic assay kits for detecting the presence, in biological
samples, of a polynucleotide that encodes a polypeptide of the
present invention, the kits comprising a first container that
contains a second polynucleotide identical or complementary to a
segment of at least 10 contiguous nucleotide bases of, as a
preferred example, in any of SEQ ID NOs:1, 3, 11 and 13.
[0241] In another embodiment, the present invention provides
diagnostic assay kits for detecting the presence, in a biological
sample, of antibodies immunoreactive with a polypeptide of the
present invention, the kits comprising a first container containing
a CPSI polypeptide, that immunoreacts with the antibodies, with the
polypeptide present in an amount sufficient to perform at least one
assay. Preferably, the CPSI polypeptide has activity in the urea
cycle, cross-reactivity on an anti-CPSI antibody, or other
biological activity in accordance with the present invention. The
reagents of the kit can be provided as a liquid solution, attached
to a solid support or as a dried powder. Preferably, when the
reagent is provided in a liquid solution, the liquid solution is an
aqueous solution. Preferably, when the reagent provided is attached
to a solid support, the solid support can be chromatograph media or
a microscope slide. When the reagent provided is a dry powder, the
powder can be reconstituted by the addition of a suitable solvent.
The solvent can be provided.
EXAMPLES
[0242] The following Examples have been included to illustrate
preferred modes of the invention. Certain aspects of the following
Examples are described in terms of techniques or procedures found
or contemplated by the present inventors to work well in the
practice of the invention. These Examples are exemplified through
the use of standard laboratory practices of the inventors. In light
of the present disclosure and the general level of skill in the
art, those of skill will appreciate that the following Examples are
intended to be exemplary only in that numerous changes,
modification, and alterations can be employed without departing
from the spirit and scope of the invention.
Materials and Methods Used in Examples 1-3
[0243] The following materials and methods are employed in each of
Examples 1-3. Additional materials and methods are also described
in each Example.
[0244] Clinical/Patient Recruitment:
[0245] More than 200 patients undergoing BMT at Vanderbilt
University Medical Center, Nashville, Tenn., have been enrolled in
the BMT-Lung Injury Following Engraftment (LIFE) Study aimed at
understanding mechanisms of acute lung injury and multiple organ
failure after transplant. Consent was sought from consecutive
patients undergoing BMT or PBSCT for treatment of malignancy.
Definitions of organ failure (including HVOD) and reversal were
prospectively defined and data was collected concurrently during
hospitalization. Plasma, cell pellets, and urine were collected at
study enrollment (before receiving chemotherapy) and on the day of
transplantation (before marrow infusion) after completing ablative
chemo-radiotherapy.
[0246] Amino Acid Analysis--
[0247] Blood and urine were immediately centrifuged after
collection. All samples were kept on ice, then stored at
-70.degree. C. until analyzed. Under these storage conditions,
glutamine, cysteine and homocysteine are known to decrease, so
these were not used in the analysis. Plasma amino acids were
measured in the Vanderbilt Diagnostic Laboratories, Vanderbilt
University, Nashville, Tenn. Briefly, a protein free extract of
plasma was prepared by protein precipitation with sulfosalicylic
acid and filtration through a 0.45 .mu.m ACRODISC.TM. 4 filter
(Gelman Sciences, Ann Arbor, Mich.). Amino acids were separated by
cation exchange chromatography using a four-component pH- and ionic
strength-graded lithium citrate buffer system on a Beckmann 7300
amino acid analyzer (Beckmann, Palo Alto, Calif.). Post column
derivatization of amino acids with ninhydrin allowed detection of
primary amine amino acids at 570 nm, and secondary amines at 440
nm. Quantification was achieved by instrument calibration with
standard reference materials (Sigma, St. Louis, Mo.).
[0248] Statistics.
[0249] Plasma amino acid values were expressed as mean.+-.SEM.
Comparisons between baseline and post-chemotherapy amino acid
values were made using Student's t-Test. Allelic frequency was
compared between patients with and without HVOD using Chi square
analysis.
[0250] Patients.
[0251] Patients were identified from those enrolled in the BMT Lift
Study at Vanderbilt University. DNA was isolated from
pre-transplant blood or spun urine samples. HVOD status was
determined using the Baltimore criteria: [0252] Bilirubin >2.0
mg/dl [0253] Hepatomegaly [0254] 2% sudden weight gain
[0255] Genotvpinq.
[0256] DNA was isolated using a QIAmp.TM. blood kit (Qiagen). The
T1405N polymorphism changes the DNA sequence as follows:
TABLE-US-00003 Normal CCT-GCC-ACC-CCA-GTG Change
CCT-GCC-AAC-CCA-GTG
[0257] The C to A transversion replaces the pyrimidine C with the
purine A which destroys a Ms/1 site. The use of a primer from
within the 35th intron of CPSI and an exotic primer from exon 36 of
the CPSI gene reliably PCR amplifies a 387 bp fragment encompassing
the region containing the change. This combination gives a robust
amplification. PCR Ready-to-Go.TM. beads are also used in
amplification (Pharmacia).
[0258] The polymorphism was detected using a non-denaturing gel to
take advantage of the secondary structures created by the C to A
transversion. This change creates enough secondary structure to
prevent reliable digestion by restriction enzymes (Msl I) to detect
the polymorphism. This change also interferes with direct sequence
analysis unless ITP is substituted for GTP in the reaction.
Non-denaturing gels take advantage of the secondary structures
created by this change. Fifteen (15) individuals were compared by
this method and sequence analysis.
[0259] To detect the DNA fragments in the gel, a silver staining
technique was adapted. This inexpensive rapid method allowed
visualization of bands shortly after electrophoresis.
[0260] Statistical Analysis.
[0261] A sufficient sample size was obtained to perform Chi Square
analysis on the results. The Hardy-Weinburg equation was used to
calculate the expected frequencies for the genotypes
(p.sup.2+2pq+q.sup.2). P values were obtained from a standard Chi
Square table using 2 degrees of freedom.
Example 1
Alleles of CPSI Exonic Polymorphism (T1405N) Are Not in
Hardy-Weinburg Equilibrium with the Presence or Absence of HVOD
[0262] In accordance with the present invention, a common
polymorphism near the 3' end of the CPSI mRNA (about 0.44
heterozygosity) has been identified. Sequence analysis of this
change revealed a C to A transversion at base 4340 changing the
triplet code from ACC to AAC. This results in a substitution of
asparagine for threonine at amino acid 1405 (referred to herein as
"T1405N"). The threonine is within the allosteric domain, preceding
the signature sequence PV(A/S)WP(T/S)(A/Q)E, a sequence that is
important in the binding of the cofactor n-acetyl-glutamate
(NAG).
[0263] In all known CPSIs activated by NAG, a threonine residue is
among the two residues that precede the signature sequence. (Rubio,
Biochemical Society Transactions 21:198-202 (1998)). On the basis
of structure-function studies, hydrogen bond formation with the
carbonyl oxygen of the acetamido group of NAG is felt to play a
role in the binding of this activator. (Stapleton et al.,
Biochemistry 35:14352-14361 (1996); Javid-Majd et al., Biochemistry
35:14362-14369 (1996)). The substitution of the threonine side
chain by asparagine is envisioned to alter the hydrogen bond
formation with NAG and results in a qualitative change in CPSI
enzymatic function and in sensitivity to the available pool of NAG.
Although applicants do not wish to be bound by any particular
theory of operation, it is speculated that based on the precedent
of the effects of other xenobiotics, that limited availability of
NAG after escalated dose chemotherapy is one of the mechanisms
promoting urea cycle dysfunction.
[0264] 126 individuals were genotyped from the BMT Life Study
group. 30 individuals manifested evidence of HVOD in this group
(24%). 70 patients were genotyped from blood samples and 56 from
urine cell pellets. Samples from 15 patients were reamplified via
PCR and sequenced to confirm the consistency of the results.
[0265] Tables 2 and 3 show the results of genotype analysis for the
T1405N polymorphism between HVOD+ and HVOD- patients. The C allele,
also referred to herein as the CPSIa allele or the threonine
encoding allele, has a frequency of 0.62 in the examined population
and the A allele, also referred to herein as the CPSIb allele or
the asparagine encoding allele, has a frequency of 0.38. The Chi
Square value for the table is 4.3 (P=0.1) indicating that the
polymorphism is probably not in Hardy-Weinburg equilibrium with the
presence of HVOD. Thus, these results provide evidence for
disequilibrium in the distribution of the T1405N alleles in BMT
patients with HVOD, indicating that the polymorphism can be used to
identify subjects who are susceptible to BMT toxicity.
TABLE-US-00004 TABLE 2 Genotype HVOD+ HVOD- CC 13 (expected 11.4)
32 (expected 36.5) AC 16 (expected 14.1) 50 (expected 45.1) AA 1
(expected 4.5) 14 (expected 14.4)
TABLE-US-00005 TABLE 3 Total alleles: Expected Frequencies: A: 96
AA: 0.15 C: 62 AC: 0.47 CC: 0.38
[0266] Additional data gathered from a study of approximately 200
patients provided additional statistical evidence supporting the
use of the polymorphism in detection of susceptibility to
sub-optimal urea cycle function. This data was subjected to the
statistical methods described above.
[0267] Bone marrow transplant toxicity results in significant
morbidity and mortality. HVOD is associated with a poor prognosis
in BMT patients. This study was undertaken to assess an association
between the CPSI enzyme and the occurrence of HVOD. The T1405N
polymorphism affects CPSI function. Its wide distribution in the
population suggests that both forms provide adequate urea cycle
function under normal conditions. The addition of metabolic
stressors (such as high-dose chemotherapy) serves to lower CPSI
efficiency below an effective threshold. Analysis of the data thus
suggests that HVOD is more likely to occur in patients with the
threonine encoding allele than those with the asparagine. The
threonine encoding allele is shared by the rodent form of CPSI.
Example 2
Biochemical and Genetic Alterations in Carbamyl Phosphate
Synthetase I in Patients with Post-Bone Marrow Transplant
Complications
[0268] Bone marrow transplantation (BMT) and peripheral blood stem
cell transplants (PBSCT) are increasingly being used as primary
therapy for selected malignancies. Use of stem cell support for
hematopoietic reconstitution allows for substantial escalation in
the dose of chemotherapy in an attempt to eradicate potentially
lethal cancers. With improvements in prophylaxis for infection and
prevention of disabling graft-versus-host disease,
chemotherapy-induced organ dysfunction remains a significant
barrier to more widespread use of this treatment.
[0269] Hepatic venocclusive disease (HVOD), a clinical syndrome of
hyperbilirubinemia (serum bilirubin >2.0 mg/dL), hepatomegaly,
and fluid retention early after BMT, is a major dose-limiting
toxicity after BMT, afflicting up to 54% of patients. Many patients
developing HVOD after BMT will also meet the criteria for acute
lung injury (ALI). Nearly half of patients with severe HVOD require
mechanical ventilation, with an attendant mortality in excess of
90%. Such data underscore the large impact on mortality of
sequential organ dysfunction, even in a young patient population,
and reinforce the clinically important association of poor
prognosis after acute lung injury in patients with hepatic
dysfunction. The mechanisms responsible for this organ interaction
remain incompletely understood.
[0270] In this Example, whether conditioning chemotherapy
administered prior to BMT might affect early enzymes in the UC and
secondarily predispose patients for hepatic dysfunction and
multiple organ failure was analyzed. The plasma amino acid analyses
supported the notions of both impaired UC function and decreased
production of nitric oxide (NO.sub.x). In light of these findings,
patients were screened for the exonic single nucleotide
polymorphism (SNP) in CPS-I disclosed herein. It was found that
homozygosity for the SNP was associated with a decreased incidence
of HVOD and enhanced early survival after BMT, consistent with a
significant pharmacogenetic interaction.
Methods
[0271] Clinical/Patient Recruitment:
[0272] Over the last three years 200 patients undergoing BMT at
Vanderbilt University Medical Center have been sequentially
enrolled in the Bone Marrow Transplant-Lung Injury Following
Engraftment (BMT-LIFE) Study, a coordinated clinical-biochemical
exploratory investigation aimed at understanding mechanisms of
acute lung injury and multiple organ failure after transplant.
Definitions of organ failure and reversal were prospectively
defined and data was collected concurrently during hospitalization
and until 60 days after BMT. Exclusion criteria included active
viral and prior escalated dose therapy with hematopoetic stem cell
support (either PBSCT or BMT).
[0273] Hepatic venocclusive disease (HVOD) was identified in
patients with bilirubin >2 mg/dL before 21 days after transplant
with either weight gain >5% of baseline or new onset of tender
hepatomegaly. Acute lung injury (ALI) was defined as bilateral
infiltrates on chest roentgenogram for three consecutive dates with
a ratio of partial pressure of oxygen in arterial blood to the
fraction of inspired oxygen concentration (PaO.sub.2/FiO.sub.2) of
less than 300 in the absence of clinical cardiac dysfunction.
Patients alive 60 days after transplant were defined as survivors.
Plasma, circulating cell pellets, and urine were collected at study
enrollment (before receiving chemotherapy) and on the day of BMT,
several days after completing high dose chemotherapy but before
marrow infusion. Samples were aliquotted, and immediately placed on
ice prior to storage at -80.degree. C. before analysis.
[0274] Amino Acid Analysis.
[0275] Amino acid analysis was performed on cryopreserved plasma
samples from days -8 and 0 (pre-treatment and day of
transplantation) in 60 patients. Patient samples were initially
randomly selected for pilot studies; subsequently analyzed samples
were specifically enriched to include extra patients with the SNP
AA genotype of CPS-I (see below) and additional patients with the
post-BMT complications of HVOD and ALI. A protein free extract of
plasma was prepared by protein precipitation with sulfosalicylic
acid and filtration through a 0.45 .mu.m Acrodisc 4 (Gelman
Sciences, Ann Arbor, Mich.).
[0276] Amino acids were separated by cation exchange chromatography
using a four-component pH- and ionic strength-graded lithium
citrate buffer system on a Beckmann 7300 amino acid analyzer
(Beckmann, Palo Alto, Calif.). Post column derivatization of amino
acids with ninhydrin allowed detection of primary amine amino acids
at 570 nm, and secondary amines at 440 nm. Quantitation was
achieved by instrument calibration with standard reference
materials (Sigma, St. Louis, Mo.). Citrulline, arginine, and
omithine were examined as measurable indices of flux of
intermediates through the urea cycle.
[0277] Measurement of Plasma Nitric Oxide Metabolites
(NO.sub.x).
[0278] Plasma NO.sub.x was measured in a subgroup of patients using
modified Griess reagents after samples were deproteinated and
incubated with cadmium beads to convert nitrate to nitrite.
[0279] Detection of T1405N Polymorphism.
[0280] Oligonucleotide primers from within the 36.sup.th exon
(CGGAAGCCACATCAGACTGG (SEQ ID NO:15) and intron
(GGAGAGTGAAACTTGACAATCATC (SEQ ID NO:16)) of CPS1 and the
polymerase chain reaction (PCR) to reliably amplify a 251 bp
fragment encompassing the region containing the change from genomic
DNA obtained from buffy coat preparations or urinary sediment. This
combination of primers gave reproducible amplification using PCR
Ready-to-Go beads (Pharmacia) and PCR cycle conditions as follows:
35 cycles of 1 minute anneal at 55.degree. C., 1 minute extension
at 72.degree. C., and 1 minute denaturation at 94.degree. C.
[0281] After formamide treatment, samples were subjected to
electrophoresis for 4 hours at 4.degree. C. in a non-denaturing
MDE.TM. gel (FMC, Rockland, Me.), then stained with silver nitrate
to detect DNA fragments. Confirmatory genotyping of 17 individuals
using both non-denaturing gel electrophoresis and direct sequence
analysis yielded identical results. Patients were classified as
having homozygous SNP genotypes of CC or AA, or as being
heterozygous (AC). For comparison, using identical methods, a
cohort of 100 patients with Alzheimer's disease was analyzed to
assess the distribution of CPSI SNP genotypes.
[0282] Statistical Analysis.
[0283] Plasma amino acid levels before and after chemotherapy, and
levels between groups of patients, were compared using Student's
T-test or Wilcoxon's Rank Sum Test (if the data were not normally
distributed). Distribution of genotypes of CPSI was compared across
groups by calculating allelic frequency for the entire group and
searching for evidence of Hardy-Weinberg disquilibrium in
specifically selected subgroups using P.sup.2 analysis.
Sensitivity, specificity, predictive values, and relative risk
assessments were generated from two-by-two contingency tables
constructed using specific amino acid values in groups of patients
divided by presence and absence of specific clinical outcomes (e.g.
HVOD, ALI, and death).
Results
[0284] Two hundred patients were enrolled in the BMT-LIFE Study.
52% underwent autologous transplant (mean age 46.+-.1 years); 48%
received allogeneic grafts (mean age 40.+-.1 years). Of the
patients undergoing allogeneic transplants, 24% received grafts
from HLA-matched unrelated donors. Nearly two-thirds of the
patients in the autologous group were women, reflecting the
increased prevalence of breast cancer in this population. The
indications for transplant were diverse, but 79% of the patients
were transplanted for breast cancer, leukemia, or non-Hodgkin's
lymphoma. The different preparative regimens used prior to BMT
included CTC (cyclophosphamide, thiotepa, carboplatin), BuCy
(busulfan, cyclophosphamide), CVP16TBI (cyclophosphamide,
etoposide, total body irradiation), CBVP16 (cyclophosphamide,
bis-chloroethylnitrosourea, etoposide) and TC (thiotepa,
cyclophosphamide).
[0285] Both morbidity and mortality are not uncommon after BMT.
While the overall 60 day mortality in the study was 14%, it was 20%
in patients receiving allografts. Complications of acute lung
injury (ALI) and hepatic venocclusive disease (HVOD) each occurred
in 19% of the patients. These complications were more than twice as
common in patients receiving allografts. In the group of patients
developing HVOD, 62% (24/38) also met criteria for ALI during
hospitalization. Only 38% (14/38) of the cases of ALI occurred in
patients who never met criteria for HVOD.
[0286] A subset (60/200) of the patients, specifically enriched
during sample selection with extra patients with CPS-I AA SNP
genotype and additional patients with post-transplant
complications, had plasma amino acid determinations before
administration of chemotherapy and on the day of transplant.
Comparison of levels of selected amino acids that participate in
the UC (citrulline, ornithine, and arginine) before and after
chemotherapy revealed significant differences. Citrulline levels
fell in virtually all patients with a mean group decrease from
23.4.+-.1.3 .mu.M to 9.1.+-.0.7 .mu.M (P<0.05). Arginine levels
rose by approximately 35% (P<0.05), and ornithine levels rose by
21% (P<0.05).
[0287] The ratio of ornithine/citrulline (O/C ratio), an index of
flux through the early steps of the UC (i.e. lower values indicate
better cycle flow), increased from 3.9.+-.0.7 at study enrollment
to 11.8.+-.1.8 after induction chemotherapy (P<0.05). Shifts
also occurred in amino acids that are not part of the UC. Levels of
glycine and alanine, two aliphatic amino acids, fell significantly
by 11% and 19%, respectively, in a pattern not consistent with
decreased flux of intermediates through the cycle simply due to
decreased protein intake (acute or chronic). Phenylalanine and
methionine levels rose by 43% and 23%, respectively, suggesting
subclinical hepatic dysfunction.
[0288] Baseline plasma levels of citrulline and the O/C ratios had
prognostic importance. Sixty day survivors of BMT had higher
baseline levels of citrulline than did nonsurvivors (24.4.+-.1.3 vs
17.7.+-.2.9 .mu.M, respectively; P<0.05). The relative risk for
death before 60 days after BMT was 2.92 for patients with an
enrollment citrulline level less than 20. The negative predictive
value for death of a plasma citrulline level greater than 20 .mu.M
was 90%. O/C ratios at enrollment were significantly lower in
patients never developing either HVOD (2.8.+-.0.2) or ALI
(2.9.+-.0.2) when compared to patients who subsequently developed
these complications (5.8.+-.1.9 and 6.5.+-.2.7, respectively;
P<0.05). Comparison of O/C ratios between 60 day survivors and
nonsurvivors of BMT at study enrollment showed a trend toward lower
values in survivors (3.3.+-.0.2 vs. 6.9.+-.3.9; P=0.06). The
negative predictive value for death within 60 days after BMT
associated with a baseline O/C ratio less than 2.5 was 92%.
[0289] Several urea cycle amino acid intermediate levels after
preparative therapy, on the day of BMT, also had significance.
Plasma arginine levels were higher in survivors (114.5.+-.5.9
.mu.M) when compared to nonsurvivors (92.3.+-.10.4 .mu.M)
(P<0.05). O/C ratios were significantly higher, suggesting more
impaired UCF, in patients who later developed ALI when compared to
those never developing severe lung dysfunction (18.4.+-.5.9 vs
9.5.+-.0.7; P<0.05). Although the negative predictive value for
development of ALI of a post-chemotherapy O/C ratio less than ten
was high (86%), the relative risk for mortality associated with
this threshold was only 1.44. There was a trend toward higher O/C
ratios in patients on the day of BMT in patients who subsequently
developed HVOD (P=0.09).
[0290] Levels of nitric oxide metabolites (NO.sub.x) in plasma were
measured in 62 patients. Plasma NO levels fell 20% after induction
therapy, from 40.+-.2 .mu.M at study enrollment to 32.+-.2 .mu.M on
the day of BMT (P<0.05). The median NO value on the day of BMT
in 20 patients developing either HVOD or ALI was 28 .mu.M; for
patients without such complications the plasma NO was 35 .mu.M. No
clear differences between plasma NO.sub.x was observed when
patients with different CPSI SNP genotypes were compared.
[0291] To assess whether certain patients might have a genetic
predisposition to develop morbid complications following induction
therapy and BMT, all patients in the study were genotyped for a
CPSI SNP. Of 200 patients, data was analyzed from 196 patients
(i.e. 2 clinical exclusions; 2 unsuccessful PCR amplifications) to
determine if the CPS-I C4340A SNP was in Hardy-Weinberg equilibrium
with the development of HVOD. The distribution of CPSI SNP
genotypes in patients undergoing BMT was identical to that of the
control group (100 patients with Alzheimer's disease): 44% CC (wild
type), 45% AC (heterozygous), and 11% AA (homozygous for the
transversion). The attack rate of HVOD in those with the CC or AC
genotype were 18% and 24%, respectively. There were no cases of
HVOD in patients with the AA genotype.
[0292] Finding that this allelic distribution was not in
Hardy-Weinburg equilibrium with the development of HVOD
(P.sup.2=5.06, P<0.05) suggests that the SNP AA genotype alters
susceptibility to hepatic toxicity following induction
chemotherapy. There were also trends toward differences in
mortality 60 days after BMT between the SNP genotypes. Nonsurvivors
constituted 15% and 20% of the AC and CC genotype groups,
respectively. Interestingly, all of the patients with the AA
genotype survived 60 days after BMT (P.sup.2=3.36; P=0.06). Of
note, almost all of the P.sup.2 score came from the AA/survivor
cell. There were no significant differences between patients with
different SNP C4340A genotypes in the attack rate of ALI (16%, 15%,
and 25% in the AA, AC, and CC groups, respectively). While ALI was
associated with significant mortality in patients with either the
AC or CC genotypes (71% and 66%, respectively), all patients with
the AA genotype who developed ALI eventually had resolution of both
bilateral pulmonary infiltrates on CXR and impaired gas exchange
and survived 60 days after BMT.
Discussion
[0293] The data presented in this Example reflect a close
association between HVOD and ALI in patients after BMT, with nearly
two-thirds of patients with HVOD meeting criteria for ALI. In this
study, 68% (26/38) of patients developing ALI required mechanical
ventilation. Rubenfelt and Crawford have reported a meaningful
survival, defined as extubation followed by discharge from the
hospital with thirty day survival, of only 6% in patients requiring
mechanical ventilation after BMT. See Rubenfeld, G. D. and
Crawford, S. W., Annals of Internal Medicine (1996) 125:625-33.
[0294] HVOD remains the major dose limiting toxicity of escalated
dose chemotherapy. It is clinically characterized by fluid
retention, jaundice, ascites, and painful hepatic enlargement
occurring within 3 weeks of BMT. Autopsy studies of those
non-surviving patients fulfilling these clinical criteria provide
histological confirmation in >80% of cases and are consistent
with the idea that enhanced local thrombosis might be an initiating
event in the pathogenesis of HVOD.
[0295] The significant fall in citrulline levels and rise in plasma
ornithine levels from patients undergoing BMT suggests a
significant disturbance in flux of carbon intermediates through the
hepatic UC in patients after induction chemotherapy. Analysis of
the patterns of other amino acids argues that this effect is not
simply due to decreased protein intake. In contrast to the patterns
seen in patients with starvation, where levels of glycine and
branched chain amino acids (BCAA) are usually significantly
elevated, we observed a fall in glycine and no significant change
in the BCAAs. Furthermore, starvation tends to increase activity of
CPSI in liver and should not lead to increases in plasma
ornithine.
[0296] The pretreatment ability of patients undergoing BMT to
maintain flow of intermediates through the UC had particular
prognostic importance. Sixty day nonsurvivors after BMT and those
patients developing HVOD or ALI had significantly lower levels of
citrulline and higher O/C ratios compared to patients who did not
develop these complications. Of interest was the observation that
nonsurvivors of BMT had lower plasma arginine values after
induction therapy when compared to surviving patients. In light of
the clustering of cells containing early UC enzymes about the
terminal hepatic venules, local concentrations of both arginine and
nitric oxide (NO) might be much higher and might play an important
role in maintaining patency of these vessels and regulating
regional hepatic blood flow. The studies showing a significant
reduction in plasma NO.sub.x levels after induction chemotherapy
support the idea that NO production is altered during BMT.
[0297] The apparent discrepancy between apparently normal plasma
levels of arginine on the day of transplant and markedly reduced
plasma NO.sub.x underscores the complex in vivo kinetics of
arginine and citrulline flux across different organ beds. Stable
isotope studies of whole body arginine homeostasis have indicated
that only about 15% of plasma arginine turnover is associated with
urea formation, and that only 1.2% of plasma arginine turnover is
associated with NO formation. Furthermore, in vitro studies have
documented substantial channeling of urea cycle intermediates, from
citrulline to arginine, that is not influenced by exogenous
provision of substrate. The ability of an individual patient to
maintain urea cycle function and hepatic NO production during the
stresses of induction chemotherapy can, in part, influence their
resistance to complications after BMT.
[0298] Since there is no gender disparity in the occurrence of
HVOD, we concentrated on potential pharmacogenetic issues related
to CPSI, an autosomally encoded gene, rather than on the X-linked
ornithine transcarbamylase gene. While characterizing the molecular
changes underlying the causes of neonatal and late-onset CPSI
deficiency, a common SNP near the 3' end of the CPSI mRNA (0.44
heterozygosity) was identifed. This C4340A transversion encodes a
predicted substitution of asparagine (AAC) for threonine (ACC) at
amino acid 1405 (T1405N). This threonine is within the allosteric
domain, preceding the sequence PV(A/S)WP(T/S)(A/Q)E important in
the binding of a cofactor, n-acetyl-glutamate (NAG), that increases
enzyme activity. Although applicants do not wish to be bound by any
particular theory of operation, it is speculated that based on the
precedent of the effects of other xenobiotics, that limited
availability of NAG after escalated dose chemotherapy is one of the
mechanisms promoting urea cycle dysfunction. Nonetheless, it
appears that the presence of the CPS-I SNP AA genotype is
associated with protection against the development of HVOD,
resolution of ALI if it occurs, and improved 60 day survival after
BMT. Thus, the data suggest that alteration in UC function plays a
role in modifying liver-lung interaction during sepsis and acute
lung injury.
[0299] In summary, this Example documents significant impairment in
hepatic UC function in patients who receive escalated dose
chemotherapy prior to BMT. Patients with more severe derangement in
cycle function are more likely to develop morbid complications
after BMT. Additionally, a significant association between a CPS-I
C4340A SNP and both post-BMT complications and short-term survival
has been found. Such data are useful in assessment of risk for
patients undergoing BMT and provide a rationale for therapeutic
attempts to support UC function during high-dose chemotherapy.
Example 3
Arginine/Citrulline Supplementation Therapy
[0300] The added decrease in urea cycle products (arginine and
citrulline) and increase in precursors (ammonia, glutamine, etc.)
resulting from the polymorphism contribute to BMT associated
toxicity. As part of the BMT Life Study, citrulline and arginine
levels were measured in 10 patients undergoing BMT.
[0301] High-dose chemotherapy used in BMT disrupts normal functions
of urea cycle enzymes and contributes to either the occurrence of
or toxicity associated with HVOD. To further evaluate this
information, an analysis of stored plasma from ten patients
undergoing BMT before treatment and after completion of induction
chemotherapy was performed. Amino acid profiles were determined
from all samples. Particular attention was paid to the urea cycle
intermediates citrulline, arginine, and ornithine. As shown in
Table 4, a marked decrease in citrulline levels of all patients
from a pre-treatment baseline mean of 24.+-.3 .mu.mol/L to a
post-treatment mean of 8.+-.1 .mu.mol/L (P<0.001). Plasma
arginine levels fell from a mean of 91.+-.6 .mu.mol/L to 70.+-.6
.mu.mol/L (P<0.05), despite the use of arginine-containing
parenteral nutrition in several patients:
TABLE-US-00006 TABLE 4 Amino Acid Pre Chemo. Post Chemo. P Value
citrulline 24 .+-. 3 .mu.M 8 .+-. 1 .mu.M <0.001 arginine 91
.+-. 6 .mu.M 70 .+-. 6 .mu.M 0.03
[0302] The fall in citrulline and arginine was similar in patients
who did and did not receive total parenteral nutrition and was the
same in males and females. The decreases in citrulline suggest that
there is a decrease in flow through the first steps of the urea
cycle (FIG. 1).
[0303] Thus, in accordance with the present invention, a method of
reducing toxicity and/or the occurrence of HVOD in a patient
undergoing BMT is provided. This method comprises administering the
BMT patient arginine and/or citrulline, with citrulline being
preferred, in an amount effective to bolster arginine and NO
synthesis in the patient. The bolstering of arginine and NO
synthesis in the patient reduces and/or substantially prevents the
occurrence of HVOD associated with BMT. Citrulline is a preferred
supplementation agent given that it is more readily converted to
NO.
Example 4
Construction of a Functional Full-Length CPSI Expression Clone
[0304] After attempting a number of strategies, a human CPSI cDNA
expression clone containing the entire coding region was
constructed. FIGS. 6 and 7 present schematic diagrams illustrating
the method used to construct the expression clone. This clone has
been completely sequenced and does not contain any changes from the
consensus CPSI sequence which has been characterized in the
art.
[0305] The ability of the clone to make CPSI protein was tested in
COS-7 cells. COS-7 cells were chosen for their lack of native CPSI
activity or production. A western blot analysis of the COS-7 cells
transfected with the flCPSI-PCDNA3.1 construct was prepared. HepG2
cell extracts were used as a control as these liver-derived cells
have retained CPSI activity. Untransfected COS-7 cells were used as
a negative control. Unlike the untransfected COS-7 cells, the HepG2
and COS-7-fICPSI cells demonstrated the expected 160 kDa band using
a rabbit anti-rat CPSI antibody. Additionally, a colorimetric assay
was performed to detect the production of carbamyl phosphate from
ammonia. As shown graphically in FIG. 8, the transected cells
demonstrated activity similar to HepG2 cells while untransfected
COS-7 cells did not.
[0306] Site-directed mutagenesis has been performed on the T1405
containing CPSI insert and a copy with the N1405 polymorphic codon
has been created. The N1405 polymorphic codon was sequenced for its
entire length and no other changes were detected. The
QuikChange.TM. (Stratagene) system, which takes advantage of the
methylation introduced into DNA by host bacteria, was used to
prepare this construct.
[0307] These constructs are used to provide a steady supply of
recombinant CPSI protein as encoded by both alleles, (T1405, N1405)
using COS cells and the respective CPSI/PC DNA 3.1 constructs as an
expression system. Enzymatically active CPSI has been produced
using this system, as shown by the graph in FIG. 8.
[0308] A component of these experiments is to determine the in
vitro effect of the T1405N polymorphism on CPSI function. As
discussed in Examples 1 and 2, this change affects the sensitivity
of the enzyme to NAG concentrations. Screening of 20 individuals
for the C to A change showed a heterozygosity rate of 50% with 25%
of the group homozygous AA. This suggests that a significant
portion of the general population has a potential qualitative
abnormality in CPSI function. This abnormality, while silent under
normal conditions, is unmasked by stressful conditions and toxins
such as high-dose chemotherapy or valproic acid administration.
[0309] Comparison of the protein products is then done in stages.
The first stage examines the physical characteristics of the
expressed mRNA and protein. Using the flCPSI insert as a probe,
Northern blots of message prepared from the expressing COS-7 cell
lines are probed. Positive controls include HepG2 and human liver
message. Negative controls were COS-7 cells transfected with empty
cassette pcDNA3.1. The expressed flCPSI derived message is somewhat
smaller than the native CPSI (4.9 kb vs. 5.7 kb) since the clone
does not contain the 1 kb 3' untranslated region.
[0310] Using the same controls, Western blot analysis of cell
lysates by SDS-PAGE are performed. Comassie blue staining is used
to examine total protein production. For specific CPSI detection, a
polyclonal rabbit anti-rat CPSI antibody is used. This antibody
detects the expressed CPSI from COS-7 cells as well as the control
samples. Finally, changes in the protein's structure are determined
by examining the mobility pattern by 2-D electrophoresis, a useful
tool to detect conformational changes. Any large changes in
confirmation likely explain the alteration in CPSI function for
that mutation.
[0311] The next stage involves measuring the functional
characteristics of the expressed enzymes. A sensitive colorimetric
assay has been modified for this purpose (Pierson, D. L., J.
Biochem. Biophys. Methods, 3:31-37 (1980)). The modified assay
allows 4-5 analyses from 20-50 mg of tissue or cells. The tissue is
first homogenized in 0.75M KCl. Small molecules, including ATP and
NAG, are removed through a SEPHADEX.TM. G25 column (Boehringer).
The reaction mix contains ammonium bicarbonate, ATP, magnesium DTT,
n-acetylglutamate (NAG), and triethanolamine. The concentration of
any reagent can be varied, and experiments on HepG2 cells show
decreased activity with both low and high concentrations of NAG
(0.50 mM). Absence of NAG in preliminary COS-7 cell expression
experiments yields no measurable enzyme activity.
[0312] Since CPSI is an allosteric enzyme, it does not follow
Michaelis-Menton kinetics under varying NAG concentrations;
however, when the amount of NAG is fixed, the production of
carbamyl phosphate is steady. As shown in FIG. 8, carbamyl
phosphate production is measured by the addition of hydroxylamine
to the solution after incubation at 37.degree. C. for varying time
periods (0, 5, 10, 20, 25, 30 minutes). This step, carried out at
95.degree. C., also serves to inactivate the enzyme and prevent
further production of carbamyl phosphate. The hydroxylamine
converts the carbamyl phosphate to hydroxyurea which is
subsequently treated with a sulfuric/acetic acid solution with
butanedione to derive a compound with peak absorption at 458 nm.
The reaction is then spun at 12,000.times.g for 15 minutes to
remove precipitated protein. Next, the 458 nm absorbance is
measured for each reaction. Activity typically begins to decrease
after 20-30 minutes of reaction.
[0313] A number of expressing cell pellets are pooled for analysis.
To ensure that activity measurements are based on consistent
amounts of enzyme, expressed CPSI is quantified by Western blot
analysis of the pooled sample using a CPSI antibody such as the
rabbit anti-rat CPSI described hereinabove. Basal activity is first
determined using fixed amounts of substrate and cofactor and a time
course analysis. Varying amounts of ammonia bicarbonate, ATP, and
NAG are then used to determine the binding efficiency for these
elements. These elements are varied from 0 to 10-fold the normal
amount. Enzyme activity is also measured after heat treatment of
the homogenate. Protein labeling (pulse-chase) experiments are
performed to determine the stability of the protein over time.
[0314] Stable CPSI protein expression is obtained using the methods
described above. The establishment of stable transfected cell lines
allows the production of sufficient quantities of both varieties of
CPSI to carry out these studies. In activity studies, changes in
activity for the N1405 as compared to the T1405 type of CPSI are
noted. A change in the enzyme activity under varying concentrations
of NAG is also noted. These results support the role of this
polymorphism of the present invention in predicting susceptibility
to sub-optimal urea cycle function and hyperammonemia and decreased
arginine production associated therewith.
Example 5
Relationship of the T1405N Polymorphism and Urea Cycle
Intermediates to the Ammonia Elevation Seen in Patients on Valproic
Acid Therapy
[0315] Valproic acid (VPA) is a commonly used seizure medication,
particularly for the treatment of absence seizures or as an adjunct
therapy of other seizure disorders. Toxicity from VPA treatment is
a complex and multi-variant process and probably reflects several
metabolic disruptions. Hyperammonemia and hepatic micro-vesicular
steatosis and necrosis are the most commonly reported serious
medical complications.
[0316] Although the development of toxic hyperammonemia involves
only a small number of patients, it carries a significant morbidity
and mortality, and several deaths have been attributed to this
complication. The development of asymptomatic hyperammonemia
(plasma ammonia level greater than 60 .mu.mol/L) occurs within one
hour of VPA administration, and is, however, relatively common.
[0317] Mechanisms of VPA-Induced Hyperammonemia.
[0318] The mechanisms by which VPA causes hyperammonemia has been
the subject of some debate, and a number of different theories
currently have support in the art. A renal model proposed that the
changed in glutamine metabolism resulted in an increased ammonia
load to the liver, while most other theories concentrate on
different aspects of urea cycle function. See, for example, Warier
et al., Revue Neurologique, 139:753-757 (1983). Since the urea
cycle is the major mechanism for the removal of ammonia in humans,
it is thought that hyperammonemia arises in some way from the
inhibitory interactions of VPA and/or its metabolites with urea
cycle function and capacity.
[0319] Evidence for urea cycle dysfunction in VPA therapy comes
from a number of experimental and clinical observations aside from
elevations in plasma ammonia described above. For example, Marrini
et al. measured a reduction in both baseline and stimulated CPSI
activity in non-nephrectomized animals following an amino acid and
VPA load (Marrini et al., Neurology 38:365-371 (1988)). Marrini et
al. also observed that nephrectomized rats injected with an amino
acid load and VPA also developed hyperammonemia. Another group,
Castro-Gago et al., measured serum amino acids in 22 epileptic
children treated with VPA, and found reduction in aspartic acid and
ornithine, implicating a decrease in urea cycle efficiency rather
than an increase in precursors (Castro-Gago et al., Childs Neurons
System 6:434-436 (1990)).
[0320] Significance of Carbamyl Phosphate Synthase I.
[0321] Mechanisms of VPA-induced urea cycle deficits typically
revolve around mitochondrial carbamyl phosphate synthetase I
(CPSI). A patient with severe toxicity following VPA overdose was
found to have 50% normal CPSI activity (Bourrier et al., Prese
Medicale 17:2063-2066 (1988)). Applicants have observed several
mild CPSI deficient patients who deteriorated when given valproic
acid with ready reversal after discontinuation.
[0322] Role of NAG.
[0323] N-acetylglutamate (NAG) is a required allosteric cofactor
for CPSI. NAGA is synthesized from glutamate and acetyl CoA in
mitochondria, with a cellular distribution that mirrors that of
CPSI (Shigesada et al., Journal of Biological Chemistry 246:
5588-5595 (1971)). It is synthesized from glutamate (from amino
acid catabolism) and acetyl CoA. There are several ways in which an
alteration of NAG availability is envisaged to reduce the activity
of CPSI. Genetic deficiencies in NAG synthetase have been observed,
and this enzyme is known to be inhibited competitively by alternate
substrates such as propionyl CoA or succinate (Bachmann et al., New
England Journal of Medicine 304:543 (1981); Kamoun et al., Lancet
48 (1987); Coude et al., J. Clin. Invest. 64:1544-1551 (1979);
Rabier et al., Biochem. And Biophys. Research Comm. 91:456-460
(1979); Rabier et al., Biochimie 68:639-647 (1986)). It has been
shown experimentally that CPSI is inhibited in a competitive manner
by the presence of increased amounts of propionyl CoA, and that VPA
therapy causes an increase in blood propionate concentration
(Coulter et al., Lancet 1 (8181): 1310-1311 (1980); Gruskay et al.,
Ped. Res. 15:475 (1981); Schmidt, R. D., Clin. Chim. Acta. 74:39-42
(1977)). VPA exposure has also been shown to decrease NAG
concentrations in intact hepatocytes, by decreasing concentrations
of both acetyl CoA and glutamine (Coude et al., Biochem. J.
216:233-236 (1983)). The decrease in glutamine concentration is
attributed to inhibition of both pyruvate dehydrogenase and
pyruvate carboxylase.
[0324] Alternatively, it has been suggested that depletion of
mitochondrial acetyl CoA occurs because CoA is diverted on VPA
therapy for the manufacture of valproyl CoA (Becker et al.,
Archives of Biochemistry & Biophysics 223:381-392 (1983)). It
is well known that VPA also disrupts fatty acid .beta.-oxidation,
with resultant diminution of acetyl CoA (Eadie et al., Med.
Toxicol. 3:85-106 (1998)). All these mechanisms could lead to a
shortage in NAG since it is synthesized from acetyl CoA. Given the
effects of VPA on NAG availability it follows that any change in
the binding properties of CPSI for NAG would affect its
activity.
[0325] Thus, this Example sets forth experimentation for
determining correlation between the presence or absence of the
polymorphism of the present invention in the CPSI gene with
susceptibility to hyperammonemia using VPA as a model agent for the
production of hyperammonemia. Initially, genomic DNA is isolated
from patients who are beginning valproic acid therapy for
genotyping for the T1405N polymorphism in accordance with the
methods described herein, such as PCR amplification and use of
non-denaturing gels. After genotyping these patients, pre- and
post-treatment amino acid and ammonia determination is performed
for these patients. Particularly, DNA is isolated from whole blood
using the QIAmp.TM. (Qiagen) kit described in Example I.
[0326] Next, plasma total VPA concentration is determined by an
enzyme-mediated immunoassay technique (EMIT.TM. Syva-Behring, San
Jose, Calif. on a Syva 30R.TM. analyzer). This technique utilizes
competitive binding for VPA antibody binding sites between VPA in
the patient plasma and that complexed with the enzyme G6PDH.
Release of the VPA enzyme complex from the antibody reactivates the
enzyme, and its activity is assessed by the rate of formation of
NADH upon addition of the substrate. NADH production is monitored
via spectroscopy at 340 nanometers (nm). Free (non-protein bound)
VPA is isolated from plasma using a centrifugal micro partition
filter device with a 3000 Dalton cut-off (CENTRIFREE, Aimcon,
Beverley, Mass.). The VPA concentration in the plasma ultra
filtrate is measured as described for total VPA.
[0327] Data collected from VPA patients is analyzed for
correlations between genotype and phenotype. Additionally, free and
conjugated VPA fractionation are compared to evaluate effects on
NAG production and availability. The latter comparison is prepared
given that there are known effects of VPA on NAG availability. For
example, VPA exposure has been shown to decrease NAG concentrations
in intact hepatocytes by decreasing concentrations of both acetyl
CoA and glutamine. See Coude et al., Biochem. J., 216:233-236
(1983). Thus, this comparison reflects that changes in the binding
properties of CPSI for NAG affect the activity of CPSI.
Example 6
Detection of Additional Polymorphisms in CPSI
[0328] Using the techniques developed for mutation analysis of CPSI
message, 10 non-CPSI deficient, unrelated patients are screened for
additional polymorphisms in the coding region. This is done using
"illegitimate" transcripts from lymphoblastoid and fibroblast cell
lines. Polymorphisms with a widespread effect on the population
should be evident in this size sample. As used herein and in the
claims, the term "polymorphism" refers to the occurrence of two or
more genetically determined alternative sequences or alleles in a
population. A polymorphic marker is the locus at which divergence
occurs. Preferred markers have at least two alleles, each occurring
at frequency of greater than 1%. A polymorphic locus may be as
small as one base pair. Provided polymorphic markers thus include
restriction fragment length polymorphisms, variable number of
tandem repeats (VNTR's), hypervariable regions, minisatellites,
dinucleotide repeats and tetranucleotide repeats.
[0329] A number of "mutation" detection techniques have been
carried out, all of which are based on detectable changes in the
mobility of non-denatured single-stranded DNA, as described by
Summar, M., J. Inherited Metabolic Disease 21:30-39 (1998).
Examples of CPSI mutations identified by these techniques are
disclosed in FIG. 3. Due to the large size of the CPSI message
(about 5,700 bases) a method to screen a large amount of DNA in a
few reactions is preferred. Restriction endonuclease fingerprinting
(REF) provides for the screening large DNA fragments, up to about
2,000 bp, with excellent sensitivity.
[0330] Reverse transcriptase reactions (RT) are carried out using 1
.mu.g of total RNA and either an oligo-dT primer or an antisense
primer from the midpoint of the CPSI message. Using the RT product
as template, PCR reactions are performed with 4 different primer
sets creating 4 overlapping fragments spanning the 4,600 base
coding region. Control PCR reactions are run with each set of
experiments, to ensure that contaminating template is not
amplified. Genomic DNA is not preferred for this study due to the
size of the gene (80,000+bp), the number of introns (36), and that
sequencing of the intron exon boundaries for CPSI has not been
completed. However, intronic locations are characterized
graphically in FIG. 9.
[0331] The 4 overlapping RT/PCR products described above are used
for mutation screening. Careful analysis of the restriction maps
leads to the selection of three restriction enzymes for each
fragment which cleave them into pieces ranging from 100-250 bp.
Fragments of this size are ideal for single strand conformation
polymorphism (SSCP) analysis. The enzymes are selected such that
each fragment can be evenly evaluated across its length. Prior to
digestion, the PCR products are purified by gel electrophoresis and
isolation from the agarose slices. After 3 hours, the digested
fragments are ethanol precipitated. These fragments are separated
in a 6% non-denaturing polyacrylamide gel at 4.degree. C. running
at a constant 35 watts. These conditions maximize the detection of
conformational changes in the single stranded fragments, as
described by Liu, Q. and Sommer, S. S., Biotechniques 18(3):470-477
(1995). DNA detection is done by silver staining and the gels are
scored for mobility shifts. Based on the location of any shifted
fragment, direct sequence analysis of the RT/PCR product is
performed using a cycle-sequencing protocol. To eliminate the
possibility of a mutation resulting from Taq polymerase errors, a
fresh RT product is amplified and sequenced in each case. The
entire 4,600 bases of coding message is rapidly screened in this
fashion Any regions containing unclear areas are sequenced, looking
for changes in the expected sequence.
[0332] The restriction digestion products of each RT/PCR fragment
are isolated. These individual fragments are then run against the
combined digestion in a non-denaturing gel as described above. By
characterizing the fragment pattern in this way, the portions of
the CPSI message involved in any observed mobility shifts are
readily identified.
[0333] Polymorphisms detected in these experiments are genotyped
against the Centre d'Etude Polymorphsim Humanise (CEPH) parents
panel to establish frequency. All changes are examined for their
effect on codon use and those resulting in mis-sense mutations are
examined using the CPSI characterization data disclosed herein.
[0334] The techniques described in Example 3 are used to express
site-directed mutants containing these changes. Using this system
the in vitro effects of the changes on CPSI production and activity
are observed.
[0335] A T344A polymorphism was detected in CPSI. Oligonucleotide
primers were used from the 10th exon
(U1119:tactgctcagaatcatggc--SEQ ID NO:17) and intron (LI10+37:
tcatcaccaactgaacagg--SEQ ID NO:18) to amplify a 91 bp fragment
containing the change. PCR cycle conditions were: 35 cycles of 1
minute anneal at 59.degree. C., 1 minute extension at 72.degree.
C., and 1 minute denaturation at 94.degree. C. Patients were
classified as having either homozygous SNP genotypes of AA or TT,
or as being heterozygous (AT). The adult population distribution of
this polymorphism is 35% AA, 44% AT, and 21% TT.
[0336] A 118-CTT polymorphism was also detected in CPSI.
Oligonucleotide primers were used from the 5' untranslated region
(U5'-74: ggttaagagaaggaggagctg--SEQ ID NO:19) and intron (L175:
aaccagtcttcagtgtcctca--SEQ ID NO:20) to amplify a 249 bp fragment
containing the change. PCR cycle conditions were: 35 cycles of 1
minute anneal at 59.degree. C., 1 minute extension at 72.degree.
C., and 1 minute denaturation at 94.degree. C. Patients were
classified as having either a homozygous genotype with the 118
trinucleotide insertion or deletion, or as being heterozygous. The
adult population distribution of this polymorphism is 34% CTT-, 43%
heterozygous, and 23% CTT+.
Example 7
Biochemical and Genetic Alterations in Carbamyl Phosphate
Synthetase I in Neonatal Patients with With Persistent Pulmonary
Hypertension
[0337] This Example investigates the role of the limitation of
endogenous NO production in the pathogenesis of persistent
pulmonary hypertension (PPHN) in the sick term neonate. Endogenous
NO is the product of the urea cycle intermediate arginine.
Production of arginine depends on the rate-determining enzyme of
the urea cycle, carbamyl phosphate synthetase (CPSI). Newborns
possess less than half the normal urea cycle function making them
particularly susceptible to minor changes in enzyme form and
function. A common exonic polymorphism (T1405N) in CPSI has been
observed which affects flow through the first step of the urea
cycle.
[0338] In this Example, it was tested whether newborns who
developed PPHN would have lower NO precursors (arginine and
citrulline) than matched controls. Whether PPHN patients have
predominantly the CC (threonine/threonine) or AC
(asparagine/threonine) CPSI genotypes which are associated with
lower function than AA (asparagine/asparagine) CPSI genotype was
also analyzed.
[0339] Methods.
[0340] Forty-seven neonates >2 kg, >35 weeks, and <72
hours old who were admitted to the Vanderbilt Neonatal Intensive
Care Unit with (n=22) and without (n=25)
echocardiographically-documented pulmonary hypertension were
enrolled. Clinically important measures of the severity of
respiratory distress were recorded. Ammonia levels and plasma amino
acid profiles were obtained. Genotypes were determined by running
PCR-amplified DNA on nondenaturing MDE.TM. gels.
[0341] Results.
[0342] Patients who developed PPHN had an average arginine of 21.5
.mu.mol/l while those who did not averaged 38.3 .mu.mol/l
(p=0.0004). The citrulline averages were 6.1 .mu.mol/l and 10.3
.mu.mol/l respectively (p=0.02). The levels of arginine and
citrulline were inversely correlated with the severity of hypoxemia
as measured by oxygenation index, days of mechanical ventilation,
and days requiring supplemental O.sub.2. Genotype analysis of PPHN
patients for T1405N showed 5CCs, 17ACs, and OAAs, whereas the
controls had 7CCs, 16ACs, and 2AAs (Chi-square p=0.005 using the
expected population allele frequency). Infants with the CC genotype
had lower arginine and citrulline means (21.5 .mu.mol/l and 5.8
.mu.mol/l) than infants with the AA genotype (31.5 .mu.mol/l and
13.5 .mu.mol/l) consistent with a functional difference between the
two forms of the enzyme.
[0343] Conclusions.
[0344] This Example shows that the development of PPHN in sick
newborns is associated with inadequate availability of the urea
cycle intermediates arginine and citrulline. The T1405N
polymorphism in the CPSI DNA leads to diminished enzyme function
and subsequent lower levels of NO precursors.
[0345] Discussion.
[0346] Carbamyl phosphate synthetase (CPS I) catalyzes the
rate-determining step in the urea cycle thereby determining tissue
levels of the urea cycle intermediates including arginine and
citrulline. As disclosed herein, a widely distributed C to A exonic
polymorphism in the CPS I gene changes a conserved threonine to an
asparagine at position 1405 near the critical N-acetyl glutamate
binding domain. Data has shown that the asparagine-containing
version of CPSI displays more efficient kinetics in enzyme function
studies.
[0347] The T1405N allele exhibits 50% heterozygosity and appears to
be a silent variant in normal healthy adults. However, consequences
of the qualitative change can be unmasked by stressful conditions.
As disclosed in Examples 1-3, adults exposed to high-dose
chemotherapy in preparation for bone marrow transplantation that
the threonine-containing enzyme produces inadequate levels of
arginine and citrulline and is associated with an increased
incidence of hepatic veno-occlusive disease, acute lung injury, and
death. As nitric oxide (NO) is generated in endothelial cells from
L-arginine by nitric oxide synthetase (NOS), decreased levels of
urea cycle intermediates could predispose to disturbances in
vascular tone by limiting endogenous NO production.
[0348] In the prospective cohort study of this Example, the
possibility that a similar process could be involved in the
pathogenesis of persistent pulmonary hypertension of the newborn
(PPHN) was investigated. Endogenously produced NO functions in
regulation of pulmonary vascular resistance and in the transition
from fetal to neonatal circulation. Lipsitz, E. C., et al. J
Pediatr Surg (1996) 31:137-140; Abman, S. H., et al. Am J Physiol
(1990) 259:H1921-H1927. Between 20 weeks gestation and term birth,
CPSI production and function are less than 50% of adult levels.
This physiologic deficiency could unmask the effect of the T1405N
gene mutation particularly if coupled with other neonatal stresses
affecting hepatic function; for instance, asphyxia or sepsis.
[0349] Patients eligible for this study included appropriately
grown neonates .gtoreq.35 weeks gestation and .gtoreq.2 kg
birthweight who were admitted to the Vanderbilt University Medical
Center neo-natal intensive care unit (NICU) between Jul. 1, 1999
and Feb. 29, 2000 for symptoms of respiratory distress. Infants
with multiple congenital anomalies, known genetic syndromes, and
anatomic causes of pulmonary hypertension (congenital diaphragmatic
hernia, Potter's syndrome, asphyxiating thoracic dystrophy, etc.)
were excluded. Parental consent was obtained for all enrollees.
Fifty-one neonates had 3 cc of blood drawn in the first 72 hours of
life for plasma amino acid profiles, ammonia and BUN levels, nitric
oxide metabolite determination, and CPS1 genotyping. Blood was
drawn prior to blood transfusion, enteral or parenteral protein
intake, inhaled nitric oxide administration, or ECMO
cannulation.
[0350] Data collected on the enrollees included (1) baseline
characteristics (birthweight, gestational age, sex, race, Apgar
scores, primary diagnosis, any pulmonary complications, and the
postnatal age at the time blood was drawn) and (2) measures of
respiratory support (FiO.sub.2, MAP, iNO, ECMO) and clinical
response (ABGs, duration of mechanical ventilation and supplemental
02, survival.) Maximum oxygenation index
[OI=FiO.sub.2.times.MAP/PaO.sub.2] was used as a measure of the
severity of respiratory distress. Predominant primary diagnoses
included (1) birth asphyxia: 5-minute Apgar score <5 with a
mixed acidosis on first ABG or cord blood gas plus evidence or
neurologic dysfunction and other end-organ injury, (2) respiratory
distress syndrome (RDS): clinical symptoms of respiratory distress
with ground-glass lung fields and air bronchograms on chest X-ray
plus combined hypercarbia/hypoxia on ABG (Note: given the
gestational age of these neonates, infants with this picture could
have had either surfactant-deficiency or congenital pneumonia;
however, in no case was a positive tracheal aspirate culture
obtained), and (3) meconium aspiration syndrome (MAS): history of
meconium-staining at delivery plus clinical symptoms of respiratory
distress, hypoxemia, and coarse infiltrates chest X-ray.
[0351] Infants were defined as having pulmonary hypertension (PPHN)
if they developed significant hypoxemia (PaO.sub.2<100 on 100%
O.sub.2>6 hours) with normal intracardiac anatomy and
echocardiographic evidence of elevated pulmonary artery pressure.
The latter was defines as (1) right-to-left or bidirectional ductal
of foramen ovale flow or (2) elevated (>35 mmHg) pulmonary
artery pressure based on Doppler estimate of the tricuspid
regurgitation jet as read by a blinded third party.
[0352] Amino acid analysis was performed on fresh plasma samples in
47 patients. A protein free extract of plasma was prepared by
protein precipitation with sulfosalicylic acid and filtration
through a 0.45 .mu.m Acrodisc 4 (Gelman Sciences, Ann Arbor,
Mich.). Amino acids were separated by cation exchange
chromatography using a four-component pH- and ionic strength-graded
lithium citrate buffer system on a Beckmann 7300 amino acid
analyzer (Beckmann, Palo Alto, Calif.). Post column derivatization
of amino acids with ninhydrin allowed detection of primary amine
amino acids at 570 nm, and secondary amines at 440 nm. Quantitation
was achieved by instrument calibration with standard reference
materials (Sigma, St. Louis, Mo.). Citrulline and arginine were
detected as measurable indices of flux of intermediates through the
urea cycle.
[0353] Measurement of Plasma Nitric Oxide Metabolites
(NO.sub.x).
[0354] Plasma NO.sub.x was measured in a subgroup of patients using
modified Griess reagents after samples were deproteinated and
incubated with cadmium beads to convert nitrate to nitrite.
[0355] SNP Detection.
[0356] Oligonucleotide primers from within the 36.sup.th exon
(U4295--SEQ ID N0:15) and intron (LI36--SEQ ID N0:16) of CPS1 and
the polymerase chain reaction (PCR) to reliably amplify a 251 bp
fragment encompassing the region containing the change from genomic
DNA obtained from whole blood preparations. This combination of
primers gave reproducible amplification using Taq polymerase
(Promega) and PCR cycle conditions as follows: 35 cycles of 1
minute anneal at 67.degree. C., 1 minute extension at 72.degree.
C., and 1 minute denaturation at 94.degree. C. After formamide
treatment, samples were subjected to electrophoresis for 5 hours at
4.degree. C. in a non-denaturing MDE.TM. gel (FMC, Rockland, Me.),
then stained with silver nitrate to detect DNA fragments. Patients
were classified as having homozygous SNP genotypes of CC or AA, or
as being heterozygous (AC). Genotyping using nondenaturing gel
electrophoresis and direct sequence analysis yielded identical
results as those disclosed above. Thus, the adult population
distribution of the T1405N polymorphism was determined to be: 45%
CC, 44% AC, and 11% AA.
[0357] An identical technique to that described above was used to
detect the T344A polymorphism. Oligonucleotide primers were used
from the 10th exon (U1119:tactgctcagaatcatggc--SEQ ID NO:17) and
intron (LI10+37: tcatcaccaactgaacagg--SEQ ID NO:18) to amplify a 91
bp fragment containing the change. PCR cycle conditions were: 35
cycles of 1 minute anneal at 59, 1 minute extension at 72 C, and 1
minute denaturation at 94 C. Patients were classified as having
either homozygous SNP genotypes of AA or TT, or as being
heterozygous (AT). The adult population distribution of this
polymorphism is 35% AA, 44% AT, and 21% TT.
[0358] An identical technique to that described above was used to
detect the 118-CTT polymorphism. Oligonucleotide primers were used
from the 5' untranslated region (U5'-74: ggttaagagaaggaggagctg--SEQ
ID NO:19) and intron (L175: aaccagtcttcagtgtcctca--SEQ ID NO:20) to
amplify a 249 bp fragment containing the change. PCR cycle
conditions were: 35 cycles of 1 minute anneal at 59.degree. C., 1
minute extension at 72.degree. C., and 1 minute denaturation at
94.degree. C. Patients were classified as having either a
homozygous genotype with the 118 trinucleotide insertion or
deletion, or as being heterozygous. The adult population
distribution of this polymorphism is 34% CTT-, 43% heterozygous,
and 23% CTT+.
[0359] Ammonia and plasma amino acid levels were compared between
groups of patients using Student's T-test. Distributions of
genotypes of CPSI were compared across groups by calculating
allelic frequency for the entire group and searching for evidence
of Hardy-Weinberg disequilibrium in specifically selected subgroups
using Chi-square analysis. Of the 51 neonates originally enrolled,
25 developed PPHN while 26 did not. There were no statistically
significant differences in the baseline characteristics of the two
groups including birthweight, gestational age, race, or the
postnatal age in hours of the infants at enrollment. There was,
however, a slight predominance of males in the control group.
[0360] The distribution of primary diagnoses was evenly
distributed. In the PPHN group, 5 infants had birth asphyxia, 9
infants had RDS, 5 infants had meconium aspiration syndrome, and 6
infants had other diagnoses, including 4 infants with primary PPHN.
In the control group, 4 infants had birth asphyxia, 8 infants had
RDS, 3 infants had MAS, and 11 infants had other diagnoses. The
other diagnoses included supraventricular tachycardia, anemia,
birth trauma, and viral sepsis. No infant in the study had a
positive bacterial blood culture.
[0361] As expected, infants who had PPHN complicate their primary
pathology did develop more severe illness than the controls by some
clinical criteria. Eight of the infants with PPHN required
treatment with inhaled NO (iNO), 2 required ECMO, and 2 died (one
infant with asphyxia and multiorgan-system failure on iNO; another
infant with alveolar capillary dysplasia was withdrawn from ECMO.)
Obviously, none of the controls were treated with iNO or ECMO; and
there was no mortality in the control group.
[0362] Three infants in the PPHN group were excluded from analysis.
The infant found to have alveolar capillary dysplasia on lung
biopsy was considered to have an anatomical etiology for pulmonary
hypertension. Another infant was mistakenly enrolled with a
congenital diaphragmatic hernia, and the third was enrolled at 119
hours of age after TPN had been initiated. One infant in the
control group was excluded from analysis after karyotype analysis
revealed the etiology of his hypotonia to be Prader-Willi
syndrome.
[0363] The infants who developed PPHN had significantly lower serum
arginine and citrulline levels on amino acid analysis. The mean
arginine level in PPHN cases was 21.5.+-.9.2 .mu.mol/l whereas the
mean arginine of the control group was 38.3.+-.18.4 .mu.mol/l
(p=0.0004). The mean citrulline in PPHN cases was 6.1.+-.3.6
.mu.mol/l compared to 10.3.+-.7 .mu.mol/l in the control group
(p=0.02). There were no significant differences in the levels of
other amino acids between the two groups, including glutamine,
glycine, alanine, lysine, valine, ornithine, and leucine. The level
of total essential amino acids (TEAA) was slightly lower in the
PPHN cases, about 537 .mu.mol/l versus about 654 Nmol/l, but this
difference was not statistically significant (p=0.08). by
birthweight, gestational age, or number of hours of postnatal life.
The level of TEAA was found to be significantly higher in the four
infants whose blood was drawn prior to six hours of age (about
1021.5 .mu.mol/l vs. about 542 .mu.mol/l, p=0.0026). This
difference is presumed to reflect the recent cessation of
parenteral protein influx in these infants from the placental
circulation.
[0364] No differences in arginine and citrulline levels were found
when the primary diagnosis categories of asphyxia, RDS, MAS, and
"other" were separately analyzed. In each group, infants with
pulmonary hypertension tended to have lower values, but the results
were not statistically significant given the small numbers of
infants in each group. For example, asphyxiated infants with PPHN
had a mean arginine of about 18.5 .mu.mol/lcompared to about 52.7
.mu.mol/l in asphyxiated controls (p=0.06) and a mean citrulline of
about 6.8 .mu.mol/l compared to about 14.3 .mu.mol/l (p=0.04).
[0365] There was an inverse relationship between the levels of
serum arginine and citrulline and the severity of hypoxemia.
Arginine and citulline values fell progressively as oxygenation
index increased, days of mechanical ventilation increased, and days
requiring supplemental oxygen increased birthweight, gestational
age, or number of hours of postnatal life. The NH.sub.3 levels in
infants with PPHN tended to be slightly higher than in controls
(54.+-.18.1 .mu.mol/l vs. 45.6.+-.12 .mu.mol/l) but these values
were not statistically significant (p=0.08). On CPS1 T1405N
genotype analysis, of the 22 infants who developed PPHN, 5 were CC
and 17 were AC. There were no AAs in the PPHN cases. In the 25
controls, there were 7 CCs, 16 ACs, and 2 AAs. These distributions
of genotypes were then compared by calculating the expected allelic
frequency for the entire group revealing evidence of Hardy-Weinberg
disequilibrium in the PPHN group. On Chi-square analysis these two
groups are significantly different from each other with a
p-value=0.005. Of the two infants with the AA genotype, one infant
had RDS while the other suffered from birth asphyxia. Neither
infant ever achieved an OI.gtoreq.15; both spent <1 week on the
ventilator and <10 days on oxygen.
[0366] Infants with the CC genotype had mean arginine levels of
21.9.+-.7 .mu.mol/l and citrulline levels of 5.8.+-.1.8 .mu.mol/l
while infants with the AA genotype had a mean arginine level of
31.5.+-.3.5 .mu.mol/l and a mean citrulline level of 13.5.+-.6.4
.mu.mol/l. Again, given the small number of AAs, this data has
difficulty reaching statistical significance with p-values of 0.1
and 0.006, respectively.
Example 8
Intravenous Citrulline Supplementation Increases Plasma Arginine
Levels in Piglets
[0367] Intravenous citrulline has not been previously used in a
clinical model. This Example assessed the safety of IV citrulline
and its effect on serum arginine levels in piglets. A total of 9
Duroc swine, aged 5-21 days, with a target minimum weight of 4 kg
were utilized. All piglets underwent anesthetic induction and
tracheostomy. Central lines were placed in the femoral artery and
femoral vein and hemodynamics monitored continuously. Citrulline
(600 mg/kg IV) was administered to 5 piglets. Saline was given to
control animals. Serum amino acids were drawn before and each hour
after citrulline administration.
[0368] Serum arginine levels peaked at 1-2 hours following IV
citrulline administration and remained sustained above baseline
three hours following, reaching significance at all time points
compared to controls (p<0.001). No hemodynamic instability was
observed.
Arginine Levels (umol/L) Following IV Citrulline
TABLE-US-00007 [0369] 1 hour 2 hours 3 hours Treatment Group (n =
5) Baseline post post post Citrulline (600 mg/kg) 131.5 535.0 559.8
498.4 Control (saline) 89.6 103.0 118.1 136.7 p-value 0.1582
<0.001 <0.001 <0.001
Mean Arterial Blood Pressures (mmHg) Following IV Citrulline
TABLE-US-00008 [0370] 1 hour 2 hours 3 hours Treatment Group (n =
4) Pre-dose post post post Citrulline (600 mg/kg) 67.0 67.4 64.8
62.2 Control (saline) 53.2 58.7 55.7 54.7 p > .05 at all time
points
[0371] Pharmacokinetics:
[0372] Based on the above data, the pharmacokinetics were
calculated for both plasma citrulline and arginie levels after the
single dose of IV citrulline. Pharmacokinetic data included plasma
half-life (t 1/2), elimination constant (Kel), volume of
distribution (Vd), and plasma clearance (CLp).
[0373] Plasma citrulline levels rapidly increased and demonstrated
a t 1/2=1.5 hrs, Kel=0.462 hr.sup.-1, Vd=2.25 L, and CLp=1.05 L/hr.
However, the effect of citrulline on plasma arginine was of
interest because it is the substrate for NO synthase. The
concentration curve of plasma arginine levels is represented in
FIG. 13. Based on this curve, the pharmacokinetics of plasma
arginine are as follows: t 1/2=18 hrs; Kel=0.039 hr.sup.-1; Vd=2.85
L; CLp=0.11 L/hr. The long half-life and slow clearance indicates
that a single dose of IV citrulline is effective at maintaining
increased plasma arginine levels over a fairly long interval
without detrimental effects on hemodynamics.
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[0491] It will be understood that various details of the invention
may be changed without departing from the scope of the invention.
Furthermore, the foregoing description is for the purpose of
illustration only, and not for the purpose of limitation--the
invention being defined by the claims.
Sequence CWU 1
1
2315761DNAHomo sapiensCDS(124)..(4626) 1gtcagcctta aacactgact
gcacccctcc cagatttctt ttacattaac taaaaagtct 60tatcacacaa tctcataaaa
tttatgtaat ttcatttaat tttagccaca aatcatcttc 120aaa atg acg agg att
ttg aca gct ttc aaa gtg gtg agg aca ctg aag 168 Met Thr Arg Ile Leu
Thr Ala Phe Lys Val Val Arg Thr Leu Lys 1 5 10 15act ggt ttt ggc
ttt acc aat gtg act gca cac caa aaa tgg aaa ttt 216Thr Gly Phe Gly
Phe Thr Asn Val Thr Ala His Gln Lys Trp Lys Phe 20 25 30tca aga cct
ggc atc agg ctc ctt tct gtc aag gca cag aca gca cac 264Ser Arg Pro
Gly Ile Arg Leu Leu Ser Val Lys Ala Gln Thr Ala His 35 40 45att gtc
ctg gaa gat gga act aag atg aaa ggt tac tcc ttt ggc cat 312Ile Val
Leu Glu Asp Gly Thr Lys Met Lys Gly Tyr Ser Phe Gly His 50 55 60cca
tcc tct gtt gct ggt gaa gtg gtt ttt aat act ggc ctg gga ggg 360Pro
Ser Ser Val Ala Gly Glu Val Val Phe Asn Thr Gly Leu Gly Gly 65 70
75tac cca gaa gct att act gac cct gcc tac aaa gga cag att ctc aca
408Tyr Pro Glu Ala Ile Thr Asp Pro Ala Tyr Lys Gly Gln Ile Leu
Thr80 85 90 95atg gcc aac cct att att ggg aat ggt gga gct cct gat
act act gct 456Met Ala Asn Pro Ile Ile Gly Asn Gly Gly Ala Pro Asp
Thr Thr Ala 100 105 110ctg gat gaa ctg gga ctt agc aaa tat ttg gag
tct aat gga atc aag 504Leu Asp Glu Leu Gly Leu Ser Lys Tyr Leu Glu
Ser Asn Gly Ile Lys 115 120 125gtt tca ggt ttg ctg gtg ctg gat tat
agt aaa gac tac aac cac tgg 552Val Ser Gly Leu Leu Val Leu Asp Tyr
Ser Lys Asp Tyr Asn His Trp 130 135 140ctg gct acc aag agt tta ggg
caa tgg cta cag gaa gaa aag gtt cct 600Leu Ala Thr Lys Ser Leu Gly
Gln Trp Leu Gln Glu Glu Lys Val Pro 145 150 155gca att tat gga gtg
gac aca aga atg ctg act aaa ata att cgg gat 648Ala Ile Tyr Gly Val
Asp Thr Arg Met Leu Thr Lys Ile Ile Arg Asp160 165 170 175aag ggt
acc atg ctt ggg aag att gaa ttt gaa ggt cag cct gtg gat 696Lys Gly
Thr Met Leu Gly Lys Ile Glu Phe Glu Gly Gln Pro Val Asp 180 185
190ttt gtg gat cca aat aaa cag aat ttg att gct gag gtt tca acc aag
744Phe Val Asp Pro Asn Lys Gln Asn Leu Ile Ala Glu Val Ser Thr Lys
195 200 205gat gtc aaa gtg tac ggc aaa gga aac ccc aca aaa gtg gta
gct gta 792Asp Val Lys Val Tyr Gly Lys Gly Asn Pro Thr Lys Val Val
Ala Val 210 215 220gac tgt ggg att aaa aac aat gta atc cgc ctg cta
gta aag cga gga 840Asp Cys Gly Ile Lys Asn Asn Val Ile Arg Leu Leu
Val Lys Arg Gly 225 230 235gct gaa gtg cac tta gtt ccc tgg aac cat
gat ttc acc aag atg gag 888Ala Glu Val His Leu Val Pro Trp Asn His
Asp Phe Thr Lys Met Glu240 245 250 255tat gat ggg att ttg atc gcg
gga gga ccg ggg aac cca gct ctt gca 936Tyr Asp Gly Ile Leu Ile Ala
Gly Gly Pro Gly Asn Pro Ala Leu Ala 260 265 270gaa cca cta att cag
aat gtc aga aag att ttg gag agt gat cgc aag 984Glu Pro Leu Ile Gln
Asn Val Arg Lys Ile Leu Glu Ser Asp Arg Lys 275 280 285gag cca ttg
ttt gga atc agt aca gga aac tta ata aca gga ttg gct 1032Glu Pro Leu
Phe Gly Ile Ser Thr Gly Asn Leu Ile Thr Gly Leu Ala 290 295 300gct
ggt gcc aaa acc tac aag atg tcc atg gcc aac aga ggg cag aat 1080Ala
Gly Ala Lys Thr Tyr Lys Met Ser Met Ala Asn Arg Gly Gln Asn 305 310
315cag cct gtt ttg aat atc aca aac aaa cag gct ttc att act gct cag
1128Gln Pro Val Leu Asn Ile Thr Asn Lys Gln Ala Phe Ile Thr Ala
Gln320 325 330 335aat cat ggc tat gcc ttg gac aac acc ctc cct gct
ggc tgg aaa cca 1176Asn His Gly Tyr Ala Leu Asp Asn Thr Leu Pro Ala
Gly Trp Lys Pro 340 345 350ctt ttt gtg aat gtc aac gat caa aca aat
gag ggg att atg cat gag 1224Leu Phe Val Asn Val Asn Asp Gln Thr Asn
Glu Gly Ile Met His Glu 355 360 365agc aaa ccc ttc ttc gct gtg cag
ttc cac cca gag gtc acc ccg ggg 1272Ser Lys Pro Phe Phe Ala Val Gln
Phe His Pro Glu Val Thr Pro Gly 370 375 380cca ata gac act gag tac
ctg ttt gat tcc ttt ttc tca ctg ata aag 1320Pro Ile Asp Thr Glu Tyr
Leu Phe Asp Ser Phe Phe Ser Leu Ile Lys 385 390 395aaa gga aaa gct
acc acc att aca tca gtc tta ccg aag cca gca cta 1368Lys Gly Lys Ala
Thr Thr Ile Thr Ser Val Leu Pro Lys Pro Ala Leu400 405 410 415gtt
gca tct cgg gtt gag gtt tcc aaa gtc ctt att cta gga tca gga 1416Val
Ala Ser Arg Val Glu Val Ser Lys Val Leu Ile Leu Gly Ser Gly 420 425
430ggt ctg tcc att ggt cag gct gga gaa ttt gat tac tca gga tct caa
1464Gly Leu Ser Ile Gly Gln Ala Gly Glu Phe Asp Tyr Ser Gly Ser Gln
435 440 445gct gta aaa gcc atg aag gaa gaa aat gtc aaa act gtt ctg
atg aac 1512Ala Val Lys Ala Met Lys Glu Glu Asn Val Lys Thr Val Leu
Met Asn 450 455 460cca aac att gca tca gtc cag acc aat gag gtg ggc
tta aag caa gcg 1560Pro Asn Ile Ala Ser Val Gln Thr Asn Glu Val Gly
Leu Lys Gln Ala 465 470 475gat act gtc tac ttt ctt ccc atc acc cct
cag ttt gtc aca gag gtc 1608Asp Thr Val Tyr Phe Leu Pro Ile Thr Pro
Gln Phe Val Thr Glu Val480 485 490 495atc aag gca gaa cag cca gat
ggg tta att ctg ggc atg ggt ggc cag 1656Ile Lys Ala Glu Gln Pro Asp
Gly Leu Ile Leu Gly Met Gly Gly Gln 500 505 510aca gct ctg aac tgt
gga gtg gaa cta ttc aag aga ggt gtg ctc aag 1704Thr Ala Leu Asn Cys
Gly Val Glu Leu Phe Lys Arg Gly Val Leu Lys 515 520 525gaa tat ggt
gtg aaa gtc ctg gga act tca gtt gag tcc att atg gct 1752Glu Tyr Gly
Val Lys Val Leu Gly Thr Ser Val Glu Ser Ile Met Ala 530 535 540acg
gaa gac agg cag ctg ttt tca gat aaa cta aat gag atc aat gaa 1800Thr
Glu Asp Arg Gln Leu Phe Ser Asp Lys Leu Asn Glu Ile Asn Glu 545 550
555aag att gct cca agt ttt gca gtg gaa tcg att gag gat gca ctg aag
1848Lys Ile Ala Pro Ser Phe Ala Val Glu Ser Ile Glu Asp Ala Leu
Lys560 565 570 575gca gca gac acc att ggc tac cca gtg atg atc cgt
tcc gcc tat gca 1896Ala Ala Asp Thr Ile Gly Tyr Pro Val Met Ile Arg
Ser Ala Tyr Ala 580 585 590ctg ggt ggg tta ggc tca ggc atc tgt ccc
aac aga gag act ttg atg 1944Leu Gly Gly Leu Gly Ser Gly Ile Cys Pro
Asn Arg Glu Thr Leu Met 595 600 605gac ctc agc aca aag gcc ttt gct
atg acc aac caa att ctg gtg gag 1992Asp Leu Ser Thr Lys Ala Phe Ala
Met Thr Asn Gln Ile Leu Val Glu 610 615 620aag tca gtg aca ggt tgg
aaa gaa ata gaa tat gaa gtg gtt cga gat 2040Lys Ser Val Thr Gly Trp
Lys Glu Ile Glu Tyr Glu Val Val Arg Asp 625 630 635gct gat gac aat
tgt gtc act gtc tgt aac atg gaa aat gtt gat gcc 2088Ala Asp Asp Asn
Cys Val Thr Val Cys Asn Met Glu Asn Val Asp Ala640 645 650 655atg
ggt gtt cac aca ggt gac tca gtt gtt gtg gct cct gcc cag aca 2136Met
Gly Val His Thr Gly Asp Ser Val Val Val Ala Pro Ala Gln Thr 660 665
670ctc tcc aat gcc gag ttt cag atg ttg aga cgt act tca atc aat gtt
2184Leu Ser Asn Ala Glu Phe Gln Met Leu Arg Arg Thr Ser Ile Asn Val
675 680 685gtt cgc cac ttg ggc att gtg ggt gaa tgc aac att cag ttt
gcc ctt 2232Val Arg His Leu Gly Ile Val Gly Glu Cys Asn Ile Gln Phe
Ala Leu 690 695 700cat cct acc tca atg gaa tac tgc atc att gaa gtg
aat gcc aga ctg 2280His Pro Thr Ser Met Glu Tyr Cys Ile Ile Glu Val
Asn Ala Arg Leu 705 710 715tcc cga agc tct gct ctg gcc tca aaa gcc
act ggc tac cca ttg gca 2328Ser Arg Ser Ser Ala Leu Ala Ser Lys Ala
Thr Gly Tyr Pro Leu Ala720 725 730 735ttc att gct gca aag att gcc
cta gga atc cca ctt cca gaa att aag 2376Phe Ile Ala Ala Lys Ile Ala
Leu Gly Ile Pro Leu Pro Glu Ile Lys 740 745 750aac gtc gta tcc ggg
aag aca tca gcc tgt ttt gaa cct agc ctg gat 2424Asn Val Val Ser Gly
Lys Thr Ser Ala Cys Phe Glu Pro Ser Leu Asp 755 760 765tac atg gtc
acc aag att ccc cgc tgg gat ctt gac cgt ttt cat gga 2472Tyr Met Val
Thr Lys Ile Pro Arg Trp Asp Leu Asp Arg Phe His Gly 770 775 780aca
tct agc cga att ggt agc tct atg aaa agt gta gga gag gtc atg 2520Thr
Ser Ser Arg Ile Gly Ser Ser Met Lys Ser Val Gly Glu Val Met 785 790
795gct att ggt cgt acc ttt gag gag agt ttc cag aaa gct tta cgg atg
2568Ala Ile Gly Arg Thr Phe Glu Glu Ser Phe Gln Lys Ala Leu Arg
Met800 805 810 815tgc cac cca tct ata gaa ggt ttc act ccc cgt ctc
cca atg aac aaa 2616Cys His Pro Ser Ile Glu Gly Phe Thr Pro Arg Leu
Pro Met Asn Lys 820 825 830gaa tgg cca tct aat tta gat ctt aga aaa
gag ttg tct gaa cca agc 2664Glu Trp Pro Ser Asn Leu Asp Leu Arg Lys
Glu Leu Ser Glu Pro Ser 835 840 845agc acg cgt atc tat gcc att gcc
aag gcc att gat gac aac atg tcc 2712Ser Thr Arg Ile Tyr Ala Ile Ala
Lys Ala Ile Asp Asp Asn Met Ser 850 855 860ctt gat gag att gag aag
ctc aca tac att gac aag tgg ttt ttg tat 2760Leu Asp Glu Ile Glu Lys
Leu Thr Tyr Ile Asp Lys Trp Phe Leu Tyr 865 870 875aag atg cgt gat
att tta aac atg gaa aag aca ctg aaa ggg ctc aac 2808Lys Met Arg Asp
Ile Leu Asn Met Glu Lys Thr Leu Lys Gly Leu Asn880 885 890 895agt
gag tcc atg aca gaa gaa acc ctg aaa agg gca aag gag att ggg 2856Ser
Glu Ser Met Thr Glu Glu Thr Leu Lys Arg Ala Lys Glu Ile Gly 900 905
910ttc tca gat aag cag att tca aaa tgc ctt ggg ctc act gag gcc cag
2904Phe Ser Asp Lys Gln Ile Ser Lys Cys Leu Gly Leu Thr Glu Ala Gln
915 920 925aca agg gag ctg agg tta aag aaa aac atc cac cct tgg gtt
aaa cag 2952Thr Arg Glu Leu Arg Leu Lys Lys Asn Ile His Pro Trp Val
Lys Gln 930 935 940att gat aca ctg gct gca gaa tac cca tca gta aca
aac tat ctc tat 3000Ile Asp Thr Leu Ala Ala Glu Tyr Pro Ser Val Thr
Asn Tyr Leu Tyr 945 950 955gtt acc tac aat ggt cag gag cat gat gtc
aat ttt gat gac cat gga 3048Val Thr Tyr Asn Gly Gln Glu His Asp Val
Asn Phe Asp Asp His Gly960 965 970 975atg atg gtg cta ggc tgt ggt
cca tat cac att ggc agc agt gtg gaa 3096Met Met Val Leu Gly Cys Gly
Pro Tyr His Ile Gly Ser Ser Val Glu 980 985 990ttt gat tgg tgt gct
gtc tct agt atc cgc aca ctg cgt caa ctt ggc 3144Phe Asp Trp Cys Ala
Val Ser Ser Ile Arg Thr Leu Arg Gln Leu Gly 995 1000 1005aag aag
acg gtg gtg gtg aat tgc aat cct gag act gtg agc aca 3189Lys Lys Thr
Val Val Val Asn Cys Asn Pro Glu Thr Val Ser Thr 1010 1015 1020gac
ttt gat gag tgt gac aaa ctg tac ttt gaa gag ttg tcc ttg 3234Asp Phe
Asp Glu Cys Asp Lys Leu Tyr Phe Glu Glu Leu Ser Leu 1025 1030
1035gag aga atc cta gac atc tac cat cag gag gca tgt ggt ggc tgc
3279Glu Arg Ile Leu Asp Ile Tyr His Gln Glu Ala Cys Gly Gly Cys
1040 1045 1050atc ata tca gtt gga ggc cag att cca aac aac ctg gca
gtt cct 3324Ile Ile Ser Val Gly Gly Gln Ile Pro Asn Asn Leu Ala Val
Pro 1055 1060 1065cta tac aag aat ggt gtc aag atc atg ggc aca agc
ccc ctg cag 3369Leu Tyr Lys Asn Gly Val Lys Ile Met Gly Thr Ser Pro
Leu Gln 1070 1075 1080atc gac agg gct gag gat cgc tcc atc ttc tca
gct gtc ttg gat 3414Ile Asp Arg Ala Glu Asp Arg Ser Ile Phe Ser Ala
Val Leu Asp 1085 1090 1095gag ctg aag gtg gct cag gca cct tgg aaa
gct gtt aat act ttg 3459Glu Leu Lys Val Ala Gln Ala Pro Trp Lys Ala
Val Asn Thr Leu 1100 1105 1110aat gaa gca ctg gaa ttt gca aag tct
gtg gac tac ccc tgc ttg 3504Asn Glu Ala Leu Glu Phe Ala Lys Ser Val
Asp Tyr Pro Cys Leu 1115 1120 1125ttg agg cct tcc tat gtt ttg agt
ggg tct gct atg aat gtg gta 3549Leu Arg Pro Ser Tyr Val Leu Ser Gly
Ser Ala Met Asn Val Val 1130 1135 1140ttc tct gag gat gag atg aaa
aaa ttc cta gaa gag gcg act aga 3594Phe Ser Glu Asp Glu Met Lys Lys
Phe Leu Glu Glu Ala Thr Arg 1145 1150 1155gtt tct cag gag cac cca
gtg gtc ctg aca aaa ttt gtt gaa ggg 3639Val Ser Gln Glu His Pro Val
Val Leu Thr Lys Phe Val Glu Gly 1160 1165 1170gcc cga gaa gta gaa
atg gac gct gtt ggc aaa gat gga agg gtt 3684Ala Arg Glu Val Glu Met
Asp Ala Val Gly Lys Asp Gly Arg Val 1175 1180 1185atc tct cat gcc
atc tct gaa cat gtt gaa gat gca ggt gtc cac 3729Ile Ser His Ala Ile
Ser Glu His Val Glu Asp Ala Gly Val His 1190 1195 1200tcg gga gat
gcc act ctg atg ctg ccc aca caa acc atc agc caa 3774Ser Gly Asp Ala
Thr Leu Met Leu Pro Thr Gln Thr Ile Ser Gln 1205 1210 1215ggg gcc
att gaa aag gtg aag gat gct acc cgg aag att gca aag 3819Gly Ala Ile
Glu Lys Val Lys Asp Ala Thr Arg Lys Ile Ala Lys 1220 1225 1230gct
ttt gcc atc tct ggt cca ttc aac gtc caa ttt ctt gtc aaa 3864Ala Phe
Ala Ile Ser Gly Pro Phe Asn Val Gln Phe Leu Val Lys 1235 1240
1245gga aat gat gtc ttg gtg att gag tgt aac ttg aga gct tct cga
3909Gly Asn Asp Val Leu Val Ile Glu Cys Asn Leu Arg Ala Ser Arg
1250 1255 1260tcc ttc ccc ttt gtt tcc aag act ctt ggg gtt gac ttc
att gat 3954Ser Phe Pro Phe Val Ser Lys Thr Leu Gly Val Asp Phe Ile
Asp 1265 1270 1275gtg gcc acc aag gtg atg att gga gag aat gtt gat
gag aaa cat 3999Val Ala Thr Lys Val Met Ile Gly Glu Asn Val Asp Glu
Lys His 1280 1285 1290ctt cca aca ttg gac cat ccc ata att cct gct
gac tat gtt gca 4044Leu Pro Thr Leu Asp His Pro Ile Ile Pro Ala Asp
Tyr Val Ala 1295 1300 1305att aag gct ccc atg ttt tcc tgg ccc cgg
ttg agg gat gct gac 4089Ile Lys Ala Pro Met Phe Ser Trp Pro Arg Leu
Arg Asp Ala Asp 1310 1315 1320ccc att ctg aga tgt gag atg gct tcc
act gga gag gtg gct tgc 4134Pro Ile Leu Arg Cys Glu Met Ala Ser Thr
Gly Glu Val Ala Cys 1325 1330 1335ttt ggt gaa ggt att cat aca gcc
ttc cta aag gca atg ctt tcc 4179Phe Gly Glu Gly Ile His Thr Ala Phe
Leu Lys Ala Met Leu Ser 1340 1345 1350aca gga ttt aag ata ccc cag
aaa ggc atc ctg ata ggc atc cag 4224Thr Gly Phe Lys Ile Pro Gln Lys
Gly Ile Leu Ile Gly Ile Gln 1355 1360 1365caa tca ttc cgg cca aga
ttc ctt ggt gtg gct gaa caa tta cac 4269Gln Ser Phe Arg Pro Arg Phe
Leu Gly Val Ala Glu Gln Leu His 1370 1375 1380aat gaa ggt ttc aag
ctg ttt gcc acg gaa gcc aca tca gac tgg 4314Asn Glu Gly Phe Lys Leu
Phe Ala Thr Glu Ala Thr Ser Asp Trp 1385 1390 1395ctc aac gcc aac
aat gtc cct gcc aac cca gtg gca tgg ccg tct 4359Leu Asn Ala Asn Asn
Val Pro Ala Asn Pro Val Ala Trp Pro Ser 1400 1405 1410caa gaa gga
cag aat ccc agc ctc tct tcc atc aga aaa ttg att 4404Gln Glu Gly Gln
Asn Pro Ser Leu Ser Ser Ile Arg Lys Leu Ile 1415 1420 1425aga gat
ggc agc att gac cta gtg att aac ctt ccc aac aac aac 4449Arg Asp Gly
Ser Ile Asp Leu Val Ile Asn Leu Pro Asn Asn Asn 1430 1435 1440act
aaa ttt gtc cat gat aat tat gtg att cgg agg aca gct gtt 4494Thr Lys
Phe Val His Asp Asn Tyr Val Ile Arg Arg Thr Ala Val 1445 1450
1455gat agt gga atc cct ctc ctc act aat ttt cag gtg acc aaa ctt
4539Asp Ser Gly Ile Pro Leu Leu Thr Asn Phe Gln Val Thr Lys Leu
1460 1465 1470ttt gct gaa gct gtg cag aaa tct cgc aag gtg gac tcc
aag agt 4584Phe Ala Glu Ala Val Gln Lys Ser Arg Lys Val Asp Ser Lys
Ser 1475 1480 1485ctt ttc cac tac agg cag tac agt gct gga aaa gca
gca tag 4626Leu Phe His
Tyr Arg Gln Tyr Ser Ala Gly Lys Ala Ala 1490 1495 1500agatgcagac
accccagccc cattattaaa tcaacctgag ccacatgtta tctaaaggaa
4686ctgattcaca actttctcag agatgaatat tgataactaa acttcatttc
agtttacttt 4746gttatgcctt aatattctgt gtcttttgca attaaattgt
cagtcacttc ttcaaaacct 4806tacagtcctt cctaagttac tcttcatgag
atttcatcca tttactaata ctgtattttt 4866ggtggactag gcttgcctat
gtgcttatgt gtagcttttt actttttatg gtgctgatta 4926atggtgatca
aggtaggaaa agttgctgtt ctattttctg aactctttct atactttaag
4986atactctatt tttaaaacac tatctgcaaa ctcaggacac tttaacaggg
cagaatactc 5046taaaaacttg ataaaatgaa atatagattt aatttatgaa
ccttccatca tgatgtttgt 5106gtattgcttc tttttggatc ctcattctca
cccatttggc taatccagga atattgttat 5166cccttcccat tatattgaag
ttgagaaatg tgacagaggc atttagagta tggacttttc 5226ttttcttttt
ctttttcttt ttttcttttt gagatggagt cacactctcc aggctggagt
5286gcagtggcac aatctcggct cactgcaatt tgcgtctccc aagttcaagc
gattctcctg 5346ctttagacta tggatttctt taaggaatac tggtttgcag
ttttgttttc tggactatat 5406cagcagatgg tagacagtgt ttatgtagat
gtgttgttgt ttttatcatt ggattttaac 5466ttggcccgag tgaaataatc
agatttttgt cattcacact ctcccccagt tttggaataa 5526cttggaagta
aggttcattc ccttaagacg atggattctg ttgaactatg gggtcccaca
5586ctgcactatt aattccaccc actgtaaggg caaggacacc attccttcta
catataagaa 5646aaaagtctct ccccaagggc agcctttgtt acttttaaat
attttctgtt attacaagtg 5706ctctaattgt gaacttttaa ataaaatact
attaagaggt aaaaaaaaaa aaaaa 576121500PRTHomo sapiens 2Met Thr Arg
Ile Leu Thr Ala Phe Lys Val Val Arg Thr Leu Lys Thr1 5 10 15Gly Phe
Gly Phe Thr Asn Val Thr Ala His Gln Lys Trp Lys Phe Ser 20 25 30Arg
Pro Gly Ile Arg Leu Leu Ser Val Lys Ala Gln Thr Ala His Ile 35 40
45Val Leu Glu Asp Gly Thr Lys Met Lys Gly Tyr Ser Phe Gly His Pro
50 55 60Ser Ser Val Ala Gly Glu Val Val Phe Asn Thr Gly Leu Gly Gly
Tyr65 70 75 80Pro Glu Ala Ile Thr Asp Pro Ala Tyr Lys Gly Gln Ile
Leu Thr Met 85 90 95Ala Asn Pro Ile Ile Gly Asn Gly Gly Ala Pro Asp
Thr Thr Ala Leu 100 105 110Asp Glu Leu Gly Leu Ser Lys Tyr Leu Glu
Ser Asn Gly Ile Lys Val 115 120 125Ser Gly Leu Leu Val Leu Asp Tyr
Ser Lys Asp Tyr Asn His Trp Leu 130 135 140Ala Thr Lys Ser Leu Gly
Gln Trp Leu Gln Glu Glu Lys Val Pro Ala145 150 155 160Ile Tyr Gly
Val Asp Thr Arg Met Leu Thr Lys Ile Ile Arg Asp Lys 165 170 175Gly
Thr Met Leu Gly Lys Ile Glu Phe Glu Gly Gln Pro Val Asp Phe 180 185
190Val Asp Pro Asn Lys Gln Asn Leu Ile Ala Glu Val Ser Thr Lys Asp
195 200 205Val Lys Val Tyr Gly Lys Gly Asn Pro Thr Lys Val Val Ala
Val Asp 210 215 220Cys Gly Ile Lys Asn Asn Val Ile Arg Leu Leu Val
Lys Arg Gly Ala225 230 235 240Glu Val His Leu Val Pro Trp Asn His
Asp Phe Thr Lys Met Glu Tyr 245 250 255Asp Gly Ile Leu Ile Ala Gly
Gly Pro Gly Asn Pro Ala Leu Ala Glu 260 265 270Pro Leu Ile Gln Asn
Val Arg Lys Ile Leu Glu Ser Asp Arg Lys Glu 275 280 285Pro Leu Phe
Gly Ile Ser Thr Gly Asn Leu Ile Thr Gly Leu Ala Ala 290 295 300Gly
Ala Lys Thr Tyr Lys Met Ser Met Ala Asn Arg Gly Gln Asn Gln305 310
315 320Pro Val Leu Asn Ile Thr Asn Lys Gln Ala Phe Ile Thr Ala Gln
Asn 325 330 335His Gly Tyr Ala Leu Asp Asn Thr Leu Pro Ala Gly Trp
Lys Pro Leu 340 345 350Phe Val Asn Val Asn Asp Gln Thr Asn Glu Gly
Ile Met His Glu Ser 355 360 365Lys Pro Phe Phe Ala Val Gln Phe His
Pro Glu Val Thr Pro Gly Pro 370 375 380Ile Asp Thr Glu Tyr Leu Phe
Asp Ser Phe Phe Ser Leu Ile Lys Lys385 390 395 400Gly Lys Ala Thr
Thr Ile Thr Ser Val Leu Pro Lys Pro Ala Leu Val 405 410 415Ala Ser
Arg Val Glu Val Ser Lys Val Leu Ile Leu Gly Ser Gly Gly 420 425
430Leu Ser Ile Gly Gln Ala Gly Glu Phe Asp Tyr Ser Gly Ser Gln Ala
435 440 445Val Lys Ala Met Lys Glu Glu Asn Val Lys Thr Val Leu Met
Asn Pro 450 455 460Asn Ile Ala Ser Val Gln Thr Asn Glu Val Gly Leu
Lys Gln Ala Asp465 470 475 480Thr Val Tyr Phe Leu Pro Ile Thr Pro
Gln Phe Val Thr Glu Val Ile 485 490 495Lys Ala Glu Gln Pro Asp Gly
Leu Ile Leu Gly Met Gly Gly Gln Thr 500 505 510Ala Leu Asn Cys Gly
Val Glu Leu Phe Lys Arg Gly Val Leu Lys Glu 515 520 525Tyr Gly Val
Lys Val Leu Gly Thr Ser Val Glu Ser Ile Met Ala Thr 530 535 540Glu
Asp Arg Gln Leu Phe Ser Asp Lys Leu Asn Glu Ile Asn Glu Lys545 550
555 560Ile Ala Pro Ser Phe Ala Val Glu Ser Ile Glu Asp Ala Leu Lys
Ala 565 570 575Ala Asp Thr Ile Gly Tyr Pro Val Met Ile Arg Ser Ala
Tyr Ala Leu 580 585 590Gly Gly Leu Gly Ser Gly Ile Cys Pro Asn Arg
Glu Thr Leu Met Asp 595 600 605Leu Ser Thr Lys Ala Phe Ala Met Thr
Asn Gln Ile Leu Val Glu Lys 610 615 620Ser Val Thr Gly Trp Lys Glu
Ile Glu Tyr Glu Val Val Arg Asp Ala625 630 635 640Asp Asp Asn Cys
Val Thr Val Cys Asn Met Glu Asn Val Asp Ala Met 645 650 655Gly Val
His Thr Gly Asp Ser Val Val Val Ala Pro Ala Gln Thr Leu 660 665
670Ser Asn Ala Glu Phe Gln Met Leu Arg Arg Thr Ser Ile Asn Val Val
675 680 685Arg His Leu Gly Ile Val Gly Glu Cys Asn Ile Gln Phe Ala
Leu His 690 695 700Pro Thr Ser Met Glu Tyr Cys Ile Ile Glu Val Asn
Ala Arg Leu Ser705 710 715 720Arg Ser Ser Ala Leu Ala Ser Lys Ala
Thr Gly Tyr Pro Leu Ala Phe 725 730 735Ile Ala Ala Lys Ile Ala Leu
Gly Ile Pro Leu Pro Glu Ile Lys Asn 740 745 750Val Val Ser Gly Lys
Thr Ser Ala Cys Phe Glu Pro Ser Leu Asp Tyr 755 760 765Met Val Thr
Lys Ile Pro Arg Trp Asp Leu Asp Arg Phe His Gly Thr 770 775 780Ser
Ser Arg Ile Gly Ser Ser Met Lys Ser Val Gly Glu Val Met Ala785 790
795 800Ile Gly Arg Thr Phe Glu Glu Ser Phe Gln Lys Ala Leu Arg Met
Cys 805 810 815His Pro Ser Ile Glu Gly Phe Thr Pro Arg Leu Pro Met
Asn Lys Glu 820 825 830Trp Pro Ser Asn Leu Asp Leu Arg Lys Glu Leu
Ser Glu Pro Ser Ser 835 840 845Thr Arg Ile Tyr Ala Ile Ala Lys Ala
Ile Asp Asp Asn Met Ser Leu 850 855 860Asp Glu Ile Glu Lys Leu Thr
Tyr Ile Asp Lys Trp Phe Leu Tyr Lys865 870 875 880Met Arg Asp Ile
Leu Asn Met Glu Lys Thr Leu Lys Gly Leu Asn Ser 885 890 895Glu Ser
Met Thr Glu Glu Thr Leu Lys Arg Ala Lys Glu Ile Gly Phe 900 905
910Ser Asp Lys Gln Ile Ser Lys Cys Leu Gly Leu Thr Glu Ala Gln Thr
915 920 925Arg Glu Leu Arg Leu Lys Lys Asn Ile His Pro Trp Val Lys
Gln Ile 930 935 940Asp Thr Leu Ala Ala Glu Tyr Pro Ser Val Thr Asn
Tyr Leu Tyr Val945 950 955 960Thr Tyr Asn Gly Gln Glu His Asp Val
Asn Phe Asp Asp His Gly Met 965 970 975Met Val Leu Gly Cys Gly Pro
Tyr His Ile Gly Ser Ser Val Glu Phe 980 985 990Asp Trp Cys Ala Val
Ser Ser Ile Arg Thr Leu Arg Gln Leu Gly Lys 995 1000 1005Lys Thr
Val Val Val Asn Cys Asn Pro Glu Thr Val Ser Thr Asp 1010 1015
1020Phe Asp Glu Cys Asp Lys Leu Tyr Phe Glu Glu Leu Ser Leu Glu
1025 1030 1035Arg Ile Leu Asp Ile Tyr His Gln Glu Ala Cys Gly Gly
Cys Ile 1040 1045 1050Ile Ser Val Gly Gly Gln Ile Pro Asn Asn Leu
Ala Val Pro Leu 1055 1060 1065Tyr Lys Asn Gly Val Lys Ile Met Gly
Thr Ser Pro Leu Gln Ile 1070 1075 1080Asp Arg Ala Glu Asp Arg Ser
Ile Phe Ser Ala Val Leu Asp Glu 1085 1090 1095Leu Lys Val Ala Gln
Ala Pro Trp Lys Ala Val Asn Thr Leu Asn 1100 1105 1110Glu Ala Leu
Glu Phe Ala Lys Ser Val Asp Tyr Pro Cys Leu Leu 1115 1120 1125Arg
Pro Ser Tyr Val Leu Ser Gly Ser Ala Met Asn Val Val Phe 1130 1135
1140Ser Glu Asp Glu Met Lys Lys Phe Leu Glu Glu Ala Thr Arg Val
1145 1150 1155Ser Gln Glu His Pro Val Val Leu Thr Lys Phe Val Glu
Gly Ala 1160 1165 1170Arg Glu Val Glu Met Asp Ala Val Gly Lys Asp
Gly Arg Val Ile 1175 1180 1185Ser His Ala Ile Ser Glu His Val Glu
Asp Ala Gly Val His Ser 1190 1195 1200Gly Asp Ala Thr Leu Met Leu
Pro Thr Gln Thr Ile Ser Gln Gly 1205 1210 1215Ala Ile Glu Lys Val
Lys Asp Ala Thr Arg Lys Ile Ala Lys Ala 1220 1225 1230Phe Ala Ile
Ser Gly Pro Phe Asn Val Gln Phe Leu Val Lys Gly 1235 1240 1245Asn
Asp Val Leu Val Ile Glu Cys Asn Leu Arg Ala Ser Arg Ser 1250 1255
1260Phe Pro Phe Val Ser Lys Thr Leu Gly Val Asp Phe Ile Asp Val
1265 1270 1275Ala Thr Lys Val Met Ile Gly Glu Asn Val Asp Glu Lys
His Leu 1280 1285 1290Pro Thr Leu Asp His Pro Ile Ile Pro Ala Asp
Tyr Val Ala Ile 1295 1300 1305Lys Ala Pro Met Phe Ser Trp Pro Arg
Leu Arg Asp Ala Asp Pro 1310 1315 1320Ile Leu Arg Cys Glu Met Ala
Ser Thr Gly Glu Val Ala Cys Phe 1325 1330 1335Gly Glu Gly Ile His
Thr Ala Phe Leu Lys Ala Met Leu Ser Thr 1340 1345 1350Gly Phe Lys
Ile Pro Gln Lys Gly Ile Leu Ile Gly Ile Gln Gln 1355 1360 1365Ser
Phe Arg Pro Arg Phe Leu Gly Val Ala Glu Gln Leu His Asn 1370 1375
1380Glu Gly Phe Lys Leu Phe Ala Thr Glu Ala Thr Ser Asp Trp Leu
1385 1390 1395Asn Ala Asn Asn Val Pro Ala Asn Pro Val Ala Trp Pro
Ser Gln 1400 1405 1410Glu Gly Gln Asn Pro Ser Leu Ser Ser Ile Arg
Lys Leu Ile Arg 1415 1420 1425Asp Gly Ser Ile Asp Leu Val Ile Asn
Leu Pro Asn Asn Asn Thr 1430 1435 1440Lys Phe Val His Asp Asn Tyr
Val Ile Arg Arg Thr Ala Val Asp 1445 1450 1455Ser Gly Ile Pro Leu
Leu Thr Asn Phe Gln Val Thr Lys Leu Phe 1460 1465 1470Ala Glu Ala
Val Gln Lys Ser Arg Lys Val Asp Ser Lys Ser Leu 1475 1480 1485Phe
His Tyr Arg Gln Tyr Ser Ala Gly Lys Ala Ala 1490 1495
150035761DNAHomo sapiensCDS(124)..(4626) 3gtcagcctta aacactgact
gcacccctcc cagatttctt ttacattaac taaaaagtct 60tatcacacaa tctcataaaa
tttatgtaat ttcatttaat tttagccaca aatcatcttc 120aaa atg acg agg att
ttg aca gct ttc aaa gtg gtg agg aca ctg aag 168 Met Thr Arg Ile Leu
Thr Ala Phe Lys Val Val Arg Thr Leu Lys 1 5 10 15act ggt ttt ggc
ttt acc aat gtg act gca cac caa aaa tgg aaa ttt 216Thr Gly Phe Gly
Phe Thr Asn Val Thr Ala His Gln Lys Trp Lys Phe 20 25 30tca aga cct
ggc atc agg ctc ctt tct gtc aag gca cag aca gca cac 264Ser Arg Pro
Gly Ile Arg Leu Leu Ser Val Lys Ala Gln Thr Ala His 35 40 45att gtc
ctg gaa gat gga act aag atg aaa ggt tac tcc ttt ggc cat 312Ile Val
Leu Glu Asp Gly Thr Lys Met Lys Gly Tyr Ser Phe Gly His 50 55 60cca
tcc tct gtt gct ggt gaa gtg gtt ttt aat act ggc ctg gga ggg 360Pro
Ser Ser Val Ala Gly Glu Val Val Phe Asn Thr Gly Leu Gly Gly 65 70
75tac cca gaa gct att act gac cct gcc tac aaa gga cag att ctc aca
408Tyr Pro Glu Ala Ile Thr Asp Pro Ala Tyr Lys Gly Gln Ile Leu
Thr80 85 90 95atg gcc aac cct att att ggg aat ggt gga gct cct gat
act act gct 456Met Ala Asn Pro Ile Ile Gly Asn Gly Gly Ala Pro Asp
Thr Thr Ala 100 105 110ctg gat gaa ctg gga ctt agc aaa tat ttg gag
tct aat gga atc aag 504Leu Asp Glu Leu Gly Leu Ser Lys Tyr Leu Glu
Ser Asn Gly Ile Lys 115 120 125gtt tca ggt ttg ctg gtg ctg gat tat
agt aaa gac tac aac cac tgg 552Val Ser Gly Leu Leu Val Leu Asp Tyr
Ser Lys Asp Tyr Asn His Trp 130 135 140ctg gct acc aag agt tta ggg
caa tgg cta cag gaa gaa aag gtt cct 600Leu Ala Thr Lys Ser Leu Gly
Gln Trp Leu Gln Glu Glu Lys Val Pro 145 150 155gca att tat gga gtg
gac aca aga atg ctg act aaa ata att cgg gat 648Ala Ile Tyr Gly Val
Asp Thr Arg Met Leu Thr Lys Ile Ile Arg Asp160 165 170 175aag ggt
acc atg ctt ggg aag att gaa ttt gaa ggt cag cct gtg gat 696Lys Gly
Thr Met Leu Gly Lys Ile Glu Phe Glu Gly Gln Pro Val Asp 180 185
190ttt gtg gat cca aat aaa cag aat ttg att gct gag gtt tca acc aag
744Phe Val Asp Pro Asn Lys Gln Asn Leu Ile Ala Glu Val Ser Thr Lys
195 200 205gat gtc aaa gtg tac ggc aaa gga aac ccc aca aaa gtg gta
gct gta 792Asp Val Lys Val Tyr Gly Lys Gly Asn Pro Thr Lys Val Val
Ala Val 210 215 220gac tgt ggg att aaa aac aat gta atc cgc ctg cta
gta aag cga gga 840Asp Cys Gly Ile Lys Asn Asn Val Ile Arg Leu Leu
Val Lys Arg Gly 225 230 235gct gaa gtg cac tta gtt ccc tgg aac cat
gat ttc acc aag atg gag 888Ala Glu Val His Leu Val Pro Trp Asn His
Asp Phe Thr Lys Met Glu240 245 250 255tat gat ggg att ttg atc gcg
gga gga ccg ggg aac cca gct ctt gca 936Tyr Asp Gly Ile Leu Ile Ala
Gly Gly Pro Gly Asn Pro Ala Leu Ala 260 265 270gaa cca cta att cag
aat gtc aga aag att ttg gag agt gat cgc aag 984Glu Pro Leu Ile Gln
Asn Val Arg Lys Ile Leu Glu Ser Asp Arg Lys 275 280 285gag cca ttg
ttt gga atc agt aca gga aac tta ata aca gga ttg gct 1032Glu Pro Leu
Phe Gly Ile Ser Thr Gly Asn Leu Ile Thr Gly Leu Ala 290 295 300gct
ggt gcc aaa acc tac aag atg tcc atg gcc aac aga ggg cag aat 1080Ala
Gly Ala Lys Thr Tyr Lys Met Ser Met Ala Asn Arg Gly Gln Asn 305 310
315cag cct gtt ttg aat atc aca aac aaa cag gct ttc att act gct cag
1128Gln Pro Val Leu Asn Ile Thr Asn Lys Gln Ala Phe Ile Thr Ala
Gln320 325 330 335aat cat ggc tat gcc ttg gac aac acc ctc cct gct
ggc tgg aaa cca 1176Asn His Gly Tyr Ala Leu Asp Asn Thr Leu Pro Ala
Gly Trp Lys Pro 340 345 350ctt ttt gtg aat gtc aac gat caa aca aat
gag ggg att atg cat gag 1224Leu Phe Val Asn Val Asn Asp Gln Thr Asn
Glu Gly Ile Met His Glu 355 360 365agc aaa ccc ttc ttc gct gtg cag
ttc cac cca gag gtc acc ccg ggg 1272Ser Lys Pro Phe Phe Ala Val Gln
Phe His Pro Glu Val Thr Pro Gly 370 375 380cca ata gac act gag tac
ctg ttt gat tcc ttt ttc tca ctg ata aag 1320Pro Ile Asp Thr Glu Tyr
Leu Phe Asp Ser Phe Phe Ser Leu Ile Lys 385 390 395aaa gga aaa gct
acc acc att aca tca gtc tta ccg aag cca gca cta 1368Lys Gly Lys Ala
Thr Thr Ile Thr Ser Val Leu Pro Lys Pro Ala Leu400 405 410 415gtt
gca tct cgg gtt gag gtt tcc aaa gtc ctt att cta gga tca gga 1416Val
Ala Ser Arg Val Glu Val Ser Lys Val Leu Ile Leu Gly Ser Gly 420 425
430ggt ctg tcc att ggt cag gct gga gaa ttt gat tac tca gga tct caa
1464Gly Leu Ser Ile Gly Gln Ala Gly Glu Phe Asp Tyr Ser Gly Ser Gln
435 440 445gct gta aaa gcc atg aag gaa gaa aat gtc aaa act gtt ctg
atg aac 1512Ala Val Lys Ala Met Lys Glu Glu Asn Val Lys Thr
Val Leu Met Asn 450 455 460cca aac att gca tca gtc cag acc aat gag
gtg ggc tta aag caa gcg 1560Pro Asn Ile Ala Ser Val Gln Thr Asn Glu
Val Gly Leu Lys Gln Ala 465 470 475gat act gtc tac ttt ctt ccc atc
acc cct cag ttt gtc aca gag gtc 1608Asp Thr Val Tyr Phe Leu Pro Ile
Thr Pro Gln Phe Val Thr Glu Val480 485 490 495atc aag gca gaa cag
cca gat ggg tta att ctg ggc atg ggt ggc cag 1656Ile Lys Ala Glu Gln
Pro Asp Gly Leu Ile Leu Gly Met Gly Gly Gln 500 505 510aca gct ctg
aac tgt gga gtg gaa cta ttc aag aga ggt gtg ctc aag 1704Thr Ala Leu
Asn Cys Gly Val Glu Leu Phe Lys Arg Gly Val Leu Lys 515 520 525gaa
tat ggt gtg aaa gtc ctg gga act tca gtt gag tcc att atg gct 1752Glu
Tyr Gly Val Lys Val Leu Gly Thr Ser Val Glu Ser Ile Met Ala 530 535
540acg gaa gac agg cag ctg ttt tca gat aaa cta aat gag atc aat gaa
1800Thr Glu Asp Arg Gln Leu Phe Ser Asp Lys Leu Asn Glu Ile Asn Glu
545 550 555aag att gct cca agt ttt gca gtg gaa tcg att gag gat gca
ctg aag 1848Lys Ile Ala Pro Ser Phe Ala Val Glu Ser Ile Glu Asp Ala
Leu Lys560 565 570 575gca gca gac acc att ggc tac cca gtg atg atc
cgt tcc gcc tat gca 1896Ala Ala Asp Thr Ile Gly Tyr Pro Val Met Ile
Arg Ser Ala Tyr Ala 580 585 590ctg ggt ggg tta ggc tca ggc atc tgt
ccc aac aga gag act ttg atg 1944Leu Gly Gly Leu Gly Ser Gly Ile Cys
Pro Asn Arg Glu Thr Leu Met 595 600 605gac ctc agc aca aag gcc ttt
gct atg acc aac caa att ctg gtg gag 1992Asp Leu Ser Thr Lys Ala Phe
Ala Met Thr Asn Gln Ile Leu Val Glu 610 615 620aag tca gtg aca ggt
tgg aaa gaa ata gaa tat gaa gtg gtt cga gat 2040Lys Ser Val Thr Gly
Trp Lys Glu Ile Glu Tyr Glu Val Val Arg Asp 625 630 635gct gat gac
aat tgt gtc act gtc tgt aac atg gaa aat gtt gat gcc 2088Ala Asp Asp
Asn Cys Val Thr Val Cys Asn Met Glu Asn Val Asp Ala640 645 650
655atg ggt gtt cac aca ggt gac tca gtt gtt gtg gct cct gcc cag aca
2136Met Gly Val His Thr Gly Asp Ser Val Val Val Ala Pro Ala Gln Thr
660 665 670ctc tcc aat gcc gag ttt cag atg ttg aga cgt act tca atc
aat gtt 2184Leu Ser Asn Ala Glu Phe Gln Met Leu Arg Arg Thr Ser Ile
Asn Val 675 680 685gtt cgc cac ttg ggc att gtg ggt gaa tgc aac att
cag ttt gcc ctt 2232Val Arg His Leu Gly Ile Val Gly Glu Cys Asn Ile
Gln Phe Ala Leu 690 695 700cat cct acc tca atg gaa tac tgc atc att
gaa gtg aat gcc aga ctg 2280His Pro Thr Ser Met Glu Tyr Cys Ile Ile
Glu Val Asn Ala Arg Leu 705 710 715tcc cga agc tct gct ctg gcc tca
aaa gcc act ggc tac cca ttg gca 2328Ser Arg Ser Ser Ala Leu Ala Ser
Lys Ala Thr Gly Tyr Pro Leu Ala720 725 730 735ttc att gct gca aag
att gcc cta gga atc cca ctt cca gaa att aag 2376Phe Ile Ala Ala Lys
Ile Ala Leu Gly Ile Pro Leu Pro Glu Ile Lys 740 745 750aac gtc gta
tcc ggg aag aca tca gcc tgt ttt gaa cct agc ctg gat 2424Asn Val Val
Ser Gly Lys Thr Ser Ala Cys Phe Glu Pro Ser Leu Asp 755 760 765tac
atg gtc acc aag att ccc cgc tgg gat ctt gac cgt ttt cat gga 2472Tyr
Met Val Thr Lys Ile Pro Arg Trp Asp Leu Asp Arg Phe His Gly 770 775
780aca tct agc cga att ggt agc tct atg aaa agt gta gga gag gtc atg
2520Thr Ser Ser Arg Ile Gly Ser Ser Met Lys Ser Val Gly Glu Val Met
785 790 795gct att ggt cgt acc ttt gag gag agt ttc cag aaa gct tta
cgg atg 2568Ala Ile Gly Arg Thr Phe Glu Glu Ser Phe Gln Lys Ala Leu
Arg Met800 805 810 815tgc cac cca tct ata gaa ggt ttc act ccc cgt
ctc cca atg aac aaa 2616Cys His Pro Ser Ile Glu Gly Phe Thr Pro Arg
Leu Pro Met Asn Lys 820 825 830gaa tgg cca tct aat tta gat ctt aga
aaa gag ttg tct gaa cca agc 2664Glu Trp Pro Ser Asn Leu Asp Leu Arg
Lys Glu Leu Ser Glu Pro Ser 835 840 845agc acg cgt atc tat gcc att
gcc aag gcc att gat gac aac atg tcc 2712Ser Thr Arg Ile Tyr Ala Ile
Ala Lys Ala Ile Asp Asp Asn Met Ser 850 855 860ctt gat gag att gag
aag ctc aca tac att gac aag tgg ttt ttg tat 2760Leu Asp Glu Ile Glu
Lys Leu Thr Tyr Ile Asp Lys Trp Phe Leu Tyr 865 870 875aag atg cgt
gat att tta aac atg gaa aag aca ctg aaa ggg ctc aac 2808Lys Met Arg
Asp Ile Leu Asn Met Glu Lys Thr Leu Lys Gly Leu Asn880 885 890
895agt gag tcc atg aca gaa gaa acc ctg aaa agg gca aag gag att ggg
2856Ser Glu Ser Met Thr Glu Glu Thr Leu Lys Arg Ala Lys Glu Ile Gly
900 905 910ttc tca gat aag cag att tca aaa tgc ctt ggg ctc act gag
gcc cag 2904Phe Ser Asp Lys Gln Ile Ser Lys Cys Leu Gly Leu Thr Glu
Ala Gln 915 920 925aca agg gag ctg agg tta aag aaa aac atc cac cct
tgg gtt aaa cag 2952Thr Arg Glu Leu Arg Leu Lys Lys Asn Ile His Pro
Trp Val Lys Gln 930 935 940att gat aca ctg gct gca gaa tac cca tca
gta aca aac tat ctc tat 3000Ile Asp Thr Leu Ala Ala Glu Tyr Pro Ser
Val Thr Asn Tyr Leu Tyr 945 950 955gtt acc tac aat ggt cag gag cat
gat gtc aat ttt gat gac cat gga 3048Val Thr Tyr Asn Gly Gln Glu His
Asp Val Asn Phe Asp Asp His Gly960 965 970 975atg atg gtg cta ggc
tgt ggt cca tat cac att ggc agc agt gtg gaa 3096Met Met Val Leu Gly
Cys Gly Pro Tyr His Ile Gly Ser Ser Val Glu 980 985 990ttt gat tgg
tgt gct gtc tct agt atc cgc aca ctg cgt caa ctt ggc 3144Phe Asp Trp
Cys Ala Val Ser Ser Ile Arg Thr Leu Arg Gln Leu Gly 995 1000
1005aag aag acg gtg gtg gtg aat tgc aat cct gag act gtg agc aca
3189Lys Lys Thr Val Val Val Asn Cys Asn Pro Glu Thr Val Ser Thr
1010 1015 1020gac ttt gat gag tgt gac aaa ctg tac ttt gaa gag ttg
tcc ttg 3234Asp Phe Asp Glu Cys Asp Lys Leu Tyr Phe Glu Glu Leu Ser
Leu 1025 1030 1035gag aga atc cta gac atc tac cat cag gag gca tgt
ggt ggc tgc 3279Glu Arg Ile Leu Asp Ile Tyr His Gln Glu Ala Cys Gly
Gly Cys 1040 1045 1050atc ata tca gtt gga ggc cag att cca aac aac
ctg gca gtt cct 3324Ile Ile Ser Val Gly Gly Gln Ile Pro Asn Asn Leu
Ala Val Pro 1055 1060 1065cta tac aag aat ggt gtc aag atc atg ggc
aca agc ccc ctg cag 3369Leu Tyr Lys Asn Gly Val Lys Ile Met Gly Thr
Ser Pro Leu Gln 1070 1075 1080atc gac agg gct gag gat cgc tcc atc
ttc tca gct gtc ttg gat 3414Ile Asp Arg Ala Glu Asp Arg Ser Ile Phe
Ser Ala Val Leu Asp 1085 1090 1095gag ctg aag gtg gct cag gca cct
tgg aaa gct gtt aat act ttg 3459Glu Leu Lys Val Ala Gln Ala Pro Trp
Lys Ala Val Asn Thr Leu 1100 1105 1110aat gaa gca ctg gaa ttt gca
aag tct gtg gac tac ccc tgc ttg 3504Asn Glu Ala Leu Glu Phe Ala Lys
Ser Val Asp Tyr Pro Cys Leu 1115 1120 1125ttg agg cct tcc tat gtt
ttg agt ggg tct gct atg aat gtg gta 3549Leu Arg Pro Ser Tyr Val Leu
Ser Gly Ser Ala Met Asn Val Val 1130 1135 1140ttc tct gag gat gag
atg aaa aaa ttc cta gaa gag gcg act aga 3594Phe Ser Glu Asp Glu Met
Lys Lys Phe Leu Glu Glu Ala Thr Arg 1145 1150 1155gtt tct cag gag
cac cca gtg gtc ctg aca aaa ttt gtt gaa ggg 3639Val Ser Gln Glu His
Pro Val Val Leu Thr Lys Phe Val Glu Gly 1160 1165 1170gcc cga gaa
gta gaa atg gac gct gtt ggc aaa gat gga agg gtt 3684Ala Arg Glu Val
Glu Met Asp Ala Val Gly Lys Asp Gly Arg Val 1175 1180 1185atc tct
cat gcc atc tct gaa cat gtt gaa gat gca ggt gtc cac 3729Ile Ser His
Ala Ile Ser Glu His Val Glu Asp Ala Gly Val His 1190 1195 1200tcg
gga gat gcc act ctg atg ctg ccc aca caa acc atc agc caa 3774Ser Gly
Asp Ala Thr Leu Met Leu Pro Thr Gln Thr Ile Ser Gln 1205 1210
1215ggg gcc att gaa aag gtg aag gat gct acc cgg aag att gca aag
3819Gly Ala Ile Glu Lys Val Lys Asp Ala Thr Arg Lys Ile Ala Lys
1220 1225 1230gct ttt gcc atc tct ggt cca ttc aac gtc caa ttt ctt
gtc aaa 3864Ala Phe Ala Ile Ser Gly Pro Phe Asn Val Gln Phe Leu Val
Lys 1235 1240 1245gga aat gat gtc ttg gtg att gag tgt aac ttg aga
gct tct cga 3909Gly Asn Asp Val Leu Val Ile Glu Cys Asn Leu Arg Ala
Ser Arg 1250 1255 1260tcc ttc ccc ttt gtt tcc aag act ctt ggg gtt
gac ttc att gat 3954Ser Phe Pro Phe Val Ser Lys Thr Leu Gly Val Asp
Phe Ile Asp 1265 1270 1275gtg gcc acc aag gtg atg att gga gag aat
gtt gat gag aaa cat 3999Val Ala Thr Lys Val Met Ile Gly Glu Asn Val
Asp Glu Lys His 1280 1285 1290ctt cca aca ttg gac cat ccc ata att
cct gct gac tat gtt gca 4044Leu Pro Thr Leu Asp His Pro Ile Ile Pro
Ala Asp Tyr Val Ala 1295 1300 1305att aag gct ccc atg ttt tcc tgg
ccc cgg ttg agg gat gct gac 4089Ile Lys Ala Pro Met Phe Ser Trp Pro
Arg Leu Arg Asp Ala Asp 1310 1315 1320ccc att ctg aga tgt gag atg
gct tcc act gga gag gtg gct tgc 4134Pro Ile Leu Arg Cys Glu Met Ala
Ser Thr Gly Glu Val Ala Cys 1325 1330 1335ttt ggt gaa ggt att cat
aca gcc ttc cta aag gca atg ctt tcc 4179Phe Gly Glu Gly Ile His Thr
Ala Phe Leu Lys Ala Met Leu Ser 1340 1345 1350aca gga ttt aag ata
ccc cag aaa ggc atc ctg ata ggc atc cag 4224Thr Gly Phe Lys Ile Pro
Gln Lys Gly Ile Leu Ile Gly Ile Gln 1355 1360 1365caa tca ttc cgg
cca aga ttc ctt ggt gtg gct gaa caa tta cac 4269Gln Ser Phe Arg Pro
Arg Phe Leu Gly Val Ala Glu Gln Leu His 1370 1375 1380aat gaa ggt
ttc aag ctg ttt gcc acg gaa gcc aca tca gac tgg 4314Asn Glu Gly Phe
Lys Leu Phe Ala Thr Glu Ala Thr Ser Asp Trp 1385 1390 1395ctc aac
gcc aac aat gtc cct gcc acc cca gtg gca tgg ccg tct 4359Leu Asn Ala
Asn Asn Val Pro Ala Thr Pro Val Ala Trp Pro Ser 1400 1405 1410caa
gaa gga cag aat ccc agc ctc tct tcc atc aga aaa ttg att 4404Gln Glu
Gly Gln Asn Pro Ser Leu Ser Ser Ile Arg Lys Leu Ile 1415 1420
1425aga gat ggc agc att gac cta gtg att aac ctt ccc aac aac aac
4449Arg Asp Gly Ser Ile Asp Leu Val Ile Asn Leu Pro Asn Asn Asn
1430 1435 1440act aaa ttt gtc cat gat aat tat gtg att cgg agg aca
gct gtt 4494Thr Lys Phe Val His Asp Asn Tyr Val Ile Arg Arg Thr Ala
Val 1445 1450 1455gat agt gga atc cct ctc ctc act aat ttt cag gtg
acc aaa ctt 4539Asp Ser Gly Ile Pro Leu Leu Thr Asn Phe Gln Val Thr
Lys Leu 1460 1465 1470ttt gct gaa gct gtg cag aaa tct cgc aag gtg
gac tcc aag agt 4584Phe Ala Glu Ala Val Gln Lys Ser Arg Lys Val Asp
Ser Lys Ser 1475 1480 1485ctt ttc cac tac agg cag tac agt gct gga
aaa gca gca tag 4626Leu Phe His Tyr Arg Gln Tyr Ser Ala Gly Lys Ala
Ala 1490 1495 1500agatgcagac accccagccc cattattaaa tcaacctgag
ccacatgtta tctaaaggaa 4686ctgattcaca actttctcag agatgaatat
tgataactaa acttcatttc agtttacttt 4746gttatgcctt aatattctgt
gtcttttgca attaaattgt cagtcacttc ttcaaaacct 4806tacagtcctt
cctaagttac tcttcatgag atttcatcca tttactaata ctgtattttt
4866ggtggactag gcttgcctat gtgcttatgt gtagcttttt actttttatg
gtgctgatta 4926atggtgatca aggtaggaaa agttgctgtt ctattttctg
aactctttct atactttaag 4986atactctatt tttaaaacac tatctgcaaa
ctcaggacac tttaacaggg cagaatactc 5046taaaaacttg ataaaatgaa
atatagattt aatttatgaa ccttccatca tgatgtttgt 5106gtattgcttc
tttttggatc ctcattctca cccatttggc taatccagga atattgttat
5166cccttcccat tatattgaag ttgagaaatg tgacagaggc atttagagta
tggacttttc 5226ttttcttttt ctttttcttt ttttcttttt gagatggagt
cacactctcc aggctggagt 5286gcagtggcac aatctcggct cactgcaatt
tgcgtctccc aagttcaagc gattctcctg 5346ctttagacta tggatttctt
taaggaatac tggtttgcag ttttgttttc tggactatat 5406cagcagatgg
tagacagtgt ttatgtagat gtgttgttgt ttttatcatt ggattttaac
5466ttggcccgag tgaaataatc agatttttgt cattcacact ctcccccagt
tttggaataa 5526cttggaagta aggttcattc ccttaagacg atggattctg
ttgaactatg gggtcccaca 5586ctgcactatt aattccaccc actgtaaggg
caaggacacc attccttcta catataagaa 5646aaaagtctct ccccaagggc
agcctttgtt acttttaaat attttctgtt attacaagtg 5706ctctaattgt
gaacttttaa ataaaatact attaagaggt aaaaaaaaaa aaaaa 576141500PRTHomo
sapiens 4Met Thr Arg Ile Leu Thr Ala Phe Lys Val Val Arg Thr Leu
Lys Thr1 5 10 15Gly Phe Gly Phe Thr Asn Val Thr Ala His Gln Lys Trp
Lys Phe Ser 20 25 30Arg Pro Gly Ile Arg Leu Leu Ser Val Lys Ala Gln
Thr Ala His Ile 35 40 45Val Leu Glu Asp Gly Thr Lys Met Lys Gly Tyr
Ser Phe Gly His Pro 50 55 60Ser Ser Val Ala Gly Glu Val Val Phe Asn
Thr Gly Leu Gly Gly Tyr65 70 75 80Pro Glu Ala Ile Thr Asp Pro Ala
Tyr Lys Gly Gln Ile Leu Thr Met 85 90 95Ala Asn Pro Ile Ile Gly Asn
Gly Gly Ala Pro Asp Thr Thr Ala Leu 100 105 110Asp Glu Leu Gly Leu
Ser Lys Tyr Leu Glu Ser Asn Gly Ile Lys Val 115 120 125Ser Gly Leu
Leu Val Leu Asp Tyr Ser Lys Asp Tyr Asn His Trp Leu 130 135 140Ala
Thr Lys Ser Leu Gly Gln Trp Leu Gln Glu Glu Lys Val Pro Ala145 150
155 160Ile Tyr Gly Val Asp Thr Arg Met Leu Thr Lys Ile Ile Arg Asp
Lys 165 170 175Gly Thr Met Leu Gly Lys Ile Glu Phe Glu Gly Gln Pro
Val Asp Phe 180 185 190Val Asp Pro Asn Lys Gln Asn Leu Ile Ala Glu
Val Ser Thr Lys Asp 195 200 205Val Lys Val Tyr Gly Lys Gly Asn Pro
Thr Lys Val Val Ala Val Asp 210 215 220Cys Gly Ile Lys Asn Asn Val
Ile Arg Leu Leu Val Lys Arg Gly Ala225 230 235 240Glu Val His Leu
Val Pro Trp Asn His Asp Phe Thr Lys Met Glu Tyr 245 250 255Asp Gly
Ile Leu Ile Ala Gly Gly Pro Gly Asn Pro Ala Leu Ala Glu 260 265
270Pro Leu Ile Gln Asn Val Arg Lys Ile Leu Glu Ser Asp Arg Lys Glu
275 280 285Pro Leu Phe Gly Ile Ser Thr Gly Asn Leu Ile Thr Gly Leu
Ala Ala 290 295 300Gly Ala Lys Thr Tyr Lys Met Ser Met Ala Asn Arg
Gly Gln Asn Gln305 310 315 320Pro Val Leu Asn Ile Thr Asn Lys Gln
Ala Phe Ile Thr Ala Gln Asn 325 330 335His Gly Tyr Ala Leu Asp Asn
Thr Leu Pro Ala Gly Trp Lys Pro Leu 340 345 350Phe Val Asn Val Asn
Asp Gln Thr Asn Glu Gly Ile Met His Glu Ser 355 360 365Lys Pro Phe
Phe Ala Val Gln Phe His Pro Glu Val Thr Pro Gly Pro 370 375 380Ile
Asp Thr Glu Tyr Leu Phe Asp Ser Phe Phe Ser Leu Ile Lys Lys385 390
395 400Gly Lys Ala Thr Thr Ile Thr Ser Val Leu Pro Lys Pro Ala Leu
Val 405 410 415Ala Ser Arg Val Glu Val Ser Lys Val Leu Ile Leu Gly
Ser Gly Gly 420 425 430Leu Ser Ile Gly Gln Ala Gly Glu Phe Asp Tyr
Ser Gly Ser Gln Ala 435 440 445Val Lys Ala Met Lys Glu Glu Asn Val
Lys Thr Val Leu Met Asn Pro 450 455 460Asn Ile Ala Ser Val Gln Thr
Asn Glu Val Gly Leu Lys Gln Ala Asp465 470 475 480Thr Val Tyr Phe
Leu Pro Ile Thr Pro Gln Phe Val Thr Glu Val Ile 485 490 495Lys Ala
Glu Gln Pro Asp Gly Leu Ile Leu Gly Met Gly Gly Gln Thr 500 505
510Ala Leu Asn Cys Gly Val Glu Leu Phe Lys Arg Gly Val Leu Lys Glu
515 520 525Tyr Gly Val Lys Val Leu Gly Thr Ser Val Glu Ser Ile Met
Ala Thr 530 535 540Glu Asp Arg Gln Leu Phe Ser Asp Lys Leu Asn Glu
Ile Asn Glu Lys545 550 555
560Ile Ala Pro Ser Phe Ala Val Glu Ser Ile Glu Asp Ala Leu Lys Ala
565 570 575Ala Asp Thr Ile Gly Tyr Pro Val Met Ile Arg Ser Ala Tyr
Ala Leu 580 585 590Gly Gly Leu Gly Ser Gly Ile Cys Pro Asn Arg Glu
Thr Leu Met Asp 595 600 605Leu Ser Thr Lys Ala Phe Ala Met Thr Asn
Gln Ile Leu Val Glu Lys 610 615 620Ser Val Thr Gly Trp Lys Glu Ile
Glu Tyr Glu Val Val Arg Asp Ala625 630 635 640Asp Asp Asn Cys Val
Thr Val Cys Asn Met Glu Asn Val Asp Ala Met 645 650 655Gly Val His
Thr Gly Asp Ser Val Val Val Ala Pro Ala Gln Thr Leu 660 665 670Ser
Asn Ala Glu Phe Gln Met Leu Arg Arg Thr Ser Ile Asn Val Val 675 680
685Arg His Leu Gly Ile Val Gly Glu Cys Asn Ile Gln Phe Ala Leu His
690 695 700Pro Thr Ser Met Glu Tyr Cys Ile Ile Glu Val Asn Ala Arg
Leu Ser705 710 715 720Arg Ser Ser Ala Leu Ala Ser Lys Ala Thr Gly
Tyr Pro Leu Ala Phe 725 730 735Ile Ala Ala Lys Ile Ala Leu Gly Ile
Pro Leu Pro Glu Ile Lys Asn 740 745 750Val Val Ser Gly Lys Thr Ser
Ala Cys Phe Glu Pro Ser Leu Asp Tyr 755 760 765Met Val Thr Lys Ile
Pro Arg Trp Asp Leu Asp Arg Phe His Gly Thr 770 775 780Ser Ser Arg
Ile Gly Ser Ser Met Lys Ser Val Gly Glu Val Met Ala785 790 795
800Ile Gly Arg Thr Phe Glu Glu Ser Phe Gln Lys Ala Leu Arg Met Cys
805 810 815His Pro Ser Ile Glu Gly Phe Thr Pro Arg Leu Pro Met Asn
Lys Glu 820 825 830Trp Pro Ser Asn Leu Asp Leu Arg Lys Glu Leu Ser
Glu Pro Ser Ser 835 840 845Thr Arg Ile Tyr Ala Ile Ala Lys Ala Ile
Asp Asp Asn Met Ser Leu 850 855 860Asp Glu Ile Glu Lys Leu Thr Tyr
Ile Asp Lys Trp Phe Leu Tyr Lys865 870 875 880Met Arg Asp Ile Leu
Asn Met Glu Lys Thr Leu Lys Gly Leu Asn Ser 885 890 895Glu Ser Met
Thr Glu Glu Thr Leu Lys Arg Ala Lys Glu Ile Gly Phe 900 905 910Ser
Asp Lys Gln Ile Ser Lys Cys Leu Gly Leu Thr Glu Ala Gln Thr 915 920
925Arg Glu Leu Arg Leu Lys Lys Asn Ile His Pro Trp Val Lys Gln Ile
930 935 940Asp Thr Leu Ala Ala Glu Tyr Pro Ser Val Thr Asn Tyr Leu
Tyr Val945 950 955 960Thr Tyr Asn Gly Gln Glu His Asp Val Asn Phe
Asp Asp His Gly Met 965 970 975Met Val Leu Gly Cys Gly Pro Tyr His
Ile Gly Ser Ser Val Glu Phe 980 985 990Asp Trp Cys Ala Val Ser Ser
Ile Arg Thr Leu Arg Gln Leu Gly Lys 995 1000 1005Lys Thr Val Val
Val Asn Cys Asn Pro Glu Thr Val Ser Thr Asp 1010 1015 1020Phe Asp
Glu Cys Asp Lys Leu Tyr Phe Glu Glu Leu Ser Leu Glu 1025 1030
1035Arg Ile Leu Asp Ile Tyr His Gln Glu Ala Cys Gly Gly Cys Ile
1040 1045 1050Ile Ser Val Gly Gly Gln Ile Pro Asn Asn Leu Ala Val
Pro Leu 1055 1060 1065Tyr Lys Asn Gly Val Lys Ile Met Gly Thr Ser
Pro Leu Gln Ile 1070 1075 1080Asp Arg Ala Glu Asp Arg Ser Ile Phe
Ser Ala Val Leu Asp Glu 1085 1090 1095Leu Lys Val Ala Gln Ala Pro
Trp Lys Ala Val Asn Thr Leu Asn 1100 1105 1110Glu Ala Leu Glu Phe
Ala Lys Ser Val Asp Tyr Pro Cys Leu Leu 1115 1120 1125Arg Pro Ser
Tyr Val Leu Ser Gly Ser Ala Met Asn Val Val Phe 1130 1135 1140Ser
Glu Asp Glu Met Lys Lys Phe Leu Glu Glu Ala Thr Arg Val 1145 1150
1155Ser Gln Glu His Pro Val Val Leu Thr Lys Phe Val Glu Gly Ala
1160 1165 1170Arg Glu Val Glu Met Asp Ala Val Gly Lys Asp Gly Arg
Val Ile 1175 1180 1185Ser His Ala Ile Ser Glu His Val Glu Asp Ala
Gly Val His Ser 1190 1195 1200Gly Asp Ala Thr Leu Met Leu Pro Thr
Gln Thr Ile Ser Gln Gly 1205 1210 1215Ala Ile Glu Lys Val Lys Asp
Ala Thr Arg Lys Ile Ala Lys Ala 1220 1225 1230Phe Ala Ile Ser Gly
Pro Phe Asn Val Gln Phe Leu Val Lys Gly 1235 1240 1245Asn Asp Val
Leu Val Ile Glu Cys Asn Leu Arg Ala Ser Arg Ser 1250 1255 1260Phe
Pro Phe Val Ser Lys Thr Leu Gly Val Asp Phe Ile Asp Val 1265 1270
1275Ala Thr Lys Val Met Ile Gly Glu Asn Val Asp Glu Lys His Leu
1280 1285 1290Pro Thr Leu Asp His Pro Ile Ile Pro Ala Asp Tyr Val
Ala Ile 1295 1300 1305Lys Ala Pro Met Phe Ser Trp Pro Arg Leu Arg
Asp Ala Asp Pro 1310 1315 1320Ile Leu Arg Cys Glu Met Ala Ser Thr
Gly Glu Val Ala Cys Phe 1325 1330 1335Gly Glu Gly Ile His Thr Ala
Phe Leu Lys Ala Met Leu Ser Thr 1340 1345 1350Gly Phe Lys Ile Pro
Gln Lys Gly Ile Leu Ile Gly Ile Gln Gln 1355 1360 1365Ser Phe Arg
Pro Arg Phe Leu Gly Val Ala Glu Gln Leu His Asn 1370 1375 1380Glu
Gly Phe Lys Leu Phe Ala Thr Glu Ala Thr Ser Asp Trp Leu 1385 1390
1395Asn Ala Asn Asn Val Pro Ala Thr Pro Val Ala Trp Pro Ser Gln
1400 1405 1410Glu Gly Gln Asn Pro Ser Leu Ser Ser Ile Arg Lys Leu
Ile Arg 1415 1420 1425Asp Gly Ser Ile Asp Leu Val Ile Asn Leu Pro
Asn Asn Asn Thr 1430 1435 1440Lys Phe Val His Asp Asn Tyr Val Ile
Arg Arg Thr Ala Val Asp 1445 1450 1455Ser Gly Ile Pro Leu Leu Thr
Asn Phe Gln Val Thr Lys Leu Phe 1460 1465 1470Ala Glu Ala Val Gln
Lys Ser Arg Lys Val Asp Ser Lys Ser Leu 1475 1480 1485Phe His Tyr
Arg Gln Tyr Ser Ala Gly Lys Ala Ala 1490 1495 15005495DNAHomo
sapiensmisc_feature(1)..(495)n is a, c, g, or t 5ctacttctca
tgttcagcaa tttcttcttc tttatgtttt aaattacatg ttccataaaa 60ataagaaatn
cactgtgata cggtaattga ttttttcatt ttaaatgcag ctgtttgcca
120cggaagccac atcagactgg ctcaacgcca acaatgtccc tgccacccca
gtggcatggc 180cgtctcaaga aggacagaat cccagcctct cttccatcag
aaagtaagaa ctaggcatac 240tgttttctga aataatttag aggattaact
ttgagaacca gtatatgaat attcaccttg 300cttgattgca agtcttttaa
aacaaattta aaaatgaata catttgtgga tgattgtcaa 360gtttcactct
ccatcactat ggaatacata acgtcatgtg tacatggtga tatgaaacgt
420gtttcaaaat acttcttagt aaggatactt tccttgacgg aaacaagtga
gagtatgaag 480aatgtaatgc agcac 495620DNAHomo sapiens 6agctgtttgc
cacggaagcc 20728DNAHomo sapiens 7cccagcctct cttccatcag aaagtaag
28820DNAHomo sapiens 8cacggaagcc acatcagact 20921DNAHomo sapiens
9ttctgatgga agagaggctt g 211024DNAHomo sapiens 10agagtgaaac
ttgacaatca tcca 24115761DNAHomo sapiensCDS(124)..(4626)
11gtcagcctta aacactgact gcacccctcc cagatttctt ttacattaac taaaaagtct
60tatcacacaa tctcataaaa tttatgtaat ttcatttaat tttagccaca aatcatcttc
120aaa atg acg agg tta ttg aca gct ttc aaa gtg gtg agg aca ctg aag
168 Met Thr Arg Leu Leu Thr Ala Phe Lys Val Val Arg Thr Leu Lys 1 5
10 15act ggt ttt ggc ttt acc aat gtg act gca cac caa aaa tgg aaa
ttt 216Thr Gly Phe Gly Phe Thr Asn Val Thr Ala His Gln Lys Trp Lys
Phe 20 25 30tca aga cct ggc atc agg ctc ctt tct gtc aag gca cag aca
gca cac 264Ser Arg Pro Gly Ile Arg Leu Leu Ser Val Lys Ala Gln Thr
Ala His 35 40 45att gtc ctg gaa gat gga act aag atg aaa ggt tac tcc
ttt ggc cat 312Ile Val Leu Glu Asp Gly Thr Lys Met Lys Gly Tyr Ser
Phe Gly His 50 55 60cca tcc tct gtt gct ggt gaa gtg gtt ttt aat act
ggc ctg gga ggg 360Pro Ser Ser Val Ala Gly Glu Val Val Phe Asn Thr
Gly Leu Gly Gly 65 70 75tac cca gaa gct att act gac cct gcc tac aaa
gga cag att ctc aca 408Tyr Pro Glu Ala Ile Thr Asp Pro Ala Tyr Lys
Gly Gln Ile Leu Thr80 85 90 95atg gcc aac cct att att ggg aat ggt
gga gct cct gat act act gct 456Met Ala Asn Pro Ile Ile Gly Asn Gly
Gly Ala Pro Asp Thr Thr Ala 100 105 110ctg gat gaa ctg gga ctt agc
aaa tat ttg gag tct aat gga atc aag 504Leu Asp Glu Leu Gly Leu Ser
Lys Tyr Leu Glu Ser Asn Gly Ile Lys 115 120 125gtt tca ggt ttg ctg
gtg ctg gat tat agt aaa gac tac aac cac tgg 552Val Ser Gly Leu Leu
Val Leu Asp Tyr Ser Lys Asp Tyr Asn His Trp 130 135 140ctg gct acc
aag agt tta ggg caa tgg cta cag gaa gaa aag gtt cct 600Leu Ala Thr
Lys Ser Leu Gly Gln Trp Leu Gln Glu Glu Lys Val Pro 145 150 155gca
att tat gga gtg gac aca aga atg ctg act aaa ata att cgg gat 648Ala
Ile Tyr Gly Val Asp Thr Arg Met Leu Thr Lys Ile Ile Arg Asp160 165
170 175aag ggt acc atg ctt ggg aag att gaa ttt gaa ggt cag cct gtg
gat 696Lys Gly Thr Met Leu Gly Lys Ile Glu Phe Glu Gly Gln Pro Val
Asp 180 185 190ttt gtg gat cca aat aaa cag aat ttg att gct gag gtt
tca acc aag 744Phe Val Asp Pro Asn Lys Gln Asn Leu Ile Ala Glu Val
Ser Thr Lys 195 200 205gat gtc aaa gtg tac ggc aaa gga aac ccc aca
aaa gtg gta gct gta 792Asp Val Lys Val Tyr Gly Lys Gly Asn Pro Thr
Lys Val Val Ala Val 210 215 220gac tgt ggg att aaa aac aat gta atc
cgc ctg cta gta aag cga gga 840Asp Cys Gly Ile Lys Asn Asn Val Ile
Arg Leu Leu Val Lys Arg Gly 225 230 235gct gaa gtg cac tta gtt ccc
tgg aac cat gat ttc acc aag atg gag 888Ala Glu Val His Leu Val Pro
Trp Asn His Asp Phe Thr Lys Met Glu240 245 250 255tat gat ggg att
ttg atc gcg gga gga ccg ggg aac cca gct ctt gca 936Tyr Asp Gly Ile
Leu Ile Ala Gly Gly Pro Gly Asn Pro Ala Leu Ala 260 265 270gaa cca
cta att cag aat gtc aga aag att ttg gag agt gat cgc aag 984Glu Pro
Leu Ile Gln Asn Val Arg Lys Ile Leu Glu Ser Asp Arg Lys 275 280
285gag cca ttg ttt gga atc agt aca gga aac tta ata aca gga ttg gct
1032Glu Pro Leu Phe Gly Ile Ser Thr Gly Asn Leu Ile Thr Gly Leu Ala
290 295 300gct ggt gcc aaa acc tac aag atg tcc atg gcc aac aga ggg
cag aat 1080Ala Gly Ala Lys Thr Tyr Lys Met Ser Met Ala Asn Arg Gly
Gln Asn 305 310 315cag cct gtt ttg aat atc aca aac aaa cag gct ttc
att act gct cag 1128Gln Pro Val Leu Asn Ile Thr Asn Lys Gln Ala Phe
Ile Thr Ala Gln320 325 330 335aat cat ggc tat gcc ttg gac aac acc
ctc cct gct ggc tgg aaa cca 1176Asn His Gly Tyr Ala Leu Asp Asn Thr
Leu Pro Ala Gly Trp Lys Pro 340 345 350ctt ttt gtg aat gtc aac gat
caa aca aat gag ggg att atg cat gag 1224Leu Phe Val Asn Val Asn Asp
Gln Thr Asn Glu Gly Ile Met His Glu 355 360 365agc aaa ccc ttc ttc
gct gtg cag ttc cac cca gag gtc acc ccg ggg 1272Ser Lys Pro Phe Phe
Ala Val Gln Phe His Pro Glu Val Thr Pro Gly 370 375 380cca ata gac
act gag tac ctg ttt gat tcc ttt ttc tca ctg ata aag 1320Pro Ile Asp
Thr Glu Tyr Leu Phe Asp Ser Phe Phe Ser Leu Ile Lys 385 390 395aaa
gga aaa gct acc acc att aca tca gtc tta ccg aag cca gca cta 1368Lys
Gly Lys Ala Thr Thr Ile Thr Ser Val Leu Pro Lys Pro Ala Leu400 405
410 415gtt gca tct cgg gtt gag gtt tcc aaa gtc ctt att cta gga tca
gga 1416Val Ala Ser Arg Val Glu Val Ser Lys Val Leu Ile Leu Gly Ser
Gly 420 425 430ggt ctg tcc att ggt cag gct gga gaa ttt gat tac tca
gga tct caa 1464Gly Leu Ser Ile Gly Gln Ala Gly Glu Phe Asp Tyr Ser
Gly Ser Gln 435 440 445gct gta aaa gcc atg aag gaa gaa aat gtc aaa
act gtt ctg atg aac 1512Ala Val Lys Ala Met Lys Glu Glu Asn Val Lys
Thr Val Leu Met Asn 450 455 460cca aac att gca tca gtc cag acc aat
gag gtg ggc tta aag caa gcg 1560Pro Asn Ile Ala Ser Val Gln Thr Asn
Glu Val Gly Leu Lys Gln Ala 465 470 475gat act gtc tac ttt ctt ccc
atc acc cct cag ttt gtc aca gag gtc 1608Asp Thr Val Tyr Phe Leu Pro
Ile Thr Pro Gln Phe Val Thr Glu Val480 485 490 495atc aag gca gaa
cag cca gat ggg tta att ctg ggc atg ggt ggc cag 1656Ile Lys Ala Glu
Gln Pro Asp Gly Leu Ile Leu Gly Met Gly Gly Gln 500 505 510aca gct
ctg aac tgt gga gtg gaa cta ttc aag aga ggt gtg ctc aag 1704Thr Ala
Leu Asn Cys Gly Val Glu Leu Phe Lys Arg Gly Val Leu Lys 515 520
525gaa tat ggt gtg aaa gtc ctg gga act tca gtt gag tcc att atg gct
1752Glu Tyr Gly Val Lys Val Leu Gly Thr Ser Val Glu Ser Ile Met Ala
530 535 540acg gaa gac agg cag ctg ttt tca gat aaa cta aat gag atc
aat gaa 1800Thr Glu Asp Arg Gln Leu Phe Ser Asp Lys Leu Asn Glu Ile
Asn Glu 545 550 555aag att gct cca agt ttt gca gtg gaa tcg att gag
gat gca ctg aag 1848Lys Ile Ala Pro Ser Phe Ala Val Glu Ser Ile Glu
Asp Ala Leu Lys560 565 570 575gca gca gac acc att ggc tac cca gtg
atg atc cgt tcc gcc tat gca 1896Ala Ala Asp Thr Ile Gly Tyr Pro Val
Met Ile Arg Ser Ala Tyr Ala 580 585 590ctg ggt ggg tta ggc tca ggc
atc tgt ccc aac aga gag act ttg atg 1944Leu Gly Gly Leu Gly Ser Gly
Ile Cys Pro Asn Arg Glu Thr Leu Met 595 600 605gac ctc agc aca aag
gcc ttt gct atg acc aac caa att ctg gtg gag 1992Asp Leu Ser Thr Lys
Ala Phe Ala Met Thr Asn Gln Ile Leu Val Glu 610 615 620aag tca gtg
aca ggt tgg aaa gaa ata gaa tat gaa gtg gtt cga gat 2040Lys Ser Val
Thr Gly Trp Lys Glu Ile Glu Tyr Glu Val Val Arg Asp 625 630 635gct
gat gac aat tgt gtc act gtc tgt aac atg gaa aat gtt gat gcc 2088Ala
Asp Asp Asn Cys Val Thr Val Cys Asn Met Glu Asn Val Asp Ala640 645
650 655atg ggt gtt cac aca ggt gac tca gtt gtt gtg gct cct gcc cag
aca 2136Met Gly Val His Thr Gly Asp Ser Val Val Val Ala Pro Ala Gln
Thr 660 665 670ctc tcc aat gcc gag ttt cag atg ttg aga cgt act tca
atc aat gtt 2184Leu Ser Asn Ala Glu Phe Gln Met Leu Arg Arg Thr Ser
Ile Asn Val 675 680 685gtt cgc cac ttg ggc att gtg ggt gaa tgc aac
att cag ttt gcc ctt 2232Val Arg His Leu Gly Ile Val Gly Glu Cys Asn
Ile Gln Phe Ala Leu 690 695 700cat cct acc tca atg gaa tac tgc atc
att gaa gtg aat gcc aga ctg 2280His Pro Thr Ser Met Glu Tyr Cys Ile
Ile Glu Val Asn Ala Arg Leu 705 710 715tcc cga agc tct gct ctg gcc
tca aaa gcc act ggc tac cca ttg gca 2328Ser Arg Ser Ser Ala Leu Ala
Ser Lys Ala Thr Gly Tyr Pro Leu Ala720 725 730 735ttc att gct gca
aag att gcc cta gga atc cca ctt cca gaa att aag 2376Phe Ile Ala Ala
Lys Ile Ala Leu Gly Ile Pro Leu Pro Glu Ile Lys 740 745 750aac gtc
gta tcc ggg aag aca tca gcc tgt ttt gaa cct agc ctg gat 2424Asn Val
Val Ser Gly Lys Thr Ser Ala Cys Phe Glu Pro Ser Leu Asp 755 760
765tac atg gtc acc aag att ccc cgc tgg gat ctt gac cgt ttt cat gga
2472Tyr Met Val Thr Lys Ile Pro Arg Trp Asp Leu Asp Arg Phe His Gly
770 775 780aca tct agc cga att ggt agc tct atg aaa agt gta gga gag
gtc atg 2520Thr Ser Ser Arg Ile Gly Ser Ser Met Lys Ser Val Gly Glu
Val Met 785 790 795gct att ggt cgt acc ttt gag gag agt ttc cag aaa
gct tta cgg atg 2568Ala Ile Gly Arg Thr Phe Glu Glu Ser Phe Gln Lys
Ala Leu Arg Met800 805 810 815tgc cac cca tct ata gaa ggt ttc act
ccc cgt ctc cca atg aac aaa 2616Cys His Pro Ser Ile Glu Gly Phe Thr
Pro Arg Leu Pro Met Asn Lys 820 825 830gaa tgg cca tct aat
tta gat ctt aga aaa gag ttg tct gaa cca agc 2664Glu Trp Pro Ser Asn
Leu Asp Leu Arg Lys Glu Leu Ser Glu Pro Ser 835 840 845agc acg cgt
atc tat gcc att gcc aag gcc att gat gac aac atg tcc 2712Ser Thr Arg
Ile Tyr Ala Ile Ala Lys Ala Ile Asp Asp Asn Met Ser 850 855 860ctt
gat gag att gag aag ctc aca tac att gac aag tgg ttt ttg tat 2760Leu
Asp Glu Ile Glu Lys Leu Thr Tyr Ile Asp Lys Trp Phe Leu Tyr 865 870
875aag atg cgt gat att tta aac atg gaa aag aca ctg aaa ggg ctc aac
2808Lys Met Arg Asp Ile Leu Asn Met Glu Lys Thr Leu Lys Gly Leu
Asn880 885 890 895agt gag tcc atg aca gaa gaa acc ctg aaa agg gca
aag gag att ggg 2856Ser Glu Ser Met Thr Glu Glu Thr Leu Lys Arg Ala
Lys Glu Ile Gly 900 905 910ttc tca gat aag cag att tca aaa tgc ctt
ggg ctc act gag gcc cag 2904Phe Ser Asp Lys Gln Ile Ser Lys Cys Leu
Gly Leu Thr Glu Ala Gln 915 920 925aca agg gag ctg agg tta aag aaa
aac atc cac cct tgg gtt aaa cag 2952Thr Arg Glu Leu Arg Leu Lys Lys
Asn Ile His Pro Trp Val Lys Gln 930 935 940att gat aca ctg gct gca
gaa tac cca tca gta aca aac tat ctc tat 3000Ile Asp Thr Leu Ala Ala
Glu Tyr Pro Ser Val Thr Asn Tyr Leu Tyr 945 950 955gtt acc tac aat
ggt cag gag cat gat gtc aat ttt gat gac cat gga 3048Val Thr Tyr Asn
Gly Gln Glu His Asp Val Asn Phe Asp Asp His Gly960 965 970 975atg
atg gtg cta ggc tgt ggt cca tat cac att ggc agc agt gtg gaa 3096Met
Met Val Leu Gly Cys Gly Pro Tyr His Ile Gly Ser Ser Val Glu 980 985
990ttt gat tgg tgt gct gtc tct agt atc cgc aca ctg cgt caa ctt ggc
3144Phe Asp Trp Cys Ala Val Ser Ser Ile Arg Thr Leu Arg Gln Leu Gly
995 1000 1005aag aag acg gtg gtg gtg aat tgc aat cct gag act gtg
agc aca 3189Lys Lys Thr Val Val Val Asn Cys Asn Pro Glu Thr Val Ser
Thr 1010 1015 1020gac ttt gat gag tgt gac aaa ctg tac ttt gaa gag
ttg tcc ttg 3234Asp Phe Asp Glu Cys Asp Lys Leu Tyr Phe Glu Glu Leu
Ser Leu 1025 1030 1035gag aga atc cta gac atc tac cat cag gag gca
tgt ggt ggc tgc 3279Glu Arg Ile Leu Asp Ile Tyr His Gln Glu Ala Cys
Gly Gly Cys 1040 1045 1050atc ata tca gtt gga ggc cag att cca aac
aac ctg gca gtt cct 3324Ile Ile Ser Val Gly Gly Gln Ile Pro Asn Asn
Leu Ala Val Pro 1055 1060 1065cta tac aag aat ggt gtc aag atc atg
ggc aca agc ccc ctg cag 3369Leu Tyr Lys Asn Gly Val Lys Ile Met Gly
Thr Ser Pro Leu Gln 1070 1075 1080atc gac agg gct gag gat cgc tcc
atc ttc tca gct gtc ttg gat 3414Ile Asp Arg Ala Glu Asp Arg Ser Ile
Phe Ser Ala Val Leu Asp 1085 1090 1095gag ctg aag gtg gct cag gca
cct tgg aaa gct gtt aat act ttg 3459Glu Leu Lys Val Ala Gln Ala Pro
Trp Lys Ala Val Asn Thr Leu 1100 1105 1110aat gaa gca ctg gaa ttt
gca aag tct gtg gac tac ccc tgc ttg 3504Asn Glu Ala Leu Glu Phe Ala
Lys Ser Val Asp Tyr Pro Cys Leu 1115 1120 1125ttg agg cct tcc tat
gtt ttg agt ggg tct gct atg aat gtg gta 3549Leu Arg Pro Ser Tyr Val
Leu Ser Gly Ser Ala Met Asn Val Val 1130 1135 1140ttc tct gag gat
gag atg aaa aaa ttc cta gaa gag gcg act aga 3594Phe Ser Glu Asp Glu
Met Lys Lys Phe Leu Glu Glu Ala Thr Arg 1145 1150 1155gtt tct cag
gag cac cca gtg gtc ctg aca aaa ttt gtt gaa ggg 3639Val Ser Gln Glu
His Pro Val Val Leu Thr Lys Phe Val Glu Gly 1160 1165 1170gcc cga
gaa gta gaa atg gac gct gtt ggc aaa gat gga agg gtt 3684Ala Arg Glu
Val Glu Met Asp Ala Val Gly Lys Asp Gly Arg Val 1175 1180 1185atc
tct cat gcc atc tct gaa cat gtt gaa gat gca ggt gtc cac 3729Ile Ser
His Ala Ile Ser Glu His Val Glu Asp Ala Gly Val His 1190 1195
1200tcg gga gat gcc act ctg atg ctg ccc aca caa acc atc agc caa
3774Ser Gly Asp Ala Thr Leu Met Leu Pro Thr Gln Thr Ile Ser Gln
1205 1210 1215ggg gcc att gaa aag gtg aag gat gct acc cgg aag att
gca aag 3819Gly Ala Ile Glu Lys Val Lys Asp Ala Thr Arg Lys Ile Ala
Lys 1220 1225 1230gct ttt gcc atc tct ggt cca ttc aac gtc caa ttt
ctt gtc aaa 3864Ala Phe Ala Ile Ser Gly Pro Phe Asn Val Gln Phe Leu
Val Lys 1235 1240 1245gga aat gat gtc ttg gtg att gag tgt aac ttg
aga gct tct cga 3909Gly Asn Asp Val Leu Val Ile Glu Cys Asn Leu Arg
Ala Ser Arg 1250 1255 1260tcc ttc ccc ttt gtt tcc aag act ctt ggg
gtt gac ttc att gat 3954Ser Phe Pro Phe Val Ser Lys Thr Leu Gly Val
Asp Phe Ile Asp 1265 1270 1275gtg gcc acc aag gtg atg att gga gag
aat gtt gat gag aaa cat 3999Val Ala Thr Lys Val Met Ile Gly Glu Asn
Val Asp Glu Lys His 1280 1285 1290ctt cca aca ttg gac cat ccc ata
att cct gct gac tat gtt gca 4044Leu Pro Thr Leu Asp His Pro Ile Ile
Pro Ala Asp Tyr Val Ala 1295 1300 1305att aag gct ccc atg ttt tcc
tgg ccc cgg ttg agg gat gct gac 4089Ile Lys Ala Pro Met Phe Ser Trp
Pro Arg Leu Arg Asp Ala Asp 1310 1315 1320ccc att ctg aga tgt gag
atg gct tcc act gga gag gtg gct tgc 4134Pro Ile Leu Arg Cys Glu Met
Ala Ser Thr Gly Glu Val Ala Cys 1325 1330 1335ttt ggt gaa ggt att
cat aca gcc ttc cta aag gca atg ctt tcc 4179Phe Gly Glu Gly Ile His
Thr Ala Phe Leu Lys Ala Met Leu Ser 1340 1345 1350aca gga ttt aag
ata ccc cag aaa ggc atc ctg ata ggc atc cag 4224Thr Gly Phe Lys Ile
Pro Gln Lys Gly Ile Leu Ile Gly Ile Gln 1355 1360 1365caa tca ttc
cgg cca aga ttc ctt ggt gtg gct gaa caa tta cac 4269Gln Ser Phe Arg
Pro Arg Phe Leu Gly Val Ala Glu Gln Leu His 1370 1375 1380aat gaa
ggt ttc aag ctg ttt gcc acg gaa gcc aca tca gac tgg 4314Asn Glu Gly
Phe Lys Leu Phe Ala Thr Glu Ala Thr Ser Asp Trp 1385 1390 1395ctc
aac gcc aac aat gtc cct gcc aac cca gtg gca tgg ccg tct 4359Leu Asn
Ala Asn Asn Val Pro Ala Asn Pro Val Ala Trp Pro Ser 1400 1405
1410caa gaa gga cag aat ccc agc ctc tct tcc atc aga aaa ttg att
4404Gln Glu Gly Gln Asn Pro Ser Leu Ser Ser Ile Arg Lys Leu Ile
1415 1420 1425aga gat ggc agc att gac cta gtg att aac ctt ccc aac
aac aac 4449Arg Asp Gly Ser Ile Asp Leu Val Ile Asn Leu Pro Asn Asn
Asn 1430 1435 1440act aaa ttt gtc cat gat aat tat gtg att cgg agg
aca gct gtt 4494Thr Lys Phe Val His Asp Asn Tyr Val Ile Arg Arg Thr
Ala Val 1445 1450 1455gat agt gga atc cct ctc ctc act aat ttt cag
gtg acc aaa ctt 4539Asp Ser Gly Ile Pro Leu Leu Thr Asn Phe Gln Val
Thr Lys Leu 1460 1465 1470ttt gct gaa gct gtg cag aaa tct cgc aag
gtg gac tcc aag agt 4584Phe Ala Glu Ala Val Gln Lys Ser Arg Lys Val
Asp Ser Lys Ser 1475 1480 1485ctt ttc cac tac agg cag tac agt gct
gga aaa gca gca tag 4626Leu Phe His Tyr Arg Gln Tyr Ser Ala Gly Lys
Ala Ala 1490 1495 1500agatgcagac accccagccc cattattaaa tcaacctgag
ccacatgtta tctaaaggaa 4686ctgattcaca actttctcag agatgaatat
tgataactaa acttcatttc agtttacttt 4746gttatgcctt aatattctgt
gtcttttgca attaaattgt cagtcacttc ttcaaaacct 4806tacagtcctt
cctaagttac tcttcatgag atttcatcca tttactaata ctgtattttt
4866ggtggactag gcttgcctat gtgcttatgt gtagcttttt actttttatg
gtgctgatta 4926atggtgatca aggtaggaaa agttgctgtt ctattttctg
aactctttct atactttaag 4986atactctatt tttaaaacac tatctgcaaa
ctcaggacac tttaacaggg cagaatactc 5046taaaaacttg ataaaatgaa
atatagattt aatttatgaa ccttccatca tgatgtttgt 5106gtattgcttc
tttttggatc ctcattctca cccatttggc taatccagga atattgttat
5166cccttcccat tatattgaag ttgagaaatg tgacagaggc atttagagta
tggacttttc 5226ttttcttttt ctttttcttt ttttcttttt gagatggagt
cacactctcc aggctggagt 5286gcagtggcac aatctcggct cactgcaatt
tgcgtctccc aagttcaagc gattctcctg 5346ctttagacta tggatttctt
taaggaatac tggtttgcag ttttgttttc tggactatat 5406cagcagatgg
tagacagtgt ttatgtagat gtgttgttgt ttttatcatt ggattttaac
5466ttggcccgag tgaaataatc agatttttgt cattcacact ctcccccagt
tttggaataa 5526cttggaagta aggttcattc ccttaagacg atggattctg
ttgaactatg gggtcccaca 5586ctgcactatt aattccaccc actgtaaggg
caaggacacc attccttcta catataagaa 5646aaaagtctct ccccaagggc
agcctttgtt acttttaaat attttctgtt attacaagtg 5706ctctaattgt
gaacttttaa ataaaatact attaagaggt aaaaaaaaaa aaaaa 5761121500PRTHomo
sapiens 12Met Thr Arg Leu Leu Thr Ala Phe Lys Val Val Arg Thr Leu
Lys Thr1 5 10 15Gly Phe Gly Phe Thr Asn Val Thr Ala His Gln Lys Trp
Lys Phe Ser 20 25 30Arg Pro Gly Ile Arg Leu Leu Ser Val Lys Ala Gln
Thr Ala His Ile 35 40 45Val Leu Glu Asp Gly Thr Lys Met Lys Gly Tyr
Ser Phe Gly His Pro 50 55 60Ser Ser Val Ala Gly Glu Val Val Phe Asn
Thr Gly Leu Gly Gly Tyr65 70 75 80Pro Glu Ala Ile Thr Asp Pro Ala
Tyr Lys Gly Gln Ile Leu Thr Met 85 90 95Ala Asn Pro Ile Ile Gly Asn
Gly Gly Ala Pro Asp Thr Thr Ala Leu 100 105 110Asp Glu Leu Gly Leu
Ser Lys Tyr Leu Glu Ser Asn Gly Ile Lys Val 115 120 125Ser Gly Leu
Leu Val Leu Asp Tyr Ser Lys Asp Tyr Asn His Trp Leu 130 135 140Ala
Thr Lys Ser Leu Gly Gln Trp Leu Gln Glu Glu Lys Val Pro Ala145 150
155 160Ile Tyr Gly Val Asp Thr Arg Met Leu Thr Lys Ile Ile Arg Asp
Lys 165 170 175Gly Thr Met Leu Gly Lys Ile Glu Phe Glu Gly Gln Pro
Val Asp Phe 180 185 190Val Asp Pro Asn Lys Gln Asn Leu Ile Ala Glu
Val Ser Thr Lys Asp 195 200 205Val Lys Val Tyr Gly Lys Gly Asn Pro
Thr Lys Val Val Ala Val Asp 210 215 220Cys Gly Ile Lys Asn Asn Val
Ile Arg Leu Leu Val Lys Arg Gly Ala225 230 235 240Glu Val His Leu
Val Pro Trp Asn His Asp Phe Thr Lys Met Glu Tyr 245 250 255Asp Gly
Ile Leu Ile Ala Gly Gly Pro Gly Asn Pro Ala Leu Ala Glu 260 265
270Pro Leu Ile Gln Asn Val Arg Lys Ile Leu Glu Ser Asp Arg Lys Glu
275 280 285Pro Leu Phe Gly Ile Ser Thr Gly Asn Leu Ile Thr Gly Leu
Ala Ala 290 295 300Gly Ala Lys Thr Tyr Lys Met Ser Met Ala Asn Arg
Gly Gln Asn Gln305 310 315 320Pro Val Leu Asn Ile Thr Asn Lys Gln
Ala Phe Ile Thr Ala Gln Asn 325 330 335His Gly Tyr Ala Leu Asp Asn
Thr Leu Pro Ala Gly Trp Lys Pro Leu 340 345 350Phe Val Asn Val Asn
Asp Gln Thr Asn Glu Gly Ile Met His Glu Ser 355 360 365Lys Pro Phe
Phe Ala Val Gln Phe His Pro Glu Val Thr Pro Gly Pro 370 375 380Ile
Asp Thr Glu Tyr Leu Phe Asp Ser Phe Phe Ser Leu Ile Lys Lys385 390
395 400Gly Lys Ala Thr Thr Ile Thr Ser Val Leu Pro Lys Pro Ala Leu
Val 405 410 415Ala Ser Arg Val Glu Val Ser Lys Val Leu Ile Leu Gly
Ser Gly Gly 420 425 430Leu Ser Ile Gly Gln Ala Gly Glu Phe Asp Tyr
Ser Gly Ser Gln Ala 435 440 445Val Lys Ala Met Lys Glu Glu Asn Val
Lys Thr Val Leu Met Asn Pro 450 455 460Asn Ile Ala Ser Val Gln Thr
Asn Glu Val Gly Leu Lys Gln Ala Asp465 470 475 480Thr Val Tyr Phe
Leu Pro Ile Thr Pro Gln Phe Val Thr Glu Val Ile 485 490 495Lys Ala
Glu Gln Pro Asp Gly Leu Ile Leu Gly Met Gly Gly Gln Thr 500 505
510Ala Leu Asn Cys Gly Val Glu Leu Phe Lys Arg Gly Val Leu Lys Glu
515 520 525Tyr Gly Val Lys Val Leu Gly Thr Ser Val Glu Ser Ile Met
Ala Thr 530 535 540Glu Asp Arg Gln Leu Phe Ser Asp Lys Leu Asn Glu
Ile Asn Glu Lys545 550 555 560Ile Ala Pro Ser Phe Ala Val Glu Ser
Ile Glu Asp Ala Leu Lys Ala 565 570 575Ala Asp Thr Ile Gly Tyr Pro
Val Met Ile Arg Ser Ala Tyr Ala Leu 580 585 590Gly Gly Leu Gly Ser
Gly Ile Cys Pro Asn Arg Glu Thr Leu Met Asp 595 600 605Leu Ser Thr
Lys Ala Phe Ala Met Thr Asn Gln Ile Leu Val Glu Lys 610 615 620Ser
Val Thr Gly Trp Lys Glu Ile Glu Tyr Glu Val Val Arg Asp Ala625 630
635 640Asp Asp Asn Cys Val Thr Val Cys Asn Met Glu Asn Val Asp Ala
Met 645 650 655Gly Val His Thr Gly Asp Ser Val Val Val Ala Pro Ala
Gln Thr Leu 660 665 670Ser Asn Ala Glu Phe Gln Met Leu Arg Arg Thr
Ser Ile Asn Val Val 675 680 685Arg His Leu Gly Ile Val Gly Glu Cys
Asn Ile Gln Phe Ala Leu His 690 695 700Pro Thr Ser Met Glu Tyr Cys
Ile Ile Glu Val Asn Ala Arg Leu Ser705 710 715 720Arg Ser Ser Ala
Leu Ala Ser Lys Ala Thr Gly Tyr Pro Leu Ala Phe 725 730 735Ile Ala
Ala Lys Ile Ala Leu Gly Ile Pro Leu Pro Glu Ile Lys Asn 740 745
750Val Val Ser Gly Lys Thr Ser Ala Cys Phe Glu Pro Ser Leu Asp Tyr
755 760 765Met Val Thr Lys Ile Pro Arg Trp Asp Leu Asp Arg Phe His
Gly Thr 770 775 780Ser Ser Arg Ile Gly Ser Ser Met Lys Ser Val Gly
Glu Val Met Ala785 790 795 800Ile Gly Arg Thr Phe Glu Glu Ser Phe
Gln Lys Ala Leu Arg Met Cys 805 810 815His Pro Ser Ile Glu Gly Phe
Thr Pro Arg Leu Pro Met Asn Lys Glu 820 825 830Trp Pro Ser Asn Leu
Asp Leu Arg Lys Glu Leu Ser Glu Pro Ser Ser 835 840 845Thr Arg Ile
Tyr Ala Ile Ala Lys Ala Ile Asp Asp Asn Met Ser Leu 850 855 860Asp
Glu Ile Glu Lys Leu Thr Tyr Ile Asp Lys Trp Phe Leu Tyr Lys865 870
875 880Met Arg Asp Ile Leu Asn Met Glu Lys Thr Leu Lys Gly Leu Asn
Ser 885 890 895Glu Ser Met Thr Glu Glu Thr Leu Lys Arg Ala Lys Glu
Ile Gly Phe 900 905 910Ser Asp Lys Gln Ile Ser Lys Cys Leu Gly Leu
Thr Glu Ala Gln Thr 915 920 925Arg Glu Leu Arg Leu Lys Lys Asn Ile
His Pro Trp Val Lys Gln Ile 930 935 940Asp Thr Leu Ala Ala Glu Tyr
Pro Ser Val Thr Asn Tyr Leu Tyr Val945 950 955 960Thr Tyr Asn Gly
Gln Glu His Asp Val Asn Phe Asp Asp His Gly Met 965 970 975Met Val
Leu Gly Cys Gly Pro Tyr His Ile Gly Ser Ser Val Glu Phe 980 985
990Asp Trp Cys Ala Val Ser Ser Ile Arg Thr Leu Arg Gln Leu Gly Lys
995 1000 1005Lys Thr Val Val Val Asn Cys Asn Pro Glu Thr Val Ser
Thr Asp 1010 1015 1020Phe Asp Glu Cys Asp Lys Leu Tyr Phe Glu Glu
Leu Ser Leu Glu 1025 1030 1035Arg Ile Leu Asp Ile Tyr His Gln Glu
Ala Cys Gly Gly Cys Ile 1040 1045 1050Ile Ser Val Gly Gly Gln Ile
Pro Asn Asn Leu Ala Val Pro Leu 1055 1060 1065Tyr Lys Asn Gly Val
Lys Ile Met Gly Thr Ser Pro Leu Gln Ile 1070 1075 1080Asp Arg Ala
Glu Asp Arg Ser Ile Phe Ser Ala Val Leu Asp Glu 1085 1090 1095Leu
Lys Val Ala Gln Ala Pro Trp Lys Ala Val Asn Thr Leu Asn 1100 1105
1110Glu Ala Leu Glu Phe Ala Lys Ser Val Asp Tyr Pro Cys Leu Leu
1115 1120 1125Arg Pro Ser Tyr Val Leu Ser Gly Ser Ala Met Asn Val
Val Phe 1130 1135 1140Ser Glu Asp Glu Met Lys Lys Phe Leu Glu Glu
Ala Thr Arg Val 1145 1150
1155Ser Gln Glu His Pro Val Val Leu Thr Lys Phe Val Glu Gly Ala
1160 1165 1170Arg Glu Val Glu Met Asp Ala Val Gly Lys Asp Gly Arg
Val Ile 1175 1180 1185Ser His Ala Ile Ser Glu His Val Glu Asp Ala
Gly Val His Ser 1190 1195 1200Gly Asp Ala Thr Leu Met Leu Pro Thr
Gln Thr Ile Ser Gln Gly 1205 1210 1215Ala Ile Glu Lys Val Lys Asp
Ala Thr Arg Lys Ile Ala Lys Ala 1220 1225 1230Phe Ala Ile Ser Gly
Pro Phe Asn Val Gln Phe Leu Val Lys Gly 1235 1240 1245Asn Asp Val
Leu Val Ile Glu Cys Asn Leu Arg Ala Ser Arg Ser 1250 1255 1260Phe
Pro Phe Val Ser Lys Thr Leu Gly Val Asp Phe Ile Asp Val 1265 1270
1275Ala Thr Lys Val Met Ile Gly Glu Asn Val Asp Glu Lys His Leu
1280 1285 1290Pro Thr Leu Asp His Pro Ile Ile Pro Ala Asp Tyr Val
Ala Ile 1295 1300 1305Lys Ala Pro Met Phe Ser Trp Pro Arg Leu Arg
Asp Ala Asp Pro 1310 1315 1320Ile Leu Arg Cys Glu Met Ala Ser Thr
Gly Glu Val Ala Cys Phe 1325 1330 1335Gly Glu Gly Ile His Thr Ala
Phe Leu Lys Ala Met Leu Ser Thr 1340 1345 1350Gly Phe Lys Ile Pro
Gln Lys Gly Ile Leu Ile Gly Ile Gln Gln 1355 1360 1365Ser Phe Arg
Pro Arg Phe Leu Gly Val Ala Glu Gln Leu His Asn 1370 1375 1380Glu
Gly Phe Lys Leu Phe Ala Thr Glu Ala Thr Ser Asp Trp Leu 1385 1390
1395Asn Ala Asn Asn Val Pro Ala Asn Pro Val Ala Trp Pro Ser Gln
1400 1405 1410Glu Gly Gln Asn Pro Ser Leu Ser Ser Ile Arg Lys Leu
Ile Arg 1415 1420 1425Asp Gly Ser Ile Asp Leu Val Ile Asn Leu Pro
Asn Asn Asn Thr 1430 1435 1440Lys Phe Val His Asp Asn Tyr Val Ile
Arg Arg Thr Ala Val Asp 1445 1450 1455Ser Gly Ile Pro Leu Leu Thr
Asn Phe Gln Val Thr Lys Leu Phe 1460 1465 1470Ala Glu Ala Val Gln
Lys Ser Arg Lys Val Asp Ser Lys Ser Leu 1475 1480 1485Phe His Tyr
Arg Gln Tyr Ser Ala Gly Lys Ala Ala 1490 1495 1500135761DNAHomo
sapiensCDS(124)..(4626) 13gtcagcctta aacactgact gcacccctcc
cagatttctt ttacattaac taaaaagtct 60tatcacacaa tctcataaaa tttatgtaat
ttcatttaat tttagccaca aatcatcttc 120aaa atg acg agg att att aca gct
ttc aaa gtg gtg agg aca ctg aag 168 Met Thr Arg Ile Ile Thr Ala Phe
Lys Val Val Arg Thr Leu Lys 1 5 10 15act ggt ttt ggc ttt acc aat
gtg act gca cac caa aaa tgg aaa ttt 216Thr Gly Phe Gly Phe Thr Asn
Val Thr Ala His Gln Lys Trp Lys Phe 20 25 30tca aga cct ggc atc agg
ctc ctt tct gtc aag gca cag aca gca cac 264Ser Arg Pro Gly Ile Arg
Leu Leu Ser Val Lys Ala Gln Thr Ala His 35 40 45att gtc ctg gaa gat
gga act aag atg aaa ggt tac tcc ttt ggc cat 312Ile Val Leu Glu Asp
Gly Thr Lys Met Lys Gly Tyr Ser Phe Gly His 50 55 60cca tcc tct gtt
gct ggt gaa gtg gtt ttt aat act ggc ctg gga ggg 360Pro Ser Ser Val
Ala Gly Glu Val Val Phe Asn Thr Gly Leu Gly Gly 65 70 75tac cca gaa
gct att act gac cct gcc tac aaa gga cag att ctc aca 408Tyr Pro Glu
Ala Ile Thr Asp Pro Ala Tyr Lys Gly Gln Ile Leu Thr80 85 90 95atg
gcc aac cct att att ggg aat ggt gga gct cct gat act act gct 456Met
Ala Asn Pro Ile Ile Gly Asn Gly Gly Ala Pro Asp Thr Thr Ala 100 105
110ctg gat gaa ctg gga ctt agc aaa tat ttg gag tct aat gga atc aag
504Leu Asp Glu Leu Gly Leu Ser Lys Tyr Leu Glu Ser Asn Gly Ile Lys
115 120 125gtt tca ggt ttg ctg gtg ctg gat tat agt aaa gac tac aac
cac tgg 552Val Ser Gly Leu Leu Val Leu Asp Tyr Ser Lys Asp Tyr Asn
His Trp 130 135 140ctg gct acc aag agt tta ggg caa tgg cta cag gaa
gaa aag gtt cct 600Leu Ala Thr Lys Ser Leu Gly Gln Trp Leu Gln Glu
Glu Lys Val Pro 145 150 155gca att tat gga gtg gac aca aga atg ctg
act aaa ata att cgg gat 648Ala Ile Tyr Gly Val Asp Thr Arg Met Leu
Thr Lys Ile Ile Arg Asp160 165 170 175aag ggt acc atg ctt ggg aag
att gaa ttt gaa ggt cag cct gtg gat 696Lys Gly Thr Met Leu Gly Lys
Ile Glu Phe Glu Gly Gln Pro Val Asp 180 185 190ttt gtg gat cca aat
aaa cag aat ttg att gct gag gtt tca acc aag 744Phe Val Asp Pro Asn
Lys Gln Asn Leu Ile Ala Glu Val Ser Thr Lys 195 200 205gat gtc aaa
gtg tac ggc aaa gga aac ccc aca aaa gtg gta gct gta 792Asp Val Lys
Val Tyr Gly Lys Gly Asn Pro Thr Lys Val Val Ala Val 210 215 220gac
tgt ggg att aaa aac aat gta atc cgc ctg cta gta aag cga gga 840Asp
Cys Gly Ile Lys Asn Asn Val Ile Arg Leu Leu Val Lys Arg Gly 225 230
235gct gaa gtg cac tta gtt ccc tgg aac cat gat ttc acc aag atg gag
888Ala Glu Val His Leu Val Pro Trp Asn His Asp Phe Thr Lys Met
Glu240 245 250 255tat gat ggg att ttg atc gcg gga gga ccg ggg aac
cca gct ctt gca 936Tyr Asp Gly Ile Leu Ile Ala Gly Gly Pro Gly Asn
Pro Ala Leu Ala 260 265 270gaa cca cta att cag aat gtc aga aag att
ttg gag agt gat cgc aag 984Glu Pro Leu Ile Gln Asn Val Arg Lys Ile
Leu Glu Ser Asp Arg Lys 275 280 285gag cca ttg ttt gga atc agt aca
gga aac tta ata aca gga ttg gct 1032Glu Pro Leu Phe Gly Ile Ser Thr
Gly Asn Leu Ile Thr Gly Leu Ala 290 295 300gct ggt gcc aaa acc tac
aag atg tcc atg gcc aac aga ggg cag aat 1080Ala Gly Ala Lys Thr Tyr
Lys Met Ser Met Ala Asn Arg Gly Gln Asn 305 310 315cag cct gtt ttg
aat atc aca aac aaa cag gct ttc att act gct cag 1128Gln Pro Val Leu
Asn Ile Thr Asn Lys Gln Ala Phe Ile Thr Ala Gln320 325 330 335aat
cat ggc tat gcc ttg gac aac acc ctc cct gct ggc tgg aaa cca 1176Asn
His Gly Tyr Ala Leu Asp Asn Thr Leu Pro Ala Gly Trp Lys Pro 340 345
350ctt ttt gtg aat gtc aac gat caa aca aat gag ggg att atg cat gag
1224Leu Phe Val Asn Val Asn Asp Gln Thr Asn Glu Gly Ile Met His Glu
355 360 365agc aaa ccc ttc ttc gct gtg cag ttc cac cca gag gtc acc
ccg ggg 1272Ser Lys Pro Phe Phe Ala Val Gln Phe His Pro Glu Val Thr
Pro Gly 370 375 380cca ata gac act gag tac ctg ttt gat tcc ttt ttc
tca ctg ata aag 1320Pro Ile Asp Thr Glu Tyr Leu Phe Asp Ser Phe Phe
Ser Leu Ile Lys 385 390 395aaa gga aaa gct acc acc att aca tca gtc
tta ccg aag cca gca cta 1368Lys Gly Lys Ala Thr Thr Ile Thr Ser Val
Leu Pro Lys Pro Ala Leu400 405 410 415gtt gca tct cgg gtt gag gtt
tcc aaa gtc ctt att cta gga tca gga 1416Val Ala Ser Arg Val Glu Val
Ser Lys Val Leu Ile Leu Gly Ser Gly 420 425 430ggt ctg tcc att ggt
cag gct gga gaa ttt gat tac tca gga tct caa 1464Gly Leu Ser Ile Gly
Gln Ala Gly Glu Phe Asp Tyr Ser Gly Ser Gln 435 440 445gct gta aaa
gcc atg aag gaa gaa aat gtc aaa act gtt ctg atg aac 1512Ala Val Lys
Ala Met Lys Glu Glu Asn Val Lys Thr Val Leu Met Asn 450 455 460cca
aac att gca tca gtc cag acc aat gag gtg ggc tta aag caa gcg 1560Pro
Asn Ile Ala Ser Val Gln Thr Asn Glu Val Gly Leu Lys Gln Ala 465 470
475gat act gtc tac ttt ctt ccc atc acc cct cag ttt gtc aca gag gtc
1608Asp Thr Val Tyr Phe Leu Pro Ile Thr Pro Gln Phe Val Thr Glu
Val480 485 490 495atc aag gca gaa cag cca gat ggg tta att ctg ggc
atg ggt ggc cag 1656Ile Lys Ala Glu Gln Pro Asp Gly Leu Ile Leu Gly
Met Gly Gly Gln 500 505 510aca gct ctg aac tgt gga gtg gaa cta ttc
aag aga ggt gtg ctc aag 1704Thr Ala Leu Asn Cys Gly Val Glu Leu Phe
Lys Arg Gly Val Leu Lys 515 520 525gaa tat ggt gtg aaa gtc ctg gga
act tca gtt gag tcc att atg gct 1752Glu Tyr Gly Val Lys Val Leu Gly
Thr Ser Val Glu Ser Ile Met Ala 530 535 540acg gaa gac agg cag ctg
ttt tca gat aaa cta aat gag atc aat gaa 1800Thr Glu Asp Arg Gln Leu
Phe Ser Asp Lys Leu Asn Glu Ile Asn Glu 545 550 555aag att gct cca
agt ttt gca gtg gaa tcg att gag gat gca ctg aag 1848Lys Ile Ala Pro
Ser Phe Ala Val Glu Ser Ile Glu Asp Ala Leu Lys560 565 570 575gca
gca gac acc att ggc tac cca gtg atg atc cgt tcc gcc tat gca 1896Ala
Ala Asp Thr Ile Gly Tyr Pro Val Met Ile Arg Ser Ala Tyr Ala 580 585
590ctg ggt ggg tta ggc tca ggc atc tgt ccc aac aga gag act ttg atg
1944Leu Gly Gly Leu Gly Ser Gly Ile Cys Pro Asn Arg Glu Thr Leu Met
595 600 605gac ctc agc aca aag gcc ttt gct atg acc aac caa att ctg
gtg gag 1992Asp Leu Ser Thr Lys Ala Phe Ala Met Thr Asn Gln Ile Leu
Val Glu 610 615 620aag tca gtg aca ggt tgg aaa gaa ata gaa tat gaa
gtg gtt cga gat 2040Lys Ser Val Thr Gly Trp Lys Glu Ile Glu Tyr Glu
Val Val Arg Asp 625 630 635gct gat gac aat tgt gtc act gtc tgt aac
atg gaa aat gtt gat gcc 2088Ala Asp Asp Asn Cys Val Thr Val Cys Asn
Met Glu Asn Val Asp Ala640 645 650 655atg ggt gtt cac aca ggt gac
tca gtt gtt gtg gct cct gcc cag aca 2136Met Gly Val His Thr Gly Asp
Ser Val Val Val Ala Pro Ala Gln Thr 660 665 670ctc tcc aat gcc gag
ttt cag atg ttg aga cgt act tca atc aat gtt 2184Leu Ser Asn Ala Glu
Phe Gln Met Leu Arg Arg Thr Ser Ile Asn Val 675 680 685gtt cgc cac
ttg ggc att gtg ggt gaa tgc aac att cag ttt gcc ctt 2232Val Arg His
Leu Gly Ile Val Gly Glu Cys Asn Ile Gln Phe Ala Leu 690 695 700cat
cct acc tca atg gaa tac tgc atc att gaa gtg aat gcc aga ctg 2280His
Pro Thr Ser Met Glu Tyr Cys Ile Ile Glu Val Asn Ala Arg Leu 705 710
715tcc cga agc tct gct ctg gcc tca aaa gcc act ggc tac cca ttg gca
2328Ser Arg Ser Ser Ala Leu Ala Ser Lys Ala Thr Gly Tyr Pro Leu
Ala720 725 730 735ttc att gct gca aag att gcc cta gga atc cca ctt
cca gaa att aag 2376Phe Ile Ala Ala Lys Ile Ala Leu Gly Ile Pro Leu
Pro Glu Ile Lys 740 745 750aac gtc gta tcc ggg aag aca tca gcc tgt
ttt gaa cct agc ctg gat 2424Asn Val Val Ser Gly Lys Thr Ser Ala Cys
Phe Glu Pro Ser Leu Asp 755 760 765tac atg gtc acc aag att ccc cgc
tgg gat ctt gac cgt ttt cat gga 2472Tyr Met Val Thr Lys Ile Pro Arg
Trp Asp Leu Asp Arg Phe His Gly 770 775 780aca tct agc cga att ggt
agc tct atg aaa agt gta gga gag gtc atg 2520Thr Ser Ser Arg Ile Gly
Ser Ser Met Lys Ser Val Gly Glu Val Met 785 790 795gct att ggt cgt
acc ttt gag gag agt ttc cag aaa gct tta cgg atg 2568Ala Ile Gly Arg
Thr Phe Glu Glu Ser Phe Gln Lys Ala Leu Arg Met800 805 810 815tgc
cac cca tct ata gaa ggt ttc act ccc cgt ctc cca atg aac aaa 2616Cys
His Pro Ser Ile Glu Gly Phe Thr Pro Arg Leu Pro Met Asn Lys 820 825
830gaa tgg cca tct aat tta gat ctt aga aaa gag ttg tct gaa cca agc
2664Glu Trp Pro Ser Asn Leu Asp Leu Arg Lys Glu Leu Ser Glu Pro Ser
835 840 845agc acg cgt atc tat gcc att gcc aag gcc att gat gac aac
atg tcc 2712Ser Thr Arg Ile Tyr Ala Ile Ala Lys Ala Ile Asp Asp Asn
Met Ser 850 855 860ctt gat gag att gag aag ctc aca tac att gac aag
tgg ttt ttg tat 2760Leu Asp Glu Ile Glu Lys Leu Thr Tyr Ile Asp Lys
Trp Phe Leu Tyr 865 870 875aag atg cgt gat att tta aac atg gaa aag
aca ctg aaa ggg ctc aac 2808Lys Met Arg Asp Ile Leu Asn Met Glu Lys
Thr Leu Lys Gly Leu Asn880 885 890 895agt gag tcc atg aca gaa gaa
acc ctg aaa agg gca aag gag att ggg 2856Ser Glu Ser Met Thr Glu Glu
Thr Leu Lys Arg Ala Lys Glu Ile Gly 900 905 910ttc tca gat aag cag
att tca aaa tgc ctt ggg ctc act gag gcc cag 2904Phe Ser Asp Lys Gln
Ile Ser Lys Cys Leu Gly Leu Thr Glu Ala Gln 915 920 925aca agg gag
ctg agg tta aag aaa aac atc cac cct tgg gtt aaa cag 2952Thr Arg Glu
Leu Arg Leu Lys Lys Asn Ile His Pro Trp Val Lys Gln 930 935 940att
gat aca ctg gct gca gaa tac cca tca gta aca aac tat ctc tat 3000Ile
Asp Thr Leu Ala Ala Glu Tyr Pro Ser Val Thr Asn Tyr Leu Tyr 945 950
955gtt acc tac aat ggt cag gag cat gat gtc aat ttt gat gac cat gga
3048Val Thr Tyr Asn Gly Gln Glu His Asp Val Asn Phe Asp Asp His
Gly960 965 970 975atg atg gtg cta ggc tgt ggt cca tat cac att ggc
agc agt gtg gaa 3096Met Met Val Leu Gly Cys Gly Pro Tyr His Ile Gly
Ser Ser Val Glu 980 985 990ttt gat tgg tgt gct gtc tct agt atc cgc
aca ctg cgt caa ctt ggc 3144Phe Asp Trp Cys Ala Val Ser Ser Ile Arg
Thr Leu Arg Gln Leu Gly 995 1000 1005aag aag acg gtg gtg gtg aat
tgc aat cct gag act gtg agc aca 3189Lys Lys Thr Val Val Val Asn Cys
Asn Pro Glu Thr Val Ser Thr 1010 1015 1020gac ttt gat gag tgt gac
aaa ctg tac ttt gaa gag ttg tcc ttg 3234Asp Phe Asp Glu Cys Asp Lys
Leu Tyr Phe Glu Glu Leu Ser Leu 1025 1030 1035gag aga atc cta gac
atc tac cat cag gag gca tgt ggt ggc tgc 3279Glu Arg Ile Leu Asp Ile
Tyr His Gln Glu Ala Cys Gly Gly Cys 1040 1045 1050atc ata tca gtt
gga ggc cag att cca aac aac ctg gca gtt cct 3324Ile Ile Ser Val Gly
Gly Gln Ile Pro Asn Asn Leu Ala Val Pro 1055 1060 1065cta tac aag
aat ggt gtc aag atc atg ggc aca agc ccc ctg cag 3369Leu Tyr Lys Asn
Gly Val Lys Ile Met Gly Thr Ser Pro Leu Gln 1070 1075 1080atc gac
agg gct gag gat cgc tcc atc ttc tca gct gtc ttg gat 3414Ile Asp Arg
Ala Glu Asp Arg Ser Ile Phe Ser Ala Val Leu Asp 1085 1090 1095gag
ctg aag gtg gct cag gca cct tgg aaa gct gtt aat act ttg 3459Glu Leu
Lys Val Ala Gln Ala Pro Trp Lys Ala Val Asn Thr Leu 1100 1105
1110aat gaa gca ctg gaa ttt gca aag tct gtg gac tac ccc tgc ttg
3504Asn Glu Ala Leu Glu Phe Ala Lys Ser Val Asp Tyr Pro Cys Leu
1115 1120 1125ttg agg cct tcc tat gtt ttg agt ggg tct gct atg aat
gtg gta 3549Leu Arg Pro Ser Tyr Val Leu Ser Gly Ser Ala Met Asn Val
Val 1130 1135 1140ttc tct gag gat gag atg aaa aaa ttc cta gaa gag
gcg act aga 3594Phe Ser Glu Asp Glu Met Lys Lys Phe Leu Glu Glu Ala
Thr Arg 1145 1150 1155gtt tct cag gag cac cca gtg gtc ctg aca aaa
ttt gtt gaa ggg 3639Val Ser Gln Glu His Pro Val Val Leu Thr Lys Phe
Val Glu Gly 1160 1165 1170gcc cga gaa gta gaa atg gac gct gtt ggc
aaa gat gga agg gtt 3684Ala Arg Glu Val Glu Met Asp Ala Val Gly Lys
Asp Gly Arg Val 1175 1180 1185atc tct cat gcc atc tct gaa cat gtt
gaa gat gca ggt gtc cac 3729Ile Ser His Ala Ile Ser Glu His Val Glu
Asp Ala Gly Val His 1190 1195 1200tcg gga gat gcc act ctg atg ctg
ccc aca caa acc atc agc caa 3774Ser Gly Asp Ala Thr Leu Met Leu Pro
Thr Gln Thr Ile Ser Gln 1205 1210 1215ggg gcc att gaa aag gtg aag
gat gct acc cgg aag att gca aag 3819Gly Ala Ile Glu Lys Val Lys Asp
Ala Thr Arg Lys Ile Ala Lys 1220 1225 1230gct ttt gcc atc tct ggt
cca ttc aac gtc caa ttt ctt gtc aaa 3864Ala Phe Ala Ile Ser Gly Pro
Phe Asn Val Gln Phe Leu Val Lys 1235 1240 1245gga aat gat gtc ttg
gtg att gag tgt aac ttg aga gct tct cga 3909Gly Asn Asp Val Leu Val
Ile Glu Cys Asn Leu Arg Ala Ser Arg 1250 1255 1260tcc ttc ccc ttt
gtt tcc aag act ctt ggg gtt gac ttc att gat 3954Ser Phe Pro Phe Val
Ser Lys Thr Leu Gly Val Asp Phe Ile Asp 1265 1270 1275gtg gcc acc
aag gtg atg att gga gag aat gtt gat gag aaa cat 3999Val Ala Thr Lys
Val Met Ile Gly Glu Asn Val Asp Glu Lys His 1280
1285 1290ctt cca aca ttg gac cat ccc ata att cct gct gac tat gtt
gca 4044Leu Pro Thr Leu Asp His Pro Ile Ile Pro Ala Asp Tyr Val Ala
1295 1300 1305att aag gct ccc atg ttt tcc tgg ccc cgg ttg agg gat
gct gac 4089Ile Lys Ala Pro Met Phe Ser Trp Pro Arg Leu Arg Asp Ala
Asp 1310 1315 1320ccc att ctg aga tgt gag atg gct tcc act gga gag
gtg gct tgc 4134Pro Ile Leu Arg Cys Glu Met Ala Ser Thr Gly Glu Val
Ala Cys 1325 1330 1335ttt ggt gaa ggt att cat aca gcc ttc cta aag
gca atg ctt tcc 4179Phe Gly Glu Gly Ile His Thr Ala Phe Leu Lys Ala
Met Leu Ser 1340 1345 1350aca gga ttt aag ata ccc cag aaa ggc atc
ctg ata ggc atc cag 4224Thr Gly Phe Lys Ile Pro Gln Lys Gly Ile Leu
Ile Gly Ile Gln 1355 1360 1365caa tca ttc cgg cca aga ttc ctt ggt
gtg gct gaa caa tta cac 4269Gln Ser Phe Arg Pro Arg Phe Leu Gly Val
Ala Glu Gln Leu His 1370 1375 1380aat gaa ggt ttc aag ctg ttt gcc
acg gaa gcc aca tca gac tgg 4314Asn Glu Gly Phe Lys Leu Phe Ala Thr
Glu Ala Thr Ser Asp Trp 1385 1390 1395ctc aac gcc aac aat gtc cct
gcc aac cca gtg gca tgg ccg tct 4359Leu Asn Ala Asn Asn Val Pro Ala
Asn Pro Val Ala Trp Pro Ser 1400 1405 1410caa gaa gga cag aat ccc
agc ctc tct tcc atc aga aaa ttg att 4404Gln Glu Gly Gln Asn Pro Ser
Leu Ser Ser Ile Arg Lys Leu Ile 1415 1420 1425aga gat ggc agc att
gac cta gtg att aac ctt ccc aac aac aac 4449Arg Asp Gly Ser Ile Asp
Leu Val Ile Asn Leu Pro Asn Asn Asn 1430 1435 1440act aaa ttt gtc
cat gat aat tat gtg att cgg agg aca gct gtt 4494Thr Lys Phe Val His
Asp Asn Tyr Val Ile Arg Arg Thr Ala Val 1445 1450 1455gat agt gga
atc cct ctc ctc act aat ttt cag gtg acc aaa ctt 4539Asp Ser Gly Ile
Pro Leu Leu Thr Asn Phe Gln Val Thr Lys Leu 1460 1465 1470ttt gct
gaa gct gtg cag aaa tct cgc aag gtg gac tcc aag agt 4584Phe Ala Glu
Ala Val Gln Lys Ser Arg Lys Val Asp Ser Lys Ser 1475 1480 1485ctt
ttc cac tac agg cag tac agt gct gga aaa gca gca tag 4626Leu Phe His
Tyr Arg Gln Tyr Ser Ala Gly Lys Ala Ala 1490 1495 1500agatgcagac
accccagccc cattattaaa tcaacctgag ccacatgtta tctaaaggaa
4686ctgattcaca actttctcag agatgaatat tgataactaa acttcatttc
agtttacttt 4746gttatgcctt aatattctgt gtcttttgca attaaattgt
cagtcacttc ttcaaaacct 4806tacagtcctt cctaagttac tcttcatgag
atttcatcca tttactaata ctgtattttt 4866ggtggactag gcttgcctat
gtgcttatgt gtagcttttt actttttatg gtgctgatta 4926atggtgatca
aggtaggaaa agttgctgtt ctattttctg aactctttct atactttaag
4986atactctatt tttaaaacac tatctgcaaa ctcaggacac tttaacaggg
cagaatactc 5046taaaaacttg ataaaatgaa atatagattt aatttatgaa
ccttccatca tgatgtttgt 5106gtattgcttc tttttggatc ctcattctca
cccatttggc taatccagga atattgttat 5166cccttcccat tatattgaag
ttgagaaatg tgacagaggc atttagagta tggacttttc 5226ttttcttttt
ctttttcttt ttttcttttt gagatggagt cacactctcc aggctggagt
5286gcagtggcac aatctcggct cactgcaatt tgcgtctccc aagttcaagc
gattctcctg 5346ctttagacta tggatttctt taaggaatac tggtttgcag
ttttgttttc tggactatat 5406cagcagatgg tagacagtgt ttatgtagat
gtgttgttgt ttttatcatt ggattttaac 5466ttggcccgag tgaaataatc
agatttttgt cattcacact ctcccccagt tttggaataa 5526cttggaagta
aggttcattc ccttaagacg atggattctg ttgaactatg gggtcccaca
5586ctgcactatt aattccaccc actgtaaggg caaggacacc attccttcta
catataagaa 5646aaaagtctct ccccaagggc agcctttgtt acttttaaat
attttctgtt attacaagtg 5706ctctaattgt gaacttttaa ataaaatact
attaagaggt aaaaaaaaaa aaaaa 5761141500PRTHomo sapiens 14Met Thr Arg
Ile Ile Thr Ala Phe Lys Val Val Arg Thr Leu Lys Thr1 5 10 15Gly Phe
Gly Phe Thr Asn Val Thr Ala His Gln Lys Trp Lys Phe Ser 20 25 30Arg
Pro Gly Ile Arg Leu Leu Ser Val Lys Ala Gln Thr Ala His Ile 35 40
45Val Leu Glu Asp Gly Thr Lys Met Lys Gly Tyr Ser Phe Gly His Pro
50 55 60Ser Ser Val Ala Gly Glu Val Val Phe Asn Thr Gly Leu Gly Gly
Tyr65 70 75 80Pro Glu Ala Ile Thr Asp Pro Ala Tyr Lys Gly Gln Ile
Leu Thr Met 85 90 95Ala Asn Pro Ile Ile Gly Asn Gly Gly Ala Pro Asp
Thr Thr Ala Leu 100 105 110Asp Glu Leu Gly Leu Ser Lys Tyr Leu Glu
Ser Asn Gly Ile Lys Val 115 120 125Ser Gly Leu Leu Val Leu Asp Tyr
Ser Lys Asp Tyr Asn His Trp Leu 130 135 140Ala Thr Lys Ser Leu Gly
Gln Trp Leu Gln Glu Glu Lys Val Pro Ala145 150 155 160Ile Tyr Gly
Val Asp Thr Arg Met Leu Thr Lys Ile Ile Arg Asp Lys 165 170 175Gly
Thr Met Leu Gly Lys Ile Glu Phe Glu Gly Gln Pro Val Asp Phe 180 185
190Val Asp Pro Asn Lys Gln Asn Leu Ile Ala Glu Val Ser Thr Lys Asp
195 200 205Val Lys Val Tyr Gly Lys Gly Asn Pro Thr Lys Val Val Ala
Val Asp 210 215 220Cys Gly Ile Lys Asn Asn Val Ile Arg Leu Leu Val
Lys Arg Gly Ala225 230 235 240Glu Val His Leu Val Pro Trp Asn His
Asp Phe Thr Lys Met Glu Tyr 245 250 255Asp Gly Ile Leu Ile Ala Gly
Gly Pro Gly Asn Pro Ala Leu Ala Glu 260 265 270Pro Leu Ile Gln Asn
Val Arg Lys Ile Leu Glu Ser Asp Arg Lys Glu 275 280 285Pro Leu Phe
Gly Ile Ser Thr Gly Asn Leu Ile Thr Gly Leu Ala Ala 290 295 300Gly
Ala Lys Thr Tyr Lys Met Ser Met Ala Asn Arg Gly Gln Asn Gln305 310
315 320Pro Val Leu Asn Ile Thr Asn Lys Gln Ala Phe Ile Thr Ala Gln
Asn 325 330 335His Gly Tyr Ala Leu Asp Asn Thr Leu Pro Ala Gly Trp
Lys Pro Leu 340 345 350Phe Val Asn Val Asn Asp Gln Thr Asn Glu Gly
Ile Met His Glu Ser 355 360 365Lys Pro Phe Phe Ala Val Gln Phe His
Pro Glu Val Thr Pro Gly Pro 370 375 380Ile Asp Thr Glu Tyr Leu Phe
Asp Ser Phe Phe Ser Leu Ile Lys Lys385 390 395 400Gly Lys Ala Thr
Thr Ile Thr Ser Val Leu Pro Lys Pro Ala Leu Val 405 410 415Ala Ser
Arg Val Glu Val Ser Lys Val Leu Ile Leu Gly Ser Gly Gly 420 425
430Leu Ser Ile Gly Gln Ala Gly Glu Phe Asp Tyr Ser Gly Ser Gln Ala
435 440 445Val Lys Ala Met Lys Glu Glu Asn Val Lys Thr Val Leu Met
Asn Pro 450 455 460Asn Ile Ala Ser Val Gln Thr Asn Glu Val Gly Leu
Lys Gln Ala Asp465 470 475 480Thr Val Tyr Phe Leu Pro Ile Thr Pro
Gln Phe Val Thr Glu Val Ile 485 490 495Lys Ala Glu Gln Pro Asp Gly
Leu Ile Leu Gly Met Gly Gly Gln Thr 500 505 510Ala Leu Asn Cys Gly
Val Glu Leu Phe Lys Arg Gly Val Leu Lys Glu 515 520 525Tyr Gly Val
Lys Val Leu Gly Thr Ser Val Glu Ser Ile Met Ala Thr 530 535 540Glu
Asp Arg Gln Leu Phe Ser Asp Lys Leu Asn Glu Ile Asn Glu Lys545 550
555 560Ile Ala Pro Ser Phe Ala Val Glu Ser Ile Glu Asp Ala Leu Lys
Ala 565 570 575Ala Asp Thr Ile Gly Tyr Pro Val Met Ile Arg Ser Ala
Tyr Ala Leu 580 585 590Gly Gly Leu Gly Ser Gly Ile Cys Pro Asn Arg
Glu Thr Leu Met Asp 595 600 605Leu Ser Thr Lys Ala Phe Ala Met Thr
Asn Gln Ile Leu Val Glu Lys 610 615 620Ser Val Thr Gly Trp Lys Glu
Ile Glu Tyr Glu Val Val Arg Asp Ala625 630 635 640Asp Asp Asn Cys
Val Thr Val Cys Asn Met Glu Asn Val Asp Ala Met 645 650 655Gly Val
His Thr Gly Asp Ser Val Val Val Ala Pro Ala Gln Thr Leu 660 665
670Ser Asn Ala Glu Phe Gln Met Leu Arg Arg Thr Ser Ile Asn Val Val
675 680 685Arg His Leu Gly Ile Val Gly Glu Cys Asn Ile Gln Phe Ala
Leu His 690 695 700Pro Thr Ser Met Glu Tyr Cys Ile Ile Glu Val Asn
Ala Arg Leu Ser705 710 715 720Arg Ser Ser Ala Leu Ala Ser Lys Ala
Thr Gly Tyr Pro Leu Ala Phe 725 730 735Ile Ala Ala Lys Ile Ala Leu
Gly Ile Pro Leu Pro Glu Ile Lys Asn 740 745 750Val Val Ser Gly Lys
Thr Ser Ala Cys Phe Glu Pro Ser Leu Asp Tyr 755 760 765Met Val Thr
Lys Ile Pro Arg Trp Asp Leu Asp Arg Phe His Gly Thr 770 775 780Ser
Ser Arg Ile Gly Ser Ser Met Lys Ser Val Gly Glu Val Met Ala785 790
795 800Ile Gly Arg Thr Phe Glu Glu Ser Phe Gln Lys Ala Leu Arg Met
Cys 805 810 815His Pro Ser Ile Glu Gly Phe Thr Pro Arg Leu Pro Met
Asn Lys Glu 820 825 830Trp Pro Ser Asn Leu Asp Leu Arg Lys Glu Leu
Ser Glu Pro Ser Ser 835 840 845Thr Arg Ile Tyr Ala Ile Ala Lys Ala
Ile Asp Asp Asn Met Ser Leu 850 855 860Asp Glu Ile Glu Lys Leu Thr
Tyr Ile Asp Lys Trp Phe Leu Tyr Lys865 870 875 880Met Arg Asp Ile
Leu Asn Met Glu Lys Thr Leu Lys Gly Leu Asn Ser 885 890 895Glu Ser
Met Thr Glu Glu Thr Leu Lys Arg Ala Lys Glu Ile Gly Phe 900 905
910Ser Asp Lys Gln Ile Ser Lys Cys Leu Gly Leu Thr Glu Ala Gln Thr
915 920 925Arg Glu Leu Arg Leu Lys Lys Asn Ile His Pro Trp Val Lys
Gln Ile 930 935 940Asp Thr Leu Ala Ala Glu Tyr Pro Ser Val Thr Asn
Tyr Leu Tyr Val945 950 955 960Thr Tyr Asn Gly Gln Glu His Asp Val
Asn Phe Asp Asp His Gly Met 965 970 975Met Val Leu Gly Cys Gly Pro
Tyr His Ile Gly Ser Ser Val Glu Phe 980 985 990Asp Trp Cys Ala Val
Ser Ser Ile Arg Thr Leu Arg Gln Leu Gly Lys 995 1000 1005Lys Thr
Val Val Val Asn Cys Asn Pro Glu Thr Val Ser Thr Asp 1010 1015
1020Phe Asp Glu Cys Asp Lys Leu Tyr Phe Glu Glu Leu Ser Leu Glu
1025 1030 1035Arg Ile Leu Asp Ile Tyr His Gln Glu Ala Cys Gly Gly
Cys Ile 1040 1045 1050Ile Ser Val Gly Gly Gln Ile Pro Asn Asn Leu
Ala Val Pro Leu 1055 1060 1065Tyr Lys Asn Gly Val Lys Ile Met Gly
Thr Ser Pro Leu Gln Ile 1070 1075 1080Asp Arg Ala Glu Asp Arg Ser
Ile Phe Ser Ala Val Leu Asp Glu 1085 1090 1095Leu Lys Val Ala Gln
Ala Pro Trp Lys Ala Val Asn Thr Leu Asn 1100 1105 1110Glu Ala Leu
Glu Phe Ala Lys Ser Val Asp Tyr Pro Cys Leu Leu 1115 1120 1125Arg
Pro Ser Tyr Val Leu Ser Gly Ser Ala Met Asn Val Val Phe 1130 1135
1140Ser Glu Asp Glu Met Lys Lys Phe Leu Glu Glu Ala Thr Arg Val
1145 1150 1155Ser Gln Glu His Pro Val Val Leu Thr Lys Phe Val Glu
Gly Ala 1160 1165 1170Arg Glu Val Glu Met Asp Ala Val Gly Lys Asp
Gly Arg Val Ile 1175 1180 1185Ser His Ala Ile Ser Glu His Val Glu
Asp Ala Gly Val His Ser 1190 1195 1200Gly Asp Ala Thr Leu Met Leu
Pro Thr Gln Thr Ile Ser Gln Gly 1205 1210 1215Ala Ile Glu Lys Val
Lys Asp Ala Thr Arg Lys Ile Ala Lys Ala 1220 1225 1230Phe Ala Ile
Ser Gly Pro Phe Asn Val Gln Phe Leu Val Lys Gly 1235 1240 1245Asn
Asp Val Leu Val Ile Glu Cys Asn Leu Arg Ala Ser Arg Ser 1250 1255
1260Phe Pro Phe Val Ser Lys Thr Leu Gly Val Asp Phe Ile Asp Val
1265 1270 1275Ala Thr Lys Val Met Ile Gly Glu Asn Val Asp Glu Lys
His Leu 1280 1285 1290Pro Thr Leu Asp His Pro Ile Ile Pro Ala Asp
Tyr Val Ala Ile 1295 1300 1305Lys Ala Pro Met Phe Ser Trp Pro Arg
Leu Arg Asp Ala Asp Pro 1310 1315 1320Ile Leu Arg Cys Glu Met Ala
Ser Thr Gly Glu Val Ala Cys Phe 1325 1330 1335Gly Glu Gly Ile His
Thr Ala Phe Leu Lys Ala Met Leu Ser Thr 1340 1345 1350Gly Phe Lys
Ile Pro Gln Lys Gly Ile Leu Ile Gly Ile Gln Gln 1355 1360 1365Ser
Phe Arg Pro Arg Phe Leu Gly Val Ala Glu Gln Leu His Asn 1370 1375
1380Glu Gly Phe Lys Leu Phe Ala Thr Glu Ala Thr Ser Asp Trp Leu
1385 1390 1395Asn Ala Asn Asn Val Pro Ala Asn Pro Val Ala Trp Pro
Ser Gln 1400 1405 1410Glu Gly Gln Asn Pro Ser Leu Ser Ser Ile Arg
Lys Leu Ile Arg 1415 1420 1425Asp Gly Ser Ile Asp Leu Val Ile Asn
Leu Pro Asn Asn Asn Thr 1430 1435 1440Lys Phe Val His Asp Asn Tyr
Val Ile Arg Arg Thr Ala Val Asp 1445 1450 1455Ser Gly Ile Pro Leu
Leu Thr Asn Phe Gln Val Thr Lys Leu Phe 1460 1465 1470Ala Glu Ala
Val Gln Lys Ser Arg Lys Val Asp Ser Lys Ser Leu 1475 1480 1485Phe
His Tyr Arg Gln Tyr Ser Ala Gly Lys Ala Ala 1490 1495
15001520DNAHomo sapiens 15cggaagccac atcagactgg 201624DNAHomo
sapiens 16ggagagtgaa acttgacaat catc 241719DNAHomo sapiens
17tactgctcag aatcatggc 191819DNAHomo sapiens 18tcatcaccaa ctgaacagg
191921DNAHomo sapiens 19ggttaagaga aggaggagct g 212021DNAHomo
sapiens 20aaccagtctt cagtgtcctc a 212115DNAHomo sapiens
21cctgccaccc cagtg 152215DNAHomo sapiens 22cctgccaacc cagtg
15238PRTHomo sapiensMISC_FEATURE(3)..(3)X is A or S 23Pro Val Xaa
Trp Pro Xaa Xaa Glu1 5
* * * * *