U.S. patent application number 09/813133 was filed with the patent office on 2002-09-26 for isolated human protease proteins, nucleic acid molecules encoding human protease proteins, and uses thereof.
Invention is credited to Beasley, Ellen M., Di Francesco, Valentina, Gan, Weiniu, Ketchum, Karen A..
Application Number | 20020137179 09/813133 |
Document ID | / |
Family ID | 25211534 |
Filed Date | 2002-09-26 |
United States Patent
Application |
20020137179 |
Kind Code |
A1 |
Gan, Weiniu ; et
al. |
September 26, 2002 |
ISOLATED HUMAN PROTEASE PROTEINS, NUCLEIC ACID MOLECULES ENCODING
HUMAN PROTEASE PROTEINS, AND USES THEREOF
Abstract
The present invention provides amino acid sequences of peptides
that are encoded by genes within the human genome, the protease
peptides of the present invention. The present invention
specifically provides isolated peptide and nucleic acid molecules,
methods of identifying orthologs and paralogs of the protease
peptides, and methods of identifying modulators of the protease
peptides.
Inventors: |
Gan, Weiniu; (Gaithersburg,
MD) ; Ketchum, Karen A.; (Germantown, MD) ; Di
Francesco, Valentina; (Rockville, MD) ; Beasley,
Ellen M.; (Darnestown, MD) |
Correspondence
Address: |
CELERA GENOMICS CORPORATION
45 West Gude Dr. C2-4#20
Rockville
MD
20850
US
|
Family ID: |
25211534 |
Appl. No.: |
09/813133 |
Filed: |
March 21, 2001 |
Current U.S.
Class: |
435/226 ;
435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
A61P 43/00 20180101;
C12N 9/6424 20130101 |
Class at
Publication: |
435/226 ;
435/69.1; 435/320.1; 435/325; 536/23.2 |
International
Class: |
C12N 009/64; C07H
021/04; C12P 021/02; C12N 005/06 |
Claims
That which is claimed is:
1. An isolated peptide consisting of an amino acid sequence
selected from the group consisting of: (a) an amino acid sequence
shown in SEQ ID NO:2; (b) an amino acid sequence of an allelic
variant of an amino acid sequence shown in SEQ ID NO:2, wherein
said allelic variant is encoded by a nucleic acid molecule that
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid
sequence of an ortholog of an amino acid sequence shown in SEQ ID
NO:2, wherein said ortholog is encoded by a nucleic acid molecule
that hybridizes under stringent conditions to the opposite strand
of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; and (d) a
fragment of an amino acid sequence shown in SEQ ID NO:2, wherein
said fragment comprises at least 10 contiguous amino acids.
2. An isolated peptide comprising an amino acid sequence selected
from the group consisting of: (a) an amino acid sequence shown in
SEQ ID NO:2; (b) an amino acid sequence of an allelic variant of an
amino acid sequence shown in SEQ ID NO:2, wherein said allelic
variant is encoded by a nucleic acid molecule that hybridizes under
stringent conditions to the opposite strand of a nucleic acid
molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of
an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein
said ortholog is encoded by a nucleic acid molecule that hybridizes
under stringent conditions to the opposite strand of a nucleic acid
molecule shown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino
acid sequence shown in SEQ ID NO:2, wherein said fragment comprises
at least 10 contiguous amino acids.
3. An isolated antibody that selectively binds to a peptide of
claim 2.
4. An isolated nucleic acid molecule consisting of a nucleotide
sequence selected from the group consisting of: (a) a nucleotide
sequence that encodes an amino acid sequence shown in SEQ ID NO:2;
(b) a nucleotide sequence that encodes of an allelic variant of an
amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide
sequence hybridizes under stringent conditions to the opposite
strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a
nucleotide sequence that encodes an ortholog of an amino acid
sequence shown in SEQ ID NO:2, wherein said nucleotide sequence
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotide
sequence that encodes a fragment of an amino acid sequence shown in
SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous
amino acids; and (e) a nucleotide sequence that is the complement
of a nucleotide sequence of (a)-(d).
5. An isolated nucleic acid molecule comprising a nucleotide
sequence selected from the group consisting of: (a) a nucleotide
sequence that encodes an amino acid sequence shown in SEQ ID NO:2;
(b) a nucleotide sequence that encodes of an allelic variant of an
amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide
sequence hybridizes under stringent conditions to the opposite
strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a
nucleotide sequence that encodes an ortholog of an amino acid
sequence shown in SEQ ID NO:2, wherein said nucleotide sequence
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotide
sequence that encodes a fragment of an amino acid sequence shown in
SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous
amino acids; and (e) a nucleotide sequence that is the complement
of a nucleotide sequence of (a)-(d).
6. A gene chip comprising a nucleic acid molecule of claim 5.
7. A transgenic non-human animal comprising a nucleic acid molecule
of claim 5.
8. A nucleic acid vector comprising a nucleic acid molecule of
claim 5.
9. A host cell containing the vector of claim 8.
10. A method for producing any of the peptides of claim 1
comprising introducing a nucleotide sequence encoding any of the
amino acid sequences in (a)-(d) into a host cell, and culturing the
host cell under conditions in which the peptides are expressed from
the nucleotide sequence.
11. A method for producing any of the peptides of claim 2
comprising introducing a nucleotide sequence encoding any of the
amino acid sequences in (a)-(d) into a host cell, and culturing the
host cell under conditions in which the peptides are expressed from
the nucleotide sequence.
12. A method for detecting the presence of any of the peptides of
claim 2 in a sample, said method comprising contacting said sample
with a detection agent that specifically allows detection of the
presence of the peptide in the sample and then detecting the
presence of the peptide.
13. A method for detecting the presence of a nucleic acid molecule
of claim 5 in a sample, said method comprising contacting the
sample with an oligonucleotide that hybridizes to said nucleic acid
molecule under stringent conditions and determining whether the
oligonucleotide binds to said nucleic acid molecule in the
sample.
14. A method for identifying a modulator of a peptide of claim 2,
said method comprising contacting said peptide with an agent and
determining if said agent has modulated the function or activity of
said peptide.
15. The method of claim 14, wherein said agent is administered to a
host cell comprising an expression vector that expresses said
peptide.
16. A method for identifying an agent that binds to any of the
peptides of claim 2, said method comprising contacting the peptide
with an agent and assaying the contacted mixture to determine
whether a complex is formed with the agent bound to the
peptide.
17. A pharmaceutical composition comprising an agent identified by
the method of claim 16 and a pharmaceutically acceptable carrier
therefor.
18. A method for treating a disease or condition mediated by a
human protease protein, said method comprising administering to a
patient a pharmaceutically effective amount of an agent identified
by the method of claim 16.
19. A method for identifying a modulator of the expression of a
peptide of claim 2, said method comprising contacting a cell
expressing said peptide with an agent, and determining if said
agent has modulated the expression of said peptide.
20. An isolated human protease peptide having an amino acid
sequence that shares at least 70% homology with an amino acid
sequence shown in SEQ ID NO:2.
21. A peptide according to claim 20 that shares at least 90 percent
homology with an amino acid sequence shown in SEQ ID NO:2.
22. An isolated nucleic acid molecule encoding a human protease
peptide, said nucleic acid molecule sharing at least 80 percent
homology with a nucleic acid molecule shown in SEQ ID NOS:1or
3.
23. A nucleic acid molecule according to claim 22 that shares at
least 90 percent homology with a nucleic acid molecule shown in SEQ
ID NOS:1 or 3.
Description
FIELD OF THE INVENTION
[0001] The present invention is in the field of protease proteins
that are related to the carboxypeptidase subfamily, recombinant DNA
molecules, and protein production. The present invention
specifically provides novel peptides and proteins that effect
protein cleavage/processing/turnover and nucleic acid molecules
encoding such peptide and protein molecules, all of which are
useful in the development of human therapeutics and diagnostic
compositions and methods.
BACKGROUND OF THE INVENTION
[0002] The proteases may be categorized into families by the
different amino acid sequences (generally between 2 and 10
residues) located on either side of the cleavage site of the
protease.
[0003] The proper functioning of the cell requires careful control
of the levels of important structural proteins, enzymes, and
regulatory proteins. One of the ways that cells can reduce the
steady state level of a particular protein is by proteolytic
degradation. Further, one of the ways cells produce functioning
proteins is to produce pre or pro-protein precursors that are
processed by proteolytic degradation to produce an active moiety.
Thus, complex and highly-regulated mechanisms have been evolved to
accomplish this degradation.
[0004] Proteases regulate many different cell proliferation,
differentiation, and signaling processes by regulating protein
turnover and processing. Uncontrolled protease activity (either
increased or decreased) has been implicated in a variety of disease
conditions including inflammation, cancer, arteriosclerosis, and
degenerative disorders.
[0005] An additional role of intracellular proteolysis is in the
stress-response. Cells that are subject to stress such as
starvation, heat-shock, chemical insult or mutation respond by
increasing the rates of proteolysis. One function of this enhanced
proteolysis is to salvage amino acids from non-essential proteins.
These amino acids can then be re-utilized in the synthesis of
essential proteins or metabolized directly to provide energy.
Another function is in the repair of damage caused by the stress.
For example, oxidative stress has been shown to damage a variety of
proteins and cause them to be rapidly degraded.
[0006] The International Union of Biochemistry and Molecular
Biology (IUBMB) has recommended to use the term peptidase for the
subset of peptide bond hydrolases (Subclass E.C 3.4.). The widely
used term protease is synonymous with peptidase. Peptidases
comprise two groups of enzymes: the endopeptidases and the
exopeptidases, which cleave peptide bonds at points within the
protein and remove amino acids sequentially from either N or
C-terminus respectively. The term proteinase is also used as a
synonym word for endopeptidase and four mechanistic classes of
proteinases are recognized by the IUBMB: two of these are described
below (also see: Handbook of Proteolytic Enzymes by Barrett,
Rawlings, and Woessner AP Press, NY 1998). Also, for a review of
the various uses of proteases as drug targets, see: Weber M,
Emerging treatments for hypertension: potential role for
vasopeptidase inhibition; Am J Hypertens November 1999; 12(11 Pt
2):139S-147S; Kentsch M, Otter W, Novel neurohormonal modulators in
cardiovascular disorders. The therapeutic potential of
endopeptidase inhibitors, Drugs R D April 1999; 1(4):331-8;
Scarborough R M, Coagulation factor Xa: the prothrombinase complex
as an emerging therapeutic target for small molecule inhibitors, J
Enzym Inhib 1998; 14(1):15-25; Skotnicki J S, et al., Design and
synthetic considerations of matrix metalloproteinase inhibitors,
Ann N Y Acad Sci June 1999 30; 878:61-72; McKerrow J H, Engel J C,
Caffrey C R, Cysteine protease inhibitors as chemotherapy for
parasitic infections, Bioorg Med Chem April 1999; 7(4):639-44; Rice
K D, Tanaka R D, Katz B A, Numerof R P, Moore W R, Inhibitors of
tryptase for the treatment of mast cell-mediated diseases, Curr
Pharm Des October 1998; 4(5):381-96; Materson B J, Will angiotensin
converting enzyme genotype, receptor mutation identification, and
other miracles of molecular biology permit reduction of NNT Am J
Hypertens August 1998; 11(8 Pt 2):138S-142S
[0007] Serine Proteases
[0008] The serine proteases (SP) are a large family of proteolytic
enzymes that include the digestive enzymes, trypsin and
chymotrypsin, components of the complement cascade and of the
blood-clotting cascade, and enzymes that control the degradation
and turnover of macromolecules of the extracellular matrix. SP are
so named because of the presence of a serine residue in the active
catalytic site for protein cleavage. SP have a wide range of
substrate specificities and can be subdivided into subfamilies on
the basis of these specificities. The main sub-families are
trypases (cleavage after arginine or lysine), aspases (cleavage
after aspartate), chymases (cleavage after phenylalanine or
leucine), metases (cleavage after methionine), and serases
(cleavage after serine).
[0009] A series of six SP have been identified in murine cytotoxic
T-lymphocytes (CTL) and natural killer (NK) cells. These SP are
involved with CTL and NK cells in the destruction of virally
transformed cells and tumor cells and in organ and tissue
transplant rejection (Zunino, S. J. et al. (1990) J. Immunol.
144:2001-9; Sayers, T. J. et al. (1994) J. Immunol. 152:2289-97).
Human homologs of most of these enzymes have been identified
(Trapani, J. A. et al. (1988) Proc. Natl. Acad. Sci. 85:6924-28;
Caputo, A. et al. (1990) J. Immunol. 145:737-44). Like all SP, the
CTL-SP share three distinguishing features: 1) the presence of a
catalytic triad of histidine, serine, and aspartate residues which
comprise the active site; 2) the sequence GDSGGP which contains the
active site serine; and 3) an N-terminal IIGG sequence which
characterizes the mature SP.
[0010] The SP are secretory proteins which contain N-terminal
signal peptides that serve to export the immature protein across
the endoplasmic reticulum and are then cleaved (von Heijne (1986)
Nuc. Acid. Res. 14:5683-90). Differences in these signal sequences
provide one means of distinguishing individual SP. Some SP,
particularly the digestive enzymes, exist as inactive precursors or
preproenzymes, and contain a leader or activation peptide sequence
3' of the signal peptide. This activation peptide may be 2-12 amino
acids in length, and it extends from the cleavage site of the
signal peptide to the N-terminal IIGG sequence of the active,
mature protein. Cleavage of this sequence activates the enzyme.
This sequence varies in different SP according to the biochemical
pathway and/or its substrate (Zunino et al, supra; Sayers et al,
supra). Other features that distinguish various SP are the presence
or absence of N-linked glycosylation sites that provide membrane
anchors, the number and distribution of cysteine residues that
determine the secondary structure of the SP, and the sequence of a
substrate binding sites such as S'. The S' substrate binding region
is defined by residues extending from approximately +17 to +29
relative to the N-terminal I (+1). Differences in this region of
the molecule are believed to determine SP substrate specificities
(Zunino et al, supra).
[0011] Trypsinogens
[0012] The trypsinogens are serine proteases secreted by exocrine
cells of the pancreas (Travis J and Roberts R. Biochemistry 1969;
8: 2884-9; Mallory P and Travis J, Biochemistry 1973; 12: 2847-51).
Two major types of trypsinogen isoenzymes have been characterized,
trypsinogen-1, also called cationic trypsinogen, and trypsinogen-2
or anionic trypsinogen. The trypsinogen proenzymes are activated to
trypsins in the intestine by enterokinase, which removes an
activation peptide from the N-terminus of the trypsinogens. The
trypsinogens show a high degree of sequence homology, but they can
be separated on the basis of charge differences by using
electrophoresis or ion exchange chromatography. The major form of
trypsinogen in the pancreas and pancreatic juice is trypsinogen-1
(Guy C O et al., Biochem Biophys Res Commun 1984; 125: 516-23). In
serum of healthy subjects, trypsinogen-1 is also the major form,
whereas in patients with pancreatitis, trypsinogen-2 is more
strongly elevated (Itkonen et al., J Lab Clin Med 1990; 115:712-8).
Trypsinogens also occur in certain ovarian tumors, in which
trypsinogen-2 is the major form (Koivunen et al., Cancer Res 1990;
50: 2375-8). Trypsin-1 in complex with alpha-1 -antitrypsin, also
called alpha-1 -antiprotease, has been found to occur in serum of
patients with pancreatitis (Borgstrom A and Ohlsson K, Scand J Clin
Lab Invest 1984; 44: 381-6) but determination of this complex has
not been found useful for differentiation between pancreatic and
other gastrointestinal diseases (Borgstrom et al., Scand J Clin Lab
Invest 1989; 49:757-62).
[0013] Trypsinogen-1 and -2 are closely related immunologically
(Kimland et al., Clin Chim Acta 1989; 184: 31-46; Itkonen et al.,
1990), but by using monoclonal antibodies (Itkonen et al., 1990) or
by absorbing polyclonal antisera (Kimland et al., 1989) it is
possible to obtain reagents enabling specific measurement of each
form of trypsinogen.
[0014] When active trypsin reaches the blood stream, it is
inactivated by the major trypsin inhibitors alpha-2-macroglobulin
and alpha-1-antitrypsin (AAT). AAT is a 58 kilodalton serine
protease inhibitor synthesized in the liver and is one of the main
protease inhibitors in blood. Whereas complexes between trypsin-1
and AAT are detectable in serum (Borgstrom and Ohlsson, 1984) the
complexes with alpha-2-macroglobulin are not measurable with
antibody-based assays (Ohlsson K, Acta Gastroenterol Belg 1988; 51:
3-12).
[0015] Inflammation of the pancreas or pancreatitis may be
classified as either acute or chronic by clinical criteria. With
treatment, acute pancreatitis can often be cured and normal
function restored. Chronic pancreatitis often results in permanent
damage. The precise mechanisms which trigger acute inflammation are
not understood. However, some causes in the order of their
importance are alcohol ingestion, biliary tract disease,
post-operative trauma, and hereditary pancreatitis. One theory
provides that autodigestion, the premature activation of
proteolytic enzymes in the pancreas rather than in the duodenum,
causes acute pancreatitis. Any number of other factors including
endotoxins, exotoxins, viral infections, ischemia, anoxia, and
direct trauma may activate the proenzymes. In addition, any
internal or external blockage of pancreatic ducts can also cause an
accumulation of pancreatic juices in the pancreas resulting
cellular damage.
[0016] Anatomy, physiology, and diseases of the pancreas are
reviewed, inter alia, in Guyton A C (1991) Textbook of Medical
Physiology, W B Saunders Co, Philadelphia, Pa.; Isselbacher K J et
al (1994) Harrison's Principles of Internal Medicine, McGraw-Hill,
New York City; Johnson K E (1991) Histology and Cell Biology,
Harwal Publishing, Media, Pa.; and The Merck Manual of Diagnosis
and Therapy (1992) Merck Research Laboratories, Rahway, N.J.
[0017] Metalloprotease
[0018] The metalloproteases may be one of the older classes of
proteinases and are found in bacteria, fungi as well as in higher
organisms. They differ widely in their sequences and their
structures but the great majority of enzymes contain a zinc atom
which is catalytically active. In some cases, zinc may be replaced
by another metal such as cobalt or nickel without loss of the
activity. Bacterial thermolysin has been well characterized and its
crystallographic structure indicates that zinc is bound by two
histidines and one glutamic acid. Many enzymes contain the sequence
HEXXH, which provides two histidine ligands for the zinc whereas
the third ligand is either a glutamic acid (thermolysin,
neprilysin, alanyl aminopeptidase) or a histidine (astacin). Other
families exhibit a distinct mode of binding of the Zn atom. The
catalytic mechanism leads to the formation of a non covalent
tetrahedral intermediate after the attack of a zinc-bound water
molecule on the carbonyl group of the scissile bond. This
intermediate is further decomposed by transfer of the glutamic acid
proton to the leaving group.
[0019] Metalloproteases contain a catalytic zinc metal center which
participates in the hydrolysis of the peptide backbone (reviewed in
Power and Harper, in Protease Inhibitors, A. J. Barrett and G.
Salversen (eds.) Elsevier, Amsterdam, 1986, p. 219). The active
zinc center differentiates some of these proteases from calpains
and trypsins whose activities are dependent upon the presence of
calcium. Examples of metalloproteases include carboxypeptidase A,
carboxypeptidase B, and thermolysin.
[0020] Metalloproteases have been isolated from a number of
procaryotic and eucaryotic sources, e.g. Bacillus subtilis (McConn
et al., 1964, J. Biol. Chem. 239:3706); Bacillus megaterium;
Serratia (Miyata et al., 1971, Agr. Biol. Chem. 35:460);
Clostridium bifermentans (MacFarlane et al., 1992, App. Environ.
Microbiol. 58:1195-1200), Legionella pneumophila (Moffat et al.,
1994, Infection and Immunity 62:751-3). In particular, acidic
metalloproteases have been isolated from broad-banded copperhead
venoms (Johnson and Ownby, 1993, Int. J. Biochem. 25:267-278),
rattlesnake venoms (Chlou et al., 1992, Biochem. Biophys. Res.
Commun. 187:389-396) and articular cartilage (Treadwell et al.,
1986, Arch. Biochem. Biophys. 251:715-723). Neutral
metalloproteases, specifically those having optimal activity at
neutral pH have, for example, been isolated from Aspergillus sojae
(Sekine, 1973, Agric. Biol. Chem. 37:1945-1952). Neutral
metalloproteases obtained from Aspergillus have been classified
into two groups, npI and npII (Sekine, 1972, Agric. Biol. Chem.
36:207-216). So far, success in obtaining amino acid sequence
information from these fungal neutral metalloproteases has been
limited. An npII metalloprotease isolated from Aspergillus oryzae
has been cloned based on amino acid sequence presented in the
literature (Tatsumi et al., 1991, Mol. Gen. Genet. 228:97-103).
However, to date, no npI fungal metalloprotease has been cloned or
sequenced. Alkaline metalloproteases, for example, have been
isolated from Pseudomonas aeruginosa (Baumann et al., 1993, EMBO J
12:3357-3364) and the insect pathogen Xenorhabdus luminescens
(Schmidt et al., 1998, Appl. Environ. Microbiol. 54:2793-2797).
[0021] Metalloproteases have been devided into several distinct
families based primarily on activity and sturcture: 1) water
nucleophile; water bound by single zinc ion ligated to two His
(within the motif HEXXH) and Glu, His or Asp; 2) water nucleophile;
water bound by single zinc ion ligated to His, Glu (within the
motif HXXE) and His; 3) water nucleophile; water bound by single
zinc ion ligated to His, Asp and His; 4) Water nucleophile; water
bound by single zinc ion ligated to two His (within the motif
HXXEH) and Glu and 5) water nucleophile; water bound by two zinc
ions ligated by Lys, Asp, Asp, Asp, Glu.
[0022] Examples of members of the metalloproteinase family include,
but are not limited to, membrane alanyl aminopeptidase (Homo
sapiens), germinal peptidyl-dipeptidase A (Homo sapiens), thimet
oligopeptidase (Rattus norvegicus), oligopeptidase F (Lactococcus
lactis), mycolysin (Streptomyces cacaoi), immune inhibitor A
(Bacillus thuringiensis), snapalysin (Streptomyces lividans),
leishmanolysin (Leishmania major), microbial collagenase (Vibrio
alginolyticus), microbial collagenase, class I (Clostridium
perfringens), collagenase 1 (Homo sapiens), serralysin (Serratia
marcescens), fragilysin (Bacteroides fragilis), gametolysin
(Chlamydomonas reinhardtii), astacin (Astacus fluviatilis),
adamalysin (Crotalus adamanteus), ADAM 10 (Bos taurus), neprilysin
(Homo sapiens), carboxypeptidase A (Homo sapiens), carboxypeptidase
E (Bos taurus), gamma-D-glutamyl-(L)-meso-diaminopimelate peptidase
I (Bacillus sphaericus), vanY D-Ala-D-Ala carboxypeptidase
(Enterococcus faecium), endolysin (bacteriophage A118), pitrilysin
(Escherichia coli), mitochondrial processing peptidase
(Saccharomyces cerevisiae), leucyl aminopeptidase (Bos taurus),
aminopeptidase I (Saccharomyces cerevisiae), membrane dipeptidase
(Homo sapiens), glutamate carboxypeptidase (Pseudomonas sp.), Gly-X
carboxypeptidase (Saccharomyces cerevisiae), O-sialoglycoprotein
endopeptidase (Pasteurella haemolytica), beta-lytic
metalloendopeptidase (Achromobacter lyticus), methionyl
aminopeptidase I (Escherichia coli), X-Pro aminopeptidase
(Escherichia coli), X-His dipeptidase (Escherichia coli),
IgA1-specific metalloendopeptidase (Streptococcus sanguis),
tentoxilysin (Clostridium tetani), leucyl aminopeptidase (Vibrio
proteolyticus), aminopeptidase (Streptomyces griseus), IAP
aminopeptidase (Escherichia coli), aminopeptidase T (Thermus
aquaticus), hyicolysin (Staphylococcus hyicus), carboxypeptidase
Taq (Thermus aquaticus), anthrax lethal factor (Bacillus
anthracis), penicillolysin (Penicillium citrinum), fungalysin
(Aspergillus fumigatus), lysostaphin (Staphylococcus simulans),
beta-aspartyl dipeptidase (Escherichia coli), carboxypeptidase Ss1
(Sulfolobus solfataricus), FtsH endopeptidase (Escherichia coli),
glutamyl aminopeptidase (Lactococcus lactis), cytophagalysin
(Cytophaga sp.), metalloendopeptidase (vaccinia virus), VanX
D-Ala-D-Ala dipeptidase (Enterococcus faecium), Ste24p
endopeptidase (Saccharomyces cerevisiae), dipeptidyl-peptidase III
(Rattus norvegicus), S2P protease (Homo sapiens), sporulation
factor SpoIVFB (Bacillus subtilis), and HYBD endopeptidase
(Escherichia coli).
[0023] Metalloproteases have been found to have a number of uses.
For example, there is strong evidence that a metalloprotease is
involved in the in vivo proteolytic processing of the
vasoconstrictor, endothelin-1. Rat metalloprotease has been found
to be involved in peptide hormone processing. One important
subfamily of the metalloproteases are the matrix
metalloproteases.
[0024] A number of diseases are thought to be mediated by excess or
undesired metalloprotease activity or by an imbalance in the ratio
of the various members of the protease family of proteins. These
include: a) osteoarthritis (Woessner, et al., J. Biol.Chem. 259(6),
3633, 1984; Phadke, et al., J. Rheumatol. 10, 852, 1983), b)
rheumatoid arthritis (Mullins, et al., Biochim. Biophys. Acta 695,
117, 1983; Woolley, et al., Arthritis Rheum. 20, 1231, 1977;
Gravallese, et al., Arthritis Rheum. 34, 1076, 1991), c) septic
arthritis (Williams, et al., Arthritis Rheum. 33, 533, 1990), d)
tumor metastasis (Reich, et al., Cancer Res. 48, 3307, 1988, and
Matrisian, et al., Proc. Nat'l. Acad. Sci., USA 83, 9413, 1986), e)
periodontal diseases (Overall, et al., J. Periodontal Res. 22, 81,
1987), f) corneal ulceration (Burns, et al., Invest. Opthalmol.
Vis. Sci. 30, 1569, 1989), g) proteinuria (Baricos, et al.,
Biochem. J. 254, 609, 1988), h) coronary thrombosis from
atherosclerotic plaque rupture (Henney, et al., Proc. Nat'l. Acad.
Sci., USA 88, 8154-8158, 1991), i) aneurysmal aortic disease (Vine,
et al., Clin. Sci. 81, 233, 1991), j) birth control (Woessner, et
al., Steroids 54, 491, 1989), k) dystrophobic epidermolysis bullosa
(Kronberger, et al., J. Invest. Dermatol. 79, 208, 1982), and l)
degenerative cartilage loss following traumatic joint injury, m)
conditions leading to inflammatory responses, osteopenias mediated
by MMP activity, n) tempero mandibular joint disease, o)
demyelating diseases of the nervous system (Chantry, et al., J.
Neurochem. 50, 688, 1988).
[0025] Aspartic Protease
[0026] Aspartic proteases have been divided into several distinct
families based primarily on activity and structure. These include
1) water nucleophile; water bound by two Asp from monomer or dimer;
all endopeptidases, from eukaryote organisms, viruses or virus-like
organisms and 2) endopeptidases that are water nucleophile and are
water bound by Asp and Asn.
[0027] Most of aspartic proteases belong to the pepsin family. The
pepsin family includes digestive enzymes such as pepsin and
chymosin as well as lysosomal cathepsins D and processing enzymes
such as renin, and certain fungal proteases (penicillopepsin,
rhizopuspepsin, endothiapepsin). A second family comprises viral
proteases such as the protease from the AIDS virus (HIV) also
called retropepsin. Crystallographic studies have shown that these
enzymes are bilobed molecules with the active site located between
two homologous lobes. Each lobe contributes one aspartate residue
of the catalytically active diad of aspartates. These two aspartyl
residues are in close geometric proximity in the active molecule
and one aspartate is ionized whereas the second one is unionized at
the optimum pH range of 2-3. Retropepsins, are monomeric, i.e carry
only one catalytic aspartate and then dimerization is required to
form an active enzyme.
[0028] In contrast to serine and cysteine proteases, catalysis by
aspartic protease do not involve a covalent intermediate though a
tetrahedral intermediate exists. The nucleophilic attack is
achieved by two simultaneous proton transfer: one from a water
molecule to the diad of the two carboxyl groups and a second one
from the diad to the carbonyl oxygen of the substrate with the
concurrent CO--NH bond cleavage. This general acid-base catalysis,
which may be called a "push-pull" mechanism leads to the formation
of a non covalent neutral tetrahedral intermediate.
[0029] Examples of the aspartic protease family of proteins
include, but are not limited to, pepsin A (Homo sapiens), HIV1
retropepsin (human immunodeficiency virus type 1), endopeptidase
(cauliflower mosaic virus), bacilliform virus putative protease
(rice tungro bacilliform virus), aspergillopepsin II (Aspergillus
niger), thermopsin (Sulfolobus acidocaldarius), nodavirus
endopeptidase (flock house virus), pseudomonapepsin (Pseudomonas
sp. 101), signal peptidase II (Escherichia coli), polyprotein
peptidase (human spumaretrovirus), copia transposon (Drosophila
melanogaster), SIRE-1 peptidase (Glycine max), retrotransposon bs1
endopeptidase (Zea mays), retrotransposon peptidase (Drosophila
buzzatii), Tas retrotransposon peptidase (Ascaris lumbricoides),
Pao retrotransposon peptidase (Bombyx mori), putative proteinase of
Skippy retrotransposon (Fusarium oxysporum), tetravirus
endopeptidase (Nudaurelia capensis omega virus), presenilin 1 (Homo
sapiens).
[0030] Carboxypeptidase
[0031] Carboxypeptidases are proteases that function in many
physiological processes. These proteases remove a wide range of
carboxyl-terminal amino acids, and in doing so are able to
activate, inactivate, and modulate enzyme and peptide hormone
activity. Many active forms of mammalian carboxypeptidases are
located in lysosomes where they regulate intracellular protein
processing, degradation and turnover. In plants and insects
carboxypeptidases play a role in posttranslational protein
modifications including mobilization of storage proteins and
hormone activation.
[0032] Carboxypeptidase activities are regulated either by
endogenous protein inhibitors or by enzymatic cleavage of a segment
of a propeptide to release the active carboxypeptidase.
Carboxypeptidases A and B (CPA and CPB, respectively) are
pancreatic zinc-containing proteolytic enzymes which catalyze the
hydrolysis of the carboxyl-terminal peptide bond in polypeptide
chains. When transcribed in rat brain and other nonpancreatic
tissues, CPA is unable to function as a protease (Normant, E. et
al. (1995) J. Biol. Chem. 270: 20543-20549). This inability to
function as a protease has been attributed to the presence of
tissue-specific, endogenous protein inhibitors such as tissue
carboxypeptidase inhibitor (TCI) or latexin (Normant, E. et al.
(1995) Proc. Natl. Acad. Sci. 92: 12225-12229; Hatanaka, Y. et al.
(1994) Eur. J. Neurosci. 6: 973-982).
[0033] Latexin and TCI are 222 and 223 amino acids in length,
respectively. They contain several potential phosphorylation sites,
but they do not show a membrane-specific signal peptide sequence
(Normant et al., supra; Hatanaka et al., supra). TCI is a
non-competitive, nearly irreversible, and potent inhibitor of CPA;
it is less potent against CPB and does not act on various other
proteases. TCI and latexin are both expressed and localized in the
cytosol of a number of tissues including brain, lung, or digestive
tract. It has been suggested that TCI or latexin may function in
regulating tissue-specific, cytosolic protein degradation (Normant
et al., supra).
[0034] Eaton et al purified a novel human plasma carboxypeptidase B
(designated pCPB protein) that has an apparent Mr of 60,000. The
deduced amino acid sequence reveals a primary translation product
of 423 amino acids that is very similar to carboxypeptidase A and B
and consists of a 22-amino acid signal peptide, a 92-amino acid
activation peptide, and a 309-amino acid catalytic domain. This
protein shows 44 and 40% similarity to rat procarboxypeptidase B
and human mast cell procarboxypeptidase. The presence of aspartic
acid at position 257 of the catalytic domain suggests that this
protein is a basic carboxypeptidase. When activated by trypsin, it
hydrolyzes carboxypeptidase B substrates, hippuryl-Arg and
hippuryl-Lys, but not carboxypeptidase A substrates, and it is
inhibited by the specific carboxypeptidase B inhibitor
(DL-5-guanidinoethyl)mercapt- osuccinic acid. Tsai and Drayna
(1992) used PCR to identify the presence of the plasma
carboxypeptidase B gene in somatic hybrid cell lines which is
called carboxypeptidase B2 (CPB2), also called carboxypeptidase U
(CPU). It is an unstable basic carboxypeptidase that circulates in
human plasma in its proenzyme form. The most likely physiologic
activator of pro-CPU is the thrombin-thrombomodulin complex.
Vanhoof et al. (1996) noted that pro-CPU exhibits affinity for
plasminogen and can be converted to its active form through the
action of thrombin and plasmin. CPB2 gene is located on human
chromosome 13. To regionalize the assignment of the gene on
chromosome 13, Vanhoof et al. (1996) used fluorescence in situ
hybridization. They found that it is localized to 13q14.11.
[0035] For a review of the carboxypeptidase, see Eaton et al., J.
Biol. Chem. 266: 21833-21838, 1991; Tsai et al., Genomics 14:
549-550, 1992; Vanhoof et al., Genomics 38: 454-455, 1996; Pascual
et al., Eur J Biochem February 15; 179(3):609-16, 1989;
[0036] The protein of the present invention has a substantial
similarity to the carboxypeptidase B as set forth above. Protease
proteins, particularly members of the carboxypeptidase subfamily,
are a major target for drug action and development. Accordingly, it
is valuable to the field of pharmaceutical development to identify
and characterize previously unknown members of this subfamily of
protease proteins. The present invention advances the state of the
art by providing a previously unidentified human protease proteins
that have homology to members of the carboxypeptidase
subfamily.
SUMMARY OF THE INVENTION
[0037] The present invention is based in part on the identification
of amino acid sequences of human protease peptides and proteins
that are related to the carboxypeptidase subfamily, as well as
allelic variants and other mammalian orthologs thereof. These
unique peptide sequences, and nucleic acid sequences that encode
these peptides, can be used as models for the development of human
therapeutic targets, aid in the identification of therapeutic
proteins, and serve as targets for the development of human
therapeutic agents that modulate protease activity in cells and
tissues that express the protease. Experimental data as provided in
FIG. 1 indicates expression in fetal brain, liver, hepatocellular
carcinoma and whole liver.
DESCRIPTION OF THE FIGURE SHEETS
[0038] FIG. 1 provides the nucleotide sequence of a cDNA molecule
or transcript sequence that encodes the protease protein of the
present invention. (SEQ ID NO:1) In addition, structure and
functional information is provided, such as ATG start, stop and
tissue distribution, where available, that allows one to readily
determine specific uses of inventions based on this molecular
sequence. Experimental data as provided in FIG. 1 indicates
expression in fetal brain, liver, hepatocellular carcinoma and
whole liver.
[0039] FIG. 2 provides the predicted amino acid sequence of the
protease of the present invention. (SEQ ID NO:2) In addition
structure and functional information such as protein family,
function, and modification sites is provided where available,
allowing one to readily determine specific uses of inventions based
on this molecular sequence.
[0040] FIG. 3 provides genomic sequences that span the gene
encoding the protease protein of the present invention. (SEQ ID
NO:3) In addition structure and functional information, such as
intron/exon structure, promoter location, etc., is provided where
available, allowing one to readily determine specific uses of
inventions based on this molecular sequence. As illustrated in FIG.
3, SNPs, including insertion/deletion variants ("indels"), were
identified at 40 different nucleotide positions.
DETAILED DESCRIPTION OF THE INVENTION
General Description
[0041] The present invention is based on the sequencing of the
human genome. During the sequencing and assembly of the human
genome, analysis of the sequence information revealed previously
unidentified fragments of the human genome that encode peptides
that share structural and/or sequence homology to
protein/peptide/domains identified and characterized within the art
as being a protease protein or part of a protease protein and are
related to the carboxypeptidase subfamily. Utilizing these
sequences, additional genomic sequences were assembled and
transcript and/or cDNA sequences were isolated and characterized.
Based on this analysis, the present invention provides amino acid
sequences of human protease peptides and proteins that are related
to the carboxypeptidase subfamily, nucleic acid sequences in the
form of transcript sequences, cDNA sequences and/or genomic
sequences that encode these protease peptides and proteins, nucleic
acid variation (allelic information), tissue distribution of
expression, and information about the closest art known
protein/peptide/domain that has structural or sequence homology to
the protease of the present invention.
[0042] In addition to being previously unknown, the peptides that
are provided in the present invention are selected based on their
ability to be used for the development of commercially important
products and services. Specifically, the present peptides are
selected based on homology and/or structural relatedness to known
protease proteins of the carboxypeptidase subfamily and the
expression pattern observed. Experimental data as provided in FIG.
1 indicates expression in fetal brain, liver, hepatocellular
carcinoma and whole liver. The art has clearly established the
commercial importance of members of this family of proteins and
proteins that have expression patterns similar to that of the
present gene. Some of the more specific features of the peptides of
the present invention, and the uses thereof, are described herein,
particularly in the Background of the Invention and in the
annotation provided in the Figures, and/or are known within the art
for each of the known carboxypeptidase family or subfamily of
protease proteins.
SPECIFIC EMBODIMENTS
[0043] Peptide Molecules
[0044] The present invention provides nucleic acid sequences that
encode protein molecules that have been identified as being members
of the protease family of proteins and are related to the
carboxypeptidase subfamily (protein sequences are provided in FIG.
2, transcript/cDNA sequences are provided in FIG. 1 and genomic
sequences are provided in FIG. 3). The peptide sequences provided
in FIG. 2, as well as the obvious variants described herein,
particularly allelic variants as identified herein and using the
information in FIG. 3, will be referred herein as the protease
peptides of the present invention, protease peptides, or
peptides/proteins of the present invention.
[0045] The present invention provides isolated peptide and protein
molecules that consist of, consist essentially of, or comprise the
amino acid sequences of the protease peptides disclosed in the FIG.
2, (encoded by the nucleic acid molecule shown in FIG. 1,
transcript/cDNA or FIG. 3, genomic sequence), as well as all
obvious variants of these peptides that are within the art to make
and use. Some of these variants are described in detail below.
[0046] As used herein, a peptide is said to be "isolated" or
"purified" when it is substantially free of cellular material or
free of chemical precursors or other chemicals. The peptides of the
present invention can be purified to homogeneity or other degrees
of purity. The level of purification will be based on the intended
use. The critical feature is that the preparation allows for the
desired function of the peptide, even if in the presence of
considerable amounts of other components (the features of an
isolated nucleic acid molecule is discussed below).
[0047] In some uses, "substantially free of cellular material"
includes preparations of the peptide having less than about 30% (by
dry weight) other proteins (i.e., contaminating protein), less than
about 20% other proteins, less than about 10% other proteins, or
less than about 5% other proteins. When the peptide is
recombinantly produced, it can also be substantially free of
culture medium, i.e., culture medium represents less than about 20%
of the volume of the protein preparation.
[0048] The language "substantially free of chemical precursors or
other chemicals" includes preparations of the peptide in which it
is separated from chemical precursors or other chemicals that are
involved in its synthesis. In one embodiment, the language
"substantially free of chemical precursors or other chemicals"
includes preparations of the protease peptide having less than
about 30% (by dry weight) chemical precursors or other chemicals,
less than about 20% chemical precursors or other chemicals, less
than about 10% chemical precursors or other chemicals, or less than
about 5% chemical precursors or other chemicals.
[0049] The isolated protease peptide can be purified from cells
that naturally express it, purified from cells that have been
altered to express it (recombinant), or synthesized using known
protein synthesis methods. Experimental data as provided in FIG. 1
indicates expression in fetal brain, liver, hepatocellular
carcinoma and whole liver. For example, a nucleic acid molecule
encoding the protease peptide is cloned into an expression vector,
the expression vector introduced into a host cell and the protein
expressed in the host cell. The protein can then be isolated from
the cells by an appropriate purification scheme using standard
protein purification techniques. Many of these techniques are
described in detail below.
[0050] Accordingly, the present invention provides proteins that
consist of the amino acid sequences provided in FIG. 2 (SEQ ID
NO:2), for example, proteins encoded by the transcript/cDNA nucleic
acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic
sequences provided in FIG. 3 (SEQ ID NO:3). The amino acid sequence
of such a protein is provided in FIG. 2. A protein consists of an
amino acid sequence when the amino acid sequence is the final amino
acid sequence of the protein.
[0051] The present invention further provides proteins that consist
essentially of the amino acid sequences provided in FIG. 2 (SEQ ID
NO:2), for example, proteins encoded by the transcript/cDNA nucleic
acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic
sequences provided in FIG. 3 (SEQ ID NO:3). A protein consists
essentially of an amino acid sequence when such an amino acid
sequence is present with only a few additional amino acid residues,
for example from about 1 to about 100 or so additional residues,
typically from 1 to about 20 additional residues in the final
protein.
[0052] The present invention further provides proteins that
comprise the amino acid sequences provided in FIG. 2 (SEQ ID NO:2),
for example, proteins encoded by the transcript/cDNA nucleic acid
sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences
provided in FIG. 3 (SEQ ID NO:3). A protein comprises an amino acid
sequence when the amino acid sequence is at least part of the final
amino acid sequence of the protein. In such a fashion, the protein
can be only the peptide or have additional amino acid molecules,
such as amino acid residues (contiguous encoded sequence) that are
naturally associated with it or heterologous amino acid
residues/peptide sequences. Such a protein can have a few
additional amino acid residues or can comprise several hundred or
more additional amino acids. The preferred classes of proteins that
are comprised of the protease peptides of the present invention are
the naturally occurring mature proteins. A brief description of how
various types of these proteins can be made/isolated is provided
below.
[0053] The protease peptides of the present invention can be
attached to heterologous sequences to form chimeric or fusion
proteins. Such chimeric and fusion proteins comprise a protease
peptide operatively linked to a heterologous protein having an
amino acid sequence not substantially homologous to the protease
peptide. "Operatively linked" indicates that the protease peptide
and the heterologous protein are fused in-frame. The heterologous
protein can be fused to the N-terminus or C-terminus of the
protease peptide.
[0054] In some uses, the fusion protein does not affect the
activity of the protease peptide per se. For example, the fusion
protein can include, but is not limited to, enzymatic fusion
proteins, for example beta-galactosidase fusions, yeast two-hybrid
GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig
fusions. Such fusion proteins, particularly poly-His fusions, can
facilitate the purification of recombinant protease peptide. In
certain host cells (e.g., mammalian host cells), expression and/or
secretion of a protein can be increased by using a heterologous
signal sequence.
[0055] A chimeric or fusion protein can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for
the different protein sequences are ligated together in-frame in
accordance with conventional techniques. In another embodiment, the
fusion gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and re-amplified to
generate a chimeric gene sequence (see Ausubel et al., Current
Protocols in Molecular Biology, 1992). Moreover, many expression
vectors are commercially available that already encode a fusion
moiety (e.g., a GST protein). A protease peptide-encoding nucleic
acid can be cloned into such an expression vector such that the
fusion moiety is linked in-frame to the protease peptide.
[0056] As mentioned above, the present invention also provides and
enables obvious variants of the amino acid sequence of the proteins
of the present invention, such as naturally occurring mature forms
of the peptide, allelic/sequence variants of the peptides,
non-naturally occurring recombinantly derived variants of the
peptides, and orthologs and paralogs of the peptides. Such variants
can readily be generated using art-known techniques in the fields
of recombinant nucleic acid technology and protein biochemistry. It
is understood, however, that variants exclude any amino acid
sequences disclosed prior to the invention.
[0057] Such variants can readily be identified/made using molecular
techniques and the sequence information disclosed herein. Further,
such variants can readily be distinguished from other peptides
based on sequence and/or structural homology to the protease
peptides of the present invention. The degree of homology/identity
present will be based primarily on whether the peptide is a
functional variant or non-functional variant, the amount of
divergence present in the paralog family and the evolutionary
distance between the orthologs.
[0058] To determine the percent identity of two amino acid
sequences or two nucleic acid sequences, the sequences are aligned
for optimal comparison purposes (e.g., gaps can be introduced in
one or both of a first and a second amino acid or nucleic acid
sequence for optimal alignment and non-homologous sequences can be
disregarded for comparison purposes). In a preferred embodiment, at
least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of
a reference sequence is aligned for comparison purposes. The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position (as used herein
amino acid or nucleic acid "identity" is equivalent to amino acid
or nucleic acid "homology"). The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences, taking into account the number of gaps, and the
length of each gap, which need to be introduced for optimal
alignment of the two sequences.
[0059] The comparison of sequences and determination of percent
identity and similarity between two sequences can be accomplished
using a mathematical algorithm. (Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a
preferred embodiment, the percent identity between two amino acid
sequences is determined using the Needleman and Wunsch (J. Mol.
Biol. (48):444-453 (1970)) algorithm which has been incorporated
into the GAP program in the GCG software package (available at
http://www.gcg.com), using either a Blossom 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment,
the percent identity between two nucleotide sequences is determined
using the GAP program in the GCG software package (Devereux, J., et
al., Nucleic Acids Res. 12(1):387 (1984)) (available at
http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight
of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or
6. In another embodiment, the percent identity between two amino
acid or nucleotide sequences is determined using the algorithm of
E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been
incorporated into the ALIGN program (version 2.0), using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4.
[0060] The nucleic acid and protein sequences of the present
invention can further be used as a "query sequence" to perform a
search against sequence databases to, for example, identify other
family members or related sequences. Such searches can be performed
using the NBLAST and XBLAST programs (version 2.0) of Altschul, et
al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches
can be performed with the NBLAST program, score=100, wordlength=12
to obtain nucleotide sequences homologous to the nucleic acid
molecules of the invention. BLAST protein searches can be performed
with the XBLAST program, score=50, wordlength=3 to obtain amino
acid sequences homologous to the proteins of the invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al. (Nucleic Acids Res.
25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used.
[0061] Full-length pre-processed forms, as well as mature processed
forms, of proteins that comprise one of the peptides of the present
invention can readily be identified as having complete sequence
identity to one of the protease peptides of the present invention
as well as being encoded by the same genetic locus as the protease
peptide provided herein. As indicated by the data presented in FIG.
3, the map position was determined to be on chromosome 13 by
ePCR.
[0062] Allelic variants of a protease peptide can readily be
identified as being a human protein having a high degree
(significant) of sequence homology/identity to at least a portion
of the protease peptide as well as being encoded by the same
genetic locus as the protease peptide provided herein. Genetic
locus can readily be determined based on the genomic information
provided in FIG. 3, such as the genomic sequence mapped to the
reference human. As indicated by the data presented in FIG. 3, the
map position was determined to be on chromosome 13 by ePCR. As used
herein, two proteins (or a region of the proteins) have significant
homology when the amino acid sequences are typically at least about
70-80%, 80-90%, and more typically at least about 90-95% or more
homologous. A significantly homologous amino acid sequence,
according to the present invention, will be encoded by a nucleic
acid sequence that will hybridize to a protease peptide encoding
nucleic acid molecule under stringent conditions as more fully
described below.
[0063] FIG. 3 provides information on SNPs that have been
identified in a gene encoding the transporter protein of the
present invention. 40 SNP variants were found, including 5 indels
(indicated by a "-") and 1 SNPs in exons, of which 5 of these cause
changes in the amino acid sequence (i.e., nonsynonymous SNPs).
SNPs, identified at different nucleotide positions in introns and
regions 5' and 3' of the ORF, may affect control/regulatory
elements.
[0064] Paralogs of a protease peptide can readily be identified as
having some degree of significant sequence homology/identity to at
least a portion of the protease peptide, as being encoded by a gene
from humans, and as having similar activity or function. Two
proteins will typically be considered paralogs when the amino acid
sequences are typically at least about 60% or greater, and more
typically at least about 70% or greater homology through a given
region or domain. Such paralogs will be encoded by a nucleic acid
sequence that will hybridize to a protease peptide encoding nucleic
acid molecule under moderate to stringent conditions as more fully
described below.
[0065] Orthologs of a protease peptide can readily be identified as
having some degree of significant sequence homology/identity to at
least a portion of the protease peptide as well as being encoded by
a gene from another organism. Preferred orthologs will be isolated
from mammals, preferably primates, for the development of human
therapeutic targets and agents. Such orthologs will be encoded by a
nucleic acid sequence that will hybridize to a protease peptide
encoding nucleic acid molecule under moderate to stringent
conditions, as more fully described below, depending on the degree
of relatedness of the two organisms yielding the proteins. As
indicated by the data presented in FIG. 3, the map position was
determined to be on chromosome 13 by ePCR.
[0066] FIG. 3 provides information on SNPs that have been
identified in a gene encoding the transporter protein of the
present invention. 40 SNP variants were found, including 5 indels
(indicated by a "-") and 1 SNPs in exons, of which 5 of these cause
changes in the amino acid sequence (i.e., nonsynonymous SNPs).
SNPs, identified at different nucleotide positions in introns and
regions 5' and 3' of the ORF, may affect control/regulatory
elements.
[0067] Non-naturally occurring variants of the protease peptides of
the present invention can readily be generated using recombinant
techniques. Such variants include, but are not limited to
deletions, additions and substitutions in the amino acid sequence
of the protease peptide. For example, one class of substitutions
are conserved amino acid substitution. Such substitutions are those
that substitute a given amino acid in a protease peptide by another
amino acid of like characteristics. Typically seen as conservative
substitutions are the replacements, one for another, among the
aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the
hydroxyl residues Ser and Thr; exchange of the acidic residues Asp
and Glu; substitution between the amide residues Asn and Gln;
exchange of the basic residues Lys and Arg; and replacements among
the aromatic residues Phe and Tyr. Guidance concerning which amino
acid changes are likely to be phenotypically silent are found in
Bowie et al., Science 247:1306-1310 (1990).
[0068] Variant protease peptides can be fully functional or can
lack function in one or more activities, e.g. ability to bind
substrate, ability to cleave substrate, ability to participate in a
signaling pathway, etc. Fully functional variants typically contain
only conservative variation or variation in non-critical residues
or in non-critical regions. FIG. 2 provides the result of protein
analysis and can be used to identify critical domains/regions.
Functional variants can also contain substitution of similar amino
acids that result in no change or an insignificant change in
function. Alternatively, such substitutions may positively or
negatively affect function to some degree.
[0069] Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, or truncation or a substitution, insertion, inversion,
or deletion in a critical residue or critical region.
[0070] Amino acids that are essential for function can be
identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham et al.,
Science 244:1081-1085 (1989)), particularly using the results
provided in FIG. 2. The latter procedure introduces single alanine
mutations at every residue in the molecule. The resulting mutant
molecules are then tested for biological activity such as protease
activity or in assays such as an in vitro proliferative activity.
Sites that are critical for binding partner/substrate binding can
also be determined by structural analysis such as crystallization,
nuclear magnetic resonance or photoaffinity labeling (Smith et al,
J. Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312
(1992)).
[0071] The present invention further provides fragments of the
protease peptides, in addition to proteins and peptides that
comprise and consist of such fragments, particularly those
comprising the residues identified in FIG. 2. The fragments to
which the invention pertains, however, are not to be construed as
encompassing fragments that may be disclosed publicly prior to the
present invention.
[0072] As used herein, a fragment comprises at least 8, 10, 12, 14,
16, or more contiguous amino acid residues from a protease peptide.
Such fragments can be chosen based on the ability to retain one or
more of the biological activities of the protease peptide or could
be chosen for the ability to perform a function, e.g. bind a
substrate or act as an immunogen. Particularly important fragments
are biologically active fragments, peptides that are, for example,
about 8 or more amino acids in length. Such fragments will
typically comprise a domain or motif of the protease peptide, e.g.,
active site, a transmembrane domain or a substrate-binding domain.
Further, possible fragments include, but are not limited to, domain
or motif containing fragments, soluble peptide fragments, and
fragments containing immunogenic structures. Predicted domains and
functional sites are readily identifiable by computer programs well
known and readily available to those of skill in the art (e.g.,
PROSITE analysis). The results of one such analysis are provided in
FIG. 2.
[0073] Polypeptides often contain amino acids other than the 20
amino acids commonly referred to as the 20 naturally occurring
amino acids. Further, many amino acids, including the terminal
amino acids, may be modified by natural processes, such as
processing and other post-translational modifications, or by
chemical modification techniques well known in the art. Common
modifications that occur naturally in protease peptides are
described in basic texts, detailed monographs, and the research
literature, and they are well known to those of skill in the art
(some of these features are identified in FIG. 2).
[0074] Known modifications include, but are not limited to,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
crosslinks, formation of cystine, formation of pyroglutamate,
formylation, gamma carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
[0075] Such modifications are well known to those of skill in the
art and have been described in great detail in the scientific
literature. Several particularly common modifications,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation, for
instance, are described in most basic texts, such as
Proteins--Structure and Molecular Properties, 2nd Ed., T. E.
Creighton, W. H. Freeman and Company, New York (1993). Many
detailed reviews are available on this subject, such as by Wold,
F., Posttranslational Covalent Modification of Proteins, B. C.
Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al.
(Meth. Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N.Y.
Acad. Sci. 663:48-62 (1992)).
[0076] Accordingly, the protease peptides of the present invention
also encompass derivatives or analogs in which a substituted amino
acid residue is not one encoded by the genetic code, in which a
substituent group is included, in which the mature protease peptide
is fused with another compound, such as a compound to increase the
half-life of the protease peptide (for example, polyethylene
glycol), or in which the additional amino acids are fused to the
mature protease peptide, such as a leader or secretory sequence or
a sequence for purification of the mature protease peptide or a
pro-protein sequence.
[0077] Protein/Peptide Uses
[0078] The proteins of the present invention can be used in
substantial and specific assays related to the functional
information provided in the Figures; to raise antibodies or to
elicit another immune response; as a reagent (including the labeled
reagent) in assays designed to quantitatively determine levels of
the protein (or its binding partner or ligand) in biological
fluids; and as markers for tissues in which the corresponding
protein is preferentially expressed (either constitutively or at a
particular stage of tissue differentiation or development or in a
disease state). Where the protein binds or potentially binds to
another protein or ligand (such as, for example, in a
protease-effector protein interaction or protease-ligand
interaction), the protein can be used to identify the binding
partner/ligand so as to develop a system to identify inhibitors of
the binding interaction. Any or all of these uses are capable of
being developed into reagent grade or kit format for
commercialization as commercial products.
[0079] Methods for performing the uses listed above are well known
to those skilled in the art. References disclosing such methods
include "Molecular Cloning: A Laboratory Manual", 2d ed., Cold
Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T.
Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular
Cloning Techniques", Academic Press, Berger, S. L. and A. R. Kimmel
eds., 1987.
[0080] Utility_Utility
[0081] The potential uses of the peptides of the present invention
are based primarily on the source of the protein as well as the
class/action of the protein. For example, proteases isolated from
humans and their human/mammalian orthologs serve as targets for
identifying agents for use in mammalian therapeutic applications,
e.g. a human drug, particularly in modulating a biological or
pathological response in a cell or tissue that expresses the
protease. Experimental data as provided in FIG. 1 indicates that
protease proteins of the present invention are expressed in the
fetal brain, liver, hepatocellular carcinoma by a virtual northern
blot. In addition, PCR-based tissue screening panel indicates
expression in whole liver. A large percentage of pharmaceutical
agents are being developed that modulate the activity of protease
proteins, particularly members of the carboxypeptidase subfamily
(see Background of the Invention). The structural and functional
information provided in the Background and Figures provide specific
and substantial uses for the molecules of the present invention,
particularly in combination with the expression information
provided in FIG. 1. Experimental data as provided in FIG. 1
indicates expression in fetal brain, liver, hepatocellular
carcinoma and whole liver. Such uses can readily be determined
using the information provided herein, that which is known in the
art, and routine experimentation.
[0082] The proteins of the present invention (including variants
and fragments that may have been disclosed prior to the present
invention) are useful for biological assays related to proteases
that are related to members of the carboxypeptidase subfamily. Such
assays involve any of the known protease functions or activities or
properties useful for diagnosis and treatment of protease-related
conditions that are specific for the subfamily of proteases that
the one of the present invention belongs to, particularly in cells
and tissues that express the protease. Experimental data as
provided in FIG. 1 indicates that protease proteins of the present
invention are expressed in the fetal brain, liver, hepatocellular
carcinoma by a virtual northern blot. In addition, PCR-based tissue
screening panel indicates expression in whole liver.
[0083] The proteins of the present invention are also useful in
drug screening assays, in cell-based or cell-free systems.
Cell-based systems can be native, i.e., cells that normally express
the protease, as a biopsy or expanded in cell culture. Experimental
data as provided in FIG. 1 indicates expression in fetal brain,
liver, hepatocellular carcinoma and whole liver. In an alternate
embodiment, cell-based assays involve recombinant host cells
expressing the protease protein.
[0084] The polypeptides can be used to identify compounds that
modulate protease activity of the protein in its natural state or
an altered form that causes a specific disease or pathology
associated with the protease. Both the proteases of the present
invention and appropriate variants and fragments can be used in
high-throughput screens to assay candidate compounds for the
ability to bind to the protease. These compounds can be further
screened against a functional protease to determine the effect of
the compound on the protease activity. Further, these compounds can
be tested in animal or invertebrate systems to determine
activity/effectiveness. Compounds can be identified that activate
(agonist) or inactivate (antagonist) the protease to a desired
degree.
[0085] Further, the proteins of the present invention can be used
to screen a compound for the ability to stimulate or inhibit
interaction between the protease protein and a molecule that
normally interacts with the protease protein, e.g. a substrate or a
component of the signal pathway that the protease protein normally
interacts (for example, a protease). Such assays typically include
the steps of combining the protease protein with a candidate
compound under conditions that allow the protease protein, or
fragment, to interact with the target molecule, and to detect the
formation of a complex between the protein and the target or to
detect the biochemical consequence of the interaction with the
protease protein and the target, such as any of the associated
effects of signal transduction such as protein cleavage, cAMP
turnover, and adenylate cyclase activation, etc.
[0086] Candidate compounds include, for example, 1) peptides such
as soluble peptides, including Ig-tailed fusion peptides and
members of random peptide libraries (see, e.g., Lam et al., Nature
354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and
combinatorial chemistry-derived molecular libraries made of D-
and/or L-configuration amino acids; 2) phosphopeptides (e.g.,
members of random and partially degenerate, directed phosphopeptide
libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3)
antibodies (e.g., polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric, and single chain antibodies as well as
Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[0087] One candidate compound is a soluble fragment of the receptor
that competes for substrate binding. Other candidate compounds
include mutant proteases or appropriate fragments containing
mutations that affect protease function and thus compete for
substrate. Accordingly, a fragment that competes for substrate, for
example with a higher affinity, or a fragment that binds substrate
but does not allow release, is encompassed by the invention.
[0088] The invention further includes other end point assays to
identify compounds that modulate (stimulate or inhibit) protease
activity. The assays typically involve an assay of events in the
signal transduction pathway that indicate protease activity. Thus,
the cleavage of a substrate, inactivation/activation of a protein,
a change in the expression of genes that are up- or down-regulated
in response to the protease protein dependent signal cascade can be
assayed.
[0089] Any of the biological or biochemical functions mediated by
the protease can be used as an endpoint assay. These include all of
the biochemical or biochemical/biological events described herein,
in the references cited herein, incorporated by reference for these
endpoint assay targets, and other functions known to those of
ordinary skill in the art or that can be readily identified using
the information provided in the Figures, particularly FIG. 2.
Specifically, a biological function of a cell or tissues that
expresses the protease can be assayed. Experimental data as
provided in FIG. 1 indicates that protease proteins of the present
invention are expressed in the fetal brain, liver, hepatocellular
carcinoma by a virtual northern blot. In addition, PCR-based tissue
screening panel indicates expression in whole liver.
[0090] Binding and/or activating compounds can also be screened by
using chimeric protease proteins in which the amino terminal
extracellular domain, or parts thereof, the entire transmembrane
domain or subregions, such as any of the seven transmembrane
segments or any of the intracellular or extracellular loops and the
carboxy terminal intracellular domain, or parts thereof, can be
replaced by heterologous domains or subregions. For example, a
substrate-binding region can be used that interacts with a
different substrate then that which is recognized by the native
protease. Accordingly, a different set of signal transduction
components is available as an end-point assay for activation. This
allows for assays to be performed in other than the specific host
cell from which the protease is derived.
[0091] The proteins of the present invention are also useful in
competition binding assays in methods designed to discover
compounds that interact with the protease (e.g. binding partners
and/or ligands). Thus, a compound is exposed to a protease
polypeptide under conditions that allow the compound to bind or to
otherwise interact with the polypeptide. Soluble protease
polypeptide is also added to the mixture. If the test compound
interacts with the soluble protease polypeptide, it decreases the
amount of complex formed or activity from the protease target. This
type of assay is particularly useful in cases in which compounds
are sought that interact with specific regions of the protease.
Thus, the soluble polypeptide that competes with the target
protease region is designed to contain peptide sequences
corresponding to the region of interest.
[0092] To perform cell free drug screening assays, it is sometimes
desirable to immobilize either the protease protein, or fragment,
or its target molecule to facilitate separation of complexes from
uncomplexed forms of one or both of the proteins, as well as to
accommodate automation of the assay.
[0093] Techniques for immobilizing proteins on matrices can be used
in the drug screening assays. In one embodiment, a fusion protein
can be provided which adds a domain that allows the protein to be
bound to a matrix. For example, glutathione-S-transferase fusion
proteins can be adsorbed onto glutathione sepharose beads (Sigma
Chemical, St. Louis, Mo.) or glutathione derivatized microtiter
plates, which are then combined with the cell lysates (e.g.,
.sup.35S-labeled) and the candidate compound, and the mixture
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads are washed to remove any unbound label, and the matrix
immobilized and radiolabel determined directly, or in the
supernatant after the complexes are dissociated. Alternatively, the
complexes can be dissociated from the matrix, separated by
SDS-PAGE, and the level of protease-binding protein found in the
bead fraction quantitated from the gel using standard
electrophoretic techniques. For example, either the polypeptide or
its target molecule can be immobilized utilizing conjugation of
biotin and streptavidin using techniques well known in the art.
Alternatively, antibodies reactive with the protein but which do
not interfere with binding of the protein to its target molecule
can be derivatized to the wells of the plate, and the protein
trapped in the wells by antibody conjugation. Preparations of a
protease-binding protein and a candidate compound are incubated in
the protease protein-presenting wells and the amount of complex
trapped in the well can be quantitated. Methods for detecting such
complexes, in addition to those described above for the
GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with the protease protein target
molecule, or which are reactive with protease protein and compete
with the target molecule, as well as enzyme-linked assays which
rely on detecting an enzymatic activity associated with the target
molecule.
[0094] Agents that modulate one of the proteases of the present
invention can be identified using one or more of the above assays,
alone or in combination. It is generally preferable to use a
cell-based or cell free system first and then confirm activity in
an animal or other model system. Such model systems are well known
in the art and can readily be employed in this context.
[0095] Modulators of protease protein activity identified according
to these drug screening assays can be used to treat a subject with
a disorder mediated by the protease pathway, by treating cells or
tissues that express the protease. Experimental data as provided in
FIG. 1 indicates expression in fetal brain, liver, hepatocellular
carcinoma and whole liver. These methods of treatment include the
steps of administering a modulator of protease activity in a
pharmaceutical composition to a subject in need of such treatment,
the modulator being identified as described herein.
[0096] In yet another aspect of the invention, the protease
proteins can be used as "bait proteins" in a two-hybrid assay or
three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et
al (1993) Cell 72:223-232; Madura et al (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent W094/10300),
to identify other proteins, which bind to or interact with the
protease and are involved in protease activity. Such
protease-binding proteins are also likely to be involved in the
propagation of signals by the protease proteins or protease targets
as, for example, downstream elements of a protease-mediated
signaling pathway. Alternatively, such protease-binding proteins
are likely to be protease inhibitors.
[0097] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for a protease
protein is fused to a gene encoding the DNA binding domain of a
known transcription factor (e.g., GAL-4). In the other construct, a
DNA sequence, from a library of DNA sequences, that encodes an
unidentified protein ("prey" or "sample") is fused to a gene that
codes for the activation domain of the known transcription factor.
If the "bait" and the "prey" proteins are able to interact, in
vivo, forming a protease-dependent complex, the DNA-binding and
activation domains of the transcription factor are brought into
close proximity. This proximity allows transcription of a reporter
gene (e.g., LacZ) which is operably linked to a transcriptional
regulatory site responsive to the transcription factor. Expression
of the reporter gene can be detected and cell colonies containing
the functional transcription factor can be isolated and used to
obtain the cloned gene which encodes the protein which interacts
with the protease protein.
[0098] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein in an appropriate animal model. For example, an
agent identified as described herein (e.g., a protease-modulating
agent, an antisense protease nucleic acid molecule, a
protease-specific antibody, or a protease-binding partner) can be
used in an animal or other model to determine the efficacy,
toxicity, or side effects of treatment with such an agent.
Alternatively, an agent identified as described herein can be used
in an animal or other model to determine the mechanism of action of
such an agent. Furthermore, this invention pertains to uses of
novel agents identified by the above-described screening assays for
treatments as described herein.
[0099] The protease proteins of the present invention are also
useful to provide a target for diagnosing a disease or
predisposition to disease mediated by the peptide. Accordingly, the
invention provides methods for detecting the presence, or levels
of, the protein (or encoding mRNA) in a cell, tissue, or organism.
Experimental data as provided in FIG. 1 indicates expression in
fetal brain, liver, hepatocellular carcinoma and whole liver. The
method involves contacting a biological sample with a compound
capable of interacting with the protease protein such that the
interaction can be detected. Such an assay can be provided in a
single detection format or a multi-detection format such as an
antibody chip array.
[0100] One agent for detecting a protein in a sample is an antibody
capable of selectively binding to protein. A biological sample
includes tissues, cells and biological fluids isolated from a
subject, as well as tissues, cells and fluids present within a
subject.
[0101] The peptides of the present invention also provide targets
for diagnosing active protein activity, disease, or predisposition
to disease, in a patient having a variant peptide, particularly
activities and conditions that are known for other members of the
family of proteins to which the present one belongs. Thus, the
peptide can be isolated from a biological sample and assayed for
the presence of a genetic mutation that results in aberrant
peptide. This includes amino acid substitution, deletion,
insertion, rearrangement, (as the result of aberrant splicing
events), and inappropriate post-translational modification.
Analytic methods include altered electrophoretic mobility, altered
tryptic peptide digest, altered protease activity in cell-based or
cell-free assay, alteration in substrate or antibody-binding
pattern, altered isoelectric point, direct amino acid sequencing,
and any other of the known assay techniques useful for detecting
mutations in a protein. Such an assay can be provided in a single
detection format or a multi-detection format such as an antibody
chip array.
[0102] In vitro techniques for detection of peptide include enzyme
linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence using a detection
reagent, such as an antibody or protein binding agent.
Alternatively, the peptide can be detected in vivo in a subject by
introducing into the subject a labeled anti-peptide antibody or
other types of detection agent. For example, the antibody can be
labeled with a radioactive marker whose presence and location in a
subject can be detected by standard imaging techniques.
Particularly useful are methods that detect the allelic variant of
a peptide expressed in a subject and methods which detect fragments
of a peptide in a sample.
[0103] The peptides are also useful in pharmacogenomic analysis.
Pharmacogenomics deal with clinically significant hereditary
variations in the response to drugs due to altered drug disposition
and abnormal action in affected persons. See, e.g., Eichelbaum, M.
(Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996)), and
Linder, M. W. (Clin. Chem. 43(2):254-266 (1997)). The clinical
outcomes of these variations result in severe toxicity of
therapeutic drugs in certain individuals or therapeutic failure of
drugs in certain individuals as a result of individual variation in
metabolism. Thus, the genotype of the individual can determine the
way a therapeutic compound acts on the body or the way the body
metabolizes the compound. Further, the activity of drug
metabolizing enzymes effects both the intensity and duration of
drug action. Thus, the pharmacogenomics of the individual permit
the selection of effective compounds and effective dosages of such
compounds for prophylactic or therapeutic treatment based on the
individual's genotype. The discovery of genetic polymorphisms in
some drug metabolizing enzymes has explained why some patients do
not obtain the expected drug effects, show an exaggerated drug
effect, or experience serious toxicity from standard drug dosages.
Polymorphisms can be expressed in the phenotype of the extensive
metabolizer and the phenotype of the poor metabolizer. Accordingly,
genetic polymorphism may lead to allelic protein variants of the
protease protein in which one or more of the protease functions in
one population is different from those in another population. The
peptides thus allow a target to ascertain a genetic predisposition
that can affect treatment modality. Thus, in a ligand-based
treatment, polymorphism may give rise to amino terminal
extracellular domains and/or other substrate-binding regions that
are more or less active in substrate binding, and protease
activation. Accordingly, substrate dosage would necessarily be
modified to maximize the therapeutic effect within a given
population containing a polymorphism. As an alternative to
genotyping, specific polymorphic peptides could be identified.
[0104] The peptides are also useful for treating a disorder
characterized by an absence of, inappropriate, or unwanted
expression of the protein. Experimental data as provided in FIG. 1
indicates expression in fetal brain, liver, hepatocellular
carcinoma and whole liver. Accordingly, methods for treatment
include the use of the protease protein or fragments.
[0105] Antibodies
[0106] The invention also provides antibodies that selectively bind
to one of the peptides of the present invention, a protein
comprising such a peptide, as well as variants and fragments
thereof. As used herein, an antibody selectively binds a target
peptide when it binds the target peptide and does not significantly
bind to unrelated proteins. An antibody is still considered to
selectively bind a peptide even if it also binds to other proteins
that are not substantially homologous with the target peptide so
long as such proteins share homology with a fragment or domain of
the peptide target of the antibody. In this case, it would be
understood that antibody binding to the peptide is still selective
despite some degree of cross-reactivity.
[0107] As used herein, an antibody is defined in terms consistent
with that recognized within the art: they are multi-subunit
proteins produced by a mammalian organism in response to an antigen
challenge. The antibodies of the present invention include
polyclonal antibodies and monoclonal antibodies, as well as
fragments of such antibodies, including, but not limited to, Fab or
F(ab').sub.2, and Fv fragments.
[0108] Many methods are known for generating and/or identifying
antibodies to a given target peptide. Several such methods are
described by Harlow, Antibodies, Cold Spring Harbor Press,
(1989).
[0109] In general, to generate antibodies, an isolated peptide is
used as an immunogen and is administered to a mammalian organism,
such as a rat, rabbit or mouse. The full-length protein, an
antigenic peptide fragment or a fusion protein can be used.
Particularly important fragments are those covering functional
domains, such as the domains identified in FIG. 2, and domain of
sequence homology or divergence amongst the family, such as those
that can readily be identified using protein alignment methods and
as presented in the Figures.
[0110] Antibodies are preferably prepared from regions or discrete
fragments of the protease proteins. Antibodies can be prepared from
any region of the peptide as described herein. However, preferred
regions will include those involved in function/activity and/or
protease/binding partner interaction. FIG. 2 can be used to
identify particularly important regions while sequence alignment
can be used to identify conserved and unique sequence
fragments.
[0111] An antigenic fragment will typically comprise at least 8
contiguous amino acid residues. The antigenic peptide can comprise,
however, at least 10, 12, 14, 16 or more amino acid residues. Such
fragments can be selected on a physical property, such as fragments
correspond to regions that are located on the surface of the
protein, e.g., hydrophilic regions or can be selected based on
sequence uniqueness (see FIG. 2).
[0112] Detection on an antibody of the present invention can be
facilitated by coupling (i.e., physically linking) the antibody to
a detectable substance. Examples of detectable substances include
various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, and radioactive
materials. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0113] Antibody Uses
[0114] The antibodies can be used to isolate one of the proteins of
the present invention by standard techniques, such as affinity
chromatography or immunoprecipitation. The antibodies can
facilitate the purification of the natural protein from cells and
recombinantly produced protein expressed in host cells. In
addition, such antibodies are useful to detect the presence of one
of the proteins of the present invention in cells or tissues to
determine the pattern of expression of the protein among various
tissues in an organism and over the course of normal development.
Experimental data as provided in FIG. 1 indicates that protease
proteins of the present invention are expressed in the fetal brain,
liver, hepatocellular carcinoma by a virtual northern blot. In
addition, PCR-based tissue screening panel indicates expression in
whole liver. Further, such antibodies can be used to detect protein
in situ, in vitro, or in a cell lysate or supernatant in order to
evaluate the abundance and pattern of expression. Also, such
antibodies can be used to assess abnormal tissue distribution or
abnormal expression during development or progression of a
biological condition. Antibody detection of circulating fragments
of the full length protein can be used to identify turnover.
[0115] Further, the antibodies can be used to assess expression in
disease states such as in active stages of the disease or in an
individual with a predisposition toward disease related to the
protein's function. When a disorder is caused by an inappropriate
tissue distribution, developmental expression, level of expression
of the protein, or expressed/processed form, the antibody can be
prepared against the normal protein. Experimental data as provided
in FIG. 1 indicates expression in fetal brain, liver,
hepatocellular carcinoma and whole liver. If a disorder is
characterized by a specific mutation in the protein, antibodies
specific for this mutant protein can be used to assay for the
presence of the specific mutant protein.
[0116] The antibodies can also be used to assess normal and
aberrant subcellular localization of cells in the various tissues
in an organism. Experimental data as provided in FIG. 1 indicates
expression in fetal brain, liver, hepatocellular carcinoma and
whole liver. The diagnostic uses can be applied, not only in
genetic testing, but also in monitoring a treatment modality.
Accordingly, where treatment is ultimately aimed at correcting
expression level or the presence of aberrant sequence and aberrant
tissue distribution or developmental expression, antibodies
directed against the protein or relevant fragments can be used to
monitor therapeutic efficacy.
[0117] Additionally, antibodies are useful in pharmacogenomic
analysis. Thus, antibodies prepared against polymorphic proteins
can be used to identify individuals that require modified treatment
modalities. The antibodies are also useful as diagnostic tools as
an immunological marker for aberrant protein analyzed by
electrophoretic mobility, isoelectric point, tryptic peptide
digest, and other physical assays known to those in the art.
[0118] The antibodies are also useful for tissue typing.
Experimental data as provided in FIG. 1 indicates expression in
fetal brain, liver, hepatocellular carcinoma and whole liver. Thus,
where a specific protein has been correlated with expression in a
specific tissue, antibodies that are specific for this protein can
be used to identify a tissue type.
[0119] The antibodies are also useful for inhibiting protein
function, for example, blocking the binding of the protease peptide
to a binding partner such as a substrate. These uses can also be
applied in a therapeutic context in which treatment involves
inhibiting the protein's function. An antibody can be used, for
example, to block binding, thus modulating (agonizing or
antagonizing) the peptides activity. Antibodies can be prepared
against specific fragments containing sites required for function
or against intact protein that is associated with a cell or cell
membrane. See FIG. 2 for structural information relating to the
proteins of the present invention.
[0120] The invention also encompasses kits for using antibodies to
detect the presence of a protein in a biological sample. The kit
can comprise antibodies such as a labeled or labelable antibody and
a compound or agent for detecting protein in a biological sample;
means for determining the amount of protein in the sample; means
for comparing the amount of protein in the sample with a standard;
and instructions for use. Such a kit can be supplied to detect a
single protein or epitope or can be configured to detect one of a
multitude of epitopes, such as in an antibody detection array.
Arrays are described in detail below for nucleic acid arrays and
similar methods have been developed for antibody arrays.
[0121] Nucleic Acid Molecules
[0122] The present invention further provides isolated nucleic acid
molecules that encode a protease peptide or protein of the present
invention (cDNA, transcript and genomic sequence). Such nucleic
acid molecules will consist of, consist essentially of, or comprise
a nucleotide sequence that encodes one of the protease peptides of
the present invention, an allelic variant thereof, or an ortholog
or paralog thereof.
[0123] As used herein, an "isolated" nucleic acid molecule is one
that is separated from other nucleic acid present in the natural
source of the nucleic acid. Preferably, an "isolated" nucleic acid
is free of sequences which naturally flank the nucleic acid (i.e.,
sequences located at the 5' and 3' ends of the nucleic acid) in the
genomic DNA of the organism from which the nucleic acid is derived.
However, there can be some flanking nucleotide sequences, for
example up to about 5 KB, 4 KB, 3 KB, 2 KB, or 1 KB or less,
particularly contiguous peptide encoding sequences and peptide
encoding sequences within the same gene but separated by introns in
the genomic sequence. The important point is that the nucleic acid
is isolated from remote and unimportant flanking sequences such
that it can be subjected to the specific manipulations described
herein such as recombinant expression, preparation of probes and
primers, and other uses specific to the nucleic acid sequences.
[0124] Moreover, an "isolated" nucleic acid molecule, such as a
transcript/cDNA molecule, can be substantially free of other
cellular material, or culture medium when produced by recombinant
techniques, or chemical precursors or other chemicals when
chemically synthesized. However, the nucleic acid molecule can be
fused to other coding or regulatory sequences and still be
considered isolated.
[0125] For example, recombinant DNA molecules contained in a vector
are considered isolated. Further examples of isolated DNA molecules
include recombinant DNA molecules maintained in heterologous host
cells or purified (partially or substantially) DNA molecules in
solution. Isolated RNA molecules include in vivo or in vitro RNA
transcripts of the isolated DNA molecules of the present invention.
Isolated nucleic acid molecules according to the present invention
further include such molecules produced synthetically.
[0126] Accordingly, the present invention provides nucleic acid
molecules that consist of the nucleotide sequence shown in FIG. 1
or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic
sequence), or any nucleic acid molecule that encodes the protein
provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists
of a nucleotide sequence when the nucleotide sequence is the
complete nucleotide sequence of the nucleic acid molecule.
[0127] The present invention further provides nucleic acid
molecules that consist essentially of the nucleotide sequence shown
in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3,
genomic sequence), or any nucleic acid molecule that encodes the
protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule
consists essentially of a nucleotide sequence when such a
nucleotide sequence is present with only a few additional nucleic
acid residues in the final nucleic acid molecule.
[0128] The present invention further provides nucleic acid
molecules that comprise the nucleotide sequences shown in FIG. 1 or
3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic
sequence), or any nucleic acid molecule that encodes the protein
provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule comprises
a nucleotide sequence when the nucleotide sequence is at least part
of the final nucleotide sequence of the nucleic acid molecule. In
such a fashion, the nucleic acid molecule can be only the
nucleotide sequence or have additional nucleic acid residues, such
as nucleic acid residues that are naturally associated with it or
heterologous nucleotide sequences. Such a nucleic acid molecule can
have a few additional nucleotides or can comprises several hundred
or more additional nucleotides. A brief description of how various
types of these nucleic acid molecules can be readily made/isolated
is provided below.
[0129] In FIGS. 1 and 3, both coding and non-coding sequences are
provided. Because of the source of the present invention, humans
genomic sequence (FIG. 3) and cDNA/transcript sequences (FIG. 1),
the nucleic acid molecules in the Figures will contain genomic
intronic sequences, 5' and 3' non-coding sequences, gene regulatory
regions and non-coding intergenic sequences. In general such
sequence features are either noted in FIGS. 1 and 3 or can readily
be identified using computational tools known in the art. As
discussed below, some of the non-coding regions, particularly gene
regulatory elements such as promoters, are useful for a variety of
purposes, e.g. control of heterologous gene expression, target for
identifying gene activity modulating compounds, and are
particularly claimed as fragments of the genomic sequence provided
herein.
[0130] The isolated nucleic acid molecules can encode the mature
protein plus additional amino or carboxyl-terminal amino acids, or
amino acids interior to the mature peptide (when the mature form
has more than one peptide chain, for instance). Such sequences may
play a role in processing of a protein from precursor to a mature
form, facilitate protein trafficking, prolong or shorten protein
half-life or facilitate manipulation of a protein for assay or
production, among other things. As generally is the case in situ,
the additional amino acids may be processed away from the mature
protein by cellular enzymes.
[0131] As mentioned above, the isolated nucleic acid molecules
include, but are not limited to, the sequence encoding the protease
peptide alone, the sequence encoding the mature peptide and
additional coding sequences, such as a leader or secretory sequence
(e.g., a pre-pro or pro-protein sequence), the sequence encoding
the mature peptide, with or without the additional coding
sequences, plus additional non-coding sequences, for example
introns and non-coding 5' and 3' sequences such as transcribed but
non-translated sequences that play a role in transcription, mRNA
processing (including splicing and polyadenylation signals),
ribosome binding and stability of mRNA. In addition, the nucleic
acid molecule may be fused to a marker sequence encoding, for
example, a peptide that facilitates purification.
[0132] Isolated nucleic acid molecules can be in the form of RNA,
such as mRNA, or in the form DNA, including cDNA and genomic DNA
obtained by cloning or produced by chemical synthetic techniques or
by a combination thereof. The nucleic acid, especially DNA, can be
double-stranded or single-stranded. Single-stranded nucleic acid
can be the coding strand (sense strand) or the non-coding strand
(anti-sense strand).
[0133] The invention further provides nucleic acid molecules that
encode fragments of the peptides of the present invention as well
as nucleic acid molecules that encode obvious variants of the
protease proteins of the present invention that are described
above. Such nucleic acid molecules may be naturally occurring, such
as allelic variants (same locus), paralogs (different locus), and
orthologs (different organism), or may be constructed by
recombinant DNA methods or by chemical synthesis. Such
non-naturally occurring variants may be made by mutagenesis
techniques, including those applied to nucleic acid molecules,
cells, or organisms. Accordingly, as discussed above, the variants
can contain nucleotide substitutions, deletions, inversions and
insertions. Variation can occur in either or both the coding and
non-coding regions. The variations can produce both conservative
and non-conservative amino acid substitutions.
[0134] The present invention further provides non-coding fragments
of the nucleic acid molecules provided in FIGS. 1 and 3. Preferred
non-coding fragments include, but are not limited to, promoter
sequences, enhancer sequences, gene modulating sequences and gene
termination sequences. Such fragments are useful in controlling
heterologous gene expression and in developing screens to identify
gene-modulating agents. A promoter can readily be identified as
being 5' to the ATG start site in the genomic sequence provided in
FIG. 3.
[0135] A fragment comprises a contiguous nucleotide sequence
greater than 12 or more nucleotides. Further, a fragment could at
least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length
of the fragment will be based on its intended use. For example, the
fragment can encode epitope bearing regions of the peptide, or can
be useful as DNA probes and primers. Such fragments can be isolated
using the known nucleotide sequence to synthesize an
oligonucleotide probe. A labeled probe can then be used to screen a
cDNA library, genomic DNA library, or mRNA to isolate nucleic acid
corresponding to the coding region. Further, primers can be used in
PCR reactions to clone specific regions of gene.
[0136] A probe/primer typically comprises substantially a purified
oligonucleotide or oligonucleotide pair. The oligonucleotide
typically comprises a region of nucleotide sequence that hybridizes
under stringent conditions to at least about 12, 20, 25, 40, 50 or
more consecutive nucleotides.
[0137] Orthologs, homologs, and allelic variants can be identified
using methods well known in the art. As described in the Peptide
Section, these variants comprise a nucleotide sequence encoding a
peptide that is typically 60-70%, 70-80%, 80-90%, and more
typically at least about 90-95% or more homologous to the
nucleotide sequence shown in the Figure sheets or a fragment of
this sequence. Such nucleic acid molecules can readily be
identified as being able to hybridize under moderate to stringent
conditions, to the nucleotide sequence shown in the Figure sheets
or a fragment of the sequence. Allelic variants can readily be
determined by genetic locus of the encoding gene.
[0138] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences encoding a peptide at
least 60-70% homologous to each other typically remain hybridized
to each other. The conditions can be such that sequences at least
about 60%, at least about 70%, or at least about 80% or more
homologous to each other typically remain hybridized to each other.
Such stringent conditions are known to those skilled in the art and
can be found in Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent
hybridization conditions are hybridization in 6.times.sodium
chloride/sodium citrate (SSC) at about 45 C, followed by one or
more washes in 0.2.times.SSC, 0.1% SDS at 50-65 C. Examples of
moderate to low stringency hybridization conditions are well known
in the art.
[0139] Nucleic Acid Molecule Uses
[0140] The nucleic acid molecules of the present invention are
useful for probes, primers, chemical intermediates, and in
biological assays. The nucleic acid molecules are useful as a
hybridization probe for messenger RNA, transcript/cDNA and genomic
DNA to isolate full-length cDNA and genomic clones encoding the
peptide described in FIG. 2 and to isolate cDNA and genomic clones
that correspond to variants (alleles, orthologs, etc.) producing
the same or related peptides shown in FIG. 2. As illustrated in
FIG. 3, SNPs, including insertion/deletion variants ("indels"),
were identified at 40 different nucleotide positions.
[0141] The probe can correspond to any sequence along the entire
length of the nucleic acid molecules provided in the Figures.
Accordingly, it could be derived from 5' noncoding regions, the
coding region, and 3' noncoding regions. However, as discussed,
fragments are not to be construed as encompassing fragments
disclosed prior to the present invention.
[0142] The nucleic acid molecules are also useful as primers for
PCR to amplify any given region of a nucleic acid molecule and are
useful to synthesize antisense molecules of desired length and
sequence.
[0143] The nucleic acid molecules are also useful for constructing
recombinant vectors. Such vectors include expression vectors that
express a portion of, or all of, the peptide sequences. Vectors
also include insertion vectors, used to integrate into another
nucleic acid molecule sequence, such as into the cellular genome,
to alter in situ expression of a gene and/or gene product. For
example, an endogenous coding sequence can be replaced via
homologous recombination with all or part of the coding region
containing one or more specifically introduced mutations.
[0144] The nucleic acid molecules are also useful for expressing
antigenic portions of the proteins.
[0145] The nucleic acid molecules are also useful as probes for
determining the chromosomal positions of the nucleic acid molecules
by means of in situ hybridization methods. As indicated by the data
presented in FIG. 3, the map position was determined to be on
chromosome 13 by ePCR.
[0146] The nucleic acid molecules are also useful in making vectors
containing the gene regulatory regions of the nucleic acid
molecules of the present invention.
[0147] The nucleic acid molecules are also useful for designing
ribozymes corresponding to all, or a part, of the mRNA produced
from the nucleic acid molecules described herein.
[0148] The nucleic acid molecules are also useful for making
vectors that express part, or all, of the peptides.
[0149] The nucleic acid molecules are also useful for constructing
host cells expressing a part, or all, of the nucleic acid molecules
and peptides.
[0150] The nucleic acid molecules are also useful for constructing
transgenic animals expressing all, or a part, of the nucleic acid
molecules and peptides.
[0151] The nucleic acid molecules are also useful as hybridization
probes for determining the presence, level, form and distribution
of nucleic acid expression. Experimental data as provided in FIG. 1
indicates that protease proteins of the present invention are
expressed in the fetal brain, liver, hepatocellular carcinoma by a
virtual northern blot. In addition, PCR-based tissue screening
panel indicates expression in whole liver. Accordingly, the probes
can be used to detect the presence of, or to determine levels of, a
specific nucleic acid molecule in cells, tissues, and in organisms.
The nucleic acid whose level is determined can be DNA or RNA.
Accordingly, probes corresponding to the peptides described herein
can be used to assess expression and/or gene copy number in a given
cell, tissue, or organism. These uses are relevant for diagnosis of
disorders involving an increase or decrease in protease protein
expression relative to normal results.
[0152] In vitro techniques for detection of mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detecting DNA includes Southern hybridizations and in situ
hybridization.
[0153] Probes can be used as a part of a diagnostic test kit for
identifying cells or tissues that express a protease protein, such
as by measuring a level of a protease-encoding nucleic acid in a
sample of cells from a subject e.g., mRNA or genomic DNA, or
determining if a protease gene has been mutated. Experimental data
as provided in FIG. 1 indicates that protease proteins of the
present invention are expressed in the fetal brain, liver,
hepatocellular carcinoma by a virtual northern blot. In addition,
PCR-based tissue screening panel indicates expression in whole
liver.
[0154] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate protease nucleic acid
expression.
[0155] The invention thus provides a method for identifying a
compound that can be used to treat a disorder associated with
nucleic acid expression of the protease gene, particularly
biological and pathological processes that are mediated by the
protease in cells and tissues that express it. Experimental data as
provided in FIG. 1 indicates expression in fetal brain, liver,
hepatocellular carcinoma and whole liver. The method typically
includes assaying the ability of the compound to modulate the
expression of the protease nucleic acid and thus identifying a
compound that can be used to treat a disorder characterized by
undesired protease nucleic acid expression. The assays can be
performed in cell-based and cell-free systems. Cell-based assays
include cells naturally expressing the protease nucleic acid or
recombinant cells genetically engineered to express specific
nucleic acid sequences.
[0156] The assay for protease nucleic acid expression can involve
direct assay of nucleic acid levels, such as mRNA levels, or on
collateral compounds involved in the signal pathway. Further, the
expression of genes that are up- or down-regulated in response to
the protease protein signal pathway can also be assayed. In this
embodiment the regulatory regions of these genes can be operably
linked to a reporter gene such as luciferase.
[0157] Thus, modulators of protease gene expression can be
identified in a method wherein a cell is contacted with a candidate
compound and the expression of mRNA determined. The level of
expression of protease mRNA in the presence of the candidate
compound is compared to the level of expression of protease mRNA in
the absence of the candidate compound. The candidate compound can
then be identified as a modulator of nucleic acid expression based
on this comparison and be used, for example to treat a disorder
characterized by aberrant nucleic acid expression. When expression
of mRNA is statistically significantly greater in the presence of
the candidate compound than in its absence, the candidate compound
is identified as a stimulator of nucleic acid expression. When
nucleic acid expression is statistically significantly less in the
presence of the candidate compound than in its absence, the
candidate compound is identified as an inhibitor of nucleic acid
expression.
[0158] The invention further provides methods of treatment, with
the nucleic acid as a target, using a compound identified through
drug screening as a gene modulator to modulate protease nucleic
acid expression in cells and tissues that express the protease.
Experimental data as provided in FIG. 1 indicates that protease
proteins of the present invention are expressed in the fetal brain,
liver, hepatocellular carcinoma by a virtual northern blot. In
addition, PCR-based tissue screening panel indicates expression in
whole liver. Modulation includes both up-regulation (i.e.
activation or agonization) or down-regulation (suppression or
antagonization) or nucleic acid expression.
[0159] Alternatively, a modulator for protease nucleic acid
expression can be a small molecule or drug identified using the
screening assays described herein as long as the drug or small
molecule inhibits the protease nucleic acid expression in the cells
and tissues that express the protein. Experimental data as provided
in FIG. 1 indicates expression in fetal brain, liver,
hepatocellular carcinoma and whole liver.
[0160] The nucleic acid molecules are also useful for monitoring
the effectiveness of modulating compounds on the expression or
activity of the protease gene in clinical trials or in a treatment
regimen. Thus, the gene expression pattern can serve as a barometer
for the continuing effectiveness of treatment with the compound,
particularly with compounds to which a patient can develop
resistance. The gene expression pattern can also serve as a marker
indicative of a physiological response of the affected cells to the
compound. Accordingly, such monitoring would allow either increased
administration of the compound or the administration of alternative
compounds to which the patient has not become resistant. Similarly,
if the level of nucleic acid expression falls below a desirable
level, administration of the compound could be commensurately
decreased.
[0161] The nucleic acid molecules are also useful in diagnostic
assays for qualitative changes in protease nucleic acid expression,
and particularly in qualitative changes that lead to pathology. The
nucleic acid molecules can be used to detect mutations in protease
genes and gene expression products such as mRNA. The nucleic acid
molecules can be used as hybridization probes to detect naturally
occurring genetic mutations in the protease gene and thereby to
determine whether a subject with the mutation is at risk for a
disorder caused by the mutation. Mutations include deletion,
addition, or substitution of one or more nucleotides in the gene,
chromosomal rearrangement, such as inversion or transposition,
modification of genomic DNA, such as aberrant methylation patterns
or changes in gene copy number, such as amplification. Detection of
a mutated form of the protease gene associated with a dysfunction
provides a diagnostic tool for an active disease or susceptibility
to disease when the disease results from overexpression,
underexpression, or altered expression of a protease protein.
[0162] Individuals carrying mutations in the protease gene can be
detected at the nucleic acid level by a variety of techniques. FIG.
3 provides information on SNPs that have been identified in a gene
encoding the transporter protein of the present invention. 40 SNP
variants were found, including 5 indels (indicated by a "-") and 1
SNPs in exons, of which 5 of these cause changes in the amino acid
sequence (i.e., nonsynonymous SNPs). SNPs, identified at different
nucleotide positions in introns and regions 5' and 3' of the ORF,
may affect control/regulatory elements. As indicated by the data
presented in FIG. 3, the map position was determined to be on
chromosome 13 by ePCR. Genomic DNA can be analyzed directly or can
be amplified by using PCR prior to analysis. RNA or cDNA can be
used in the same way. In some uses, detection of the mutation
involves the use of a probe/primer in a polymerase chain reaction
(PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as
anchor PCR or RACE PCR, or, alternatively, in a ligation chain
reaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080
(1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter of
which can be particularly useful for detecting point mutations in
the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682
(1995)). This method can include the steps of collecting a sample
of cells from a patient, isolating nucleic acid (e.g., genomic,
mRNA or both) from the cells of the sample, contacting the nucleic
acid sample with one or more primers which specifically hybridize
to a gene under conditions such that hybridization and
amplification of the gene (if present) occurs, and detecting the
presence or absence of an amplification product, or detecting the
size of the amplification product and comparing the length to a
control sample. Deletions and insertions can be detected by a
change in size of the amplified product compared to the normal
genotype. Point mutations can be identified by hybridizing
amplified DNA to normal RNA or antisense DNA sequences.
[0163] Alternatively, mutations in a protease gene can be directly
identified, for example, by alterations in restriction enzyme
digestion patterns determined by gel electrophoresis.
[0164] Further, sequence-specific ribozymes (U.S. Pat. No.
5,498,531) can be used to score for the presence of specific
mutations by development or loss of a ribozyme cleavage site.
Perfectly matched sequences can be distinguished from mismatched
sequences by nuclease cleavage digestion assays or by differences
in melting temperature.
[0165] Sequence changes at specific locations can also be assessed
by nuclease protection assays such as RNase and S1 protection or
the chemical cleavage method. Furthermore, sequence differences
between a mutant protease gene and a wild-type gene can be
determined by direct DNA sequencing. A variety of automated
sequencing procedures can be utilized when performing the
diagnostic assays (Naeve, C. W., (1995) Biotechniques 19:448),
including sequencing by mass spectrometry (see, e.g., PCT
International Publication No. WO 94/16101; Cohen et al., Adv.
Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.
Biotechnol. 38:147-159 (1993)).
[0166] Other methods for detecting mutations in the gene include
methods in which protection from cleavage agents is used to detect
mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al.,
Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988);
Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et
al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144
(1993); and Hayashi et al., Genet. Anal. Tech. Appl. 9:73-79
(1992)), and movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed
using denaturing gradient gel electrophoresis (Myers et al., Nature
313:495 (1985)). Examples of other techniques for detecting point
mutations include selective oligonucleotide hybridization,
selective amplification, and selective primer extension.
[0167] The nucleic acid molecules are also useful for testing an
individual for a genotype that while not necessarily causing the
disease, nevertheless affects the treatment modality. Thus, the
nucleic acid molecules can be used to study the relationship
between an individual's genotype and the individual's response to a
compound used for treatment (pharmacogenomic relationship).
Accordingly, the nucleic acid molecules described herein can be
used to assess the mutation content of the protease gene in an
individual in order to select an appropriate compound or dosage
regimen for treatment.
[0168] Thus nucleic acid molecules displaying genetic variations
that affect treatment provide a diagnostic target that can be used
to tailor treatment in an individual. Accordingly, the production
of recombinant cells and animals containing these polymorphisms
allow effective clinical design of treatment compounds and dosage
regimens.
[0169] The nucleic acid molecules are thus useful as antisense
constructs to control protease gene expression in cells, tissues,
and organisms. A DNA antisense nucleic acid molecule is designed to
be complementary to a region of the gene involved in transcription,
preventing transcription and hence production of protease protein.
An antisense RNA or DNA nucleic acid molecule would hybridize to
the mRNA and thus block translation of mRNA into protease protein.
FIG. 3 provides information on SNPs that have been identified in a
gene encoding the transporter protein of the present invention. 40
SNP variants were found, including 5 indels (indicated by a "-")
and 1 SNPs in exons, of which 5 of these cause changes in the amino
acid sequence (i.e., nonsynonymous SNPs). SNPs, identified at
different nucleotide positions in introns and regions 5' and 3' of
the ORF, may affect control/regulatory elements.
[0170] Alternatively, a class of antisense molecules can be used to
inactivate mRNA in order to decrease expression of protease nucleic
acid. Accordingly, these molecules can treat a disorder
characterized by abnormal or undesired protease nucleic acid
expression. This technique involves cleavage by means of ribozymes
containing nucleotide sequences complementary to one or more
regions in the mRNA that attenuate the ability of the mRNA to be
translated. Possible regions include coding regions and
particularly coding regions corresponding to the catalytic and
other functional activities of the protease protein, such as
substrate binding.
[0171] The nucleic acid molecules also provide vectors for gene
therapy in patients containing cells that are aberrant in protease
gene expression. Thus, recombinant cells, which include the
patient's cells that have been engineered ex vivo and returned to
the patient, are introduced into an individual where the cells
produce the desired protease protein to treat the individual.
[0172] The invention also encompasses kits for detecting the
presence of a protease nucleic acid in a biological sample.
Experimental data as provided in FIG. 1 indicates that protease
proteins of the present invention are expressed in the fetal brain,
liver, hepatocellular carcinoma by a virtual northern blot. In
addition, PCR-based tissue screening panel indicates expression in
whole liver. For example, the kit can comprise reagents such as a
labeled or labelable nucleic acid or agent capable of detecting
protease nucleic acid in a biological sample; means for determining
the amount of protease nucleic acid in the sample; and means for
comparing the amount of protease nucleic acid in the sample with a
standard. The compound or agent can be packaged in a suitable
container. The kit can further comprise instructions for using the
kit to detect protease protein mRNA or DNA.
[0173] Nucleic Acid Arrays
[0174] The present invention further provides nucleic acid
detection kits, such as arrays or microarrays of nucleic acid
molecules that are based on the sequence information provided in
FIGS. 1 and 3 (SEQ ID NOS:1 and 3).
[0175] As used herein "Arrays" or "Microarrays" refers to an array
of distinct polynucleotides or oligonucleotides synthesized on a
substrate, such as paper, nylon or other type of membrane, filter,
chip, glass slide, or any other suitable solid support. In one
embodiment, the microarray is prepared and used according to the
methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT
application W095/11995 (Chee et al.), Lockhart, D. J. et al. (1996;
Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc.
Natl. Acad. Sci. 93 : 10614-10619), all of which are incorporated
herein in their entirety by reference. In other embodiments, such
arrays are produced by the methods described by Brown et al., U.S.
Pat. No. 5,807,522.
[0176] The microarray or detection kit is preferably composed of a
large number of unique, single-stranded nucleic acid sequences,
usually either synthetic antisense oligonucleotides or fragments of
cDNAs, fixed to a solid support. The oligonucleotides are
preferably about 6-60 nucleotides in length, more preferably 15-30
nucleotides in length, and most preferably about 20-25 nucleotides
in length. For a certain type of microarray or detection kit, it
may be preferable to use oligonucleotides that are only 7-20
nucleotides in length. The microarray or detection kit may contain
oligonucleotides that cover the known 5', or 3', sequence,
sequential oligonucleotides which cover the full length sequence;
or unique oligonucleotides selected from particular areas along the
length of the sequence. Polynucleotides used in the microarray or
detection kit may be oligonucleotides that are specific to a gene
or genes of interest.
[0177] In order to produce oligonucleotides to a known sequence for
a microarray or detection kit, the gene(s) of interest (or an ORF
identified from the contigs of the present invention) is typically
examined using a computer algorithm which starts at the 5' or at
the 3' end of the nucleotide sequence. Typical algorithms will then
identify oligomers of defined length that are unique to the gene,
have a GC content within a range suitable for hybridization, and
lack predicted secondary structure that may interfere with
hybridization. In certain situations it may be appropriate to use
pairs of oligonucleotides on a microarray or detection kit. The
"pairs" will be identical, except for one nucleotide that
preferably is located in the center of the sequence. The second
oligonucleotide in the pair (mismatched by one) serves as a
control. The number of oligonucleotide pairs may range from two to
one million. The oligomers are synthesized at designated areas on a
substrate using a light-directed chemical process. The substrate
may be paper, nylon or other type of membrane, filter, chip, glass
slide or any other suitable solid support.
[0178] In another aspect, an oligonucleotide may be synthesized on
the surface of the substrate by using a chemical coupling procedure
and an ink jet application apparatus, as described in PCT
application W095/251116 (Baldeschweiler et al.) which is
incorporated herein in its entirety by reference. In another
aspect, a "gridded" array analogous to a dot (or slot) blot may be
used to arrange and link cDNA fragments or oligonucleotides to the
surface of a substrate using a vacuum system, thermal, UV,
mechanical or chemical bonding procedures. An array, such as those
described above, may be produced by hand or by using available
devices (slot blot or dot blot apparatus), materials (any suitable
solid support), and machines (including robotic instruments), and
may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or
any other number between two and one million which lends itself to
the efficient use of commercially available instrumentation.
[0179] In order to conduct sample analysis using a microarray or
detection kit, the RNA or DNA from a biological sample is made into
hybridization probes. The mRNA is isolated, and cDNA is produced
and used as a template to make antisense RNA (aRNA). The aRNA is
amplified in the presence of fluorescent nucleotides, and labeled
probes are incubated with the microarray or detection kit so that
the probe sequences hybridize to complementary oligonucleotides of
the microarray or detection kit. Incubation conditions are adjusted
so that hybridization occurs with precise complementary matches or
with various degrees of less complementarity. After removal of
nonhybridized probes, a scanner is used to determine the levels and
patterns of fluorescence. The scanned images are examined to
determine degree of complementarity and the relative abundance of
each oligonucleotide sequence on the microarray or detection kit.
The biological samples may be obtained from any bodily fluids (such
as blood, urine, saliva, phlegm, gastric juices, etc.), cultured
cells, biopsies, or other tissue preparations. A detection system
may be used to measure the absence, presence, and amount of
hybridization for all of the distinct sequences simultaneously.
This data may be used for large-scale correlation studies on the
sequences, expression patterns, mutations, variants, or
polymorphisms among samples.
[0180] Using such arrays, the present invention provides methods to
identify the expression of the protease proteins/peptides of the
present invention. In detail, such methods comprise incubating a
test sample with one or more nucleic acid molecules and assaying
for binding of the nucleic acid molecule with components within the
test sample. Such assays will typically involve arrays comprising
many genes, at least one of which is a gene of the present
invention and or alleles of the protease gene of the present
invention. FIG. 3 provides information on SNPs that have been
identified in a gene encoding the transporter protein of the
present invention. 40 SNP variants were found, including 5 indels
(indicated by a "-") and 1 SNPs in exons, of which 5 of these cause
changes in the amino acid sequence (i.e., nonsynonymous SNPs).
SNPs, identified at different nucleotide positions in introns and
regions 5' and 3' of the ORF, may affect control/regulatory
elements.
[0181] Conditions for incubating a nucleic acid molecule with a
test sample vary. Incubation conditions depend on the format
employed in the assay, the detection methods employed, and the type
and nature of the nucleic acid molecule used in the assay. One
skilled in the art will recognize that any one of the commonly
available hybridization, amplification or array assay formats can
readily be adapted to employ the novel fragments of the Human
genome disclosed herein. Examples of such assays can be found in
Chard, T, An Introduction to Radioimmunoassay and Related
Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands
(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,
Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3
(1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays:
Laboratory Techniques in Biochemistry and Molecular Biology,
Elsevier Science Publishers, Amsterdam, The Netherlands (1985).
[0182] The test samples of the present invention include cells,
protein or membrane extracts of cells. The test sample used in the
above-described method will vary based on the assay format, nature
of the detection method and the tissues, cells or extracts used as
the sample to be assayed. Methods for preparing nucleic acid
extracts or of cells are well known in the art and can be readily
be adapted in order to obtain a sample that is compatible with the
system utilized.
[0183] In another embodiment of the present invention, kits are
provided which contain the necessary reagents to carry out the
assays of the present invention.
[0184] Specifically, the invention provides a compartmentalized kit
to receive, in close confinement, one or more containers which
comprises: (a) a first container comprising one of the nucleic acid
molecules that can bind to a fragment of the Human genome disclosed
herein; and (b) one or more other containers comprising one or more
of the following: wash reagents, reagents capable of detecting
presence of a bound nucleic acid.
[0185] In detail, a compartmentalized kit includes any kit in which
reagents are contained in separate containers. Such containers
include small glass containers, plastic containers, strips of
plastic, glass or paper, or arraying material such as silica. Such
containers allows one to efficiently transfer reagents from one
compartment to another compartment such that the samples and
reagents are not cross-contaminated, and the agents or solutions of
each container can be added in a quantitative fashion from one
compartment to another. Such containers will include a container
which will accept the test sample, a container which contains the
nucleic acid probe, containers which contain wash reagents (such as
phosphate buffered saline, Tris-buffers, etc.), and containers
which contain the reagents used to detect the bound probe. One
skilled in the art will readily recognize that the previously
unidentified protease gene of the present invention can be
routinely identified using the sequence information disclosed
herein can be readily incorporated into one of the established kit
formats which are well known in the art, particularly expression
arrays.
[0186] Vectors/Host Cells
[0187] The invention also provides vectors containing the nucleic
acid molecules described herein. The term "vector" refers to a
vehicle, preferably a nucleic acid molecule, which can transport
the nucleic acid molecules. When the vector is a nucleic acid
molecule, the nucleic acid molecules are covalently linked to the
vector nucleic acid. With this aspect of the invention, the vector
includes a plasmid, single or double stranded phage, a single or
double stranded RNA or DNA viral vector, or artificial chromosome,
such as a BAC, PAC, YAC, OR MAC.
[0188] A vector can be maintained in the host cell as an
extrachromosomal element where it replicates and produces
additional copies of the nucleic acid molecules. Alternatively, the
vector may integrate into the host cell genome and produce
additional copies of the nucleic acid molecules when the host cell
replicates.
[0189] The invention provides vectors for the maintenance (cloning
vectors) or vectors for expression (expression vectors) of the
nucleic acid molecules. The vectors can function in prokaryotic or
eukaryotic cells or in both (shuttle vectors).
[0190] Expression vectors contain cis-acting regulatory regions
that are operably linked in the vector to the nucleic acid
molecules such that transcription of the nucleic acid molecules is
allowed in a host cell. The nucleic acid molecules can be
introduced into the host cell with a separate nucleic acid molecule
capable of affecting transcription. Thus, the second nucleic acid
molecule may provide a trans-acting factor interacting with the
cis-regulatory control region to allow transcription of the nucleic
acid molecules from the vector. Alternatively, a trans-acting
factor may be supplied by the host cell. Finally, a trans-acting
factor can be produced from the vector itself. It is understood,
however, that in some embodiments, transcription and/or translation
of the nucleic acid molecules can occur in a cell-free system.
[0191] The regulatory sequence to which the nucleic acid molecules
described herein can be operably linked include promoters for
directing mRNA transcription. These include, but are not limited
to, the left promoter from bacteriophage .lambda., the lac, TRP,
and TAC promoters from E. coli, the early and late promoters from
SV40, the CMV immediate early promoter, the adenovirus early and
late promoters, and retrovirus long-terminal repeats.
[0192] In addition to control regions that promote transcription,
expression vectors may also include regions that modulate
transcription, such as repressor binding sites and enhancers.
Examples include the SV40 enhancer, the cytomegalovirus immediate
early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus LTR enhancers.
[0193] In addition to containing sites for transcription initiation
and control, expression vectors can also contain sequences
necessary for transcription termination and, in the transcribed
region a ribosome binding site for translation. Other regulatory
control elements for expression include initiation and termination
codons as well as polyadenylation signals. The person of ordinary
skill in the art would be aware of the numerous regulatory
sequences that are useful in expression vectors. Such regulatory
sequences are described, for example, in Sambrook et al., Molecular
Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., (1989).
[0194] A variety of expression vectors can be used to express a
nucleic acid molecule. Such vectors include chromosomal, episomal,
and virus-derived vectors, for example vectors derived from
bacterial plasmids, from bacteriophage, from yeast episomes, from
yeast chromosomal elements, including yeast artificial chromosomes,
from viruses such as baculoviruses, papovaviruses such as SV40,
Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses,
and retroviruses. Vectors may also be derived from combinations of
these sources such as those derived from plasmid and bacteriophage
genetic elements, e.g. cosmids and phagemids. Appropriate cloning
and expression vectors for prokaryotic and eukaryotic hosts are
described in Sambrook et al., Molecular Cloning: A Laboratory
Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., (1989).
[0195] The regulatory sequence may provide constitutive expression
in one or more host cells (i.e. tissue specific) or may provide for
inducible expression in one or more cell types such as by
temperature, nutrient additive, or exogenous factor such as a
hormone or other ligand. A variety of vectors providing for
constitutive and inducible expression in prokaryotic and eukaryotic
hosts are well known to those of ordinary skill in the art.
[0196] The nucleic acid molecules can be inserted into the vector
nucleic acid by well-known methodology. Generally, the DNA sequence
that will ultimately be expressed is joined to an expression vector
by cleaving the DNA sequence and the expression vector with one or
more restriction enzymes and then ligating the fragments together.
Procedures for restriction enzyme digestion and ligation are well
known to those of ordinary skill in the art.
[0197] The vector containing the appropriate nucleic acid molecule
can be introduced into an appropriate host cell for propagation or
expression using well-known techniques. Bacterial cells include,
but are not limited to, E. coli, Streptomyces, and Salmonella
typhimurium. Eukaryotic cells include, but are not limited to,
yeast, insect cells such as Drosophila, animal cells such as COS
and CHO cells, and plant cells.
[0198] As described herein, it may be desirable to express the
peptide as a fusion protein. Accordingly, the invention provides
fusion vectors that allow for the production of the peptides.
Fusion vectors can increase the expression of a recombinant
protein, increase the solubility of the recombinant protein, and
aid in the purification of the protein by acting for example as a
ligand for affinity purification. A proteolytic cleavage site may
be introduced at the junction of the fusion moiety so that the
desired peptide can ultimately be separated from the fusion moiety.
Proteolytic enzymes include, but are not limited to, factor Xa,
thrombin, and enteroprotease. Typical fusion expression vectors
include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New
England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway,
N.J.) which fuse glutathione S-transferase (GST), maltose E binding
protein, or protein A, respectively, to the target recombinant
protein. Examples of suitable inducible non-fusion E. coli
expression vectors include pTrc (Amann et al., Gene 69:301-315
(1988)) and pET 11d (Studier et al., Gene Expression Technology:
Methods in Enzymology 185:60-89 (1990)).
[0199] Recombinant protein expression can be maximized in host
bacteria by providing a genetic background wherein the host cell
has an impaired capacity to proteolytically cleave the recombinant
protein. (Gottesman, S., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990)119-128).
Alternatively, the sequence of the nucleic acid molecule of
interest can be altered to provide preferential codon usage for a
specific host cell, for example E. coli. (Wada et al., Nucleic
Acids Res. 20:2111-2118 (1992)).
[0200] The nucleic acid molecules can also be expressed by
expression vectors that are operative in yeast. Examples of vectors
for expression in yeast e.g., S. cerevisiae include pYepSec1
(Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kujan et al.,
Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123
(1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).
[0201] The nucleic acid molecules can also be expressed in insect
cells using, for example, baculovirus expression vectors.
Baculovirus vectors available for expression of proteins in
cultured insect cells (e.g., Sf 9 cells) include the pAc series
(Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL
series (Lucklow et al., Virology 170:31-39 (1989)).
[0202] In certain embodiments of the invention, the nucleic acid
molecules described herein are expressed in mammalian cells using
mammalian expression vectors. Examples of mammalian expression
vectors include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC
(Kaufman et al., EMBO J. 6:187-195 (1987)).
[0203] The expression vectors listed herein are provided by way of
example only of the well-known vectors available to those of
ordinary skill in the art that would be useful to express the
nucleic acid molecules. The person of ordinary skill in the art
would be aware of other vectors suitable for maintenance
propagation or expression of the nucleic acid molecules described
herein. These are found for example in Sambrook, J., Fritsh, E. F.,
and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed.,
Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989.
[0204] The invention also encompasses vectors in which the nucleic
acid sequences described herein are cloned into the vector in
reverse orientation, but operably linked to a regulatory sequence
that permits transcription of antisense RNA. Thus, an antisense
transcript can be produced to all, or to a portion, of the nucleic
acid molecule sequences described herein, including both coding and
non-coding regions. Expression of this antisense RNA is subject to
each of the parameters described above in relation to expression of
the sense RNA (regulatory sequences, constitutive or inducible
expression, tissue-specific expression).
[0205] The invention also relates to recombinant host cells
containing the vectors described herein. Host cells therefore
include prokaryotic cells, lower eukaryotic cells such as yeast,
other eukaryotic cells such as insect cells, and higher eukaryotic
cells such as mammalian cells.
[0206] The recombinant host cells are prepared by introducing the
vector constructs described herein into the cells by techniques
readily available to the person of ordinary skill in the art. These
include, but are not limited to, calcium phosphate transfection,
DEAE-dextran-mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection,
lipofection, and other techniques such as those found in Sambrook,
et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold
Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1989).
[0207] Host cells can contain more than one vector. Thus, different
nucleotide sequences can be introduced on different vectors of the
same cell. Similarly, the nucleic acid molecules can be introduced
either alone or with other nucleic acid molecules that are not
related to the nucleic acid molecules such as those providing
trans-acting factors for expression vectors. When more than one
vector is introduced into a cell, the vectors can be introduced
independently, co-introduced or joined to the nucleic acid molecule
vector.
[0208] In the case of bacteriophage and viral vectors, these can be
introduced into cells as packaged or encapsulated virus by standard
procedures for infection and transduction. Viral vectors can be
replication-competent or replication-defective. In the case in
which viral replication is defective, replication will occur in
host cells providing functions that complement the defects.
[0209] Vectors generally include selectable markers that enable the
selection of the subpopulation of cells that contain the
recombinant vector constructs. The marker can be contained in the
same vector that contains the nucleic acid molecules described
herein or may be on a separate vector. Markers include tetracycline
or ampicillin-resistance genes for prokaryotic host cells and
dihydrofolate reductase or neomycin resistance for eukaryotic host
cells. However, any marker that provides selection for a phenotypic
trait will be effective.
[0210] While the mature proteins can be produced in bacteria,
yeast, mammalian cells, and other cells under the control of the
appropriate regulatory sequences, cell-free transcription and
translation systems can also be used to produce these proteins
using RNA derived from the DNA constructs described herein.
[0211] Where secretion of the peptide is desired, which is
difficult to achieve with multi-transmembrane domain containing
proteins such as proteases, appropriate secretion signals are
incorporated into the vector. The signal sequence can be endogenous
to the peptides or heterologous to these peptides.
[0212] Where the peptide is not secreted into the medium, which is
typically the case with proteases, the protein can be isolated from
the host cell by standard disruption procedures, including freeze
thaw, sonication, mechanical disruption, use of lysing agents and
the like. The peptide can then be recovered and purified by
well-known purification methods including ammonium sulfate
precipitation, acid extraction, anion or cationic exchange
chromatography, phosphocellulose chromatography,
hydrophobic-interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography, or high
performance liquid chromatography.
[0213] It is also understood that depending upon the host cell in
recombinant production of the peptides described herein, the
peptides can have various glycosylation patterns, depending upon
the cell, or maybe non-glycosylated as when produced in bacteria.
In addition, the peptides may include an initial modified
methionine in some cases as a result of a host-mediated
process.
[0214] Uses of Vectors and Host Cells
[0215] The recombinant host cells expressing the peptides described
herein have a variety of uses. First, the cells are useful for
producing a protease protein or peptide that can be further
purified to produce desired amounts of protease protein or
fragments. Thus, host cells containing expression vectors are
useful for peptide production.
[0216] Host cells are also useful for conducting cell-based assays
involving the protease protein or protease protein fragments, such
as those described above as well as other formats known in the art.
Thus, a recombinant host cell expressing a native protease protein
is useful for assaying compounds that stimulate or inhibit protease
protein function.
[0217] Host cells are also useful for identifying protease protein
mutants in which these functions are affected. If the mutants
naturally occur and give rise to a pathology, host cells containing
the mutations are useful to assay compounds that have a desired
effect on the mutant protease protein (for example, stimulating or
inhibiting function) which may not be indicated by their effect on
the native protease protein.
[0218] Genetically engineered host cells can be further used to
produce non-human transgenic animals. A transgenic animal is
preferably a mammal, for example a rodent, such as a rat or mouse,
in which one or more of the cells of the animal include a
transgene. A transgene is exogenous DNA which is integrated into
the genome of a cell from which a transgenic animal develops and
which remains in the genome of the mature animal in one or more
cell types or tissues of the transgenic animal. These animals are
useful for studying the function of a protease protein and
identifying and evaluating modulators of protease protein activity.
Other examples of transgenic animals include non-human primates,
sheep, dogs, cows, goats, chickens, and amphibians.
[0219] A transgenic animal can be produced by introducing nucleic
acid into the male pronuclei of a fertilized oocyte, e.g., by
microinjection, retroviral infection, and allowing the oocyte to
develop in a pseudopregnant female foster animal. Any of the
protease protein nucleotide sequences can be introduced as a
transgene into the genome of a non-human animal, such as a
mouse.
[0220] Any of the regulatory or other sequences useful in
expression vectors can form part of the transgenic sequence. This
includes intronic sequences and polyadenylation signals, if not
already included. A tissue-specific regulatory sequence(s) can be
operably linked to the transgene to direct expression of the
protease protein to particular cells.
[0221] Methods for generating transgenic animals via embryo
manipulation and microinjection, particularly animals such as mice,
have become conventional in the art and are described, for example,
in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al.,
U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used
for production of other transgenic animals. A transgenic founder
animal can be identified based upon the presence of the transgene
in its genome and/or expression of transgenic mRNA in tissues or
cells of the animals. A transgenic founder animal can then be used
to breed additional animals carrying the transgene. Moreover,
transgenic animals carrying a transgene can further be bred to
other transgenic animals carrying other transgenes. A transgenic
animal also includes animals in which the entire animal or tissues
in the animal have been produced using the homologously recombinant
host cells described herein.
[0222] In another embodiment, transgenic non-human animals can be
produced which contain selected systems that allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage P1. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al PNAS
89:6232-6236 (1992). Another example of a recombinase system is the
FLP recombinase system of S. cerevisiae (O'Gorman et al. Science
251:1351-1355 (1991). If a cre/loxP recombinase system is used to
regulate expression of the transgene, animals containing transgenes
encoding both the Cre recombinase and a selected protein is
required. Such animals can be provided through the construction of
"double" transgenic animals, e.g., by mating two transgenic
animals, one containing a transgene encoding a selected protein and
the other containing a transgene encoding a recombinase.
[0223] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut,
I. et al. Nature 385:810-813 (1997) and PCT International
Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell,
e.g., a somatic cell, from the transgenic animal can be isolated
and induced to exit the growth cycle and enter G.sub.o phase. The
quiescent cell can then be fused, e.g., through the use of
electrical pulses, to an enucleated oocyte from an animal of the
same species from which the quiescent cell is isolated. The
reconstructed oocyte is then cultured such that it develops to
morula or blastocyst and then transferred to pseudopregnant female
foster animal. The offspring born of this female foster animal will
be a clone of the animal from which the cell, e.g., the somatic
cell, is isolated.
[0224] Transgenic animals containing recombinant cells that express
the peptides described herein are useful to conduct the assays
described herein in an in vivo context. Accordingly, the various
physiological factors that are present in vivo and that could
effect substrate binding, protease protein activity/activation, and
signal transduction, may not be evident from in vitro cell-free or
cell-based assays. Accordingly, it is useful to provide non-human
transgenic animals to assay in vivo protease protein function,
including substrate interaction, the effect of specific mutant
protease proteins on protease protein function and substrate
interaction, and the effect of chimeric protease proteins. It is
also possible to assess the effect of null mutations, that is
mutations that substantially or completely eliminate one or more
protease protein functions.
[0225] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the above-described modes for carrying out
the invention which are obvious to those skilled in the field of
molecular biology or related fields are intended to be within the
scope of the following claims.
Sequence CWU 1
1
4 1 1625 DNA Human 1 gaaaattgct gttgggatga agctttgcag ccttgcagtc
cttgtaccca ttgttctctt 60 ctgtgagcag catgtcttcg cgtttcagag
tggccaagtt ctagctgctc ttcctagaac 120 ctctaggcaa gttcaagttc
tacagaatct tactacaaca tatgagattg ttctctggca 180 gccggtaaca
gctgacctta ttgtgaagaa aaaacaagtc catttttttg taaatgcatc 240
tgatgtcgac aatgtgaaag cccatttaaa tgtgagcgga attccatgca gtgtcttgct
300 ggcagatgtg gaagatctta ttcaacagca gatttccaac gacacagtca
gcccccgagc 360 ctccgcatcg tactatgaac agtatcactc actaaatgaa
atctattctt ggatagaatt 420 tataactgag aggcatcctg atatgcttac
aaaaatccac attggatcct catttgagaa 480 gtacccactc tatgttttaa
aggtttctgg aaaagaacaa gcagccaaaa atgccatatg 540 gattgactgt
ggaatccatg ccagagaatg gatctctcct gctttctgct tgtggttcat 600
aggccataat cgaatgtgga gaaagaaccg ttctttctat gcgaacaatc attgcatcgg
660 aacagacctg aataggaact ttgcttccaa acactggtgt gaggaaggtg
catccagttc 720 ctcatgctcg gaaacctact gtggacttta tcctgagtca
gaaccagaag tgaaggcagt 780 ggctagtttc ttgagaagaa atatcaacca
gattaaagca tacatcagca tgcattcata 840 ctcccagcat atagtgtttc
catattccta tacacgaagt aaaagcaaag accatgagga 900 actgtctcta
gtagccagtg aagcagttcg tgctattgag aaaattagta aaaataccag 960
gtatacacat ggccatggct cagaaacctt atacctagct cctggaggtg gggacgattg
1020 gatctatgat ttgggcatca aatattcgtt tacaattgaa cttcgagata
cgggcacata 1080 cggattcttg ctgccggagc gttacatcaa acccacctgt
agagaagctt ttgccgctgt 1140 ctctaaaata gcttggcatg tcattaggaa
tgtttaatgc ccctgatttt atcattctgc 1200 ttccgtattt taatttactg
attccagcaa gaccaaatca ttgtatcaga ttatttttaa 1260 gttttatccg
tagttttgat aaaagatttt cctattcctt ggttctgtca gagaacctaa 1320
taagtgctac tttgccatta aggcagacta gggttcatgt ctttttaccc tttaaaaaaa
1380 attgtaaaag tctagttacc tactttttct ttgattttcg acgtttgact
agccatctca 1440 agcaagtttc gacgtttgac tagccatctc aagcaagttt
aatcaatgat catctcacgc 1500 tgatcattgg atcctactca acaaaaggaa
gggtggtcag aagtacatta aagatttctg 1560 ctccaaattt tcaataaatt
tctgcttgtg cctttaaaaa aaaaaataaa aaaaaaaaaa 1620 tacat 1625 2 386
PRT Human 2 Met Lys Leu Cys Ser Leu Ala Val Leu Val Pro Ile Val Leu
Phe Cys 1 5 10 15 Glu Gln His Val Phe Ala Phe Gln Ser Gly Gln Val
Leu Ala Ala Leu 20 25 30 Pro Arg Thr Ser Arg Gln Val Gln Val Leu
Gln Asn Leu Thr Thr Thr 35 40 45 Tyr Glu Ile Val Leu Trp Gln Pro
Val Thr Ala Asp Leu Ile Val Lys 50 55 60 Lys Lys Gln Val His Phe
Phe Val Asn Ala Ser Asp Val Asp Asn Val 65 70 75 80 Lys Ala His Leu
Asn Val Ser Gly Ile Pro Cys Ser Val Leu Leu Ala 85 90 95 Asp Val
Glu Asp Leu Ile Gln Gln Gln Ile Ser Asn Asp Thr Val Ser 100 105 110
Pro Arg Ala Ser Ala Ser Tyr Tyr Glu Gln Tyr His Ser Leu Asn Glu 115
120 125 Ile Tyr Ser Trp Ile Glu Phe Ile Thr Glu Arg His Pro Asp Met
Leu 130 135 140 Thr Lys Ile His Ile Gly Ser Ser Phe Glu Lys Tyr Pro
Leu Tyr Val 145 150 155 160 Leu Lys Val Ser Gly Lys Glu Gln Ala Ala
Lys Asn Ala Ile Trp Ile 165 170 175 Asp Cys Gly Ile His Ala Arg Glu
Trp Ile Ser Pro Ala Phe Cys Leu 180 185 190 Trp Phe Ile Gly His Asn
Arg Met Trp Arg Lys Asn Arg Ser Phe Tyr 195 200 205 Ala Asn Asn His
Cys Ile Gly Thr Asp Leu Asn Arg Asn Phe Ala Ser 210 215 220 Lys His
Trp Cys Glu Glu Gly Ala Ser Ser Ser Ser Cys Ser Glu Thr 225 230 235
240 Tyr Cys Gly Leu Tyr Pro Glu Ser Glu Pro Glu Val Lys Ala Val Ala
245 250 255 Ser Phe Leu Arg Arg Asn Ile Asn Gln Ile Lys Ala Tyr Ile
Ser Met 260 265 270 His Ser Tyr Ser Gln His Ile Val Phe Pro Tyr Ser
Tyr Thr Arg Ser 275 280 285 Lys Ser Lys Asp His Glu Glu Leu Ser Leu
Val Ala Ser Glu Ala Val 290 295 300 Arg Ala Ile Glu Lys Ile Ser Lys
Asn Thr Arg Tyr Thr His Gly His 305 310 315 320 Gly Ser Glu Thr Leu
Tyr Leu Ala Pro Gly Gly Gly Asp Asp Trp Ile 325 330 335 Tyr Asp Leu
Gly Ile Lys Tyr Ser Phe Thr Ile Glu Leu Arg Asp Thr 340 345 350 Gly
Thr Tyr Gly Phe Leu Leu Pro Glu Arg Tyr Ile Lys Pro Thr Cys 355 360
365 Arg Glu Ala Phe Ala Ala Val Ser Lys Ile Ala Trp His Val Ile Arg
370 375 380 Asn Val 385 3 55827 DNA Human 3 tcgaatatta cattcagcta
aactagtact tgaaagtgaa ggcaaaagaa agttattgtt 60 aaagatacag
agcataaaag attttatcac ctgtagactt ttgctatagg aacttttaaa 120
agattgcttc agcaataaga aatgtaattt aaaatttatt gttttttatg cactctgttt
180 cttttgtatc ctgtttctgt ttccccagag aggaaacagg acataaaata
aagaagaaac 240 acagatacaa aataagtagc acaaaaattg atagaattta
ttagcatatt ttaactattt 300 tgactgttta ttttaaagtt aacttttatg
ttaaaaagat aaggtaaaag ttacttgggt 360 tagtttttct ttctctcctt
cagtgtgatt atgttattca tttgaaacac aggttcgttt 420 ttgtttgtat
tattttttaa aatttatttg tttgcttgtt ttaagtacat atgtgaaaag 480
aacatggttc taaaattcag agtagttcta aagttcagaa ctattcaaaa cacttcaccc
540 aaagaagcgt ccctccctgt ctcttctacc ctgtcttttc cagtgtgttt
ccactcacct 600 cccgtggata accagtctca ttgatttcta atctatcctt
cttatgtttc tttctccaca 660 tatgagcaga cacacacata ttttcttatt
tcttcttctt tcttatacaa caagtggtta 720 cagtggaggt cactttaatt
cattaaatat cattcaatag ttttaaatct caaaaggaaa 780 agtttgaaat
ctcaatcatt ttcttctggc caggcacgat ggctcacgcc tgtattccca 840
gcactttgga aggcagaggc aggtggatct cctgagctca ggagtttgag accatccagg
900 gcaacatggt gcaaccctgt ctctactaaa aatacaaaaa aaattaaccg
ggtgtggtgg 960 ggcacacctc tagtcccagc tacttgggag gctgaggcag
gagaattgct tgagccccag 1020 aggtgaaggt tgcagtgagc caagatcacg
cctctgcact ccagcttggg ctacagagtg 1080 agactctgtc tcaaaaaaaa
aaaaaaaaga aaagaaaaaa gaaaaaaaat cattttcttc 1140 tcagaagtta
attgtgggca ggctgattta ttttgcaaat ttgccaattc tgacttcaag 1200
aacattcaag tgcattaacc aatgggaatg taggggaaga gggctccact cacttacaga
1260 gggtaggata tggcctcata ctagacaaaa tgttatttga tgctactttc
aagatgatag 1320 gggatgggcc tggatttaat tgatggctat tatggtgacc
tttaaataaa tgagattcaa 1380 agtaacctga tgtctttact gcttgaacca
gcttccatga aatagtattc ctattggggg 1440 tgggcctatc attccatatg
gtcaaggaaa catctttttg aacagagatc ctgtaatcat 1500 ccttacaaac
tgcacttcaa cattggattg gattagccag atttgaggaa ctcacttttt 1560
acgtcttcat aaatttaaaa tgttgaaaaa gtcagaggca agggaagaca tttatagtac
1620 ttcacggtag atctccctca acatgggcta tatatccatt agtcaatatt
ctatagctat 1680 tgttctgcaa taaaccagac aagatcctac tgtattacta
cccttttatt cttggcccta 1740 ccttccccaa ggagttacac attttctaga
tagtctaaat taagagcaac tctcatcata 1800 ctctttttga gtgtttaatt
atcaagcaac agcctaacta agccaataat atttctcttt 1860 ttgggagtgg
aaatggaagc taagttgatt gacccacagg aacaagaggg aacatgccgt 1920
tatattttaa ccagtgtgta aagaaggctg ttatgcaatc aatgatctgg gtttttctct
1980 tcagagaaat ttgttgtaca gaaaattgct gttgggatga agctttgcag
ccttgcagtc 2040 cttgtaccca ttgttctctt ctgtgagcag catgtcttcg
cgtttcagag gtaacccaat 2100 agaatcttag actgtggtgg gccactctcc
tcacttgttt gcctcatgtc gtgtcaagtc 2160 agtgcactga gctggtggac
aaaatggtaa actttgaagg ccaggtcttt cagaactttc 2220 caagttgccc
tgacaaataa gtagacttta gcacaatggg ctatcactaa agacagggtc 2280
ttttttcttt cctggctctg gttttattat tgggagaacc ttggatgata cgcatatcca
2340 gtgactatgg agattcaaga aattaaatct tttataaacg taactattta
tactctaact 2400 tgatgtatga ttcatattct tcctgtcttc acataaaaaa
agttaactat ggatcattta 2460 ttttcccctt gtacatggaa cataggagga
agaagagggt gaagtgttaa ataggaggtt 2520 tggatcatgc atgattattt
agcatggaat atgaaaggaa gaagagttgt gtgataaaga 2580 actattatct
gattcttatt ttgcttagta gattccctta ggataaacta tctagaagaa 2640
cacaaatgaa ttcatgctat agcacatgca atgcatggag aaaatagttc cagggtatat
2700 gtaatgtaat ttattaagta gtcaattttt aggctttaaa acattgatat
tgtttccttt 2760 ggaattatct tattttttcc cctttgtttt ggttctatga
tcgctttctc ctccaattat 2820 ctttgagaca gatccctctc ctcatgttag
taaatgacaa agaaagaaga gacatagggc 2880 aaaggaatat accagtgaca
aggaacattc taccaccaaa aaaatgttca cggtcataaa 2940 taaccatagg
acaatggttt ggaaaataga tcttgacttg tgagcctgaa gctgtgtttg 3000
tacatgatca ctgaactgat tatagttgat tgatcttctt ttgttcaaca tgattgtcga
3060 atgtcgagca acaaattcta tcataaaatg atattatttt tgttatttaa
ttgacgtggg 3120 ggtcaagatt gctgcaatga tcagtgactt atgtctttct
ctgtatttta tcggtgaatc 3180 atatggtcag gatttctaag gttcttgcta
gttctaatat tccataactt gataattggc 3240 ttcagttaag ggaaaggggg
agaagagaaa aattggtatc aacatgtcca acttggctac 3300 tgtacacagt
ggcagtacca ttgacagtta ggggaaaggg aggaaacctc tgcttattta 3360
gtgcctgtgt ttgtgccagg cactgaacta gtcacttaga aatgttatct ctttaaatgc
3420 ataaaatcct acatgctagg aatctttact gacattttac aaaggaggaa
actgagcctc 3480 aggaagaata aataattggc ccaagatcaa acagtaaatg
tagagcttgg attcaaaccc 3540 actttagcct catttcaact ccatgcactg
gacagcattg cctccataaa atctggaaat 3600 taggaagaga gccagtttga
aggaaggtca gatttagtca aagggagttg caggcagcag 3660 ttggtttgga
aagtagcttg gaagagaggt tcgggattag aggttcagtc tcatggttct 3720
cacccactag cagatctaat catggccttg gcgtcagccc agtgcaatta tcctcagctg
3780 gttgttgcag aggttggcgg gcaggtgggc tcactgcaga ccgccatctt
gatcgtagag 3840 taacccaaac tcttggatag gataatcaat agcaaaacac
actaaaagct ttagcacatc 3900 tcttcaaatg agtacgtgta tagcagctta
gtgacactaa atataacgca aatagaagaa 3960 gtagccaaca ataaaatagt
aaaaaaatga gtgagaacat atcttcatgc atgggctttg 4020 ttactatttg
ttgcttcagc ttatactctg aaatctgact gatacttatg cttgaaaaaa 4080
ggaatgagaa tgtgactata ttttaaccaa agaatatcac attaaaaata tttaatactt
4140 ttgcatactg cgagggtccc tttgcagagg agaggaggta ggaggacctc
agtattgtag 4200 acagatgaat atctgaatcc tggttcccat cccttcactg
gaaataacat tgcaaactac 4260 tctttctgtg agtaaaaata aattttttta
ccaaatgttt ctgtgctcca cttttccagg 4320 aatggcctat tcctgaagct
aaaaaggaaa tctaatttca ttcagggcaa cagactttga 4380 taaattgttg
ctggggttca gaatatcaac ccttctaaaa aaaaaaaaaa aaaactaaca 4440
gtctggcttt ttcttaaagc tgttctttgt tttttttttt ttttttttgt cataatcatt
4500 ttcctactaa cagtttttat tcatgcagtc tcttagtggc tgatttgtag
gttcattttg 4560 ataaatttca tcagtgaaat gccctggaac aacaacaagt
tttaaaggca taaatatcat 4620 atgccaaagg gaaaggcagc caaaaaatca
tgactccata ttcatttgct tttaaaagcc 4680 aaacactata aagggtaaaa
ataaaatact agcaagaatc ttgtaaacag aatcagtaat 4740 tgtattgtgc
agtgattacc taaatgcagc ctgccagccc agactatttg gaaagaggaa 4800
gtaagagaca ctaggaagaa gacttaggaa ttagagagtg gaggagggtt gaggataaag
4860 ggcttctgaa ttattaatag accacaggaa gtgttcctct gttgacttca
catactgttt 4920 gggtacctgg agaccagttt actctctttc actttgttcc
tactgatgta ttgttttcat 4980 ctcaaagaac aggccaccag tggccttaaa
acactgtaat gtgtgcaaca aaattgcagc 5040 cttgggctat gttccattgt
tcagagacat cttgccagct ttttaaattc aaaataatct 5100 ttcagaatgg
tgaaagtgtg aaccctcccc tgtaaaccat agcaggggat acaccccaat 5160
gaacataatg acattctcag aagggaagga acagaggaag tgttgcatag gtattaaaag
5220 ctcaggatct ggattcgagc cccagatctg ctacttatca cccatgcaga
cttgggcaat 5280 ttgctcgtcc cctttcagcc tttacttttt ttgtaaagtg
acctgttact tcactgtgct 5340 tgtacttctc attcgatttt tggtgcaagg
ctgttctttt ttctcaagtg gttattgtgt 5400 aagtgctata atcgtatcat
tcagagacgc agttgaaaca cagctttagt ttttgtctcc 5460 cattgcccca
tgacattttg cgtagtgggg ttatctatca ctgctctcgc atggaaagtt 5520
agaaaatttc aaggcttttt agcctgcttt taagtgacag tccttgggtc ctgctaaaaa
5580 tacaaatagc ctcaatttag aaattagaat gtcacctcca accaaggtat
tgttcaaata 5640 tccccatctt tgttgttaaa agaaaatctt taaaagaatt
atatttagca aaatttaatt 5700 gaacaaagaa caattttcta atcaagtaac
cctcaaaaac gaaagaagtt cagagagttc 5760 tgctcagcaa agtgggcagg
cagcacttat aaacagcaaa tggaaatgag gtccagaagc 5820 agcttgagta
gttacaggtg agcagttgtc ttactgggca taggctgatc agttggccac 5880
atgggattgg ctgtagcttg gctgctgtga ttggctgaga ctcacctcgt tagtacaaaa
5940 aaaaaatact cctaagttag gttgcagttt gttatgtagc gactcaagtt
acgaggcatc 6000 ctcagaccaa atttagttta atttaacatt atttatagga
aaacaactgc ctcacctctt 6060 ccacaaacac accttactct ttttcttgtt
agtctttttc tcgagttcta acttcttaga 6120 gttgtgtgag acatctttat
tggggaagcc tctggaccag gacagatgct tctttgtcta 6180 ggttttcact
tgcgactcca tccttccccg ctaagagtct tgcttctacc tctgggctct 6240
tgttgttgag aactttccat ccctttaggt ggccctattg gatggcatct aacattaagt
6300 gtttcttttc attttaacta ctactatcta gccaactaga gaccagccac
atgcaggttt 6360 agctttatca ggagaagcca ggcaccagtc tttgtgtctg
taaatttgag gaaacatcca 6420 actctctcat tatctcctgg aagtccccct
actaggctga ggtaagggga gtgcaccccg 6480 aaacttcatc cctttgggag
ggtggtgact tacagaacca taaaaacatg ctaaaaaaaa 6540 aattcacaaa
tcctctccct ctttccactc tgacagcttt ttatatagcc tgtttatgac 6600
taagtaaggg gaagcagtca tgaaaccagt ttccaaaaat agagtgatct gactgaccct
6660 catcccatta cctaactctg ttgtgttagc actttgctca aattctgcat
aagaagagtc 6720 tgttcactac aagctgaact tggacatatc aataaatttt
tggtgaattt ttaacttcat 6780 aattttactc actatttcct aacttatttt
ttgaatttcc tttatttttt cttcttaaga 6840 ggtctcattt ggataacata
catttttacc tttatatttt ctttctttct ctgcttgttg 6900 actaattttt
atacttttct ccttctttaa tacattaggt ttttttttaa tttaatactg 6960
cccactcaac attttttgtt cattattttc tttctttctt ttgagaccta gtcatgctct
7020 cttacccagg ctggagtcca gtggtgtgat tttggctcac tgtaacctcc
acctcgtggg 7080 ttcaagtgat tctcgtgctt cagccttctg agtagctggg
actacaggtg tgagctacta 7140 tgccacgctt atttttgtat ttttagtaaa
gacagggttt caccatgttg gtcaggctgg 7200 tcttgaactc ctgacctcag
gtgatccacc cacctcggcc tcccaaagtg ctgggattac 7260 aggcgtgagc
cactgtgcct ggcccattat tttcaatata atagattatc taccatactg 7320
ccttgtgagg attaaataag aatacctgta aagcacttag cacaatatcc aagttactaa
7380 atatcagtaa aaaagaagaa aagtcccccc agacatatta tgctctagtc
aacacaagac 7440 ttcctctaca tggacttgaa attcagcatc tctttagata
atgaagagct cattgcttga 7500 taaggtgtcc tatctcatgg ttagctcaaa
ttgttagaag ttcacactga aattacagtg 7560 atttaatgat atgaacctcc
acttctctat actttacatg aaaaggaagc tttgagtttg 7620 ccacatcttt
tgctacaact cccaaaatca tgcccaacca acttttaagt aagggccaca 7680
atcttgaccc cagcatttaa gacccttaac aatcaggtcc taccctgcca ttcgtcctgg
7740 ctttatttcc tggtatatct ctatataggc cccatatttc tgcccagctg
gatcacttct 7800 ccttccttga gctctgattt tacttttcta cttgtgcacc
tgcatttatg atgtttcatc 7860 tccaattcgt ttagcaaaat tctgcctatg
ttagtcttat accatctcat cttcccttca 7920 cctattgaat cctagtatct
cagaagtcca actcagaata tctccattct ctgactacgt 7980 aagtcaaaaa
tgatacctga ctttgtattc ctgtagcaga atatttatac cactcgcatt 8040
gtacttttag tgttttatct cacagaatag gcatttgtat cagttgcagg ttagtttccc
8100 caggaaacag actcctgaga tgaagattgc atggaggaag tttactggaa
aggaatctca 8160 ggatcagcat ctgtggagga atgaaggaag aaggcttggg
cagaggagaa actgacctgt 8220 gatgtaatca caactatggc ctcacctgtt
cctgtggaga gccttgaggc tgggttggcc 8280 tggtaaagtt gtcccaaact
ggggcaagca ggcatgcctt tgtaacccct gtttattagt 8340 cactgggtgt
ggattgtacc ttggaggagg catcatgttg ggcagcacag ctctcttcag 8400
gcaagggcaa gtcctagaaa gggactcagg tgagaatatc agctgccaac cctcccagaa
8460 gctgaggaga caaagaagct gaggaaataa gagttcatcc ctggatggag
atctaggcag 8520 caaaacgtga catctactac agtccaaccc tttgtgtcac
tcaggtcaat tttcttcata 8580 aaataagctt tgagatcaac tcttctgggg
ttttgcttgg tccctcttcc taaaggaaat 8640 atacaagagg aacgttagtg
aaataaacta caaccttcac agctacagct agtctcgagg 8700 ccataaccaa
tacttgtcat tcctcttctc ccctacccgt tctgttttcc ccaaagaggt 8760
gccctttgct agcacctctt ctaatctagg tggtttacct ggtgagacaa cccagaccct
8820 caatcctcaa ggatctgagt cataatcact gggccctctt gggccatggc
tgctgcaatt 8880 gtctgtatac atcaaatttg gacaagggaa tattacagaa
cgcctaagct ggattccaaa 8940 catattcttc cctgccacat tcagtatgta
agtgcagccc tcaatgtcct tctgattatt 9000 ggagtcaatt acccttcctg
catggctaga aaacccatgg taactcctcg ccttcctgat 9060 aatcaatcag
catgaggaaa ctgaaatgac tggacagtaa caacagcttt cagtttaaag 9120
gaactcttcc tatgctctct ggaaaccgga atttttataa gtacagagtc catatttgta
9180 gatttaaagt actaattctc cgagtgggtt acttggagtg acggtatatg
gaaacactcc 9240 aacttggttc ctggaaccat gtatactacc tagtagggac
acagcaccac atgagtattg 9300 atttaaagtg tgcactggag gatgttgcct
tccaagttag cacctcaatt gatccttcaa 9360 caaaccattt ttatttcagt
aacaggatag cagcaactag gtattctggt atgtgagagg 9420 ctaagtggat
tccatggtaa tggacccatt tctgcaattc ccttgttgta aagtggagcc 9480
catgatctga taggatgtta tgtgagatgt tttatcagtg gtcaaacact ccgtaagccc
9540 ttgtatggtg atgcttgcct gaggccctac aggcaggcga gataaaccca
tacccagagt 9600 ggaagaggac agatgtagtc aacttaccac ccagtgacta
gctggtctct atgaggaatg 9660 gtgctgattc cggggctcaa ctttggtctc
cgttgctggc aagttagaca tggggcagca 9720 acagtagcta gatcagcctt
aatgagagga gtccgtgctg ctggagccat gcacacttca 9780 tctctgccat
caggctattc tgttctagtg ccttttgtgc cagcattggg gtggctgttg 9840
acacaggctg actgacatca actggctgag tcatttgttt atgcagctgt ttaatgtcta
9900 ttctgttgtg ggtgctcctg gttaggcatt aataaatgat acaaagatct
tcacactttc 9960 tgcccactct catagttcca ttcacatccc cctttctcca
atctctttgt ctccaatctg 10020 tcaagatttc ttcttccagg ttcttgaggg
gttttccagt catgtcactt gccactctcc 10080 atgaattcct gcatattcta
acactggaaa caccttttcc acccaaggtg tatgatgaga 10140 tgcaacactg
aagctctgcc tatttgggac gatttccctc tctgctctct ttcggtcacc 10200
cgagtgagtc cataatgcag caccacttca cttttttttt tcttttttga gatggagtct
10260 ccctctgtcg cccaggctag agtgcagtgg tgcgatatta gctcactaca
acctctgcct 10320 cctgggttca agcaattctc ctgcctcagc ctcccaagta
gctggcatta caggtgcccg 10380 ccaccatgcc cagctaattt tttgtatttt
tagtagagat gggatttcac catattggcc 10440 aggctcgtct cgaactcctg
acctcaggtg atccacccac ctcggcctcc caaagtgctg 10500 ggattacaag
cgtgagccac cgtgcccagc ccacttcatt ttttacttgt acccatgcac 10560
tatcaacttg actgtccatg tatcagactt cctcttccta cttgtgtcag ttatgatcct
10620 ctgagaagca gacaccaaga tgggattagc tgtgcaagag gtgtgttgag
gaaaatgctt 10680 gggtgcaaga aatggggaga gggctggagg aggctgggag
agccatgaga ctgcaatgca 10740 agcttaaccc ctgtggagga gagaggaaag
gaaggaagca ggtagggaac atttcaggtc 10800 gtagtgcagt tctaagaaag
ttttggcaaa accaaccaag agtcctggcc aggcacggtg 10860 gctcacacct
gtaatcccag cactttgaga ggccgaggtg tgtggattac ctgaggtcag 10920
gagttcgaga ccagccttac caatatggtg aatccccgtt
tttactaaaa atacaaaaat 10980 tagccaggtg tggtggtgcg tgcctgtaat
cccagttact cgagaggctg aggcaggaga 11040 atcacttgaa cccacgaggc
agaattgtag tgagccaaga ttgctccatt gcactccagc 11100 ctgggtgaca
agagcaaaac tctgtctcaa aaaaaaaaaa aaaaaaaaaa agtcctatag 11160
gcagagtcac acatcagaag agtttccagt tttgtagaaa gagcctccct tagtatcccc
11220 accatactca gttattagct gcaagaagcc agtgggaaat gtggtattag
cactaacaca 11280 gggacagatt tcagagcaca gtagctgggg gcttatatca
agtacacatc ttgcagctgg 11340 agagtgagaa agttaattaa agctgaggca
agactgtaaa tatgcactgg tgtctgtccc 11400 aagtggatgt taactgttct
gatgcttttt ccgactgaca tatccagcgc aatagctgaa 11460 taccatatgc
ctgagactct accccggcaa agatgccaca tcaagcacta tggctgcaat 11520
tgagattgtt gcttggttga gtttgattgt ttgctgtcgt tttccaggat ccatctggtt
11580 tttgtgggat ccagatggca aattaaatgt ggttttgatg ggatctatca
tccctgcatc 11640 ttttaggtct ttaagggtgg tactgatatt tgtcatttcc
cctcaaggat gaattttttt 11700 tttttaattt tgatatcttg gctgggaggt
tgggcaattt cagaggtttc cttttggctt 11760 ttcccactat gatagctctg
gtcttacagt caaggaaaca atgtggaggt tctgccaact 11820 acctagtatg
ttcatgtcaa ttatacattt ggtgaccagg gaaataatga tgggggaatc 11880
cattaacatg gtgcacccgc tatgagctag tcttaggcta gggctccaga tacccaagtt
11940 tcaaaatcaa cttggatagt gaccctgcat ccaacacacc tgaaaatatt
tgagtattac 12000 cctttcccca gggtgcagac ttacctgagg aaatttccat
aggtctcttt gggaaaggac 12060 tgaaggagtc atgatctttt tagattcttt
ttttatacag ttgcagggtc tttccttgtg 12120 gggacctgac tcctccttca
ggcaagaaat tctgggtcta agaacagctc agatctggaa 12180 aagggcaatg
gattatgact tttgattagg atagctgtcc tcagcctctc tcattatcca 12240
gctttgattt atttttattg taaagattga gcaatccttt tgttggctgc ttctctatct
12300 tgcccctaag aactctgtgt tctcctaacc gactccacaa ttttctaagg
gtcatggtcc 12360 tctggctgcc actccgacct tactgctcat tgtaataact
gtgcccaact tgttactggt 12420 ggttaagccc tcccgcctgg cttctctata
cagggatctt aacatctcca ttggtatcag 12480 agagctcagt tctgtaatgg
catgtcctgt agttagccct caggatctat tcccacctcc 12540 cctcctggcc
ttcaagccaa taacctaggg tagtcacaac ataagctggc tgtggaagtg 12600
ctgggcatgt taacaaagga aagggactat accccaaagg aggtgcactc acccatgcta
12660 cagaatttaa cattctagtt agttccttga gaggtggtac aaacatgctg
ttagggtagt 12720 ttttggaagc ttggagaaag catggcctgc accatctgag
ctagaaatgt cagagcgaat 12780 gtggcagatg gtgaaaaaag agatagagca
cactagaata aaaatgctat gtaaggctag 12840 aaaacccact gggtgactat
gctactccaa gggcccagag aacattctgt ttatgaaaat 12900 aataaggaat
gtgctggtga gatgagaggg ttaccagcat cactgagaag cttgttagtg 12960
gctttttcac agtcaaagga gatttttctt taaatttaat tttcttgaat aaatacaaga
13020 ataggagttc ttttgggttt aaaaagtaaa caatacagaa aagcataaaa
ttagggagaa 13080 aatactaaaa tttcaccatc ctggtgaaaa tatgaacatg
tttgtgatca tcctttcata 13140 catttctcca catagttata cctccctggg
tataattgta tactagttca atgttgtatc 13200 tcctattggt actatagaaa
cctttctttt aaaaagaatc tcatttgttc ttcccctgcc 13260 acttacccaa
ggcttcaatt gccatttccc accctccaaa tcaaagctaa caatgtgttg 13320
tttatttatg tataattttc tcctgatttt aacacatata atttttcttt atctttctaa
13380 cttttttcca aaaataggat catatttcat aaagttctcc atatcttgct
tttctccctt 13440 aatatgccat ttaaagcccc caagttaact gttataaatc
aaactcattt tttataatga 13500 ctgaaaagca ttctagaatg tggagacact
accaacattc gacaattctg ttactgctga 13560 gcattcacat ggtttttagg
ttttgttact atgaataatg ccgtaataca catccttgaa 13620 catgtatctt
taatcagtgg tttaatagtt tatgctaaac ttgtaccaga gattgacata 13680
aaatttctca gtctagctac ttttcccctc ttctaataag caagtctctc catagactta
13740 tttccagaat tcagaatatt ttactcagga tttccaaaat aaagccaccc
tccacccttg 13800 ttaaagttat ccttggtggg cgcggtggct cacgcctgta
atcccagcat tttgggaggc 13860 tgaggcgggt ggattgtctg aggtcaggag
ttcaagacca gcctgaccaa catggtgaaa 13920 ccccatctct actaaaaata
caaaaattag ccgggcatgg tggtgcacgc ctgtaatccc 13980 agctactcag
gaggctaagg caggagaatc gcttgaaccc agaaggtgga ggttgcagtg 14040
agccaagatc acgccattgc actccagcct gggtgacaga gcgagactcc atctcaaaaa
14100 caaacaaaca aacaaaaaca aacaaaggaa aacaaataaa attatcccta
taaatcacag 14160 ctcaaatgtt acctttctaa cttctaattg cctacaagat
aaagtccaaa tttctcagca 14220 tgcattcaag accttctcta gggaaggatg
aacataactt cccacactca tttctgttta 14280 gctcccattc ttctcttgct
ttaaacaccc gtatcctata cttggcaaca atgaacaaga 14340 gccatttttc
caaaaatgcc ctttatctct tgctattgtg cctttaccca ctcttagaca 14400
ttcttacaca cccagacatc ccttctatga agcctttgct aataatgaca aacagaagtt
14460 atcataacct cttttgtgct ttgagagctc ttggtacatg gttttcttaa
ataagatgat 14520 ttatttggaa tatttttaga tttacagaaa agttgccaat
tgtaatacaa ctgtataccc 14580 ctcacccaat ataccctaat gttaacattt
tatattatca tgatgtatta gtaaaaactg 14640 agaaatcaac attgttatat
tactattaac taaactccag acttttttgg atttcaccag 14700 ttttcccact
aaagctcttt ttctgttccg agatctaatc cagaacacca tgttgcattt 14760
agtcataatg ttgctgttgt ctaatgtctt gagtgctgtt gttccataca ttttgtccag
14820 gtttttagtt atttcataaa ggagggtata actggaagca aagtctaaga
ttagttttaa 14880 ataaagcaag aggaagcatt ttttctaatt taaaatatat
ctatcgtcat atttcaaggg 14940 caatatttgt ttgaaaataa aagaaaaatc
tcgttcagtt aaaaaaaagg gggggctcag 15000 agctggcaaa tgccaccaac
atgcttaatt ttaatttaaa taaaatagtt cttgtgaggt 15060 tactcagtgg
tatactggaa acctgagaat gccattgccg ttaacagagt ccacaatccc 15120
tcacctcact gcttcctttc tctccttatc acttacctat aaaactggat ggagagctgc
15180 agaaatgagg acatttgcta agaaattctt tcttttctaa gtggtatgtg
aaaataaagt 15240 aaattcatgt tgagtcacat taatctattg ccttggctgt
gtaagaatca ccaagaattc 15300 tcacaacctt agcaacagtt gcaaaataga
aatacaacaa agcaaaggtg agaaaaccaa 15360 ccaagtgtct gctttttaaa
caatctattg atataattca ccacattaag atattaagcc 15420 agaaaaccca
tatgctcatc ttatagaaag catttaaaat ccacacttat tcatcataaa 15480
aactcttaac aaagagcaag gagtttttta aaactgataa aagacatcta ccaaaaatct
15540 acaacaagca ttctaatggt aaaatatttt aaggtttttc ttaaaaatca
ggaatcatgc 15600 attttatctc cacttctatt caatgttgta ctgaagtccc
aggcaacaca gcaagacaaa 15660 aaggaagggg aaaaggggct ctataagcat
tgaaattaaa gaagcagaac gtattaaaag 15720 tacattaagt acattaaaag
taacggcaaa aaccgcaatt acttttgcac caacctaata 15780 gtgtgtgcag
atgtaatgat tacttgcaaa gagaaatatc cccccaaata tctataccaa 15840
aattatcaaa actaccaaga gagctaaata gaaaatcaac accaaaaatc atttttattt
15900 ctatatctta gcaaaaaaga gcttagaggt ggcatgttaa aagttaccat
ttactaacga 15960 aaaggcaaat ttgttagaag aaaacataat ttaaaaatgt
gcagccgggc acggtggctc 16020 acgcctgtaa tcccagcact ttgggaggct
gaggcaggag gatcacgagg tcaggagttc 16080 gagaccagcc tgaccaacat
ggtgaaaccc agtctctact aaaaatacaa aaaattagcc 16140 aggtgtgttg
gtgtgcacct gtaatcccag ctactcagga ggcttgaggc aggaaaatcg 16200
cttgaaccag ggacgtggag gttgcagtga gccgagatgg cgccactgca ctccagtctg
16260 ggcaaaagag cgagactccg tctcaaaaag aaaaaaaaaa aagtgcaaca
tctttatgga 16320 taaaattgta aaacttttgg aaaggcatta aagaatagat
aaatgggctg tgtgcagtgg 16380 ctcacacctg taatcccagc actttgggag
gctgaggcgg gtggatcacg gggtcaggag 16440 ttcgagacca gcctgactaa
catggtgaaa ccacgtctct actaaaaaat acaaaaatta 16500 gccagccatg
gtggtgtgca cctgtaatcc cagctactca ggaggccgag tcaggagaat 16560
tgcttgaacc tgggaggcgg aggttgccct gagccaagat cgctccattg cactcctgcc
16620 tgggtgacag agtgagactc catctccaaa aaaaaaaaaa aaaaagaata
gacaaataga 16680 caaattcact gtatttatta ataatgacac tcagaatcgt
gagtatatct gttctttcca 16740 aattattaat ctattgatcc aatataattc
taatgaaaat ttcatttttt tcatgaaaca 16800 taacaagctg atttttaaaa
attatgtgaa aaagcaaagg atcaagacaa gaggcttgta 16860 aaaaaaaaag
aattgggcag ggcagagggg aagcaagagt ttgttctcta agatattagg 16920
atgtaatatg aagctaccat cactaagatg agtagtattg gctcaagggt agacaaatat
16980 atcaatagaa cataatagag aactaagaaa tagagcacat tatattagca
agggtaatcc 17040 ttgattatgc tataatcact aataaaacct gaaacagctt
tacttaatac aatatagggt 17100 taattctgtc ttagtacgtt tgggcagtta
taacaaaaaa taccttaagt ggtgtagctt 17160 ataaacaaca gaaatttatt
ctcacggttc tggaggctgg gaagttcatg acaaagcacc 17220 agctaattct
tgtgtttggg gagggactat cttccgcata gacagaacct tcccgctata 17280
tattcacatg gtggaagggg gaggggatct cttttttaag gtcactaatc ccattcatga
17340 agcctctccc cttatgacct aatcacctcc caaaaccctc atctcctaat
accttggagg 17400 ttaggatttc aacataggaa ttttgggggg acacaaacat
tcagatcaga gcaatttctc 17460 actcataata ctaattgatg aaggtcatag
aactctccct ggtgctctcc ttcaacttgg 17520 agatcttggc tgcttccatt
atgcaactcc acatttgagg ctctttgctt ctggccgcag 17580 gatgagaggg
ggcacgtgca catgaagaca cacctactct taggcaccta taacactccc 17640
acgcattccc attggcaaag ctcaggcact ggctctcaac acaaccacac ggaaggctgg
17700 gtaatgaagt ttttctgtct atgcaggaag aggcagtggt gttgatgaac
caacattttc 17760 tctgccaaac agtatggaaa cttgatatat gacaaaattg
acatagtgga tccctgggaa 17820 aaatatagat gatgtaaaaa atcataataa
taaacgatgc taagaagaaa aaaaaagaag 17880 tctatttctt ctttacactg
cacccaacca aataattttc aattgaattt aaaatttaaa 17940 taagaaggac
agaagaagta tatgagaata tctttatatc ccagatatct agaaagacat 18000
cttaaaccac acaaacctgg aaggaaatga ttaaaaaaaa aaaagcacat catcaagaaa
18060 gagaaaagac aaattacaaa ctgctagaag atatttgcaa tacatataac
tgacaagaaa 18120 ttagtattta gaatatataa agaaattata caaattaact
actacaaaaa tacaagtaaa 18180 ttagaaaaaa atgggcagtt gatctgaata
aacataacat ataagcaaaa atatgaatgg 18240 ccaataatca aatataaaca
actgtacttc attaataagt cagaaaatga aaatcaaaat 18300 cacaagaaaa
tttcatttca taaaaatttg attgcaaaag ttgaaaagtc agagagcatc 18360
aggtattggc aaggaggaag aacaacagga aatcttttcc actgctactg acaatataaa
18420 ttgatacaaa aaacttgagg actgatatga cactatccta tgaaattaaa
actgtgcata 18480 tcccatgaaa tggcaattcc acttgtaaga aaatgctttc
atgtgtgaac tgtggctagc 18540 tacttcaaca cagatgataa tcttgagaat
acaatactga acaaaagaaa aacaagattc 18600 agaagaatac atatagtata
ccatttttat aaagttgaat caggcaaatc taagggtatt 18660 gtttcagaat
tcacacatac atgtgtttaa aaatccatgc tataaagaaa aacaagggaa 18720
tgagcaaaag tcaaaattta aggtagagga tacctctggt gatgtggcaa gggggtagga
18780 cagagaggag cacacaagga tcttcaggat gtcaaggaag ctggactttt
taagtggggt 18840 gatgggttca caggcattca ttttattgta taattaacta
ggatcagcat aaatattccc 18900 ttatgcatca aatatttaat ttttaaaatt
aaaacacaca tgcacgcaca agaaaaagga 18960 aagaagtaaa tactctgtaa
actgaccccc agtcaagaga gctgttgatt ttgcaattgc 19020 ttaggagcat
aaagactgag agtatatgtt ctcttattac actgaatctg tagtaagatc 19080
ctctgtccta ataacatttt aaattttgtt tcccttttgc aattacctaa aagctcctca
19140 cagtataata tattccatct ttactcttta tttaatatca aaatcctctt
ttattttttt 19200 ccccagtggc caagttctag ctgctcttcc tagaacctct
aggcaagttc aagttctaca 19260 gaatcttact acaacatatg aggtaatttc
tccctaattt atgtttatat tggtttcact 19320 ttgtataagc actgggtgtt
gagtttcctc ctgtatgttg tctggcttac atgtatctgg 19380 tatgaactct
tcttctcata gtcttctctc ccttctcata atcacatgat tttgttggtt 19440
ccccaaatca acacttcttc acttgtgcta ttggctttcc agccaatttc ataatagtac
19500 cttgggatat aaagtgtgca cttacaaaga ggctacagta acagaaatta
aaatatttat 19560 aaataaaacc ttactcatga aacaatggtt cttaaccaag
gatgcaccag aaagacagag 19620 tacatttatt aaaattctca cccaggcacc
catctcgaca taatgtctaa gatgtagaaa 19680 attgacaaga attacagaat
attaatggca gtggcggccc atctagagcg gctgctgcca 19740 tgcgggaggc
acggctgggg ctgtgtgctc cacggagcca gcagaagcca ggaggaggta 19800
aaagtcccgc ccccttctgt gatggcaggg cggcagcctc atgctcccca ggcgcagctg
19860 cagctgcccg ctgcagctac agacctggac atccctgtgc tcttgggggc
caggagcagg 19920 caggagccct gccctcctgg gcgcagctgc agctgcccag
ctggggttgc agacccaggc 19980 atttctgcac tctcagtgtt ctgagaagga
ccctcattcc cctacaggct cggaagtgcc 20040 tgctcccact gtctggtctc
tccgagttcc tggtgctcac tccaatcttg gagcaaaatt 20100 gaggctgagc
ctaggtgttg tcacaacctg gctggctgtg tgcatgatca gagcggtact 20160
gacatgctag ccccctgctg cctcagcccc ctctggactt tgggtactga cgagcacagg
20220 agggaagcca agggggtggc tgagggcttc tcggcactgg cctgcaggcc
ccctcagctg 20280 gaaaatcctg ggtgccataa atagccgtag gaggcagaca
ggatcctagg cagaaaaggg 20340 cgggtccctg gtgaagcccc accttcaagc
ccaggaaggc tgccagtccc gtggaccgca 20400 gtgggaactg acggtgattt
ttccgcaccc gcctatggcc acccatgaac caatcagcac 20460 tcacttcctc
ccatctgaag cccatagaaa tcccccggat tcagccagac tcttctggag 20520
agacatgggg aggaccagct gtagagagga gccacccact ccagggtctc ctctctgctg
20580 agaactaaca ctcatcagga caccctggct gcagagagga gctacccacc
gcgagtctcc 20640 tctgagctgt tctattactc agtaaagctc ctcttcacct
tgctcaccct ccgcttgtcc 20700 acgtacgtca ttgttcccgg gcgctgaacc
tgccaaatgg tggaggtgaa agagctctaa 20760 cacaaacagg gctgaaacac
gccccttgct tgccacgttg tgggtgacaa gaaggagaga 20820 agagctgcag
ccttttgggg agctcagacc taagaactcc ccgaggcagg actatgacac 20880
cctctttagg gctctgtggt tcctgacgtc tccaaacttc tgggtgccac caccttcccc
20940 ggtgccagcc attgaagctg cttgagggac acctggtcca gccacagcct
tgcagggagc 21000 cgaaagatgc ccaccctgcc gcagccagca tgcctcgctg
tgtgtagtag ctggacccca 21060 cacctgctca ctcacacacc cctcaccgct
cagctcgccc ttggcacgaa tgagacccaa 21120 gccagtagca cgagatgagt
gcagcctgcc aggccgagtg ggctcagcgg gcctgagcaa 21180 agcttaggca
aaggcgccac tgaccacaga ggtttctgct ggtgaagcga ccccagggat 21240
cctgtaacaa tatcatggta caaaattgat ggctccttgt ttgttagtgt tttccaagaa
21300 gcagaggtca agactagact ggaggagcaa gcgatgaggg gaatgctgtt
ggaggataga 21360 ggcgggagct ggagaaggca gagagcatca tcagaccatg
tcatagctct gacacctctg 21420 cagaaagggg aattttgttt ggagaatctc
agactatagg gcaggtccaa agaaggctgg 21480 gctaggtcag tctcagtctg
gcaagaatgg gcctgcatta acacttccac aggactcggt 21540 tactggctgg
atgcagccct gagaccacat ggcctcagct tctagtgggt caccagggca 21600
gccactgaaa acaccagcca actgtatttc tctcaaccga agagctaaat ggtgcatatt
21660 cacgaccacc acatcatggt aaagaggaaa tactacaaga ggaagcatct
gagatttaga 21720 attctagttc ttgttctgtc atttctaggt gtatgatttt
agatgtcagg tatgaacctt 21780 aatttcttca cctgaacaat gcaaataata
acacctgcct agtctatatc aaagcgttat 21840 aaatatcaaa ggaaatgagt
gtgaaagtgc tttgaaaaag tacgtgtagt ggctcgtgcc 21900 tgtaatccca
gcactttgga gggccgaggt gggcggatca cgaggtcagg agatcgagac 21960
catcctggct aacacggtga aaccccgtct ctactaaaaa tacaaaaaat tagccgggcg
22020 tggtggcggg cgcctgtagt cccagctacc cgggaggctg aggcaggaga
atggcgagaa 22080 ctcgggaggc agagcttgca gtgagccaag atcgcgccac
tgcactccag cctgggagac 22140 agccagactc cgtctcaaaa aaaaaataaa
aaataaaaaa taaataaata aataaataaa 22200 aagcacatta agagagaaaa
aatgtaaatc ttattggaag cctttttaaa aaaaggaaca 22260 atgacatgat
gataattaca agaacatgaa atttttatta aataaaatca atgtttaatc 22320
aactttcttt ctagaaaaaa ttttgtttcc tttcaaatat ctgatgtaca catgcaattt
22380 tacagttaag ccatgaatat agtcattcat tcatcattgt ctcatcaaat
atttatggat 22440 tatcttgtat attccaggcc cttttatttt attttttttt
agcaactaga gttatagaaa 22500 ggaattttaa aaaactcact gcaaaataaa
tgtttatatt accatgtgtg tggatgggga 22560 ccagcaccag ggagtgtcct
tttcatactc cttatagata aaactgtcat ggctctagct 22620 acagatgaga
atgatgtgaa caactctttt ttaattttat caattttgcc ccttaaactg 22680
tagattgttc tctggcagcc ggtaacagct gaccttattg tgaagaaaaa acaagtccat
22740 ttttttgtaa atgcatctga tgtcgacaat gtgaaagccc atttaaatgt
gagcggaatt 22800 ccatgcaggt aggcaccgtt caatacgtat tgagtagtta
ttataaacac ttactatgca 22860 cttgactagg gtatggtata attgcttcct
ggaaaaataa aatgtattaa ccatggcagc 22920 atagaagtct ctgactggac
caaatggact ggtgataaag cctaaggtcc agctctgtga 22980 tcttggataa
atggttcaac ccctcatgac ctccgtccct tatctaaaat gcaggttaga 23040
ctcagtgatt ggtaaaggct ctcatagttc ctttttctct gactctgtac ccagactcag
23100 ggagcaaaac tgtcatttgc cttggtaggc tttttgatat ctcctgaaaa
agcagcttcg 23160 ggaggggatt tagcttctgc taattcttct tcacaaagac
agtgaccatt tctgaatgtc 23220 tggctttaaa aagtgtaaca ggtggttgga
ctctgcagag acctcgggtt agtctggcac 23280 tgccccttac cacctatatg
accctggggg aattattcac ctctctgctc ccaagttttg 23340 tatattaagg
gtaaaaacag cacctaccct gtggattaga aatgatttcc ttttcttaaa 23400
aagtgtatca ggtacaattt ctgctcacag tctagccttc ttcttatgga gtctcctaat
23460 atctcccctc catatccact gcccaactgc cagtaccttc ctggtggcct
ggccccttga 23520 gaccatgctc tcttctgtgt atcaatgggt gccccctgga
taatatgcta tgttaattat 23580 tagtaatata ttatagagta tattataggt
gtgtactgtt ttccaggaac tgtgctgaac 23640 ctttctatta attgacattg
tgtctattaa tctttattta accccgtgaa gtagatgcaa 23700 ccccattata
tagatgaaaa aatatcctta cttataaagg aatttttcag ggtaaatcag 23760
aaagaacatg gcagagttag gagtcgaact tagacctttc tgatgtcaac actgcggctt
23820 ttatttattg gcctaaataa aagtaaagaa ccctttatta gtatgatagc
taactttcaa 23880 cttgtccatc tcaggcgata gaatgcctga attcagctaa
aatatttgcc tggttaacaa 23940 atgtggtgct ctgaagagaa cttgaatgag
atgcctttcc tgtacttccc ttttcctgtt 24000 ctatttcttt ggctctgcag
aacatctgat gcaggtcaat gggggaaaaa ataagaaaaa 24060 aaaaaaaaga
aagaaaaggc ttttctgctt cttcttcctc tttaactgaa aacagcataa 24120
tacagtgtta gtctggattg aacaaaggta cattaatcca tatattcata taaaagacac
24180 tgaagaatca ccattgagta atgttggtaa tggtgggaaa cggtggtttt
tatggaggtc 24240 ctgaaaatat acctaatagg agctactttt tctctagtgc
ccatgtaggc tctactgaaa 24300 gggtttgtca accagtttac cacaatgcga
gatgtcttac ttttaccttg atgaaatgct 24360 tatgaagttt cttagtgatt
ttttttcttc atgctcacct gctgtgcctg caatgggcca 24420 tgtgggaaga
tccaccctct gcttggaaac tagctcactc tctgtttcat cacctagtgt 24480
cttgctggca gacgtggaag atcttattca acagcagatt tccaacgaca cagtcagccc
24540 ccgagcctcc gcatcgtact atgaacagta tcactcacta aatgaagtaa
gccatcacac 24600 agctcttcaa agctactatt ttcatttaac cagtattgcc
atttcaatca ggggaatatt 24660 caagaatcat aattggtgga agatggtaaa
aaataaaaca caaacacact taggttaatt 24720 aaatggtggt cattcatttt
ttggtagatc tcttccctga gaagactgca tcatatttgg 24780 taaactgcag
gatgtttgtc tacagctaag aatatctcta actgctggga ataacacttt 24840
atgctatgga acaacagaaa ttaaagaatt ggggctttta attaaaactg ccaccaaaaa
24900 attaccagtc caattaatca tgtctctttg gaccattacc ctaattttac
taattaccag 24960 attagctcac tgaattaaag gaatatattc acttatattt
aatacactat aactaattgc 25020 attttattcc ttagaaggaa gctatttaaa
ctaataataa taataatgcc tttgttttaa 25080 tctgtaagaa attggatttt
ttttctatca gtacttacag gttccactcc ttctagagag 25140 aacttgagta
agatgttgat gtgcaggtga gacctcagca agctttcaca taatccacta 25200
aaagccattc cctgtatttg ttagttgaaa gaataaattc gcaggaggac tttctttttt
25260 atatgatatt ctccaagtag taaaaatacc ttgatgcctt tttatgagta
tgcagctata 25320 ttgcctaata taactatttt tgtcatcttt gactaagtgc
ccagaaacta ttagggacca 25380 tatccatatt tttaagacat ctaagactta
ggtaatgaga atcaatttta tgtatataat 25440 ctttaaaagc atctgttcct
tcccagttaa ttaagccaga gtcagtatgc ttctagaatg 25500 tgtgcctggt
tgattgaggg ggccttaaaa ttgcaccccc ccttttttaa tctctcctac 25560
atctatccaa cttagaccac ctctctccag catccatcag cacgactgca tgagcaaact
25620 tgatgcagag aggcttcata ggtgggattt caccttcata gaaggtgaaa
ctgtcactgc 25680 tgtgataagt ttggtgggga gaggggaatg ccgtaaacag
aagtattttt aaatatttgt 25740 taaaacatat tttaattatt ttgttcaaaa
aagttatgtt ttcttacgat atgttcagga 25800 aagagttgga atgacacagg
aggaaaaaat aagcacatgg ctctattagt tttctagggc 25860 tgtggtaata
aaataccaca gactgtgtag ctgaaatcac agaaatttgt tttctcatga 25920
ttctagaggc tagaagatca aggtgtcagt aggtttggtt tctactgagg cctctttcct
25980 cagcttgtag gtagttgcca tctcacagcg ttcttcctca
tatgcctttt ctttcctttt 26040 tttttttttt ttttttgaga cagagtttct
ctctgtcacc caggctggag tgcgatagca 26100 tgattttggc tcactgcaac
ctgcgcctct tgggttcaag caattctcct gcctcagcct 26160 ccagagtagc
tgggactaga ggcgcatacc accacgctca gctaattttt tgtattttta 26220
gtagagatgg gatttcacca tgttggacag gctggtcttg aattcctgag ctcaagtgat
26280 ctgctcgcct tggcctccca aaattctagg attacaggcg tgagccacca
tgcccagcct 26340 catatgacct tttgtttgtg cacatgcatc cctggtctct
ctctgtatat cttaatctcc 26400 tcttcatata aggacaccag tcagattgga
ttcgtgccca ctctaagggc ctcatgttga 26460 cttgatcatc tctttaaagg
ccctatctcc aaatacagtc actttctaac ctactgggag 26520 ttagggattg
aacatatgaa ttggagaaag gggtacaaca tctactcctt aactatgaca 26580
ttatagaaaa tgtcttgtgc ttctctttgc acccccgccc ctattatttt ctaacaggtt
26640 cataggaacc ataagcattt tgctctcaga atattcctct aagtgcttct
ttccctttga 26700 tcggtggtct cttgatcagc cctacctaca agatggactg
gtgggcagca gaggttattt 26760 tgtcattgac tcacaccagg agatcttaaa
tgatccggtg tagggagaaa gaaacaaatg 26820 gccaaaaatt acttcttaga
agaaatggtg agagaaaaga gttcttcaaa ggatgttaca 26880 ttattacccc
agcttagttt gagaaatgaa taaagtctgt cggttaaact gccttcatat 26940
tatacagcct ctcctgttag aggaaatcta ctgaagtatc aatgcataaa ttattttttt
27000 gtggtagctt tctcagatgt atttatgcct agaagagtaa cacaggaaat
ggagattcaa 27060 ttaggaaatt gctgacagtt acatttctga caccccagac
actgacaggg tcggtacttg 27120 gtggcaggtg ggcaggagcc cttaattctc
agcatgggga caaccactca cacctaccac 27180 tcatgctggt tatgtgatct
cagagaaccc aaggataaat ggtgctccag tttttaccag 27240 ctaggattgc
tatttgaaat cacctctaga aaagttccca gagataaagc cagggtttga 27300
ttgcttctgt ttcagaaggc atcagagttt aagaatggac cctggaaagt tggtccaaat
27360 taaaacataa cccagttcaa tcccagcaat cccaaaccag acaataattc
aatgtttgct 27420 ttgaagtggg tgctagccta aagtcagaat ttttttcttt
tcttttcttt tcttttcttt 27480 tttttttttt tttttgcttt ttccttcccc
tattatcttg acagaacctc aaatacaact 27540 ggacttccac ccaagagaga
ggtccagaat cgaactactt cttgggggga taattgagtt 27600 tgtttgtttt
tcctccagat ggtccccacc tttgcctctc atcattgtgc caatctcact 27660
gtgcttgcac aggtctttag tgggaaacaa tgatgcttcc atttatcctg catgaagaca
27720 gtgctaaggg ctcccttcat cttgaaaagt gcatttttaa aaaagtctca
tataaaagtg 27780 aacttttgaa tgaatgagaa caagaatttc atacacaggg
gcagtgactc aatgtgatga 27840 ctttaaaagt aactttcagg ggccatagtt
tatagattaa cttttcctac ctcattataa 27900 gtatcttagc actttttcac
tctttctcaa aaccttgaca cttatcaaaa ctttaatttt 27960 attaatttcc
ctaaacagca gaagaaacac cctgccctaa gtgctttagg tcctcgtgca 28020
ttccacatac agaggttttc ctttctctga agaagttgtc tgcttgcttt ggtcagggaa
28080 atgctttgaa cttggcttcg tgactaacct ctggtttcca ttttgctaga
tctattcttg 28140 gatagaattt ataactgaga ggcatcctga tatgcttaca
aaaatccaca ttggatcctc 28200 atttgagaag tacccactct atgttttaaa
ggtatgttgt ggggaaagtt gttgatcttt 28260 cactgtgagg ggagggatta
attctccagt cgtgtttgtt aaaacttgag tttgtttcct 28320 ttgagttctg
aaaatatttg cattacaagt gttcctcaac tttaatacct ggctatttag 28380
gggttggtta tttttcccat taataatata gtcttgtcct ggtctgtatg tcctaatctc
28440 ctcccacaag gacaccagtc agtctggatt agtacacacc ctaagggcct
catgttaact 28500 taatcacctc tttaaaggcc ctatctccaa atacagtctc
tagggggtta aggcttcaat 28560 tctagatgaa tcccagttct agaattaact
ctgtttctgt ttatgtgaca ttagataagc 28620 catttaacat ttccataaaa
tgaaggaagt ggtgtttatt tttttcaagt ccttgtttta 28680 ttttcgttag
tggacaaaca ctatttctgt taggggacaa acactaacag aaaataaaac 28740
agggacttga aaaaataaaa ttaaaaatta aaaaaagtgg tgcagctttt tgatgttaat
28800 ttttaaaaat tgatacataa taattgtaca tgtttctggg gtacatgtga
ttttttttct 28860 ccctccctcc tcacatgtga tgttttgata catgcataca
atgtgtaaat cagggtattt 28920 gggatatcca tcacttcaaa catttatcat
ttctttgtgt tgggaacatt ttaagttcat 28980 cttctagcta ttttgaaata
ttgttgattc tcgtcaccct actgtgctac tacacactag 29040 aacttattcc
ttctatctat tttgtaccca ctaattaatc tctctttatc ctcctttccc 29100
agcctctggt aatcaccatt ctactctcta cctctatgag atcaacgttt tccactcccc
29160 atatcagtga gaacatgtag tatttgtctt tccctacata gcttatttca
gggcatgttg 29220 ctgcaaatga taggatttta ttccttttaa tgcctgagta
atattccatt tgttatccac 29280 attttccaca tgcatatcca cattttcttt
atccacatcc acattttctt tatccattca 29340 tctgttgaag aacacttagg
ttgattctat atcttgacta ttgtgaatgg tgctgtaata 29400 aacatgggag
tgcaggtatc tttttgatat actgatttcc tttcttttgg atacataccc 29460
aataatagga ctgctggctt atatggtagt tccattttta gttttttgag gaacctccac
29520 atggtttttc atagtggctg aactaattta cattcctacc aacagtgtac
aagggttccc 29580 ctttctccac atcctctcta gcattcgtaa ttgcctgtct
tttggataaa agccatttta 29640 actggaatga gatgacattg cattgtggtt
ttaattcaca tttccctgat gattagtgat 29700 gttgaggatt ttttcatata
cctgttgccc atttgtgtgt cttcttttga gaaatgtctg 29760 ttcagattcc
tttctcattt ttaaaattgg attatttgtt tttttccttt tgaattgttt 29820
gcgttcctta tatattctgg ttattaagtc cctgttggct ggatagcttg cacatatttt
29880 ctcccatttt tttcttttca cgctgttatt tcctttgctg tgcagaagca
aattttcagt 29940 ttgatgtaat cccctttatc tatttttgct tctgttgact
gtgcttttga gatcttaccc 30000 aaaaaatctt tgctgagacc aaagtcctga
attgttttcc caatgttttc ttctagtagt 30060 tttatagttt taggtattac
atttaattct aatctgtttt tagttgattt tatatataag 30120 gcgagagata
ggcatttagt ttgaatttta tgaataaaat ttttcccaat accatttatt 30180
gacaagactg tccttttccc aatgtatgtt cttggtgcct ttgttgaaaa tgagttaact
30240 gtagatctgt ggatttattt ctgggttctc tattctaatc cattggtcca
tgtgtctgtt 30300 tttatgccag taccatgctg ttttagtact ccagctttgc
tcattctgtt cagcattgct 30360 ttggattttc aagatctctt atggctccat
atgaatttta gaattttttt tctctttcta 30420 tgaagagtat attgatattg
acagggattg cattgaatct gtagattcca ttcggtagta 30480 tggacatttt
aacaatatta attcttaagc ccgtgagcat gaggtatctt ttcatttttt 30540
tgtgtgttct cttcagtttc tttcatcagt gttctatggt cttaattgta ggtctttcac
30600 ttctttggtt agatttatta caggtttttt ttttttggtc attgtaaatg
gtatttcttt 30660 cttgattttt cttttaggtt gtctgctatt gttgtatata
aatgctactg attttgtgtg 30720 ctgattttat aacctgcaat ttactgaatt
tatcagttct aacacagttt tttggtggag 30780 gctaggtttt tctaaatata
agatcgtgtc atctaaaacc aaggataatt tgatttttcc 30840 cttccaattt
agatgccttt tatttctttc tcttacctgt ttgctctggt tagtacttcc 30900
tggtacagct tttgaaacta aagtaagacc aggacaacaa atcccagcga gggacaaaca
30960 gccggacaag gctgaagtcc tttgcagtag ggttcttatg atggtttcta
ctccaatttc 31020 cacccatttg gttatttatt ttcagtcgca aaatattatg
caagagaaat tgattaacct 31080 aacttggatt ggatgtcttc tctcttgaat
aaattgacct tagtaaaggt cagtgaacat 31140 agccacagcc aattgttttc
agaactagga aacaactcta tagttctgtt ttctacctct 31200 ctctcttaaa
aaaaattttt tttaaagctc tggaaaataa tgtagtcact aaaaatgtac 31260
atttaattta gtaacatata atttatgcac agtatcccaa tattatctaa attgtgatag
31320 gtgagcctct tcagtcattc aaagataaga ctttgggtta ggacttctca
attttaatct 31380 gtcgtttaca agaacttaca gtgcagactc aaggcagaca
tatgaaatgt tgggtcccct 31440 tggttattga gttggtcaat cagattggat
ccatgtatca tggcatatcc acccatgaca 31500 tttgctttca gccatgttgt
gtgtagtcct tggaacatac ttatctggaa cctgtacacg 31560 ttgaaaaatc
atgcattctg gatggtttgg tcctactctt acttgatcaa ggatgtgcag 31620
ataatgtgag tctctgggat tttgccaact tttcggtgtc agaaccagtg ccaagaaaat
31680 tggcccagga cttagaaagg tcaagtaaag taatgaatcc agacaactta
agattttctt 31740 tgcattgagt agattaagct aggtagttct ctttgactat
acaatttgac gattagtggc 31800 caatgccatt gggctttctc acttactatc
ctgttaaata ttgctagctc caagttagga 31860 aaaaacctcc tggagtggtt
caaatgacaa tctaaatatc taactctttc tttttcttat 31920 tttggaattg
caagtctaca tatttgtttg attttacaac agtcttctcc cttccctcta 31980
taccagtggt cctcaacccc tgggctgcag acaggtacca gtccatggcc tgttaggaac
32040 gaggccacgc aactggagat gaatagccag cgagcaagca ttactgcctg
agcaatgctt 32100 cctgtcagat cagtggcagc gttagattct cataggagca
caaaccccac tgtgaaccgc 32160 gcatgcaaag gatttaggtt gcatgctcct
tatgagaatc taatgcctga tgatctgagg 32220 tggaacagtt ttatccccaa
accatcccca ccactgattc caccccaact ctgccccatc 32280 catggaaaaa
ttgtcttcca tgaaactggt ccctggtgcc aaaaaggttg gggaccactg 32340
ctctataccc taaactgtgt tgtagctgac ttttaaaggc aaatacatta tgattaattt
32400 tggaggtgtt cttgataatt cttctaaaga catcaaaggc tattattgag
aaaaggttga 32460 tgattcttat tccagagtta gcagcttgtg ttagcccacc
atactgggaa aaaagcctct 32520 gtccctggat ttgctggtaa gttcgtgaga
ggttagatgt atgcttcttt ttgtgtgaaa 32580 taaagaaata atccacataa
aaaaatatgc actcaggaaa atcttgaggg agtttttgct 32640 ccgggtgtgt
ctccacacct cccggggaag attgccatcc aactcacacc catttacctc 32700
taaatgaagc atgaagatac agcccaaatc attagttctc tggtctcttc tttgaaactt
32760 ccacatgcag ctctgacatg actgcataat tgtggaggat aaaaacagtt
ttaaatcaaa 32820 gagtcctggc ttcaaacttc agtttcaatt cacaccagct
ttgctacctt aactaatgtc 32880 acttagtatc accagtgttt aaatttccct
tgagaatttt caaagaaatg cagaacaatg 32940 catatctcag agatttgctg
aaactattaa atataagcac tatataaatg aaagttatta 33000 tcctgaagct
tattgttact gtttttgcta cttttggggt ttctttgagc aggtttctgg 33060
aaaagaacaa gcagccaaaa atgccatatg gattgactgt ggaatccatg ccagagaatg
33120 gatctctcct gctttctgct tgtggttcat aggccatgta agtattcaca
ttctcttaac 33180 cctatttctc aaaatggtgc ccaagatcac ctgtgtcaga
ctcaactggg ctatttatta 33240 aaatgcattt tcctaggtca catcatgaag
cttgggaatc tacaattttc acaagtttcc 33300 caggtgactt ttatgcatta
gtaagttgaa gaacatgact tcaagcattt aaatcaccca 33360 aaatattttt
ggtcttttct acattaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaga 33420
aatagtacat tgattataat atgtttacta agtagaggaa aggactgaag gagttatcta
33480 agttggggcc caattaattt atttctcttt tggttttaat tatccagaca
tcttttgcca 33540 cctttgccct tggaaattga acataaagca caacattaca
gaggtgaaac agaatatgtt 33600 ttctcatatg tcataatagg gaattttctt
cctgaagaag ggttttgcat caaaaaagcc 33660 atatataaga caaactgtat
gttggaaaag taaaagatat aacgactatt aacctccctg 33720 atgaatgaaa
acagtaaaaa ttatgcttca aatcctataa aatgggcata tatgttctct 33780
acactgattt ctacaaagaa tcatagccac tggaaaaata actcaaaata tgtgtctttc
33840 tgataatgat ttttgcagtc tttgcattca cagatacata gtaacagaat
aaatgagtta 33900 ggaaattaag atattggcac ttattaagta catcaagata
aaactgtttc tgtctttgct 33960 tgaccttgac aaatgcaaca tccctatttg
ccttcattca ctgtgaatct tctatcctcc 34020 attctccatg gatgggagct
gcaacctccc tgagtctact ggaaattccc aaacaccatt 34080 ggtactacct
tggccaaatg aggatgattc caactgatca gacccttaac atatcccagt 34140
cacatcatga tctggaacct tccccaagtt gagtagtttg gttattttag gtagtgaagg
34200 gagcagcagt taatagagaa aggtccaaag taggagagta agaacattta
ttttccattc 34260 tatcactgaa catgagaagg gccaagaaga aacctccatt
catattgact ctaatttata 34320 tcggtgaggt tgtccctaat gactgccctt
ctcaccctga cacctctgcc ctcctattag 34380 acatcctacc ctctacccaa
actgcttgct gaatctttgt aagattaaat tatttaatcc 34440 acaaatattt
ataaattgcc tatgatgttt cagatcctgg aaatacaagg atgaacaaaa 34500
tatagcccaa ggatcttata gctgagtatt ttgctccaac aatgtgaacc tgatttgtgt
34560 agcccaaaga aacataatca ataagggctt tttaaatcga catttaaact
ccattcttgc 34620 ctgcctaaaa ctaattcaga tcatctgact ctcttagtac
ttcaaagcac tggaggaggg 34680 aaagtaaaat aaaatattta cctttcaaca
attgtgaagg caggttttat attcaaaaac 34740 taaaccaccc aaaggcaaat
taaaatctta gcttttaagt ctctcactct tttctacaac 34800 tcaataagga
tttcaaaaat cttataatct agtctcagtg gaaatccact acactacact 34860
ttgagaagct tgaagccagt catttctttc taagcttctc attcatgtac tctcgggagg
34920 caaatttaga tccttctctt tccgcaaagg cagagctgag accaatttgt
gcatgactgc 34980 atcaccaagc caaaatccgg cacagggctg gcacatcata
ggacccagtg aatatatgtt 35040 aaccatcaca acttgccaag tactttttct
gccaaatggc ttttctcact gctaacctcc 35100 tgccaaacct ctgccctaga
aaactctcat ctaattgcac acaaagttag agctctacaa 35160 cctcagggcc
ttcacagaat tatctctgcc cctcctcacc acagctgaca catgacctaa 35220
ggacactgct ccctggtggc tccttcaagt agaggggctg ctcttttttt cacatcacca
35280 tgtgctgaga ggcctggtgg agtggatcag cattctcttc tcctgatact
accaatgatc 35340 cttctcttct cagaaactta cacaaactgg ttgcactctt
attttattgc tatcgtgcac 35400 tgaccttcag ataatttcct ggtatccggt
tcatgattct ttattcccct ccaactcttg 35460 ccatcattct gagtgaattc
aaagtccatg tgtgagagtc acctaacaat gtatcttcac 35520 agttccttgt
tctctgttct actaaacctc atctcaactc ctctttagca gatttctcct 35580
gtagccatcc tctggatctc agaagtaatg ttttgctgat ccttagaccc agaatgtggc
35640 catggacagc aacaaggaat gttagaagaa gccatctagc aatgtaactt
cttaatttcc 35700 tgtcttctct catttctcac ccctactatg actgcttttt
tttcaacctt agcatatttc 35760 tagttcctac acagatctat atcattttaa
tttatcagtc cctttccagg aacactttct 35820 tcactaattg gtcccatcac
aaattcatcc gaaccctcaa tttcttgctc ccttgacctg 35880 ctcttctgga
gttccaaccc caaacactcc acagaattaa ccatcctttt tctgagccac 35940
cttgtacatc ttgccattgt ttattatcat acttatatta atagcattga actgctgctt
36000 ttccctttcc aacttatcac ttctattagc tttctgaagg cagagaccaa
gtctaaagta 36060 atttttttgt tccccataac ctggtatatt gtttggtcaa
cataattggt gctcaatatc 36120 cccttgtgga atttgaaatt taaattaatg
ttgcaggttt aggctgacat acaattttgg 36180 gttgcagaga gtatctaaac
agtacctact gttgggataa atactttatt gtcattggct 36240 acagttcaaa
ctatacatac atatatagag attggagtaa aaactgagac agatagctct 36300
ctgatatatt tgtaatggta atgaaaatga cattttgttt taaaattttc ccttcatgtg
36360 tcttatattt ttttttagca accccattaa ctgacctata tgtcgttatg
tactaattta 36420 ttatctctca aatggtcatt ggttaattcc taggcaggaa
ttgttgttgt tgttgttgtt 36480 gtttagggcc acattaaagg caaagcttga
gtgcacccca ggcaaagtga gaggaagagc 36540 tgagtaatca ttgaccacag
gccagctgat gggaatcaat cccaccctct catcactcag 36600 tcttacactc
ttctccattc tcctctattc tcatcttctt tttcttttat acagaggctt 36660
ctcaatttat ggtggggtta tgtctcaata tacccaataa acaatcacaa ctgaaaatat
36720 tctaagtaaa aaatgcattt aatataccta atctaccgag tactgaatat
catagcctaa 36780 ccttccttaa atgtgctcag aacacttacg ttagcctaca
gtagggcaaa atcttctaac 36840 acagagccta atttataata aattgctgaa
tatccatatc acttattaaa tactgcacta 36900 aaactgaaaa atggaatggt
ggcatgggta tggtttctac tgaatatgca ttgcttttgc 36960 gctattgtaa
agtcaaaaaa tcataagtga aatcattgta tattgcagac catctctagt 37020
aggacaggat ttcaattatg ttacttgcca tgttggtaaa tcgtaccttc aacaaatatt
37080 tatttgtcgt caggcaaaat ttcttcagcc actttgaaaa acaatgtgga
agttcctcaa 37140 aagattaagt atagagttac catatgaccc agcagttcta
ctcctaggtg tatacccaag 37200 acaagtgaac acatatgttg acaaatgatt
atagcaatat tattcataat agtcaaaaag 37260 tggaaacaac tcaaatttcc
atcaacttat gagcagaaaa acaaaatgca gcatattcat 37320 gcaatgaaac
atcaatcagc aatcaaaagg aatgaagtac tgattcatgc tacaacatag 37380
atgaatcttg aaaatactat gctaagtaaa agaaaccaga tacaaaatgc cacatatatt
37440 attccattta tatgaaatgt ccagaatggg caaatccaca gagacagaaa
gttcattagt 37500 gattgtcaga ggcttgggga aatggcagga gggaaagggg
agtgagttat aatgggcaca 37560 ggcatgggga ttttttatga tgaaatgttc
caaaaatcag atactagtga tggttgcaaa 37620 actctatgaa tacaccaaaa
accgctgaat ttcacacttt aaaatggtga atttctggaa 37680 tgtaaattat
atctcaataa gctgttaaag aaaaaatggg caccccttcc ttcgggattg 37740
tagctatagc cacacttgaa ggtgtggctt ggcacacagc acagactgta tttcagccct
37800 cactcactcc ttctgtctgg agtcctacct attagataaa gaaataggta
acattgttct 37860 gggcctaaca tcggtaatct ctcagagcat aactttttgt
agaaagattc ccatccaacc 37920 agaggtaaat gtaggaagga aatttaaaaa
gtgaagcaga aaaagaaatt catatgctgc 37980 atctattaaa agtttggccc
atgttgtaga aatgaaaatg agaaatgctt tattatttgc 38040 tttattattt
taaaaggaac aggctctcct aatatttttc taataatgaa tgctacatta 38100
ttactgaaaa gtgatgctaa cataaattta taaattcgta gcataaaaat gtatttaact
38160 ggttgctcga ctgtttaaaa catggcttcc ctggaaacca tcattctcag
caaactaaca 38220 caagagcaga aaaccaaaca ccacatgttc tcactcataa
gtgggagctg aacaatgaga 38280 acacatggac acagggaggg gaacatcaca
caccggggcc tgtcgggggt ggggggctag 38340 gggagggaga gcattaggag
aaatacctaa tgtagatgac gggttgatgg gtgcagctaa 38400 ccaccgtggc
acgtgtataa ctatgtaaca aacctgcatg ttctgcacat gtatcccaga 38460
acttaaagta taataaaaaa caaacaaaca aaaaaacaaa acatggcttc cttcattcta
38520 caaattttgc ttccttttca ttaacctttt atttctgacc tacagtagat
tttaaaataa 38580 cttttttctt ttctttctct ccgatttcat aagtatttat
tcatggcaaa gatttttaat 38640 gtgactcttg tgattgttct agggaaatat
gaatataata ttttaaacgt ttaaagggaa 38700 aatagtaaag tttataaaag
gcttgttttt attttgtcaa taatgaaaaa gacatttctt 38760 aacaatgtca
tgagtatgct ttaaggcaac aaacaattat aaactaaatt aaatatttaa 38820
tgtaattaaa tgtgaattaa attaaaatat agcaatgttg ccacaaatta agattttgaa
38880 ccaaaagctt tgtcctagat gaaacgattt gaccagctaa aatttgtctt
tatagttctc 38940 ctgcctgtac attttgtcat tttggggtaa acttctcagt
caccaaattt ggatgccatt 39000 ggatcacact gcaatatgtg ccactaagct
ggatgactct aaagtagaga ggaacaagtt 39060 tgagatgatg tccgttagga
attcatagcc agttcctagg aaaagctacc ctaattctac 39120 agctagatga
tcaaagcctt gggaaacaca ctcaattcta gcaaaacttg agctccacaa 39180
gttctaagga caatgtagcc aatatcatgt aatcacatct ggggataaaa catggtaggt
39240 agtttaagct ctgatgaaca tgaattacag aaaaaggagc taaactaaat
ctaggttttt 39300 gtttccttaa atcttcttag tgggctctat ggctttaata
aagaattaat tttatttttt 39360 aaggaaaatt tagaaagttt atggttcgat
tgtctgcctt cattaactag gaatactgga 39420 ccacgtgtaa ggcatttatc
accacttcgt agcaccctaa gttcagttct tttgaggaat 39480 tagcactctt
tctgaaagtt aaatctgcaa atctaaacat gccaaatgac aaattaaaaa 39540
aaagaaaaag aaacacacta agtttagaag aacttaaaac atctaattaa atatacttgg
39600 tttaatttgc agataactca attctatggg ataatagggc aatataccaa
tctcctgagg 39660 cttgtggatt tctatgttat gccggtggtt aatgtggatg
gttatgacta ctcatggaaa 39720 aaggtaggag aaaaggcaaa gaagacaaat
catgttctcc ttggggatat aggatataca 39780 ggttgaatta ttcatagaat
tctggatcta ggcacaatgg ctttattatt aatttttttt 39840 taacttttat
tgtggaaaat gtcaaatata tatataagtg cacataattg tgtagtaaac 39900
ttctatctac ccatcataga gcttcaacaa taattaactc atgaccagtc ttgtttcatc
39960 tgtattctct ctacccactt ctaccttact cattttattt tgaagtaaat
cctaggtacc 40020 atatcacttc atcaataaat atttcagtat gcatttctaa
aaaaaaaaga actctgaaaa 40080 aaataattat agtataatta taccttaaaa
actagctgtt tctgaatacc ataaaatatt 40140 gccagtattt tcaattgtat
aataactttt tttttttttt ttttttgaga tggtgtcttg 40200 ctctgtcacc
caggctggag tgcagtggca cgatctcggc tcactgcaac ctccacctcc 40260
cgggttcaag agatttttct gcctcagcct cctgagccac tgggactacg ggtgcctgcc
40320 accatgcccg gctaagcttt ttgtattttt agtagagaca gggtttcacc
atattggcca 40380 ggctggtctc aaactcctgc ccttgtgatg cacctgcctc
ggcctcccaa cgtgctgaga 40440 ttacaggcat gagccacctc gcccggcctc
ctaacttttt aaaaagtatg tttctttgat 40500 tctggatcca aataaggctc
ttacattatg attggtttat atgtctttta attctatttt 40560 aatccatgaa
ctcacattcc atcttttgct ctttctctct cttctttttt tccttgcaat 40620
ttatttgtca aagaaaagag tttcccatta tcaggatttg ctaattgcat taccatcttg
40680 tagtttaaca tgctcttctg tctgtatttt ctggttactc aacattgtga
ttcatgtaaa 40740 attactcaag caatatgaaa tactctgctt tctaatttaa
agaggggcac atagaaacat 40800 aactaggtat atataaattt agaaaaacct
acttgagtag cacatataaa tactaagagg 40860 aataagatta gttggtgtga
ttggaaacat ggaattacac atgaattatt tcatgtagga 40920 ggtaatttat
gcagaagata tggaaatggc acaggagatt gaggaaaaag tcatctctgg 40980
tgagaggaat actgtaactg aaaattttgt aggtggaggt gggcaaatgc caaactaagt
41040 aaatgagaat tacctagcat aatgcctaac acaaatttgg
tgtccaatga atggtcatat 41100 ctgtaaactg gtaataaagt atatttacac
cttaacctga atcacagtgg aattcagtca 41160 ccctttagat ttccagcttc
ccaactgttc tttgtatcat taccctatta ttaattccca 41220 cagtttgaga
acttgatatc cccagggcct attgttgcca cggaaccaca ggcctgggag 41280
tggtaacagg ctggaaggct tggcggaggg ttggtgagag taggagaaaa gggtgctaca
41340 tcatcccaaa ctcagaactt aaatgaagta tgtgcaactc tttttttttt
tttttttttt 41400 tttgagacgg agtcttgctc tgtcgcccag gctggagtgc
agtggcaaga tctcggctca 41460 ctgcaagctc cgcctcccag gttcatgcca
ttctcttgcc tcagcctccc gagtagctgg 41520 gactacaggt gcccgccacc
acgcctggct aattttttcg tatttttagt agagatgggg 41580 tttcaccgtg
ttagccagaa tggtctcgat ctcctgacct cgtgatccgc ccgcctcggc 41640
ctcccaaagt gctgggatta caggcgtgag ccaccgcgcc cggcctgtgc aactctttac
41700 atgaccaaac tttcccagtt taccccaaga acccaatagg gaatttgctt
tatatttaaa 41760 aaccagagtc aaatcagcac aatcgaagaa gtcatcagat
taaaggtgtc ttcacatctc 41820 caccttttct agctttgaaa ggggagtggt
gaattctacc taaagagagc attttaactt 41880 atgactcagc gttcagttga
gacacaaagt tattttgctt ttcttcgaag gagctcagaa 41940 tgaccctgtg
cataaaatta atgtaaagga aacaagacta aacaagaagg ctaataagca 42000
gcctagtgga atgaaaggaa atctttattt gtatcagtca aaattgatca aatattacca
42060 ttatgtttgg ttcaactaaa atagtctgag tggatgtgat tgaaacccgg
atagcaatag 42120 ggaccgtgca aaggaatatt gcaacaacag tgatgtgatg
aagccatgca aggtatggga 42180 ttgaaggaga gaaaggcaat tctggcttca
tggactttca aatgcatgtc tttcctcagg 42240 ccttgaacgt ggctacccag
gttgtctgtt tgtattttgt ttatgtagaa tcgaatgtgg 42300 agaaagaacc
gttctttcta tgcgaacaat cattgcatcg gaacagacct gaataggaac 42360
tttgcttcca aacactggtg tggtaggttg ttggctttat ttcttgcaat gtctcttcac
42420 tgaaagggtg atgttcacag ggaaaggccc atgaattcaa attaaataca
gagctggcct 42480 gtctgaatca gggaataatt taaatgataa atgcttaggt
aaatgtaatg ctgcgactgt 42540 tggccagagt cagcaaatca ctttggcctc
tcctctctcc tgtttcccta tctttaaaat 42600 aagaaagttg aatcagtttt
ttaagatccc ttctagcttc aaaattctaa aatctattat 42660 cttggaataa
taaagaagtg acagttaaag atcctatttt aataaacaaa aacattcatc 42720
attagaatat caaagacctg agatgggggg gaggacctct cttttttttt tgagacagag
42780 tcttgctgtg ttgaccaggc tggagtgcag tggcacaatc ttggctcact
gcagcctctg 42840 cctcctaggt tcaaggattc tcctgcctca gcctcccaag
tagctgggac tacaggcata 42900 tgccaccgtg cctggctaat ttttgtattt
tttttagtac agatgggatt tcaccatgtt 42960 ggccaggctg gtctcaaact
cctggcctta agtgatccgc ccacctcggc ctcccaaagt 43020 ggctcacagg
agtgagccac tgtgcctggc ctggacccct catttttaat tgcacaagta 43080
aatgtttact tctatagtgt ttgaagacat ttttttcact attcactttc ttaatttctt
43140 taataaataa tataaagaaa atataaaaat attaaaaata gtataaaaag
cagcacagtg 43200 ggaatttatt atttcttaat tcgaatgagt taaggcattc
gatgatgttg agttatgcat 43260 tcaagaacag tctgctttca ggagtttgaa
gattttttaa agaactaaaa gtagaattac 43320 tatttgactc agcaatctca
tcactgagta tatactcata ggaaaatgaa tcgatctacc 43380 caaaagacac
atgcaatcat atgttcattg cagcactatt cacaagagca aagacatgga 43440
atcaatctag gtgtctgtca atggcggatt ggataaagaa aatgtggtaa atatacatca
43500 tggaatacta cacagccata aaaaagaaca aaattatgtc ctttacagca
acatggatgc 43560 agctggaagg cattgtccta agtaaattaa cacagaaaca
gaaaatcaaa tactgtatgg 43620 tctcacttat aagtaaaagc taaacactga
gtatacacag acataaagat gggaatagac 43680 actggggact caaaaaaggg
gcagggaagg agagagaggg gaaagagttg aaaaagtacc 43740 taagtggtac
tgtgttcacc atttgggtga tgggttcaat agaatcccaa acctcagcat 43800
cacacaatat atccatggaa aaaacctgca catgtacccc ctgaacccga agacaaagaa
43860 gtttgctttt aggggggtag gtgttagttc actctttctt cccacccact
caacattatt 43920 tttcatagta ctacatttca gaaacagcta cgaaaataaa
ctaaccctga caaggagtat 43980 gcatcatcta tatttttggg ctccatgggg
cccataaggg agagaagcta ttgtatccac 44040 agaaacatct tcttcctccc
agacctggac cctatacaat cctatgcaca taattttgcc 44100 tatttccttt
aaaaaggtaa aatttcatga ttttaaacat tttatcaaaa tcccagaata 44160
cctattaaaa cctcacaaca ttcagcctgg gaaagctgat tgctaaaaca aaagaaaacc
44220 aaacctcaca acaaagcact taccttattt ccttattttt ttccctgtct
aggttagaaa 44280 ctccatgcag acagaaacca atacccatta tctagtgcag
tgcctggcac aaggagggtc 44340 ctcataaaat attaactaaa tgagtccatg
aatgaattta gttgctctga gagctacaga 44400 tatggtagga actcagagga
agaagcagtt catcccgact taggttccag ggaatcattt 44460 agtggtttct
ccctaaaaaa ccactcgtgt tcccagaggc ccaaagtttg ctgcggcact 44520
aataacatgc caggggctca caggaacagc agccatgtaa aaagaatcta agtaaataga
44580 gctgacagtt actcagcgct gagccattga catagttcat cttccagatt
tcattatcta 44640 tgaatcatag atggagaaac ccgggctgaa aacagttaag
tcccttcctc aagggcacgt 44700 agcaagtatg tgcaagtacg tggcagagct
gggctataaa cccaagttat cagttccctt 44760 ttggaagttt ttattttatc
ttcaagctct tttggtgctt gattttactt aatatttttc 44820 ttggtgaagt
cagtgttatt taatttggat agccaagtag tcaaaatata ttctgttatt 44880
gtcatcaaga aatgtctcag tcccctcttg ggcatggtgc tatattgtta cgtatcataa
44940 gagtgaaaaa cagaaacaga agcagcaagc atatgggttt ttaactaaaa
aaaaaaaaaa 45000 aaaaccaaat aaaaagtaat tgtaaggaac tgtccttatt
accaactgtt ccagtatcta 45060 ttctgtacta tgtaagcaag acagtgagaa
agaagaattt aatcttttct catccctaca 45120 actagaatgt gcccctatga
ttctttatat aaaggatcca aaaacacctc acttattaac 45180 aggaagtgac
atatcaaacc tacttactca ttttatgctc ctctgtatta aaattttttg 45240
tgtgtgtgtg ttggagatga gagtggaggg taggttgtag gggtgtcttt gtcttctcag
45300 gctgctataa ccaaatacca taggttgggt ggcttaaaca acagaaattt
attttctcac 45360 agttgaggat tgggagtcca aaatcaaggt accagcagag
tgaggtcttc ctgaggattc 45420 tctcattggc ttgtagatgg ctgccttctc
tctgtgccct cacatggcct tctctttctg 45480 cacaaacctc cctggtgtct
ctctttattc ctataagggc accagtcaca ttggattaga 45540 gccccatgcc
tatgacttca tttcactttg tctccttaaa ggccttatct ttaaatacag 45600
tcacattggg gcttagggct tcaacatagg aatttgggag gatgcaattc agttcataac
45660 aggagtacat tatgagaacc tttggtctca aacttcctaa gatagcacca
cacattttct 45720 aaaacactga gttcaactac aaagtttttg caactggctt
gaatggaaaa ttctttattt 45780 ctttttctag gagactatag tgttttttaa
aattattttt tattatgata aaatacatgc 45840 aatataaaat ttgccatttt
aacaattttt aattgtacag ttcagtggca ttaagtacat 45900 tcacaattac
tactatctat tactaaaatt ttttaattgt cccaaagaga gatttttact 45960
tattgtaacc agtaggcaat atctcctcat ccctaccttc tccccagccc ttggtaattt
46020 cttatctact ttctgtctct atgaatttgc ctattataga tatctcacag
tgtgcttggt 46080 tccatgtcta tagatcaaag aatgcttgag cttggaggga
tccagtggcc caagttcctt 46140 cctggtacag atgaggcccc tgaggctgag
acgatgaagt agttgcccaa attaacatga 46200 ctgcttaatg gtaaagcaga
gtctcgacct caagtttcct gcctcttcag ggctctttcc 46260 actaaaatgc
ttgaaatctc tagaatgaca atcatagaat gagaatctga ggctcactgt 46320
ccagcatagt agccactaac cacatgtggc tatccagtgc ttaaaatgta gcttgtctga
46380 attgagacat acactgagtg ttaaatacac accagaattt gaaggcttag
tatgaaaaaa 46440 gtaacataaa atatttcaag aataattttt atattggtta
cacttgaaat gatactttgt 46500 acatatttgg ttaaataaat tacaatattg
aattaatctc acatatttct ttttgtgtgt 46560 gtgtgtgtgg cagggtcttg
ttctgtcatc caggctggag tgcagtgtca tgatctcagc 46620 tcactgcaac
ctctgcctcc tgggttcaag caagtttcgt gcctcagcct cccaagtagc 46680
tgggattaca ggcgtgcacc actatgcctg gctatttttg tatttttggt agagacgggg
46740 tttcaccatg ttggcctcgc tggtgtcaaa ctcctgatct caagtgatcc
gcctgcctca 46800 gcctcccaaa gtgctgggat tataggtgtg agccaccgtg
cctggccttc tttttatttt 46860 tcttaaagtg gtaacttgaa aatttaaagt
aaatatgtga cttgcattat atttctatta 46920 aacaaccctg gtctgaggat
tcatattagg gcaccacctc tctatttagt ggttatgtct 46980 tccccgacct
ccatacccaa tatataatct ctattctcta agaattatat atcacataaa 47040
agggcaggaa tattcaaagg tgaccgaact atcaaaaatg gtttatccaa tcaccttatt
47100 ggttaaaaat gaaatacttg ggaagacctt agatgttcac atttcctctc
agggaaacaa 47160 ttttttaaca aacattaatg ttgtgtttgt ataataacag
gaataaagca gaatgagctt 47220 aattaagaaa agcaggctct gtaaggatag
tgagtagcct cagccatgga ctcctgaggc 47280 agagatgcag ctggactcag
aaacagaaag gaactgggcc tggagcccta gagaggctca 47340 gtgaatcctt
cctctcccct tctcatctct gtgatgcaca ctggcttctt tcaggtctca 47400
gtccacatga tgatgatgat gatgatgatg atgatgatga tgatgatgaa cagcaacagt
47460 tatgaaatgc atactacatg ccaggcactg tgcaaagcac tttgtatgaa
ctagctcatt 47520 taattctcat tcaatcagca tttaatgtat aatttttcaa
ttttgcagat aaggaaattg 47580 agatacagat agatttttaa aaatttaccc
aaagccatat agctaataaa tggtagtcaa 47640 gatttagaat caagtaattg
ggtgcttaac aatatgctgt atagcctctt attctgaaga 47700 atggttacca
ccaagaatat ccagattgca tctcctaaaa tgacagtatt tacttcatag 47760
ggctgctgta agaattacat gagatgtggc aaaaatctta gcagagttcc tgacgtacag
47820 catgtgctcc acaggtgtca gctggtagta ttactatttt tactgtctgt
tcaagagagc 47880 agctagactg agactagact cttagtattg atttcaagtt
atctttgaag ggattcagat 47940 tggcaagcac aagagtcaga ccctatcctg
agccctcaac tgtttgcagg aaggaataat 48000 ctcttgtgtc acatgcagct
tgctggggct tcaccttgtg aattagggac agggggagaa 48060 gtgttggaag
gcagcctacc atctcaagtg atgcaaatta taatctacca aaggaatgaa 48120
tgaacgttgg tctggcaaca aatatcacca tcccatttta tttactaaac ttactaaacc
48180 actttagcaa gttaaaagta gcactgaagg cagatttaca tattctgagc
tctgaagtga 48240 ggcttttctt ttatgggcta tattgatggt agctttaaaa
ctacaaatat cagaaaaact 48300 aaatttacag tggattaagg aaaatggggt
ttattttttc tcccataacc ataagtctgg 48360 agacagggct ggcatctctg
aggatctctt agccactttc tcaaggttgc aagggagaca 48420 ggtgctggga
atgactgtta gaaggtcagc tatgtgagca gataagtatt tgacttcaaa 48480
agaaacataa cacttagtgg aactatgttc tttgcagagc cctacctaat ccattcatct
48540 aaaagtgttg caacataggt aggagaatac gttgtctggg aaaccacaaa
ttacagtact 48600 atgtgcatcc cctcataatt tcaccttaac aatttcgtta
cagaggaagg tgcatccagt 48660 tcctcatgct cggaaaccta ctgtggactt
tatcctgagt cagaaccaga agtgaaggca 48720 gtggctagtt tcttgagaag
aaatatcaac cagattaaag catacatcag catgcattca 48780 tactcccagc
atatagtgtt tccatattcc tatacacgaa gtaaaagcaa agaccatgag 48840
gaactggtaa gtgctactta attatttttc tcattagcat tttggaaata aaataatact
48900 tagttgaaga atcaaaaact gggaaaaatt ttggcctcta gaaggcaaat
gatagatgtt 48960 ttaaatcatg gtgtgatcct gttgagagtc accctgggtc
agtgttctct aagggaatat 49020 aaagaacgtg ccttacccta acaacacaca
ctttattcta gcacgtgggc ttcctaagaa 49080 aatgtcagac aaattccttg
aaggttagga aggaactact actacacttg acctgatctg 49140 catgtgaagc
ggtataagca aggatgagta tggaatcatg cgacagcttt gtggtcacta 49200
gcttcctaca acagcacacc acagattaag tctcaacaca gcactcattg ttttggtatt
49260 agcagcagga attgttcctg ccctgacttc cttaaccctc agggttttgg
tcctattaaa 49320 gtacctccaa ttttagcatt gaggagagag tctgtttttt
ggaacataac agacaataca 49380 ggaaattcaa agaggactca cacaatttga
tactccctta gcacttttta gtccaagata 49440 ctgtatgttt gggttcatgg
caaaagatgc aaggattctt gaaggattgt agctaggctt 49500 tgacaaatcc
tcatcccaga tgctctccag acagtggaag tgttacatca acagccccat 49560
tcttgggaag ggactaattt ttaggtagta gcttgtttct tagtgactca tttttttttc
49620 tggctctctt aacagaataa aatatagtca cattacagga gctagcaatt
gctgatgaca 49680 aatataagat tatttgcatt ctctgaaaat agcccattta
gaacataaat gtacttgata 49740 cttgagcttt tttcttctca agggaaaact
gttaaggaaa gcacctttca aaaatattat 49800 ctttgaagaa ataaaaggaa
atttatcatg atttgggaag tagaattagt ctaattatgc 49860 tttttttttt
ttgcatcact gccagcacac atatatgttg agagccatta cgtgtaaaat 49920
accttgtcaa tggatgttta aagaagcatt aggtaaaatc ctgcccttta agagaatgtg
49980 ttatggttag ggagctcaac cattagcaaa tgttacaaat agttgtactc
taaggcgaca 50040 tagagtaact actaaatacg tggcacagac agtacaactc
acttctaact agaatatcaa 50100 gggatggctt cactaatgca ttcagaggga
aatgctgaga taagtgagga gataaagtag 50160 ttactgtcct tgaggaattt
acaatctatt aaggggggga aaaactacaa ataataaagt 50220 gctgttgatg
tcaaagatca gctacatttt agacaggcat tgaaagagga tttctatagg 50280
cagacaggga aggaaggacc ttccaagcaa agaagttggt gttcaccata agaggatgca
50340 aaagtggagg gtgatagcat cagaaagtag attaggttgg cttctgaagg
ggtgtgactg 50400 tcagataaat ttgtatttca ttatgtagac aatggggtta
cattaaaact tattttttga 50460 acaatgagat ggcataaaat aatatccgct
gataaatctc ttgagttttt caagaaggta 50520 acagtgtata ccatgatgct
agttccaatt tccgaaaagt tccagataag tgagaacttc 50580 agaatagatt
tgacaaaatg aatatcaaca gacaaaatga agtcaaatgg gggtcttagt 50640
tattatcctg ctccatacca gaggcataat cttttttgat ttgatgaatc tatggaagtc
50700 attagacatt ttacacaaga agaaaataga agttgtgaga aggataagaa
gtgagtcatg 50760 catgcattag gtgtttgtat gtgtttagaa aggttggatt
taaagtttgg tgataatttt 50820 gttcagaaat ggagtacctc taagcctttg
agatgtagtt atacttcatt ttccataata 50880 aatgagttcc caaaaaggca
tgtgataatt tttttctgca aattaatata tttatttata 50940 taaattattt
caatatattg aaatagttta tgtttaaagc cacccaattg tgattgccat 51000
aaagtgcaca tattttaaat taatttgttt accttattta tttgcctttt agatgaatct
51060 agattttcta cctgtatact ttgattcaat taatgtatga ttatttttta
gaaacttcta 51120 cttgtcatgt ttcaaagctg cacattaact gaaattctat
atctttttgc ttccagtctc 51180 tagtagccag tgaagcagtt cgtgctattg
agaaaactag taaaaatacc aggtatacac 51240 atggccatgg ctcagaaacc
ttatgtaagt atttcttctt atgatcttag agaactttga 51300 gctactaaag
aaatctgtgt gatctgtttt tctttgtgta tttaattttt ctgaattaaa 51360
tagggtcaca tgtaatacaa ctgaattgta ataattagga acagaagcat aatagctatg
51420 acaatgctga acaaagctat attaataaat gagttactaa aaagaagcca
aaatcctatt 51480 taagaaatca tatttatcac aatcaagtag gaattacaga
attggcatca tactagttga 51540 gtgaagcaga aaagttcata aaacttttgc
atgattccca gggccaccat ggaaggttgt 51600 gcaggttgta cactacacta
atctagggca tgccatttgc atcaagtgtt ttttagtgtt 51660 agcctgttcc
caagagtata gctcataaca cattacagtt gattgtcttt aatatatatt 51720
acacacacaa aacttgtgac aaactcttaa caaaaagttt tgattaattt ttgctgaaag
51780 atatttagtg agtaactcct atctacacac agtgggagga cagactgatt
ttgccctttt 51840 gaagtttgaa gggagatggg aaaagaggag cataaaataa
acctgtaacc aggcatcaga 51900 aaactacagc ctgaaggcca aatccaggtt
tttccatttt tttttttttt aatgattaga 51960 aaaaaacaaa aagaggccag
gtgctgtggc tcatgcctgt aataccagca ctttgggagg 52020 ctgaggcagg
aggatcactt gaggtcagga gttcgagacc agactggcca acatggtgaa 52080
accctgtctc tactaaaaat acaaaaatta gctgggcatg gggccttcca tgtaatgcca
52140 gctacttggt tggctaatgc ataagaatta cttgaacctg ggaggtgggg
gtggcaatga 52200 gctgagattg tgcaactgca ctctagcctg ggtgacagag
tgagactcca tctcaaaaaa 52260 ggtcgaaact gtatttatca tgaacactaa
aatatgtaca cattttagtt aacatgcatt 52320 aaactgtaac aagtcttctg
gcaattgtag ctttcatgag atgcttccca aactgtatta 52380 gatagatgct
aaaattataa attaaaattt tgggtcagac tttgccataa acctggactc 52440
aatttagcac ccccccaaaa aaagtcagat tattcaatta atgcggttgg aaaacctaac
52500 aagttaccta gaaaaaaatt aattggatta ttaacatgtc tttcaccaaa
gtaaattcca 52560 ggtacagcat atattttcat atgaaaaccc tgcataaacc
aagttgaaat ctcagtaagg 52620 agaaaaaatt cttgtgaaag gagaaatgaa
tgaaaggaga aaaaaaggtc tacatgccaa 52680 acaaagctaa taacactaat
gtcgttttta taagcaattg ataaaatgaa ccaagtagac 52740 aaatgaggaa
ggacaattga taggaaatat aaagatagcc aataaatatg ccaaacaaat 52800
gtgcaactca ctgataatca aaaaacataa attaagacag ttggatatta tttttcgccc
52860 ataaaattat cagaattcat aattcctatt gatggtatgg gaaggggaaa
tgggcaaatt 52920 cataccctgc ttgtggaagt ataaatgaat tcagttcttt
tgacgtccat ttgggaacat 52980 gccgtaattg caaaaagtac agagccttag
actagcaaat ctattctagg gaagaatatt 53040 ctaaagagac aaagaagcaa
ttatgtataa acaagggtac tcattgtaaa gttgtttata 53100 ttagttaaaa
actgaaaaaa atctaaaggt atacaaacaa ataaacattt aaatcaaaca 53160
attcccagtt tgtaaattaa tttgaaacgt ctgtatttca acaatttctt tcttcttctt
53220 ttagacctag ctcctggagg tggggacgat tggatctatg atttgggcat
caaatattcg 53280 tttacaattg aacttcgaga tacgggcaca tacggattct
tgctgccgga gcgttacatc 53340 aaacccacct gtagagaagc ttttgccgct
gtctctaaaa tagcttggca tgtcattagg 53400 aatgtttaat gcccctgatt
ttatcattct gcttccgtat tttaatttac tgattccagc 53460 aagaccaaat
cattgtatca gattattttt aagttttatc cgtagttttg ataaaagatt 53520
ttcctattcc ttggttctgt cagagaacct aataagtgct actttgccat taaggcagac
53580 tagggttcat gtctttttac cctttaaaaa aaattgtaaa agtctagtta
cctacttttt 53640 ctttgatttt cgacgtttga ctagccatct caagcaactt
tcgacgtttg actagccatc 53700 tcaagcaagt ttaatcaatg atcatctcac
gctgatcatt ggatcctact caacaaaagg 53760 aagggtggtc agaagtacat
taaagatttc tgctccaaat tttcaataaa tttctgcttg 53820 tgcctttaga
aatacaacca tgcattccgt ttgctccacg gtaattaggc gatggcccag 53880
aaaggggagg ggtgtcaaaa acgacaaaca tagcctctca ttccagctca gctgctcaat
53940 aaacactgtt gaacgaatga atgagtggct ctaggtactg tcaacaaatg
ccgcattttg 54000 cgcatttaca acagctgttt atggtaagga attatgtaat
aaaaagagaa aactcactta 54060 aattcacttt taattgggaa ttttagttct
cccgggctcc cagtttcctt tcctaggatc 54120 tctcacagag cacagattcg
atttccaagt cccgccgcac tcttaccgct cgcatggaac 54180 cttacgccta
gagggcgtgt ccacgaaggg tggtgtctgc gcactgacga ctaatctgac 54240
ggccggaagc tgcctgggtc tacagaggaa cagggcaaac ctctgacttc cggcggcatt
54300 ttgaggcggt cctcctagcg gcctggtagt gtttttgttg ccttttctta
atctacaatc 54360 tcttcgttat ttttcttcct gcgacccagt ttcgcttgac
cctggagagg cggcgggcgg 54420 gttggttctg cttctcagcc atcccggggg
ctcctcgcta gccaagagcc ggttcccggg 54480 agccgcgcgc gcatcgcttt
ctcctcgtcg tcgtcctcct gggtccaggc gcggggacag 54540 agtcgcctcc
cccgctcctc ggagcggcgg cggcggtggt gcctccggac tgcacttgcg 54600
aagggagctt ggggaggaag taagcgttct gtgaattggt gtgggtatct ggggaaggca
54660 ttgagcggac ccgtaatgcg gaggcccggg ttaccccccc ccgtctttgc
ttgagtcact 54720 gggattttga gctttccttg agcatcccac ccttaactct
gcaatagccc cctgtgctca 54780 ggcgtaattt ctcactctga ttatgattct
ggcatttgtc taagggcgat aagtagactc 54840 agacaatagg ctgtacccct
cgttaccatt tgatgtaagc acgggaaccc ttgtatggtg 54900 ttcgtatttg
tgtgcgatgg aagggtgcag caatttgggc ttaaatttag aatcttcctc 54960
tatactcatt ccagatctgt tagagaaaaa catcttactt gtgattggtc ttgttttttt
55020 tttttttttt ttttccctca gcagtgataa cgatttaggt cctgggaatt
gagtgctact 55080 ttatcttcac aagccttagg taggtagttt tggcaactgt
cagaaacggg ggaaagtgga 55140 atagaaagaa gagagtctgt ttggcggcat
tatctctctg taataggcta acgcaattta 55200 tgtggtttga aaattattta
gagttgataa tacttgaatt atgttggtaa gatgttgttt 55260 gtgaagggta
gtcttaaggt atttggttat actatggggc tttcaggtaa ttcgaactac 55320
tttgaaaatt atgggagtat gaagtctctt aagatttttg gatttttaaa gtagttttaa
55380 aaatttggaa aacatcttta cacctcaagt tttcgaagtc cgcgataccg
ttggagaata 55440 aatacttatg cagttcagtc tatgggtata tggtgccagt
tagcggggtc tagttctgta 55500 acatttgaaa ttactggctt tagtacaata
tattggagcg ttttgtgaat acaatctata 55560 gattttcaaa taatttttaa
tttcttaatg aactatttac attataacag atgacagttt 55620 caactagaga
ctagcaaagt tgatgcaagc ttgtaacaat tgcggcttta aaaatagttg 55680
cactctgaaa ctaaggcttt cactctgtgc atctggtagg attcagtttt atcaaatgta
55740 tgcctcttac tggcttcctg attactggtc attctaaatg aacattgcat
attttgagat 55800 ttgcaagctt atgtgatttt catattt 55827 4 423 PRT
Human 4 Met Lys Leu Cys Ser Leu Ala Val Leu Val Pro Ile Val Leu Phe
Cys 1 5 10 15 Glu Gln His Val Phe Ala Phe Gln Ser Gly Gln Val Leu
Ala Ala Leu 20 25 30 Pro Arg Thr Ser Arg Gln Val Gln Val Leu Gln
Asn Leu Thr Thr Thr
35 40 45 Tyr Glu Ile Val Leu Trp Gln Pro Val Thr Ala Asp Leu Ile
Val Lys 50 55 60 Lys Lys Gln Val His Phe Phe Val Asn Ala Ser Asp
Val Asp Asn Val 65 70 75 80 Lys Ala His Leu Asn Val Ser Gly Ile Pro
Cys Ser Val Leu Leu Ala 85 90 95 Asp Val Glu Asp Leu Ile Gln Gln
Gln Ile Ser Asn Asp Thr Val Ser 100 105 110 Pro Arg Ala Ser Ala Ser
Tyr Tyr Glu Gln Tyr His Ser Leu Asn Glu 115 120 125 Ile Tyr Ser Trp
Ile Glu Phe Ile Thr Glu Arg His Pro Asp Met Leu 130 135 140 Thr Lys
Ile His Ile Gly Ser Ser Phe Glu Lys Tyr Pro Leu Tyr Val 145 150 155
160 Leu Lys Val Ser Gly Lys Glu Gln Ala Ala Lys Asn Ala Ile Trp Ile
165 170 175 Asp Cys Gly Ile His Ala Arg Glu Trp Ile Ser Pro Ala Phe
Cys Leu 180 185 190 Trp Phe Ile Gly His Ile Thr Gln Phe Tyr Gly Ile
Ile Gly Gln Tyr 195 200 205 Thr Asn Leu Leu Arg Leu Val Asp Phe Tyr
Val Met Pro Val Val Asn 210 215 220 Val Asp Gly Tyr Asp Tyr Ser Trp
Lys Lys Asn Arg Met Trp Arg Lys 225 230 235 240 Asn Arg Ser Phe Tyr
Ala Asn Asn His Cys Ile Gly Thr Asp Leu Asn 245 250 255 Arg Asn Phe
Ala Ser Lys His Trp Cys Glu Glu Gly Ala Ser Ser Ser 260 265 270 Ser
Cys Ser Glu Thr Tyr Cys Gly Leu Tyr Pro Glu Ser Glu Pro Glu 275 280
285 Val Lys Ala Val Ala Ser Phe Leu Arg Arg Asn Ile Asn Gln Ile Lys
290 295 300 Ala Tyr Ile Ser Met His Ser Tyr Ser Gln His Ile Val Phe
Pro Tyr 305 310 315 320 Ser Tyr Thr Arg Ser Lys Ser Lys Asp His Glu
Glu Leu Ser Leu Val 325 330 335 Ala Ser Glu Ala Val Arg Ala Ile Glu
Lys Ile Ser Lys Asn Thr Arg 340 345 350 Tyr Thr His Gly His Gly Ser
Glu Thr Leu Tyr Leu Ala Pro Gly Gly 355 360 365 Gly Asp Asp Trp Ile
Tyr Asp Leu Gly Ile Lys Tyr Ser Phe Thr Ile 370 375 380 Glu Leu Arg
Asp Thr Gly Thr Tyr Gly Phe Leu Leu Pro Glu Arg Tyr 385 390 395 400
Ile Lys Pro Thr Cys Arg Glu Ala Phe Ala Ala Val Ser Lys Ile Ala 405
410 415 Trp His Val Ile Arg Asn Val 420
* * * * *
References