U.S. patent application number 11/074697 was filed with the patent office on 2005-08-18 for isolated human protease proteins, nucleic acid molecules encoding human protease proteins, and uses thereof.
This patent application is currently assigned to APPLERA CORPORATION. Invention is credited to Beasley, Ellen M., Di Francesco, Valentina, Ketchum, Karen A., Webster, Marion.
Application Number | 20050181414 11/074697 |
Document ID | / |
Family ID | 26929010 |
Filed Date | 2005-08-18 |
United States Patent
Application |
20050181414 |
Kind Code |
A1 |
Webster, Marion ; et
al. |
August 18, 2005 |
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: |
Webster, Marion; (San
Francisco, CA) ; Ketchum, Karen A.; (Germantown,
MD) ; Di Francesco, Valentina; (Rockville, MD)
; Beasley, Ellen M.; (Darnestown, MD) |
Correspondence
Address: |
CELERA GENOMICS
ATTN: WAYNE MONTGOMERY, VICE PRES, INTEL PROPERTY
45 WEST GUDE DRIVE
C2-4#20
ROCKVILLE
MD
20850
US
|
Assignee: |
APPLERA CORPORATION
Norwalk
CT
|
Family ID: |
26929010 |
Appl. No.: |
11/074697 |
Filed: |
March 9, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11074697 |
Mar 9, 2005 |
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10060333 |
Feb 1, 2002 |
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10060333 |
Feb 1, 2002 |
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09734675 |
Dec 13, 2000 |
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6365391 |
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60235557 |
Sep 27, 2000 |
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Current U.S.
Class: |
435/6.16 ;
435/226; 435/320.1; 435/325; 435/69.1; 530/350; 536/23.2 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 25/28 20180101; A61P 29/00 20180101; A61P 35/00 20180101; C12N
9/6424 20130101; A61P 9/10 20180101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/226; 435/320.1; 435/325; 536/023.2; 530/350 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/64; C07K 016/40 |
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:1 or
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 serine protease 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 1999 November;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 1999 April;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 1999 Jun. 30; 878:61-72; McKerrow J H, Engel J C,
Caffrey C R, Cysteine protease inhibitors as chemotherapy for
parasitic infections, Bioorg Med Chem 1999 April;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 1998 October;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 1998 August;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] Trypsin-like serine proteases have been isolated from
patients with chronic airway diseases and may play a role in
respiratory diseases and host defense systems on the mucous
membranes of the respiratory system (see Yamaoka et al., J. Biol.
Chem. 273: 11895-11901, 1998 and Yasuoka et al., Am. J. Resp. Cell
Molec. Biol. 16: 300-308, 1997). Therefore, novel human serine
protease proteins, and encoding genes, may be useful for screening
for, diagnosing, preventing, and/or treating disorders such as
respiratory diseases. For example, serine protease genes/proteins
may be useful in drug development, such as by serving as novel drug
targets for respiratory disease, and SNPs in serine protease genes
may be useful markers for diagnostic kits for respiratory
diseases.
[0012] Trypsinogens
[0013] 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).
[0014] 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.
[0015] 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).
[0016] 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.
[0017] 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.
[0018] Metalloprotease
[0019] 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.
[0020] 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.
[0021] 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).
[0022] 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.
[0023] 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).
[0024] 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.
[0025] 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. Natl. 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. Natl. 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).
[0026] Aspartic Protease
[0027] 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.
[0028] 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.
[0029] 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.
[0030] Examples of the aspartic protease family of proteins
include, but are not limited to, pepsin A (Homo sapiens), HIV 1
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).
[0031] Proteases and Cancer
[0032] Proteases are critical elements at several stages in the
progression of metastatic cancer. In this process, the proteolytic
degradation of structural protein in the basal membrane allows for
expansion of a tumor in the primary site, evasion from this site as
well as homing and invasion in distant, secondary sites. Also,
tumor induced angiogenesis is required for tumor growth and is
dependent on proteolytic tissue remodeling. Transfection
experiments with various types of proteases have shown that the
matrix metalloproteases play a dominant role in these processes in
particular gelatinases A and B (MMP-2 and MMP-9, respectively). For
an overview of this field see Mullins, et al., Biochim. Biophys.
Acta 695, 177, 1983; Ray, et al., Eur. Respir. J. 7, 2062, 1994;
Birkedal-Hansen, et al., Crit. Rev. Oral Biol. Med. 4, 197,
1993.
[0033] Furthermore, it was demonstrated that inhibition of
degradation of extracellular matrix by the native matrix
metalloprotease inhibitor TIMP-2 (a protein) arrests cancer growth
(DeClerck, et al., Cancer Res. 52, 701, 1992) and that TIMP-2
inhibits tumor-induced angiogenesis in experimental systems (Moses,
et al. Science 248, 1408, 1990). For a review, see DeClerck, et
al., Ann. N.Y. Acad. Sci. 732, 222, 1994. It was further
demonstrated that the synthetic matrix metalloprotease inhibitor
batimastat when given intraperitoneally inhibits human colon tumor
growth and spread in an orthotopic model in nude mice (Wang, et al.
Cancer Res. 54, 4726, 1994) and prolongs the survival of mice
bearing human ovarian carcinoma xenografts (Davies, et. al., Cancer
Res. 53, 2087, 1993). The use of this and related compounds has
been described in Brown, et al., WO-9321942 A2.
[0034] There are several patents and patent applications claiming
the use of metalloproteinase inhibitors for the retardation of
metastatic cancer, promoting tumor regression, inhibiting cancer
cell proliferation, slowing or preventing cartilage loss associated
with osteoarthritis or for treatment of other diseases as noted
above (e.g. Levy, et al., WO-9519965 A1; Beckett, et al.,
WO-9519956 A1; Beckett, et al., WO-9519957 A1; Beckett, et al.,
WO-9519961 A1; Brown, et al., WO-9321942 A2; Crimmin, et al.,
WO-9421625 A1; Dickens, et al., U.S. Pat. No. 4,599,361; Hughes, et
al., U.S. Pat. No. 5,190,937; Broadhurst, et al., EP 574758 A1;
Broadhurst, et al., EP 276436; and Myers, et al., EP 520573 A1.
[0035] Protease proteins, particularly members of the serine
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 serine subfamily.
SUMMARY OF THE INVENTION
[0036] 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 serine protease 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 humans in testis, placenta, fetal
lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in
cancers.
DESCRIPTION OF THE FIGURE SHEETS
[0037] FIG. 1 provides the nucleotide sequence of a cDNA molecule
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 humans in testis,
placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone
marrow, and in cancers.
[0038] 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.
[0039] 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 indicated in FIG.
3, SNPs, including insertion/deletion polymorphisms ("indels"),
were identified at 69 different nucleotide positions in and around
the gene encoding the serine protease protein of the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0040] 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 serine protease 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 serine protease 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 serine protease subfamily and the
expression pattern observed. Experimental data as provided in FIG.
1 indicates expression in humans in testis, placenta, fetal lung,
fetal kidney, fetal heart, fetal brain, bone marrow, and in
cancers. 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 serine 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 serine
protease 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 humans in testis, placenta, fetal lung,
fetal kidney, fetal heart, fetal brain, bone marrow, and in
cancers. 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%, 460%, 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):444453 (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. The gene provided by the present invention
is located on a genome component that has been mapped to human
chromosome 4 (as indicated in FIG. 3), which is supported by
multiple lines of evidence, such as STS and BAC map data.
[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. The gene provided by the present invention is
located on a genome component that has been mapped to human
chromosome 4 (as indicated in FIG. 3), which is supported by
multiple lines of evidence, such as STS and BAC map data. 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 the gene encoding the protease protein of the present
invention. SNPs, including indels (indicated by a "-"), were
identified at 69 different nucleotide positions. Non-synonymous
cSNPs were identified at position 30496. The changes in the amino
acid sequence caused by these SNPs is indicated in FIG. 3 and can
readily be determined using the universal genetic code and the
protein sequence provided in FIG. 2 as a reference. SNPs outside
the ORF and in introns 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. The gene
provided by the present invention is located on a genome component
that has been mapped to human chromosome 4 (as indicated in FIG.
3), which is supported by multiple lines of evidence, such as STS
and BAC map data.
[0066] FIG. 3 provides information on SNPs that have been
identified in the gene encoding the protease protein of the present
invention. SNPs, including indels (indicated by a "-"), were
identified at 69 different nucleotide positions. Non-synonymous
cSNPs were identified at position 30496. The changes in the amino
acid sequence caused by these SNPs is indicated in FIG. 3 and can
readily be determined using the universal genetic code and the
protein sequence provided in FIG. 2 as a reference. SNPs outside
the ORF and in introns 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. Enzynol. 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] 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 humans
in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal
brain, bone marrow, and in cancers. Specifically, a virtual
northern blot shows expression in cancers. In addition, PCR-based
tissue screening panels indicate expression in testis, placenta,
fetal lung, fetal kidney, fetal heart, fetal brain, and bone
marrow. A large percentage of pharmaceutical agents are being
developed that modulate the activity of protease proteins,
particularly members of the serine 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
humans in testis, placenta, fetal lung, fetal kidney, fetal heart,
fetal brain, bone marrow, and in cancers. Such uses can readily be
determined using the information provided herein, that which is
known in the art, and routine experimentation.
[0081] 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 serine 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 humans in testis, placenta, fetal lung,
fetal kidney, fetal heart, fetal brain, bone marrow, and in
cancers. Specifically, a virtual northern blot shows expression in
cancers. In addition, PCR-based tissue screening panels indicate
expression in testis, placenta, fetal lung, fetal kidney, fetal
heart, fetal brain, and bone marrow.
[0082] 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 humans in
testis, placenta, fetal lung, fetal kidney, fetal heart, fetal
brain, bone marrow, and in cancers. In an alternate embodiment,
cell-based assays involve recombinant host cells expressing the
protease protein.
[0083] 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.
[0084] 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.
[0085] 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).
[0086] 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.
[0087] 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.
[0088] 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 humans in testis, placenta, fetal lung,
fetal kidney, fetal heart, fetal brain, bone marrow, and in
cancers. Specifically, a virtual northern blot shows expression in
cancers. In addition, PCR-based tissue screening panels indicate
expression in testis, placenta, fetal lung, fetal kidney, fetal
heart, fetal brain, and bone marrow.
[0089] 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.
[0090] 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.
[0091] 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.
[0092] 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 microtitre
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.
[0093] 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.
[0094] 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 humans in testis, placenta, fetal
lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in
cancers. 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.
[0095] 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 WO94/10300),
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.
[0096] 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., GAL4). 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.
[0097] 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.
[0098] 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
humans in testis, placenta, fetal lung, fetal kidney, fetal heart,
fetal brain, bone marrow, and in cancers. 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.
[0099] 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.
[0100] 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.
[0101] 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.
[0102] 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.
[0103] 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 humans in testis, placenta, fetal lung,
fetal kidney, fetal heart, fetal brain, bone marrow, and in
cancers. Accordingly, methods for treatment include the use of the
protease protein or fragments.
[0104] Antibodies
[0105] 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.
[0106] 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.
[0107] 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).
[0108] 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.
[0109] 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.
[0110] 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).
[0111] 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, .alpha.-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.
[0112] Antibody Uses
[0113] 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 humans in
testis, placenta, fetal lung, fetal kidney, fetal heart, fetal
brain, bone marrow, and in cancers. Specifically, a virtual
northern blot shows expression in cancers. In addition, PCR-based
tissue screening panels indicate expression in testis, placenta,
fetal lung, fetal kidney, fetal heart, fetal brain, and bone
marrow. 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.
[0114] 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 humans in testis, placenta, fetal
lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in
cancers. 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.
[0115] 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 humans in testis, placenta, fetal lung, fetal kidney,
fetal heart, fetal brain, bone marrow, and in cancers. 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.
[0116] 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.
[0117] The antibodies are also useful for tissue typing.
Experimental data as provided in FIG. 1 indicates expression in
humans in testis, placenta, fetal lung, fetal kidney, fetal heart,
fetal brain, bone marrow, and in cancers. 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.
[0118] 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.
[0119] 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.
[0120] Nucleic Acid Molecules
[0121] 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.
[0122] 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.
[0123] 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.
[0124] 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.
[0125] 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.
[0126] 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.
[0127] 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.
[0128] 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.
[0129] 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.
[0130] 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.
[0131] 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).
[0132] 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.
[0133] 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.
[0134] 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.
[0135] 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.
[0136] 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.
[0137] 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.
[0138] Nucleic Acid Molecule Uses
[0139] 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 indicated in FIG.
3, SNPs, including insertion/deletion polymorphisms ("indels"),
were identified at 69 different nucleotide positions in and around
the gene encoding the transporter protein of the present
invention.
[0140] 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.
[0141] 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.
[0142] 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.
[0143] The nucleic acid molecules are also useful for expressing
antigenic portions of the proteins.
[0144] 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. The gene provided by the
present invention is located on a genome component that has been
mapped to human chromosome 4 (as indicated in FIG. 3), which is
supported by multiple lines of evidence, such as STS and BAC map
data.
[0145] The nucleic acid molecules are also useful in making vectors
containing the gene regulatory regions of the nucleic acid
molecules of the present invention.
[0146] 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.
[0147] The nucleic acid molecules are also useful for making
vectors that express part, or all, of the peptides.
[0148] The nucleic acid molecules are also useful for constructing
host cells expressing a part, or all, of the nucleic acid molecules
and peptides.
[0149] The nucleic acid molecules are also useful for constructing
transgenic animals expressing all, or a part, of the nucleic acid
molecules and peptides.
[0150] 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 humans in testis, placenta, fetal lung, fetal kidney,
fetal heart, fetal brain, bone marrow, and in cancers.
Specifically, a virtual northern blot shows expression in cancers.
In addition, PCR-based tissue screening panels indicate expression
in testis, placenta, fetal lung, fetal kidney, fetal heart, fetal
brain, and bone marrow. 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.
[0151] 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.
[0152] 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 humans in testis, placenta,
fetal lung, fetal kidney, fetal heart, fetal brain, bone marrow,
and in cancers. Specifically, a virtual northern blot shows
expression in cancers. In addition, PCR-based tissue screening
panels indicate expression in testis, placenta, fetal lung, fetal
kidney, fetal heart, fetal brain, and bone marrow.
[0153] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate protease nucleic acid
expression.
[0154] 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 humans in testis,
placenta, fetal lung, fetal kidney, fetal heart, fetal brain, bone
marrow, and in cancers. 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.
[0155] 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.
[0156] 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.
[0157] 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 humans in
testis, placenta, fetal lung, fetal kidney, fetal heart, fetal
brain, bone marrow, and in cancers. Specifically, a virtual
northern blot shows expression in cancers. In addition, PCR-based
tissue screening panels indicate expression in testis, placenta,
fetal lung, fetal kidney, fetal heart, fetal brain, and bone
marrow. Modulation includes both up-regulation (i.e. activation or
agonization) or down-regulation (suppression or antagonization) or
nucleic acid expression.
[0158] 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 humans in testis, placenta, fetal
lung, fetal kidney, fetal heart, fetal brain, bone marrow, and in
cancers.
[0159] 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.
[0160] 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.
[0161] 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 the
gene encoding the protease protein of the present invention. SNPs,
including indels (indicated by a "-"), were identified at 69
different nucleotide positions. Non-synonymous cSNPs were
identified at position 30496. The changes in the amino acid
sequence caused by these SNPs is indicated in FIG. 3 and can
readily be determined using the universal genetic code and the
protein sequence provided in FIG. 2 as a reference. SNPs outside
the ORF and in introns may affect control/regulatory elements. The
gene provided by the present invention is located on a genome
component that has been mapped to human chromosome 4 (as indicated
in FIG. 3), which is supported by multiple lines of evidence, such
as STS and BAC map data. 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.
[0162] Alternatively, mutations in a protease gene can be directly
identified, for example, by alterations in restriction enzyme
digestion patterns determined by gel electrophoresis.
[0163] 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.
[0164] 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)).
[0165] 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.
[0166] 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.
[0167] 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.
[0168] 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
the gene encoding the protease protein of the present invention.
SNPs, including indels (indicated by a "-"), were identified at 69
different nucleotide positions. Non-synonymous cSNPs were
identified at position 30496. The changes in the amino acid
sequence caused by these SNPs is indicated in FIG. 3 and can
readily be determined using the universal genetic code and the
protein sequence provided in FIG. 2 as a reference. SNPs outside
the ORF and in introns may affect control/regulatory elements.
[0169] 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.
[0170] 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.
[0171] 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 humans in
testis, placenta, fetal lung, fetal kidney, fetal heart, fetal
brain, bone marrow, and in cancers. Specifically, a virtual
northern blot shows expression in cancers. In addition, PCR-based
tissue screening panels indicate expression in testis, placenta,
fetal lung, fetal kidney, fetal heart, fetal brain, and bone
marrow. 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.
[0172] Nucleic Acid Arrays
[0173] 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).
[0174] 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 WO95/11995 (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.
[0175] 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.
[0176] 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.
[0177] 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 WO95/251116 (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.
[0178] 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.
[0179] 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 the gene encoding the protease protein of the present
invention. SNPs, including indels (indicated by a "-"), were
identified at 69 different nucleotide positions. Non-synonymous
cSNPs were identified at position 30496. The changes in the amino
acid sequence caused by these SNPs is indicated in FIG. 3 and can
readily be determined using the universal genetic code and the
protein sequence provided in FIG. 2 as a reference. SNPs outside
the ORF and in introns may affect control/regulatory elements.
[0180] 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).
[0181] 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.
[0182] In another embodiment of the present invention, kits are
provided which contain the necessary reagents to carry out the
assays of the present invention.
[0183] 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.
[0184] 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.
[0185] Vectors/Host Cells
[0186] 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.
[0187] 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.
[0188] 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).
[0189] 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.
[0190] 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.
[0191] 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.
[0192] 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).
[0193] 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).
[0194] 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.
[0195] 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.
[0196] 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.
[0197] 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 67:3140 (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 Enymology 185:60-89 (1990)).
[0198] 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)).
[0199] 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 (Kurjan et al.,
Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123
(1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).
[0200] 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)).
[0201] 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)).
[0202] 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.
[0203] 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).
[0204] 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.
[0205] 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).
[0206] 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.
[0207] 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.
[0208] 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.
[0209] 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.
[0210] 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.
[0211] 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.
[0212] 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.
[0213] Uses of Vectors and Host Cells
[0214] 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.
[0215] 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.
[0216] 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.
[0217] 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.
[0218] 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.
[0219] 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.
[0220] 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.
[0221] 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.
[0222] 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.
[0223] 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.
[0224] 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 1225 DNA Homo sapien 1 cgcccttatg ctgaagccat ggatgattgc
cgttctcatt gtgttgtccc tgacagtggt 60 ggcagtgacc ataggtctcc
tggttcactt cctagtattt gaccaaaaaa aggagtacta 120 tcatggctcc
tttaaaattt tagatccaca aatcaatttc aatttcggac aaagcaacac 180
atatcaactt aaggacttac gagagacgac cgaaaatttg gtggatgaga tatttataga
240 ttcagcctgg aagaaaaatt atatcaagaa ccaagtagtc agactgactc
cagaggaaga 300 tggtgtgaaa gtagatgtca ttatggtgtt ccagttcccc
tctactgaac aaagggcagt 360 aagagagaag aaaatccaaa gcatcttaaa
tcagaagata aggaatttaa gagccttgcc 420 aataaatgcc tcatcagttc
aagttaatgc aatgagctca tcaacagggg agttaactgt 480 ccaagcaagt
tgtggtaaac gagttgttcc attaaacgtc aacagaatag catctggagt 540
cattgcaccc aaggcggcct ggccttggca agcttccctt cagtatgata acatccatca
600 gtgtggggcc accttgatta gtaacacatg gcttgtcact gcagcacact
gcttccagaa 660 gtataaaaat ccacatcaat ggactgttag ttttggaaca
aaaatcaacc ctcccttaat 720 gaaaagaaat gtcagaagat ttattatcca
tgagaagtac cgctctgcag caagagagta 780 cgacattgct gttgtgcagg
tctcttccag agtcaccttt tcggatgaca tacgccggat 840 ttgtttgcca
gaagcctctg catccttcca accaaatttg actgtccaca tcacaggatt 900
tggagcactt tactatggtg gggaatccca aaatgatctc cgagaagcca gagtgaaaat
960 cataagtgac gatgtctgca agcaaccaca ggtgtatggc aatgatataa
aacctggaat 1020 gttctgtgcc ggatatatgg aaggaattta tgatgcctgc
aggggtgatt ctgggggacc 1080 tttagtcaca agggatctga aagatacgtg
gtatctcatt ggaattgtaa gctggggaga 1140 taactgtggt caaaaggaca
agcctggagt ctacacacaa gtgacttatt accgaaactg 1200 gattgcttca
aaaacaggca tctaa 1225 2 405 PRT Homo sapien 2 Met Leu Lys Pro Trp
Met Ile Ala Val Leu Ile Val Leu Ser Leu Thr 1 5 10 15 Val Val Ala
Val Thr Ile Gly Leu Leu Val His Phe Leu Val Phe Asp 20 25 30 Gln
Lys Lys Glu Tyr Tyr His Gly Ser Phe Lys Ile Leu Asp Pro Gln 35 40
45 Ile Asn Phe Asn Phe Gly Gln Ser Asn Thr Tyr Gln Leu Lys Asp Leu
50 55 60 Arg Glu Thr Thr Glu Asn Leu Val Asp Glu Ile Phe Ile Asp
Ser Ala 65 70 75 80 Trp Lys Lys Asn Tyr Ile Lys Asn Gln Val Val Arg
Leu Thr Pro Glu 85 90 95 Glu Asp Gly Val Lys Val Asp Val Ile Met
Val Phe Gln Phe Pro Ser 100 105 110 Thr Glu Gln Arg Ala Val Arg Glu
Lys Lys Ile Gln Ser Ile Leu Asn 115 120 125 Gln Lys Ile Arg Asn Leu
Arg Ala Leu Pro Ile Asn Ala Ser Ser Val 130 135 140 Gln Val Asn Ala
Met Ser Ser Ser Thr Gly Glu Leu Thr Val Gln Ala 145 150 155 160 Ser
Cys Gly Lys Arg Val Val Pro Leu Asn Val Asn Arg Ile Ala Ser 165 170
175 Gly Val Ile Ala Pro Lys Ala Ala Trp Pro Trp Gln Ala Ser Leu Gln
180 185 190 Tyr Asp Asn Ile His Gln Cys Gly Ala Thr Leu Ile Ser Asn
Thr Trp 195 200 205 Leu Val Thr Ala Ala His Cys Phe Gln Lys Tyr Lys
Asn Pro His Gln 210 215 220 Trp Thr Val Ser Phe Gly Thr Lys Ile Asn
Pro Pro Leu Met Lys Arg 225 230 235 240 Asn Val Arg Arg Phe Ile Ile
His Glu Lys Tyr Arg Ser Ala Ala Arg 245 250 255 Glu Tyr Asp Ile Ala
Val Val Gln Val Ser Ser Arg Val Thr Phe Ser 260 265 270 Asp Asp Ile
Arg Arg Ile Cys Leu Pro Glu Ala Ser Ala Ser Phe Gln 275 280 285 Pro
Asn Leu Thr Val His Ile Thr Gly Phe Gly Ala Leu Tyr Tyr Gly 290 295
300 Gly Glu Ser Gln Asn Asp Leu Arg Glu Ala Arg Val Lys Ile Ile Ser
305 310 315 320 Asp Asp Val Cys Lys Gln Pro Gln Val Tyr Gly Asn Asp
Ile Lys Pro 325 330 335 Gly Met Phe Cys Ala Gly Tyr Met Glu Gly Ile
Tyr Asp Ala Cys Arg 340 345 350 Gly Asp Ser Gly Gly Pro Leu Val Thr
Arg Asp Leu Lys Asp Thr Trp 355 360 365 Tyr Leu Ile Gly Ile Val Ser
Trp Gly Asp Asn Cys Gly Gln Lys Asp 370 375 380 Lys Pro Gly Val Tyr
Thr Gln Val Thr Tyr Tyr Arg Asn Trp Ile Ala 385 390 395 400 Ser Lys
Thr Gly Ile 405 3 38844 DNA Homo sapien 3 ttatattcat aaaagtaggc
agtaagttga agatttattc atataggatt tagtagctgc 60 agctttaacc
tgtggcttct gtagcttttg taatctggca gtgcgcatct gctatattat 120
ctaaatgttt cctcaaaagg agaaacactc taacaactta tcaccctagt ctgctggcca
180 ccattttccc tcagatgctc acagcttctt ccgtgggatt tgaagatatg
acttccatga 240 cacttgatca gtatgtcaat gggtattgaa ccactcttca
gctctgatcc cacggttcag 300 ttcctttcag tgtgactatg tgtcttggtg
gtgggagatg tgattctttt atctactttc 360 tccatttatc ttactcagag
gaactgtgct ctaataggga aatagattga aagcttataa 420 atttccttga
gttttaactt ttctcctttg gtcttttttt cttttcaaat gacttgaaga 480
cacattgata agattctatg agaaaatgaa gagttgaaca aattgaatat gtatgagtga
540 atgaatagat taatacataa atgataaatt tattaaataa tttgaacgaa
atcaatcgag 600 aggcaccgag aataaatttg tgtcctagaa gtaagaagac
ctgagtttga gataactagt 660 agttctatta tactggagaa attacttaat
catcactgga cttcattttt ctcatatgga 720 aagtaattca atcacactaa
acaatcttta aggtctcctt cacttataaa tgtatgtttt 780 aagccattta
ggaggttaaa taatgtcatg tcccatggga cttctgtttg ttgttctatt 840
caagcatgtt agcttgtttc tatcacagga cctgctgcct ttccgcagcc agttctctag
900 attattttta atcagtcggt gcacacatgg tcaatattta ctcaatagaa
ttcaggtttc 960 ccaaattcca tgaggattct tgattaattt tattacttat
gccaaaacta ttatcttctt 1020 aactatttta ggtccaaaca gttttaactt
ttatcctggc atttatatat aaaaaacttt 1080 tgtaagaccg ggtgcagtgg
ctcatgcctg taatcccagc actttgggag gccgaggtgg 1140 gtggatcacc
aggtcaggag atggagacca tcctggctaa caccatgaaa ccctgtttct 1200
actaaaaata caaaaaatta gccgggcgtg gtggtggacg cctttagtcc cagctattca
1260 ggaggctgag gcaggagaat ggcgtgaacc tgggaggcag agcttgcagt
gagcagagat 1320 cacaccactg cactccagcc tggcagcctg gatgacacag
cgagactccg tctcaaaaaa 1380 aaaaaaaaaa aaaagaaaaa aactgtttta
tagtcaaaag aaaaactttc tataaatcaa 1440 ccaatcctgt gaagaaaata
tgaaaaatat cctctgtttc caaaaaaatt taggctatca 1500 atatatacac
ataaagagat aaactctgat aaattggata aataaaattc actataatag 1560
caagttttag agaacaagca cgggagttag tcgacctggg cccttaaaca gatatcctct
1620 ctctcatcct gtgttatttc ctgtgtaatg ttggtatcat tcctgcctga
ctctcataga 1680 tttatatgat tcctactctg tccaggtgcc ttattgggtc
ttagcggtaa aaagatgaac 1740 aaggctaatg cagcccattg agaagctatc
tgtaagtgaa catacatgca aactaatact 1800 tgattcaatg tgagaagcac
tgttgctgat cataggtgcc agaagaacag caaagagtta 1860 ttttttcctc
caaaattgtg gaaaaatttt tatccccggt gtgatgcaat ataaaataca 1920
cagcaccacc tttgaagtat tcttgccaaa tgaatttaac caaaatctaa tcaagacttc
1980 agagctaaag aaaatctaaa ggtaatccaa tttataggaa atgagggata
taaaagaaca 2040 agttaaataa taccacagga aagcattcag acaagtccag
aaagtaagat attctaaagg 2100 atgtttagct tgatctcttc aacagtcaat
gtcattaaaa actaaaaaag aagcaggact 2160 cttttagatt aaaagagatt
aaaaaggcat aacaaacaag tgcactgcat ggtcctcgat 2220 tatgtcttgg
cttttacaaa tcatgtgtaa ttataatgaa accatggagg gaacttgaag 2280
atggactggg tattagatga tatggcagaa atatcattaa ttttttagga gtgttaagag
2340 tatcatggtt atgttggata tatcctaatt gtctataata atgatttggt
aaaaagtcac 2400 gatgttttat ttcacattaa aatatagcag cagaaaaaat
aaatgagcca aatacagtaa 2460 aattttcaac aattgatata ataatgtgat
atatatatgg atgttcaatt atactattct 2520 tagtaatttt ttatgtctga
acattttcat aatacttaaa aataaaagat aaaagataaa 2580 aataaatgag
ataatagatt taaaatcact ttgtaaactc taaaaggata gacagataaa 2640
agagataaca aagtgctgga gaaaggagga atggtccctt ttcaagcatg tatgccacct
2700 tggaccatgc tgctaagaga aaccattcct gaccaccaca aagaggccac
caaatgcctc 2760 taaaatagaa agcaggagca acattaggat tcccagatcc
tgatattttt tttttaacac 2820 atcttctcag accaagatga cattgaacaa
aattaaagac ctttttgcag ggaaaggtag 2880 gctacagcaa cttgaacttg
tctaaggaga gctggaaaac ctgcaagcat tgctatctga 2940 gagtaaccag
tgggcccttc cttttctcag gacagtggga tttggcaccc gaagcagaaa 3000
tgctgaagcc atggatgatt gccgttctca ttgtgttgtc cctgacagtg gtggcagtga
3060 ccataggtct cctggttcac ttcctagtat ttggtaggta aaattaaaga
tttcactcta 3120 tttgatttta tttttctgca aagctccatt tacatatatg
taaatgtaac ttcatctaaa 3180 aaattgcaca tttaccttca aatttccaca
gagtatattt aactgtttca gtcatttcat 3240 caacaaacaa gtactaaatt
cttattatat gtgagtactt ttctggatat tcaagataca 3300 gctttaagca
aagtagacag atttctaatt tccttagagc tctcaaccca gaattctttt 3360
gagaatctac acaaaaagat caaaaattgt aattgtctga aacttactag taattataat
3420 aaacaactca tcacttatta tatattaaaa tgaaaagcta tgataaatta
gttattaaaa 3480 ttggctcttt tactcatgaa ccatcatttt ctgtccaaca
tttctaaggc aaaagaaaaa 3540 cacttgtcta ataaaataag gaatttcaaa
atgattgaaa acctatacgt atgacacaat 3600 attatcattt atttttagag
aaaaaaaatt ttactctttc caaaacaata ttcagggatt 3660 atatttttat
caactaatat atttgtaatt acacaaataa tgcacttcaa gattctcttt 3720
ttacattcag tctctttctg gggagaatgc aagccattta cattttttca caaatctcta
3780 caatgtgact ctcacatgga tgtatgtgat aaaacaaata actcaggctg
ctcactttaa 3840 cgctcttatc tgctgtcacc ttcacagagt caatggggga
gcaaagactc tacttggagc 3900 cttaaagggc ttaagatcat agtcctaggc
cttatatgat aaccccagct gtagtttata 3960 ccattggcaa aagattctca
ggtcacttta tttggttgca taaaagtctc tttacaatga 4020 gagtaaggtt
tgttaacagt atggattata tgggtaagta atcaggatgt ccaaaaatgt 4080
attacaaggt ccagagattt cccacttaag acatatgcct tcctgatatc cctgtttctt
4140 tccttggttt gtagtctcga aacccactcc ctcttccctg agccaggctt
ctcaaggatt 4200 gaggttgttt tgtatttttc ccattctcta tctttaactc
tgtatctttc ttactccctc 4260 tgggccttac tcctcagatt accaaattcc
ttaggagtct caactgcttt cctttcttac 4320 atttcctaat agatttatcc
ctgtttcatg ctcgtcttgt cttcaatctc agacagctct 4380 tctctacact
ttcttttcag gtttttctta gtgtgcctgg ctctcttgtt aaaaatcaaa 4440
attcacaagg acattcactt atctctactt ccactagagt gtatgatggt acacatttca
4500 actcagcaag gagcaatgta gcaatgaaat gttcaagctc tacagctaga
ctggatttaa 4560 aacttggaca ggccacctac tagttacaga acaatttact
taatgcctct gtgccttaat 4620 ttccttatct gtaaaatgaa ggtgatacca
atcttagaga gctggtgtgg ggattaaatg 4680 ggctaataca taaaaagtgc
acaggacagt gcctgccata ttgtagaaac tcaataaatg 4740 gcagctatta
taattgatat aaaacattaa ctgttatttt ttaaataaaa ctcaattatg 4800
aagaggctca gggacatatt caagatttat attggcccca ttgtaattga gttctgaaat
4860 ctttgtccaa accatttagt ttcctatttt tcatttccat tgcagaccaa
aaaaaggagt 4920 actatcatgg ctcctttaaa attttagatc cacaaatcaa
taacaatttc ggacaaagca 4980 acacatatca acttaaggac ttacgagaga
cgaccgaaaa tttggtgagt caggtaaact 5040 tctttttatc atagaataat
gcaagtggaa gggattttgt ggatcatttc tccatttcta 5100 aaaacatgat
tttcagaccg ccaacattag aatcatcttg cagattgcta ggccccatcc 5160
cagacctgct taatcagagt atgatgagat gggtaggtgg ggagaggaga gtaagggaat
5220 ctgcatgtct aacaaatggg tgattctaat aagcctctct ttctaactca
gctaccttat 5280 ttaaaggtaa gagaattgag gccaagatat cctagcccgt
ttcttcccca attccaccac 5340 gtttcccctg tagaaaagcc taatcatacc
aaaactagtt tttataagtc cacacacttg 5400 tttgtaagac cacattttaa
gattttgagt attttcagaa tttacgttca tcttgtaagt 5460 atattgataa
agacaaaaaa ccagacttat tttgtagtaa tcaagtcaaa tgctaataat 5520
tttgttaaag ctaaagtgca agactgctcc caaaaagaaa aaaagcacac tcagttgtat
5580 aatcattcca ctcagaatgc ccatgaactc tcactcaaaa actaggttca
aattaatttt 5640 tctaacaagg aagcacagaa gcagagactt attttaaaaa
gaaagaaatg acaaatgtat 5700 tggtttgttt taatcaaaga accattttta
agacactttc tttcccaaat catctaccat 5760 tttttcctgt catcatttgc
tctttgtcca tagtatacct aatggcatca tatttacaat 5820 aatattgtag
agtttataat ctctattttc agttaacatt aaatcattca caatttctta 5880
attttgtggt ttcatctttc ccaaccaata attaatgtct acagattgat atagattctg
5940 cattctttca catgcagagc atcttataaa agagcatttg caatcagttc
ttaagttatg 6000 ctaggatgaa cggggagcct gcaccaatac acccaaatac
cttctctact cctccagtcc 6060 taagtgactc cacataacct cctcgatgca
aaaagagaaa actcttaact tgccttagtt 6120 aaaaagataa acacaccttt
gaatgatgga aaatgttaca atttactggg aaattttgaa 6180 atttgtttca
tttatatttt atggccaaca ttactgctac tgttgttgtt gtaagttaac 6240
taggcaattc tgtctttact gaagtaaacg gacaagaatg caataggtct taaaagaagt
6300 gagagaaatg cagaggtgca tgttgaacag aaactctatt taaaagtgga
gttttaagtt 6360 tcacctaagc atgtgttcct tcaaaggcta aggctaagtt
aagtaaggac acattatcat 6420 catgggtacc tgcaaggccc ttctctggtt
gtcattattt atttatcctc ctttatcacc 6480 atagcataag cccttaccct
ccccccttgc aggaaatcat tctatgtttc atgtggtatt 6540 cttttgtttg
tattcattct tacaaaaata tgttttgcta ttttgcgtac acttgctttt 6600
aacttacatt ttgtgttata aatcactttt gtttcatctc tttttactga gaacttttta
6660 aaagatatat gttactaaat atacctttag tttattgctg ttagctgcta
attcatagtg 6720 tgtatcttcc atatttacct gcctgtcatg ccaagaaatg
ccacactaaa cagactccta 6780 cttaccccct tatagaccta tgcaagtact
tctggaagca gaattactag gtcattgaat 6840 gtacatatac ttaacttgac
caattggtgc aggtttgctc ttcaaaatgg ctgactcagt 6900 gtgcacgccc
atctacaatg catgaggatt tctatgtccc cacatctaac caacacttag 6960
tgtcttagta tgtttaggct actacaacaa aaaataccat aggctgggta tcttaaacaa
7020 caaacaatta tttctcatag ttctggaggc tgaagattcc aagatgaaga
tgatcaaggc 7080 tctagcagat gtctggtgag agcctgcttc ctggttcata
gaataccatc ttgctgtgtc 7140 cctcatggca gaagccataa gagaactttc
ttttgtaagg acactaatga ctttcatgag 7200 aactccaccc tcatgaccta
actatcctcc aaaggcccca tctcctctat catcggtttg 7260 ggagttaagg
tctcaaaata taaatttcag gggaacacaa acattcagtc cacagcactt 7320
ggtattattt ggctttctaa atttgccacc ctaatatgta taaagtagta ttttatttgt
7380 gatttaattt gcatgtttct aattactaat gagtttgtgc attgttacgt
ataattatta 7440 actttttgga ctttcatttc tataaattgc ctgtacatat
tatttgccta tttttctgtt 7500 aaacttgctt tttcacctta tttgtattgc
tttgcagaag ttctttacat tttctggata 7560 ttgatagtgt gttggttgtg
gacactgcgc ttatccattc tgtcttctac taatatggac 7620 cgtgttgttc
tttatgaaac cgaaatctgt aactgaagta atcatttttt cactgttttg 7680
ccttatgatt gtattttgaa gcttttcttt aagaagtcct tcttcccttc taagacataa
7740 aaatatttta ctatgttact tattaacctt atagttttat cttttacatt
aggtctccaa 7800 tacatgtgga atccaccttt ggatgtgtta ggtagattca
gttttttaat tcatatagtg 7860 agccagtttt tgaatataac tagttaaaat
atcttggctt ttcctaatat atggtattat 7920 tattgagttc attgcatgca
tttcttggca cctgggtctt gcagaaaagg aaacatgaat 7980 ctgtctcctc
aaattgcttc caatcttttt ggaaagatgt gagtaacaca catggaattg 8040
aatatcatga catgatataa ttaagggcta aattacatgt tgaggacagt aagtacagaa
8100 aaacttcaaa accaaacaag ggttcccatg gtcagaaaag gctttatatt
attttacctt 8160 tgtttaaatg agacaggtgt ttttctcctc ccatcccgca
ccaggttagc tttagaagaa 8220 ttacaggaag agtttatgcc tcatcctgag
ccacacctgt ttgttgttgc taaatcccaa 8280 tgaatacaac cagattcttc
tctctgtcct atatgggtgc taattagaca accaaggaag 8340 aacaggttgc
acgtcctgtt cttcctcaca ttgggcttta ctgatttgaa tgcaaattga 8400
gatgcaaaag taaaaatgag ttcatattta gatattgcta taatccgccc ctgttccctg
8460 agatagtgga gcagacatat ctcatctctc atatcattct tcagagaagg
gtccattaat 8520 cagacattac tgatgtctga ttactgccgg ctggccatcc
tgcaggtgga gaagcatggc 8580 atccagcaga aactgacagc atgcactttg
agggagggaa ggataagcca ggaatttatg 8640 ctgaataagc tgcctaagta
tacatgttca ataagttcta ggggaagtca caaatactta 8700 tgaaaggaga
aacataacta tgtgcaattg agctttatgt ctcttcatgt gttgcatgtt 8760
caaaaaatgg tggcattagc atgatccaag ggtggagttt tcagccattt gatgttcaaa
8820 ggtgaagcag aggacacaaa acccttacta tgcatcctct gtgagtcagc
caaaaccagt 8880 ctggactgct agctagatta acaaagaaaa aaagagaaag
aagatacaaa taagcacgat 8940 cagaaatgat agaggtaaca ttacaaccaa
tcccacagaa atacaaaaga tcgtctgaga 9000 ctcttatgaa cacttctatg
tagataaact agaaaatcta gaggaaatgg gtaaattcct 9060 ggaaaaacac
aatcttccaa gattgaatca gaaagaaatt gaaaccctga acagaccaat 9120
attgagttca tacttaaatc agtaatttaa aaaacttacc agccaaaagg aaaaaaaaag
9180 gcccaaacta gatggattca cagccaaatt ctaccagacg tacaagaaat
agctaggacc 9240 aattctagtg aaactattcc aaagaattga gaagagactt
cttcttaaat cattctatga 9300 agtcagcatt accctaacgc caaaacctca
caaagacaga atgaaaaaag aaaattacag 9360 gccaatatcc ctgatgaaca
tagatataaa aatcctcaac caaataccag caaaccaaat 9420 ccagcagcac
atcaaaaagt taattttcca aaatcaagta ggctttattt ctgtgatgca 9480
agactggttc aacatatgta aatcaataaa tgcgatttac cacataaacc gaattaaaaa
9540 caaaaatcat acaattagcc aggcatggtg gctcacactt gtaatcccag
cactttggga 9600 gaccatggtg ggcaaattac ctgaggtcag aagttcgaga
ccaacctggc caacatggtg 9660 aaaccccatc tgtattaaaa atacgaaaat
tagccgggca tggtggcagg tgcctgtaat 9720 cccagctact cggagggctg
aggcaggaga atcacttgaa cccaggaggc agaggttgca 9780 gtgagccgag
atcgtgccat tgcactccag cctgggtgac agagcaaaaa tccatctcaa 9840
aaaaattaaa aatttaagaa aattaaaatc atacaatcat ctcaatatat gtagaaaaag
9900 cttttgataa aattaaacat cccttcataa taaaaacact tagactaggc
atcgaagaaa 9960 catacttcaa aataataaga gccatctgtg acaaacccac
agccatcatc acactgaatg 10020 ggcaaaagct ggaggcacta tccttaagaa
cagggaaaaa gacaagaatg ttcactctca 10080 ctactcctat tcaacatagt
actagaagtt ctagaaagag caatcgagca ggagaaagaa 10140 ggaaaatgca
tccaaatacg aaaagaggaa gtcaaattat ctctctttac tgacaatatg 10200
attatatgcc tagaaaaccc taaagacttt acaaaaagtt tccaaaactg ataaacaact
10260 tcagtaaagt ttcaggatac aaaatcaatg tacaaaattc agtagcattt
ctaaacaata 10320 atgtccaagc tgagaaccaa atcaagaaca caatcccatt
ttcaatagcg acacacacac 10380 acaaatgaaa tacctaggaa tacatctaac
caaggaggta aaagatctct ataaggagaa 10440 taaaaaaaca ctattgaaag
aaatcggaga tgacacaaat gaatgcaaaa acattccatg 10500 ctcatggatt
ggaagaatca atattgttaa aatgtcccta ctgcccagag caatctacag 10560
attcaatgct attcctatca aactaccaac ataattttcc acacaaagtt agaaaaagct
10620 tttgtaaatt tcatatggta caaaaaaaaa aagccccaat agccaaagga
ctcctaataa 10680 aaaagaacag agccagaggc ctcacattat ctgacttcaa
actatacttt aaggctacag 10740 taatcaaaac agaatggcat tggtcaaaaa
cagacatata aaccaataga acagaataga 10800 gaacccagaa ataaagccac
acatctacag ccatcagata ttcaataaaa ttaacaaaaa 10860 taagcaatgg
ggagagaact ttctattcaa taaatggtgc tggaatagct agctagtcag 10920
aagcagaaaa atgaaattgg actcctatca ctaaatacaa aaactaactc aagatgcagt
10980 aaagaattaa atgtaagacc acaaacaatt aatacaagaa ccctagaaga
aaacctagga 11040 aatactgttg tagacatcag tcttggcaca gaatttagga
ctaagtcctc aaaagcaact 11100 gcaacaaaaa caaaaattga taagttggac
ctaattaaac taaagaactt ctgcacaata 11160 aaagaaacta tcaacagagt
aaacaaacaa cctacagact gggagaaaat atttgcaaac 11220 tatgcatctg
aaaaggtcta atgtccagaa
tctgtaaaga acttaaacaa ctcaacaagc 11280 aaaagaaacc aagtaacgcc
attaaaaagt aggcaaagaa catgaacaga tgcttcacaa 11340 aagaagacat
acaacgcagt caagaaacat atgaacaaat gctccacatc actaattatc 11400
caagtaatgc aaatcaaaac tacagtgaga taatatctca taccagttac aatggctatt
11460 attaaagatt aaaaaaataa catgctgatg agactgcgga ggaaagagaa
tgcttaaata 11520 ctgttggaaa cgtaaatggg ttcagccact gtggaaagca
gtttggagac ttctcaaagt 11580 acttaaaatg gaactactat tcaacctagc
aatcctactt actgggtgta tacccaaagg 11640 agtataaact tttttcccag
aaagacagct gcactctcac attaattacc acagtattca 11700 caatagcaaa
gatgtggaat caacctagat atccatcaat ggtggattgg acaaagaaac 11760
tgtgagatat atatgtatat atatctatat ataccatgga atactatgta gccataaaaa
11820 aggatgaaat catgtccttt gcagcaacat ggatgtaaca ccacaaggaa
ggcactttta 11880 tctcctcttt acaggtaaga gaaccaagct tctgaaatta
aggtccatag ctggaaaatg 11940 atggagggga gatttgaagt catctaggca
actccacaca tgtgctcttt ccactaaatt 12000 gttctactgt caggaaggga
ctcagctaag acagaagata aaattattaa aatctaaatc 12060 aattcttctc
tcatttcatt ttttaaatcc atgaagatta taaatcctct atgctgtgct 12120
agctaacttt ttcttgacag atacattagg tatacttatt agagaaaaat attctctttc
12180 tcatttccct gtatcagttt ttggtgagga aggcaaaggt aggaggaact
gtaatagaga 12240 aagatgaagg aagctgatgg atatattgac atgtgtatgt
acatctagtg tgaacaatct 12300 atagttggaa gaaaggtgtg gatgggtatg
ctttttgagg gaagtttttg agaaaagaag 12360 taatatgaac tatttctaaa
tttcctgata aagttgtaaa tacagcatag tcttcacagg 12420 agaatctatt
tagtttatca tcatcattca gcaaatacag catgatgtta ggcactataa 12480
aaggctaaga aaaatgattc tctctctctc ataaactaat ccaatttaga gatttagaag
12540 acaacaaatc tggagaggac atgaaccttc taaataatga ccttcccttg
ctttgggtat 12600 cctggtttta aatattttta gtacagcttt aaatagatcc
aaatgagata ttttcctctt 12660 ttacaaaagc aattcaaaga tctaggtttt
tgttgtacac tgagaattaa tacttttttc 12720 tttaaaatcc ttaattgcaa
atctttaaat tctataaata ttttgccttg tgatctcaga 12780 aatataagcc
aatttgggat atggatatct aatatattgc tacttgttac acgtgagtag 12840
tgacagatgt ctgtccattt ctttctgaca ttccacaaag aaacactgaa gaaggaccag
12900 tgcaatcaaa gaaatgactg atggcatcac aaaatatcac atcccatttg
atgatctgat 12960 tacctttttg tttagggtga tcagaaagtc acagtttcat
ggcaccctcc acacccacac 13020 accttgtatg acactggatc caactgcttt
ctccaataga cacagcactt aaagatgtgg 13080 cagttaggct tgaccccaag
aaggccaaaa agccttctgt gagcatcact cagtgctcag 13140 gttgactaag
ctctatccag gcttgagaga atggttcata gctgacttct tggatccaaa 13200
aaaaaaaaaa aaacacctag agttttatac agatatgata cgaacttaaa aggactgcac
13260 taaaaactac caagattatg attcttattt ttggagagta aagaaaatag
gctgcctttg 13320 gagaggggtg caacagtttc tgatcctctt acaaactgct
tgctgcccat cagtgggtag 13380 gaggtcttag tgagaaccta cctgcatgct
catcctgagg taggcactgt gaaggcgtta 13440 acaggctctg aagctacatg
gccctggttt cagtgaactc tgtggtgtca acttgggcaa 13500 gtcacttcct
cttctatgaa acgtgaataa tcatagtact caccttagag ggctgatttg 13560
aaagcaaatg agctcaaaca caatgacatc tgtgcttggt gcatatatgg cagacaacag
13620 tgattcccac tattataatt attacagtct taccaaggag gagctttcca
caaataatca 13680 attacctaaa atgtccaaaa acaggaaaaa aaaatctctt
ccgataattc atgtgtaatt 13740 ttcttttttc tctaggagca ttgatctcaa
cctgatgtaa agcaagcact ttaaaaagtc 13800 ttataaaatt ttcctggtaa
atgcaaaact ttctgataaa taaattctca cctttttatc 13860 aatttgttaa
ttcaacaaaa atatactaca taccaacagc atgcaaagca ctatgctaga 13920
ttttatagac tatgaaaaga taaattgcca tctctatgca taaagggttt gccatttaat
13980 aaaagagact atatatttgc ataaatatat agtgaatata ttgcataaat
atataatata 14040 tgtttacatt aaagaataaa aggtataaga gggataagaa
aaattgagac agagggaaga 14100 caggtcagtt tgagattaac gaatatcccc
aaagaaggta ttatctgaga ttggccttga 14160 aggatagttg tgattcagga
acacagaact tgcagaatga gaaggttgtt acagaccaaa 14220 ggaacagcct
gagaggcgtg agtatgcagg aaaatgaggg ccatgcctga aagtactggt 14280
ggtgttgaag atggagccag gcaagttggt cacagaggga gaggaccttg aatgtctaac
14340 attgtggaca gaggctcaaa ggctcaaatt ccctattttt accttgagtt
caatccttgt 14400 ggcaatgaaa cctcagtgaa gctttattta aggctaaaag
tgtcttttaa aaatccctct 14460 tatataatat cctttgcatg ttactcttgt
tgtaattagg agaaagcaat aggatctaaa 14520 gttttttttc acagcatggt
tttggtttct ttaattctaa ggagctcacc tggtgttacg 14580 ttggaaaaaa
cagcttttat atctcattta tattccatat gccagtctgc agtgacatat 14640
ctatctgagg tttacagtgt tagccacaaa acactcccta agtgaataca ttgactgctg
14700 taaggggagc cagtcaggaa gcacctgcag agaaaagcag gcaacatgta
taaacagagt 14760 taattcagga atgaaagctg aatggctggg cgagtctgtt
tgtttgagtt gacagcctct 14820 ccctcactct ttcattaaat atccaactaa
ccttcaattg ccctcttgga acttaatctc 14880 agtgtaattt ccagcatgtc
aaaattatca agcagaaaga gatactaccc tgaaagaggg 14940 tcttttgttc
aatgctagga gacaaactcc aactacaaaa ttctagaaat gccctaaaga 15000
gagagatagg atagatttac aaattgctaa tgctattagg ttgtatagat aacaatagat
15060 ttataacaac ctggcacaca gctttaaata tataagtttc tctgaaactt
ctgggaactt 15120 ggaatgccag aacgttggca aaaagaatgc ttctaataat
gaaagccatc atctgccatg 15180 gaaacaattt cagggtcttt agaaagctag
tttatacata agctccattc tacaataaaa 15240 cttatgttca tgttttttct
gattttcctc ctgctgtaaa ttcattttat cagaattctt 15300 tttaccagtc
cctctgcccc atttctcaaa gcgttgtcct cagactacct gtatcaccta 15360
aagattctaa ggcctcctcc gatgtagtaa atgagacttt tctagagaga gagtcctaga
15420 attttataaa gaaggatcct ttttattatt gtgatcacca aagttacttc
tgcctagatt 15480 cttctcatgt tatttttaca gctcctatct tcccagacaa
cctaacaatt caaagataaa 15540 attggtgctt ggtttagaca ttcatagcag
gcacggtgcc agattgatga tgtcatccag 15600 agtcaaaaac ttcatccaat
gccttcacca aaaagttaca aatggccagg aatcaaatgt 15660 ggttgaactt
attcagaggg taattacaaa acaaacttct ttaaataccc aactgctatt 15720
tgcttttttc cttctaaatt gtatcacttc tctccctgtt ccattttgtt tgccttttta
15780 ttttttggaa tccctcacct ccatactgag tagtagagct ggctgtgggt
gatgagagag 15840 aaattgttat aacaaagtca ccctttcaaa aacatgtctt
ccaaaagaat tttgtttcta 15900 gcagataaac cccacaccac ctcagctaaa
tggggctttc tttatttaag taccaataaa 15960 gacatatttt ggatactagc
aatttatttt ccaaatgcta tctttgatct taagtttaag 16020 gctattacca
aatctatatc tctacaagtt ttatacttta ggtcaataaa ttacttgata 16080
acttattact atgtgttcta caaaagaaac cgaagtaaaa tttacatcac atttaacagg
16140 gtggttgtgt gattgagtgg gaagaggcgg accctacaga tagaagactt
gggtttcagt 16200 cccagcttac tagtatctgc gtgatgccag ggaaattcac
ataatgcctc tgagtcacag 16260 atttctaaca ggaatgaaga tacttcttcg
cagaattgtc attagagtta aagaagataa 16320 caaataatgt ggttcctgat
gaggtattta tgaattcctg agcatgctaa ggaagttata 16380 acttgtttct
tgatccctga aacagctttc cctatatttg tgtgtgtgtg tgtgtgtgtg 16440
tttcagtcat gcaagttggt ttttcttctc attccttgag aatttaggat attttgtgcg
16500 cacatttggt tcttctgtcc aacatgaact gtagtacctt acccacattg
agatgacact 16560 atttctacca agtgagtgct aggggatact gcaagccgaa
tgccaggtgt gagagaccac 16620 agcatcacaa taccgtggca gtagattaaa
gctgtgcata tggactaaaa gcagtggctt 16680 tgcttctcct accttggtga
cataaactga gtaacaaatt tgacctaata ctggaatacc 16740 acctaattct
tttttcctcc ctgatttacc ctagagtcca caattgacaa taatttaaaa 16800
attttggctc tctcttaaat ccctaatgcc tcctccttac accttacaag caaagacctg
16860 cagagctaag acctgtaatg ccaggatgga ggctagagga ccatcagcaa
ttaactacca 16920 aaacttaccc aacattttat atctgtttaa ccttcatagc
cttatgagta gcagatcaat 16980 atctttgttt tacaggttag aaaactgagg
ctcaaattga ttcagtaact ttgccaagat 17040 tgcccagttt gggaaaagta
gtatacgctc aaatccagga ctgaggcagg gttttctttg 17100 tcaccactca
aagcctctct gaatatccta tctctgctct gtatctctct gctactcctt 17160
ctatggtgtt ttagcaagat atcttctact ccagaaacct actctagcac agtagaatta
17220 cttgggtagg ttttttaaaa atatgagtgc ctaggtcccc tctagaccaa
tcgaaaccaa 17280 aattcttgga gaggatccct ggcatccata aattttttta
attcatcaaa tgattctgtt 17340 gcactgtgaa agctgagatc caccaattta
aataatgatg ttagttctgt gaaaaaattt 17400 ttgattgctt taacatttaa
tcaaggatat attcctatta taaaatatat tattaacaca 17460 tagtttctct
cttgttgtgt aacaggtgga tgagatattt atagattcag cctggaagaa 17520
aaattatatc aagaaccaag tagtcagact gacgtatgta tgtttgggca aaggtggaat
17580 cacaagactg gagggaaaag gaacaaagga gacagggact ctcatgtatt
gtatgtctcc 17640 atggactagg cttttggcta gaatttttca taaacattac
ctttaaagca gtcttgaagt 17700 atagggctga ccaccgtttt gtcaacaaaa
agactaagat tcaggaaggg taagaaatat 17760 gttcaaagtt caccaactga
cagtttccca aagtgacaga accaggaatc aaaccccatt 17820 aacttattgt
gaggcctgga acctaccaga acccatgacg tggggaaaac ccagcagctt 17880
gtcgttgcat gcaccaagtt atattatgtt gacaattata ttatttcaac cacgttaagc
17940 aggcaaactt ggctataaaa tgggttcaca aattttacct gtaatgtaac
cgaatgacat 18000 aaggcatgcc taaacaaaaa gatattcctg ttgtaataaa
ttttctttct gtcatggtgg 18060 agggggaaga ctcatatcag ttgcagatat
tgctcagaag tttcaattgt gttattttga 18120 aaaactacat agcagaacac
gcatgtcata tacacaaatc catgagcctg tatgactcat 18180 atttcttaaa
gataaagaaa aataatatat tcagattttg atttatttga agaaaataat 18240
tatccctttc tcaccaatag actaataatg ctttgttggc aggtgtactc aaagttctct
18300 atgtcttgac tgagtaacta gtgacttccg taaggatttt ataacataaa
ttgggtaatt 18360 cctacaatac ttaggaggga aaaagcatat aaatgctaga
actttctaga tttcatgttt 18420 tctgttttca aattctcctt taccatatta
ttgtagcaac attattatac tcctgtgaac 18480 tcctttggat ggtagccatc
actatataat acctggtaaa aatgttaatt cctcagattt 18540 aagaagtaaa
attagtcatc tgtttgccaa tttgacataa aattctagtt atttagatct 18600
ttatattcca gagcctaaat gaacaaaaat acataaattg tctcagaatt tccttttagc
18660 caaaagattc agggagatgg gcctctagag tttttcacag tttttttttt
ttttgtaaaa 18720 aaaaaaaaaa aaaaaaaaag gagagataac agatcaatat
atattagttt caaggttttt 18780 tgtttttttt tttaaacaaa aacctgtaat
tgcttttcct attttaacag tatttaaaag 18840 tttagttcct caggtaacag
aacttgaacc tgtttatatg atcaaagttc aagaaattgg 18900 gcatgtttaa
tttggagaag actcggggac cacaatattg ttgtcttcaa atatttgggc 18960
tagaggagga aattatttta tgtatgttcc aactggtaga cctaagcctt atggaatggg
19020 agatataggg agacatattt caactcaaaa tgatgaactc ttaaaagcag
agctgaccaa 19080 agagaaacaa gcctctttag aaaattaaac ttactatctt
tttaattact gcactgtcat 19140 tagagggcca attgtcatgg accctgtaga
agtgattcag gtatcaaata tacaattgat 19200 tagcctaaga aaacatgaag
gcttcttcta actctcagag cttgtaattt tgatgatgat 19260 tttttatatc
tgtcattcct agctgctgta acaatccttc aaattaatgg gggaaatgca 19320
ctgaaaacat aatgaaagct agaagaggga acatatgaaa tgaccttggg tcagaatgac
19380 atgagaggat cagcacttga cactctcagc aactgaggga tcattcaggg
gaggaagata 19440 caggtaagac tgaaggacaa ttccaggtgt attctttgaa
aatgtacctt tcttttgtgt 19500 gtcacagtcc agaggaagat ggtgtgaaag
tagatgtcat tatggtgttc cagttcccct 19560 ctactgaaca aagggcagta
agagagaaga aaatccaaag catcttaaat cagaagataa 19620 ggaatttaag
agccttgcca ataaatgcct catcagttca agttaatggt aaggaggtcc 19680
ccttctatgt gatatgaagt tgtctattag gtccatgttt tgacgaatct caaatttatt
19740 tgtcattatt tccatttcaa ataatagcta gaattcagat gaaaaaattc
aagttaaaga 19800 tgtgacattt caaggtgtat tagtctctaa cgtaagcatg
tctgaagtta gtcatccagt 19860 ggttttcccg acagtaattg attggcactc
atcccaaaat ataggcaagc atttacaact 19920 aacagagagt taatcccacc
caggcactgc ctccatgact aagcaagtga aaatactagg 19980 ggtttagcaa
taattgtttt tctgggtggg accttcctaa aacacaaatt catgtgttgc 20040
catactttta ttgatagttt ctatatatgg tgatatacaa tttttgttag ctttttttcc
20100 tatgggcatt tgggaaaatg gcaagccaac tttgaagttg ttagagtcat
tttaccatta 20160 atgctttaaa aatcacagtc taggaaaaca tcactgaaac
tatgtgtaca ttgttccact 20220 tttctctttt tttttgttca cccttagccc
attataccat tatcacttcc ctcaattaag 20280 gagaacaaac ctttatcaag
gtctatctct atggccttta ccttaagtaa ctaatttctt 20340 tttatattcc
agtgacgtac gcaaattcac ctttatagaa gtgaaattca cacaaaaaga 20400
gttgaggaat tcagtaatta aaaggagcta agaatcaaat ttaaatctct aatttcttaa
20460 aaggctccaa ttaaaaaagg tttctatagt caaacacatc ttaaaaattc
tggctttgat 20520 actcgtttct tggaaattct tccttatagt gtcatattaa
aaattctaag gcagccagct 20580 agagagaaac ttgtttaccc tcgtccgcta
agctgtttgc acagcatctt cttccaacag 20640 acaagtatag atttctccta
caaatttcaa tggataccag acctaagtgt tacagaagag 20700 attcagggca
agcgattttt atcagacatg aaacaggaca ctctgccctt gtaagggtct 20760
agctgacact tcaagaggaa accagataag gaagtaaaaa atgtgaggta atggaatggg
20820 cagatgtttg ctgatgtgag aacgagtcag ctacttaggg aataaagctg
aggacctctc 20880 ccagccagaa gggaggaacc tgacaagtgc ttaatccatc
ttctttgtta gatggggaag 20940 caaatgaata gaagttgtga aacaatgggc
attctgataa tttacatgat gctttctgtg 21000 taatttccaa taaatagtta
atttgtcagg aatgtaaaag cctgaactat ctgaaaccag 21060 agtaaagcat
aaattgttca ttggctgcct ggtctttttg ttttttgtag gctcagcttc 21120
taaacttcag cttattttaa taattgtact aaattaaatg gtaggatatg ctaatggaga
21180 acctgatttg agagtcacct gaggctgggc atggtggctc aagcctataa
ttccagcact 21240 ttgggaggcc gaggcgggtg gatcacctga ggtcaggagt
tcaagaccag cctggccaat 21300 atggtgaaac cccgtctctt ctaaaaatac
aaaatattag tcaggcctgg tgacgggcac 21360 ctgtaatccc agctacttgg
gagactgagg gggaagaatc acttgaaccc gggaggcgga 21420 ggttgcagtg
agccaagatc gcgccactgc actcaagcct gggcttgaca gagcaagact 21480
ccatctccaa aaaaataaaa aataaaagag ttacctgacc aattctaact ccactaagtc
21540 accacaggac cacccaaata attggctcat gcctttgtct tcattttctc
atctgtaaaa 21600 ttccaatggt aatgtttgtt cttcctgaaa tcacagagag
attataacga tatacaagga 21660 aatagaaaac acaatgtgaa ataaagaggc
tgttactaat gagaaaacta ttatgttgtg 21720 catatgcttt ggaaacctga
aatcattaat ttgagtgatt gactagtagc agaaagatag 21780 atccttgaaa
gtttcagaat gttcaatgta gaaagaacag tgtttgttag tgatatggga 21840
gcctaggggg tgttgctttt ctggccagaa acctctgtgg ccagtggttg gtgcctttgc
21900 ccaagttttg ctctggccca ctgggcttgt tctgcccact tgacctggca
gactgtgccc 21960 accttccgct accagcctgg atcccatgcc caccaaggcc
aacccaggca tggagctgtg 22020 agggttgtct gagcgagcac agggtctggc
cactgcccac agccaggcac actggctgca 22080 gcatgacggg cagctccagg
cactggcaca ggtgtgctgt ctctctgtga ggctgtggct 22140 ggacaaagct
cactgcaagc agcttccctg gcaggcacct gggaatgtgg tggcacccag 22200
gaagcttgga gatgccagga actgcagggt cccaaagagg gagtcacaac cctggcttgg
22260 ggagctccca ggtctgggat ccctaaaggg ctgcagcttt tctctctttt
tacccacaat 22320 gtggccagca aggggtatgt ttcattcctg tttgtgttac
agctctttta gtcttgctat 22380 ttggcaggtc ctgagttctt gtcctgagac
caagaagaat gaggtatgca gacaagtgga 22440 gggtgagcaa gacgaagaaa
ggtttactga gcaagagaac agctcacagg agacccacag 22500 tgggcagctc
ctcttcatag ccagggtgtc ccaacaagtg tccagctcct agcaaagagg 22560
aggccctgga ggtagaagct cctctctgca ggcaggttgt cctgttgagt gttcagcttt
22620 cagcacacag taggcagtag gccctagagt ggtctatctc ctctctgcag
gcaggtagtc 22680 ccatggtctc ccagtcacct ctccatctgc aagggtccaa
tgctgcctcc agcacctctc 22740 tgcccacccc tccgtgcctg accaagctgc
tcccccacca gtgggcaact cagcccagcc 22800 ccattgtggt agctcccagg
gtggcaggct ctggggggct cccagggatg ggctccaagg 22860 actgtccacc
ttctccccac gccctccctg cagtggccat ggtcaagaat ggcaatgtgg 22920
ggccaggttc cggagcagga gaggctccag gcctgggagc aggtcctgcc tggtcacgtg
22980 aggttggggg tggcacagtc ggctgcctca gggatgtggg acacagggga
cccaccacca 23040 tcactgctac tcccgcatcc gctcctgcta ccactgctcc
agacagcctg tagctgccat 23100 cactagcact taagaaaggc acattcagtg
gacagctcag gaaaatcttt acgtcaattt 23160 tttataggca aaaacattgt
ttcctgggca aacaaaattt atggactacc aataaataga 23220 aaactgtaga
gattctagat taagtctaga aataatcctg tagcccaaga tttatttata 23280
atttgtcaag aatctgtatt ttgttttgac aaaaaaaaaa ctgtgtggtg tgggtccttc
23340 aggagacaca gtgtgacaaa gcaaagctaa aatcaacttc tttgcattgc
aaacaccaag 23400 gctgtagtca agcagctcac tgcctatgtg tcagatgact
ttgcttcatt tttcatcatg 23460 atacttgtag tctatagagc cctgaatatt
aactagcttt ctcccaactc agaaccgtgt 23520 taggaggtgg ttgctttcaa
aactaaagtg ttaatgttta tttccatttc tataccagga 23580 aagtaaaaat
ctttggtcaa aattagaaat ctttaacaac tagttacttg tgtattgaca 23640
gtttgtttcc aggtgtaatc attctccctt aaaatccggt tatattcacg accattatac
23700 ttatcctggt atcattcctg gaaatggcta acttgcatcc tgctcagact
aagttgacaa 23760 agtttcaatt gaagaattct aactttatgc tattttccac
tttattgcat tacaaaggac 23820 aaaatatata gttttcttaa aaatgaaata
aatttactgc cttaaactac atttgacggt 23880 aaactgagtt ccttccatag
aataaccact aacagcaatc gatggtcctg agcaattgac 23940 tcttcaccat
acaatgattt gggatgcctt taagggtata tttgaattga atattttcaa 24000
aagctcccac tttgtagagt ttatcatcac tagtttcccc agtggaattt gtagaaagtt
24060 agtagaatga aacaatctta ttttgtataa tgaggaatag aatactgaga
atgtgtctga 24120 gaaacatggc actggtagga aaaagtaaac agtttattct
catctgctca ataagctaag 24180 tcattttaac ttgaaaatca tcaaaatttt
catgaaacct tccaccaact ttatttttcc 24240 ccagctttag taagatataa
ttgacaaata aaaattgtat actgtataca acatgatgct 24300 ttgatacatg
tatacaagtt taaatatttg tgtttcctta gtcaaactcc tcactttttt 24360
ggaagttgac agaatttaat cttggattgt gtccaataac tagcttttac cactattcag
24420 tatattttgg ataagaaaca cataacagtt tattctttaa aaaagcaatt
ttactattta 24480 ggaactgtgt ttaaaaagca ttttaaatat catttatgca
agagttttca aggttttttc 24540 attctaaacc ctttaaccaa aaaaaaaaaa
aaaaagattt atgtgaaatt cgaagtaaat 24600 agaagagatc aaagcagatc
tgttctggct gaggctgagt ttgagacctg taagacagtc 24660 tacttgccat
atggcttggc tgtgtcccca cccaaatctc atctcgaatt gtagccccca 24720
taattcccac atgttgtgag agggacctgg tgggagataa attaaatcat gggtgcagtt
24780 tcccccatac tgttctatgg tagtgaatga gatctgatgg ttttataaga
ggcttcccct 24840 ttcacttggc tcacattctc tgacttgctt gccaccatgt
aagacatgcc ttttgccttc 24900 ctccatgatt gtgaggcctc cccagccaca
tggaactctg agtccattaa acctcttttt 24960 ctttataaat tacccagtct
cagatatgtc tttatcagca gtgtgaaaac aaactaatat 25020 aacctgtttc
ctctgtccca tttatccatc ttctgaagtg gaatgcaaag aagctttacc 25080
ccgaactgct ggaaaaccat agttctctat taatacaaac tatttgtggg ctttagtcat
25140 ccactatttg tgccttactc acccattgct tgtgatagta tccacctaat
tagaggctgc 25200 ctataagtct ctacaaaaac tgtacacaga tgttgttata
tcagatagcc attctcctaa 25260 ttaatctata tgttcaactg tctagaatcc
atatatggtc agtatcctct gattattcct 25320 ggtcattgag accaaccagg
aaaatatcaa attatcacta tttgttttat cttctttttc 25380 agcaatgagc
tcatcaacag gggagttaac tgtccaagca agtaagtcaa gttagcttat 25440
ataaacaagt tcaattttca catcagaaag gacattttca aatatttgct catacttgcc
25500 catctgtcct ccagattttc tttgagagat aataactatt tgtacgatag
atttaaatac 25560 attttttttc taactcatgg actgatcttt tagtcatgtt
caagaaaaaa attgccatgg 25620 taaccttctg gggcaatttg aagaaagcat
ttatttttga ttgggaatat tggacttgtt 25680 tttctaattt ttaaaaatgc
cataaaatgt actttctgct acaaaataaa ataataagaa 25740 agtaatcaat
aggaaggaca taaaacccat tgtctgtgac tgacaatttg tctgtgaaat 25800
atgctaaggt caggagttcg agaccagcct gaccaacatg gagaagaaaa cccatctcta
25860 ttaaaaatac aaaaattagc caggtgcggt ggcaggtgcc tgtagtccca
gctacttggg 25920 aggctgaggc aggagaatca cttgaacctg ggaggcagag
gttgcagtga gccaagattg 25980 caccactgca ctccagcctc agcgacagag
tgagactcca tctcaaaaaa gaagaaaaaa 26040 atatgcttaa tagattcatc
ttaatcgcta acagtggctt cattaaatca cttcaaatca 26100 ctgtggccta
aattttgaaa gattttacaa aaaacagtga tgaatttgag caatgatgtt 26160
catgcatttg cctctgtgac ttgcaaacac cctaagtatt tttatccatg tgtttattca
26220 ttcaacaata tcttttaaca tctaccaagt gccagaaatt agaccaggag
ttggtggtac 26280 cattgtgaat aaaacatgat ccctgctcta
aaattagaat tccaaagtag agaaagatat 26340 aaataaatca ggaagtatga
aaataatgtg attaatgcta tgacagagga agtgcatagt 26400 gctatgagag
ttgatcagag agtcagctaa cctgttctca cacagtaaga aagtgaaccc 26460
tgaaatgtga gagagaagag gccatgaatc cagtgacagg tggggtaagt gtcctgggca
26520 ggaggagtag tatacgaaaa tgtcttcagg caagtaagaa tggggtcatt
tcctgtaatt 26580 acaagatgtt tcttataact taatgatctc atcttttttc
aggttgtggt aaacgagttg 26640 ttccattaaa cgtcaacaga atagcatctg
gagtcattgc acccaaggcg gcctggcctt 26700 ggcaagcttc ccttcagtat
gataacatcc atcagtgtgg ggccaccttg attagtaaca 26760 catggcttgt
cactgcagca cactgcttcc agaagtaagt tattgacctt aagttagaac 26820
ccacttctgc taaaaagccc tgagttttgt catattcttg gtaacaatta atgtctcaaa
26880 tattactgaa gtaaaataag aaaaagttat ttcaggttct tttctaaaat
aatgttacac 26940 ttgcatactt aatcagaaat ttgatgggaa taagtaacag
tcattatcct agtatccatc 27000 aatcatttcc tcaaagtttt taataaggaa
actgtgtaaa gaaatcagaa ctattttgtg 27060 acatcctaac acaaaatatt
cactaataac atgtaccatt aatcttttgt caaacaatgc 27120 tctccactta
aaactagtgt ctgtttctgc caaacacttg ggccagtctc atactgatct 27180
taaataatca aactaattcc aaagtaaaat ggaaattttc aataaatgcc ggaagttggt
27240 aaccgtgatg atggagaact gcagatcaaa tttagagcat tgacatatga
agatctgtgg 27300 aatcagaaca gtttacaacc aaaatgagag attgctagca
tgataaagac aggcacttca 27360 aaagagattc ctcggagtat caaaggattc
atagaggccc ttgggccact caatgtgacc 27420 ttcccataat agagcatctc
ttcacaatag tgacacaaaa gacaaagctg aagtgaagaa 27480 tagcaaattg
tgctatccta taattgtttc tgaatgcata cattttatta aatatatgat 27540
taaatgactt tttataactt ttaatcttac ttttcaagat aataaccagt catttttatc
27600 actattacat ttagaatttt agatttgttt ctaagtagat taactgtatc
gcctttcttc 27660 ttcattgcca attattacag taataacaaa gacttcttga
gtatctctat ataataggtg 27720 gcagcaggat ttagtgggaa aaatatgtcc
caggcagttg gagagctggg caaattattg 27780 aaccttagtg tattaggtaa
tagataggct agatcttttc acattctttt tgacctataa 27840 aattctaact
tttgttacta taataaattt catttgccta ggagcataaa tctttataga 27900
gactcttaat attccaaaga atatacatat taagaatcta ggcttggcat ggtggctcat
27960 gcctgtaatc ccagcatttt gggaggccga ggcaagagga ccacttgagc
tcaggagttc 28020 aagaccagct tgggcaagat agtgaaaccc cattgggcat
ggtggtgcat acctatcatc 28080 ccagctactt gggaggctaa cgcaggagga
tcccttaagc ccaggagttt gaggctcctg 28140 caagctatga ttgcaccact
gcactccagc ctgagtgaca atgcaagacc ccatcttaaa 28200 aaaatagtaa
tatattttta aaaataatct acataaattc ttaatgtttg aaagatgtga 28260
gagctcagta agctgatata ttagaaagcc agaaatccct tatgctggtg tctggttttt
28320 caaagtaatg ggaaacttac tttgccaaag ttagccattt ttgtggtaga
tagttctatt 28380 tttgcaaata tctttatagc attgaacacc aaatctatac
tctattaact tctaccatca 28440 atatttgttt ttcttttaat ctggaacaac
aggaaccaat tttatttctt cattcatata 28500 acagctattc tttagtttct
ctttttcaga ccaaacataa aatgagggag aatatccaaa 28560 ccataagtga
aaataaatat cattactgtg agctttagtt tgctaaggat aatgacctcc 28620
agccctatcc atgtccctgc aaagggcatg attttgttct ttttatggct gcatagcatt
28680 cccatggtgt atgtatacca cattttcttt atccagtcta tcactaatgg
gcatttaggt 28740 tgattctatg tctttgctat accgaagagt gctagaggga
gaggatcagg aaaaataact 28800 aatgggtact aggcttaata cctgggtgat
gaaataatat gtacaacaaa accccatgac 28860 acaagtttac ctgtgtaaca
aacctgcaca tgtaaccctg aactttgaaa aaagtatata 28920 tatgcacaca
catatatatg catacatata tatgtgtgta tatatatgca tatatgtgtg 28980
tgtgtatata taaaaaaaaa tatatatata tatatatata tataattacc tcatttttcc
29040 agaaccaact tccagatgcc ctaccacatt ggttcttatt ctctgaacat
tcgagacttt 29100 gtcagtgtct tccttaaaat atgcttccaa taactaaata
caccaagaca gatgtgtgac 29160 tagtgtcaca cataacaaaa taaagcagga
agtcttctga aaaatacaaa taatgtaaat 29220 tggtgggaga cagtgtttta
taaagggaag agcagagaga ggcaggcaga tatgtgatgt 29280 gaatcaaata
gtttaaccta tccaggcttt attttcctta agtataaaac acagtcttta 29340
ctagatgatc tttcattgct actaaatgat ttttccgatt cctgtatgta ccataatcca
29400 cccattgccc aagcccacaa gctagaagtc aaccgcattt accacatttg
atcatctctc 29460 aaaggactat gcagtcatct aatagacttt accacatcca
ttcttgacct tcaagaatct 29520 actccccaga aagaacaaac atgtttttta
aaaatgtaaa tgagactaca ttattctctg 29580 gcttaattat ccagtagatt
cccatatcac ttcaataaaa tttaagcact ttatcatgac 29640 ctataaaaca
ctctaaaatc tagtccctgc ttacctctcc aagctcaccc ccaaccattc 29700
tttcccttgt gttctgactg cagcccatcc aacccaagac cttgggattt ttgcctggaa
29760 acttgtttcc ctcatctcct cacactgacc ctcttttact atgtcttagc
ccaaatgcgt 29820 tatcaaaata atcataatga cctgttagta ctctattccg
ttaccctatt ttattttgtt 29880 catagccttt atcaatgttt aagattattt
atctatttgt ttgcttgctt tgatcctttt 29940 ccttctctgg aatcttatac
tcctgtgagc aggcacctta ggtcctgttc atcactttat 30000 ccccagcagt
tcagataagg ctcagcacac agatgctcag taaatatttg tggaagggat 30060
aaatgaatga tattttatgt gtattacagt tctaaaattc aatagttttg tattaaatat
30120 cagttctaat atggcattta tatgatttta tctttcaaaa cattagcaat
agattatatt 30180 taaatgataa aagaaaacta taactgcagc caagtattct
caggattgta tttctcttat 30240 attagcctaa atgcaattaa tctagctcat
atactttggg cagcttatat atattctgtt 30300 aatttctaac cttttccagg
tataaaaatc cacatcaatg gactgttagt tttggaacaa 30360 aaatcaaccc
tcccttaatg aaaagaaatg tcagaagatt tattatccat gagaagtacc 30420
gctctgcagc aagagagtac gacattgctg ttgtgcaggt ctcttccaga gtcacctttt
30480 cggatgacat acgccagatt tgtttgccag aagcctctgc atccttccaa
ccaaatttga 30540 ctgtccacat cacaggattt ggagcacttt actatggtgg
tgggtatctc aggatagcta 30600 acagagcgct aagccctgtc taaggcaatg
tgatttcatc tccatcaata ttatcctgac 30660 agccatttcc acacagtctg
gttggattag ttagggttct tactttgtgt gacagaaatt 30720 caattcacat
taaccagtgc agaataaaaa acaaagaaac aaaaacttcc acaaatttgg 30780
ctcatgtaat ttggaagtca aaaaagtgta gtaagtttca cttcagacac aggggtttat
30840 atgatgtcat ctggctctgt gtctctgaat ttgaattttt tgccccttct
tttctctatg 30900 ttggcttcat tcagagggat gctagcttca cctagtgtca
gaggtggcta acaacacctc 30960 aacacatcat cctcaacaaa gaaaaaatac
atagaaagga atatttattt cttttctttg 31020 ccagaattca cattaatttc
tattgttcca gctgtgtcta ggaggactca gattgagtgg 31080 ctaactcaaa
tattctttat gcctatgtag caaaatttgc ttcagtactg aagaagctaa 31140
tttaagtgtg atggtgaata agaatagtgt agagataaat tgtcaaacta tttgtcccct
31200 ctaaaagtat tcaacttgat atactaactt agtcttgtaa gaaataatga
tgatttagtt 31260 actgaatgtt ctaggcaatc ttagtgagac acgctctgga
ttctaacatg tggtccaggt 31320 acatatgtat aacaaagcta gaaagtttct
ttaacactgg gcttgagaaa atgcaaaagg 31380 gctttctgag aatgactaaa
tctatttgca ggattctata caatttattt acatacaaga 31440 aattataaag
aataagcttt tgattctcag tctaccatta aggaactagg aataaccttt 31500
cactcacata ggcaggaatc ggttttaggg tctctagatt ttttccagat gtcccatgtg
31560 gttttgtttt atcttataca gagtgagaca tgcattgctt tctttaaggt
tgtattacca 31620 atcacagaaa atattaccta tggtttatta attctagtag
atccagtgct gctgtaagcc 31680 tgacacctcc ctaggtctgc actctcttgg
atggattttc tctgaagata gggcttgcat 31740 tctctgcttc atagtggtgg
gaaagacatc acaaatcccc tttggcttgg tgggaaaaat 31800 cactttcagg
agtttgagac tggcacagaa acatacctgt cataatgcgc tgtgagtggc 31860
aacagaatct gacacttata gagcactcca ccctacttga acacggcctc tcttggtgag
31920 tgacccacag gtgcttttaa tctattaaat agattaaatt aacctatcat
tcttaatctg 31980 ttaagtacat taatagatta aaagcagcca ttcgttactc
accaagagag gctatattca 32040 agtctgtaaa gcaaacctta agaagttttt
taaaattgaa attgtacaaa gtatattctc 32100 tgatcataat ggaatctaac
tagacatcag taacagaaag ataacataaa aatccccaaa 32160 tgcttaccaa
ttaaaaaaca tatgtaaata aagagaatat ctcgaagaaa tttgtaaaaa 32220
caaatagaac taaatgaaaa caaaaatata taaatatatg ccagatgctg ctaaaatagt
32280 gtagaaaggg aaatttatag aaaatgcata ttataaggaa agatatcaaa
tcaataatta 32340 agttctcact tcaagaaact agaaaaataa aaaataaacc
taaaacaaac ataaggaagg 32400 aaataataag aataagaata gaaatgaata
aaattaaaaa taaactatag aaaattgata 32460 aataaaaagc tgattatttg
ataaaatcaa tattttgcta gaaatgtcat taagcatttt 32520 tacagaagat
gagatatagc tcagggatgt ccagaattta tgggctatgc ttttcatgac 32580
ttggaataca ttttaccaac cagtttagtt tgctgaagaa gttgtggatt tgcactgtca
32640 cctacttaca atacttagat tgtcagtttc accttactct tctcaccatt
attttatttt 32700 tatttttatt tttattttta ttttgaaaca gagtctcgct
ctgtctccca ggctggagtg 32760 cagtggcgtg atctcggctc actgcaaact
ccgcctcccg ggttcacgcc attctcctgc 32820 ctcagcctcc cgagtagctg
ggactgcagg cgcccaccac catgcccggc taattgtttt 32880 gtagttttag
taaagaaggg gtttcaccgt gttagccagg atggttttga tctcctgacc 32940
tcgtgatcca cctgcctcgg cctcccaaag tgctgggatt acaggcgtga gccaccgcgc
33000 gccaggccat gaatgttttt aattgatgat atagtaggca atataaatgt
gtgtgtgtgt 33060 gtgtgtgtgt gtgtataata tatataaacc aattgtattc
aaataacaga ataatttgaa 33120 aaatctctta gcatatttct gagttacaca
cttaaatctt ccgagcactt ttaaatatgt 33180 gtttacaaac atttcttcag
aaataaatct tggaaatcgt cttctaaaga aactggtgta 33240 ttagggtttt
ttcaaatgta cttagttttt tttttaattg atgtataaaa ttgcatgtac 33300
ttaccatgtg caacataatg tgttgaagta tagtatatgt acactgtgag tgttaaatct
33360 agttaactaa gaagcgtctt attttacata attatcattt ttgtggcaag
aacacttaat 33420 atctactctt gtagcgtttc tcaagaatac gatatatcaa
cagtaggcaa ccagaagctg 33480 ggggtcttta caggggaagg agttagggag
atgctggtca acaaattcat atttgcagtt 33540 aggaagaaaa agttcaagag
atctctcatc catcatggtg actatagctg atgatatatc 33600 gtattcttgt
attagttttt tataaatgtg taacaaataa tcacaaacag ttaaaacagc 33660
actcatttat ttttatctca ctgttttcat gagtcagacg ttcagacaca gcttagttga
33720 gtcctcttct cagggtctca ccaaactgta atcaaggtgt cagctggggt
tgtggccaca 33780 tctgtggctc ctttgaaggt ctcctcaagg tttgctggca
gaattccttt actcgcagct 33840 gtagaatgca tgccagcttg ctgctttaac
tctttaggaa agtgtctcaa ctccagcaag 33900 gctcgccctt tttgaaatgg
ctcagctgat taggtcaggc ccacctttga taatctcctt 33960 ttgatgaatt
caaagtcaaa ctcattagag gtcttaatcg catctgtaaa attccctcat 34020
cttggccata taacataacc taatcatgag aatggcatcc ctcatattca cagatcctgc
34080 ccatatttgg gaggagggga atcacacagg aatcttgggg actatcctag
aattctgcca 34140 accatggggt catggtttcc caatcaatat atggtttggt
ataaagaatc cctgaatgct 34200 tgtgctattc ttagttttct acgtagcctg
ccataataat ggtttctaaa actcagaacc 34260 tagcttacag tctgcagcca
ccaacttgta atacattgga agtgaaatca ttgccgttta 34320 atgcatttat
atatatatga tgtataatat atgtatattt cacatatatc ttatatatgt 34380
gaaagctcat cataaacttt aaataataaa ataaatgtac atagtattat aggcatttta
34440 tcaagccaat ggagaaaacc atctaggcat gcagagtttc tgggaacaat
ctggaaccca 34500 caaataaaag ctttacaaaa gataaaaggc cttcctgaaa
tatataagct gattattttt 34560 aaggttagat tttaccagga aaaagaatcc
aaatggcttt cttgctttga gaagttttta 34620 taaaaatgtg attggacaat
aattatcgtt agatgtgcca gatttaacca gaaattcttt 34680 tttctagaaa
ctgcttatat taacttcatt ctgtattgac aattttacca tgaaaaaaat 34740
attaggaaag tcttctcact tcactctagc caaagatgct gattgtaaat actagaataa
34800 ctctattttt ccttaagggg aatcccaaaa tgatctccga gaagccagag
tgaaaatcat 34860 aagtgacgat gtctgcaagc aaccacaggt gtatggcaat
gatataaaac ctggaatgtt 34920 ctgtgccgga tatatggaag gaatttatga
tgcctgcagg gtaagttgga gggatttttt 34980 tatattacta actcaaaaat
ttgtatctgg cttagaatat attatatgtt ctttacataa 35040 ggacaaaaca
tagatatcat gtcagctcaa aaaagttaca aatgcaaatt tcacagcaca 35100
aaatactttt aaatgtttta ttaagataaa tgaagtaaga gtttctctga tgctatcaaa
35160 caaacaaaat tagaatttct taaccagaaa tccaaagatt aataaagcag
tttattttct 35220 caagcggctc acattcaaga aagaaaataa tcataaacag
agaagtataa agtgatgtta 35280 tgaataatat aatgaaaagc aaatattttt
cttgaaggaa acatttttgg aacaagtatc 35340 agagagatga gacgtaaata
aggcctgaag aataaataac atccaatttc agaataagaa 35400 aataatgtta
tagaaaagac aaaaagcata gccaaaatta tgaaggtgtg aaattacaat 35460
tcatatctga gggaactcca agtaattggt tgggtctcag catgaggagg atgagaagag
35520 aaacaagtag ataaccatga gaaggtggat taggccatgt tgtgattcca
tgggccctcc 35580 ccagtgccct catctgcctt ctaacatgga tgttttccag
cgaaggtacg tttcttcctg 35640 gagacacttg ctttttaaca tgagatactt
tagaactcta aggaggccac tctatgtgga 35700 aatgatggaa tggtattgat
atcaggtggc agaaagtcct gtccagagtc ccacaaactg 35760 taccacatgt
gcgacctcta tcagaaaagg agcagggacc tatgtgacat agaggctggg 35820
caaaagcagg atctggtcca cagccagcct cggttgctaa taatgtggag ggaggcaggc
35880 agaatttagg gattccaaca aaaggtccat accacgggga acaggtggaa
ggtgcaggag 35940 tcttggagca gacaggaccg gggaattcag gtgaaccatg
acattactga aaagccttag 36000 gagggattgg tggtcataga gatgcttcac
tggattgggg agcagaggta aacttgctgc 36060 ctaactgtgc aaagtaagtg
ataaaacaag gctttagtca tagaaaaata cagtaagtta 36120 tcagggcagc
ggttcaggta caaggatcca agacaggaat acagtgattg taattggggc 36180
acatggtgag gggcctagtc tgatacaaca gaagtgcaag caccaccaac acctcgtctt
36240 tctccataag tctttctctc cagagccctc atgacctaat cacctcttct
taagtcccat 36300 ctctcaacac tattgtattg gagattaagt ttccccaacc
tatgaactct tgggctcaca 36360 ttcaaaccat agcaccaccc agcacaaaag
cacagagctt ccaatctggt ttctagctcc 36420 ataccctaga accaaacagt
aagaatcacc tctggaaatg tagcaataat ataatcataa 36480 tttttaaaat
ccagtggaag gattggaaga taaaatcaag gaaatctctc agaaagaaca 36540
acaacaacaa aaaagacaca gaggagaaaa ataatcagaa aaattaagaa aactagagga
36600 taagctcagg agatccaaca ccaaatgaat aggagctctg aaaacataaa
acgcgagtgt 36660 acaatataaa aaaaaataaa gaatgctcct agttctgaag
cttacatgca tcctattgaa 36720 gaaaaggtcc aagtagtgct gggcacaata
aatgaagtac ttctttccaa gacataccat 36780 cataaagggt cagaagccag
ggataaggag aacaatctta aaactttgaa ggaagaacca 36840 tcagaactac
atagaactcc tcaacagtaa ctctagaagg tagacgatgg tggaaaacac 36900
attcaaattt caaagggaag attatttcaa cctagattcc tacccatgct aactaaatat
36960 caactgtgag ggtggaatta agaagtttag acaagcaatg actgaaaaaa
atgtacttct 37020 gataccctac ttcttaggaa actacttgag agggtacctc
agcaaaatga gggaataaat 37080 caagaaagtg gaagacgtaa gacctgaaac
tgttagtcca acactaaaga gtggtatcag 37140 ataatcccaa caccatagct
ctgcaccagg cttaaagtaa ccagctcgaa tttgagcaga 37200 agtaagaaaa
gattgtgtgt atgtgtatgt gtatgtgtgt atgtgtgtgt gtgtgtgtgt 37260
gtgtgttgat atggtggaac agcttcagag gaagtaaaag aactaacaag ctatctgatg
37320 tccttgaaca ttagtaaaca ttattgtgag gtgttggtag atcttttgga
gcattcagca 37380 tttaccaggt acatagaaaa ctatccacat gaaaaaaaga
gttgtgttat taattctagg 37440 aaagcaaaaa aagatttctg taatccaaat
atgttacttg actcttcaat taataaaatt 37500 tacacactgg tactaaatgt
aggctgttaa tttaaccaaa aatagagatg ctataatgta 37560 aagatgtggt
gtggaaaagt tgcaaagaag ttgtaaaaca actaaatccc taactacgta 37620
agagaaaata aatatttact gtctaaacct agaagctgta atttgagcat attatctagt
37680 gataaggagt tagatactat aagaaatcat taaacaagca tgaagtggct
acctcttgga 37740 gaacagcttg cgtgaggtaa catgggacat aactgctttt
caagcctctt catgtttttt 37800 cgtttttgcc ttttttaact aagtgctgtt
tactctaaca aaataaattt tattttttaa 37860 atgtgaaagt tgaaccttaa
ggctctttgt aatattaaaa tccatgtctc aattaattat 37920 tctgtgttga
tagtctatac atgtactgtc tagtaacaaa atatgtgatt catcaaaata 37980
tcttaaataa tgagctttat gtttagctaa ttttctttct tttttcttat gtttttattt
38040 ttagggtgat tctgggggac ctttagtcac aagggatctg aaagatacgt
ggtatctcat 38100 tggaattgta agctggggag ataactgtgg tcaaaaggac
aagcctggag tctacacaca 38160 agtgacttat taccgaaact ggattgcttc
aaaaacaggc atctaattca cgataaaagt 38220 taaacaaaga aagctgtatg
caggtcatat atgcatgaga attcaactat ttagtgggtg 38280 tagtacaaca
aagtgatatt aaattactgg atctagtaac atgaaacaca caacgtaagt 38340
tatttagaat cactttaatc aaccaataat ccttagccaa tttataaggg acttttattt
38400 gtaaagtaat ggatctggct tgaaaaatac ggtagagata cttagctctt
taaatcacga 38460 atgttgaagt accagtgaga ctcaatacat atttttgaag
atagtccatg ggatttttag 38520 aatgtcgttg tcaagggtct ccttttaact
gagaaacttt ttgaactcac aaagtgttca 38580 agaaaccctt gtataattcc
ctacatttct ctcgagctca caaatacttt tttttctttt 38640 tccttattca
atcagatttt ccaaagtacc tttccaccat aagaaatgaa ttttctactt 38700
ctacacccat ttgagagaca ccaataaaag aaagtcatat gtaggaaaca aagtctgata
38760 gtaaaacaag ccagagatct tctaactttt tttagttata aaacctctaa
tttttggtga 38820 cttttctaca cacacacaca cata 38844 4 407 PRT Homo
sapien 4 Glu Pro Trp Val Ile Gly Leu Val Ile Phe Ile Ser Leu Ile
Val Leu 1 5 10 15 Ala Val Cys Ile Gly Leu Thr Val His Tyr Val Arg
Tyr Asn Gln Lys 20 25 30 Lys Thr Tyr Asn Tyr Tyr Ser Thr Leu Ser
Phe Thr Thr Asp Lys Leu 35 40 45 Tyr Ala Glu Phe Gly Arg Glu Ala
Ser Asn Asn Phe Thr Glu Met Ser 50 55 60 Gln Arg Leu Glu Ser Met
Val Lys Asn Ala Phe Tyr Lys Ser Pro Leu 65 70 75 80 Arg Glu Glu Phe
Val Lys Ser Gln Val Ile Lys Phe Ser Gln Gln Lys 85 90 95 His Gly
Val Leu Ala His Met Leu Leu Ile Cys Arg Phe His Ser Thr 100 105 110
Glu Asp Pro Glu Thr Val Asp Lys Ile Val Gln Leu Val Leu His Glu 115
120 125 Lys Leu Gln Asp Ala Val Gly Pro Pro Lys Val Asp Pro His Ser
Val 130 135 140 Lys Ile Lys Lys Ile Asn Lys Thr Glu Thr Asp Ser Tyr
Leu Asn His 145 150 155 160 Cys Cys Gly Thr Arg Arg Ser Lys Thr Leu
Gly Gln Ser Leu Arg Ile 165 170 175 Val Gly Gly Thr Glu Val Glu Glu
Gly Glu Trp Pro Trp Gln Ala Ser 180 185 190 Leu Gln Trp Asp Gly Ser
His Arg Cys Gly Ala Thr Leu Ile Asn Ala 195 200 205 Thr Trp Leu Val
Ser Ala Ala His Cys Phe Thr Thr Tyr Lys Asn Pro 210 215 220 Ala Arg
Trp Thr Ala Ser Phe Gly Val Thr Ile Lys Pro Ser Lys Met 225 230 235
240 Lys Arg Gly Leu Arg Arg Ile Ile Val His Glu Lys Tyr Lys His Pro
245 250 255 Ser His Asp Tyr Asp Ile Ser Leu Ala Glu Leu Ser Ser Pro
Val Pro 260 265 270 Tyr Thr Asn Ala Val His Arg Val Cys Leu Pro Asp
Ala Ser Tyr Glu 275 280 285 Phe Gln Pro Gly Asp Val Met Phe Val Thr
Gly Phe Gly Ala Leu Lys 290 295 300 Asn Asp Gly Tyr Ser Gln Asn His
Leu Arg Gln Ala Gln Val Thr Leu 305 310 315 320 Ile Asp Ala Thr Thr
Cys Asn Glu Pro Gln Ala Tyr Asn Asp Ala Ile 325 330 335 Thr Pro Arg
Met Leu Cys Ala Gly Ser Leu Glu Gly Lys Thr Asp Ala 340 345 350 Cys
Gln Gly Asp Ser Gly Gly Pro Leu Val Ser Ser Asp Ala Arg Asp 355 360
365 Ile Trp Tyr Leu Ala Gly Ile Val Ser Trp Gly Asp Glu Cys Ala Lys
370 375 380 Pro Asn Lys Pro Gly Val Tyr Thr Arg Val Thr Ala Leu Arg
Asp Trp 385 390 395 400 Ile Thr Ser Lys Thr Gly
Ile 405
* * * * *
References