U.S. patent application number 10/612012 was filed with the patent office on 2004-12-16 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, Guegler, Karl, Ketchum, Karen A., Shao, Wei, Webster, Marion, Yan, Chunhua.
Application Number | 20040253705 10/612012 |
Document ID | / |
Family ID | 26942293 |
Filed Date | 2004-12-16 |
United States Patent
Application |
20040253705 |
Kind Code |
A1 |
Guegler, Karl ; et
al. |
December 16, 2004 |
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: |
Guegler, Karl; (Menlo Park,
CA) ; Webster, Marion; (San Francesco, CA) ;
Yan, Chunhua; (Boyds, MD) ; Shao, Wei;
(Frederick, MD) ; Ketchum, Karen A.; (Germantown,
MD) ; Di Francesco, Valentina; (Rockville, MD)
; Beasley, Ellen M.; (Darnstown, MD) |
Correspondence
Address: |
CELERA GENOMICS CORP.
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: |
26942293 |
Appl. No.: |
10/612012 |
Filed: |
July 3, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10612012 |
Jul 3, 2003 |
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10160187 |
Jun 4, 2002 |
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6620607 |
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10160187 |
Jun 4, 2002 |
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09741150 |
Dec 21, 2000 |
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6436689 |
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60252410 |
Nov 22, 2000 |
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Current U.S.
Class: |
435/226 |
Current CPC
Class: |
G01N 2500/04 20130101;
G01N 33/573 20130101; C12N 9/64 20130101; G01N 2333/96433 20130101;
Y10T 436/143333 20150115 |
Class at
Publication: |
435/226 |
International
Class: |
C12N 009/64 |
Claims
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 ATP-dependent protease subfamily (a type of
mitochondrial Ion protease homolog 1 precursor), 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 functionng 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 prothrombiase 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] Trypsinogens
[0012] The trypsinogens are serine proteases secreted by exocrine
cells of the pancreas (Travis J and Roberts R. Biochemistry 1969;
8: 2884-9; Mallory P and Travis J, Biochemistry 1973; 12: 2847-51).
Two major types of trypsinogen isoenzymes have been characterized,
trypsinogen-1, also called cationic trypsinogen, and trypsinogen-2
or anionic trypsinogen. The trypsinogen proenzymes are activated to
trypsins in the intestine by enterokinase, which removes an
activation peptide from the N-terminus of the trypsinogens. The
trypsinogens show a high degree of sequence homology, but they can
be separated on the basis of charge differences by using
electrophoresis or ion exchange chromatography. The major form of
trypsinogen in the pancreas and pancreatic juice is trypsinogen-1
(Guy C O et al., Biochem Biophys Res Commun 1984; 125: 516-23). In
serum of healthy subjects, trypsinogen-1 is also the major form,
whereas in patients with pancreatitis, trypsinogen-2 is more
strongly elevated (Itkonen et al., J Lab Clin Med 1990; 115:712-8).
Trypsinogens also occur in certain ovarian tumors, in which
trypsinogen-2 is the major form (Koivunen et al., Cancer Res 1990;
50: 2375-8). Trypsin-1 in complex with alpha-1-antitrypsin, also
called alpha-1-antiprotease, has been found to occur in serum of
patients with pancreatitis (Borgstrom A and Ohlsson K, Scand J Clin
Lab Invest 1984; 44: 381-6) but determination of this complex has
not been found useful for differentiation between pancreatic and
other gastrointestinal diseases (Borgstrom et al., Scand J Clin Lab
Invest 1989; 49:757-62).
[0013] Trypsinogen-1 and -2 are closely related immunologically
(Kimland et al., Clin Chim Acta 1989; 184: 31-46; Itkonen et al.,
1990), but by using monoclonal antibodies (Itkonen et al., 1990) or
by absorbing polyclonal antisera (Kimland et al., 1989) it is
possible to obtain reagents enabling specific measurement of each
form of trypsinogen.
[0014] When active trypsin reaches the blood stream, it is
inactivated by the major trypsin inhibitors alpha-2-macroglobulin
and alpha-1-antitrypsin (AAT). AAT is a 58 kilodalton serine
protease inhibitor synthesized in the liver and is one of the main
protease inhibitors in blood. Whereas complexes between trypsin-1
and AAT are detectable in serum (Borgstrom and Ohlsson, 1984) the
complexes with alpha-2-macroglobulin are not measurable with
antibody-based assays (Ohlsson K, Acta Gastroenterol Belg 1988; 51:
3-12).
[0015] Inflammation of the pancreas or pancreatitis may be
classified as either acute or chronic by clinical criteria. With
treatment, acute pancreatitis can often be cured and normal
function restored. Chronic pancreatitis often results in, permanent
damage. The precise mechanisms which trigger acute inflammation are
not understood. However, some causes in the order of their
importance are, alcohol ingestion, biliary tract disease,
post-operative trauma and hereditary pancreatitis. One theory
provides that autodigestion, the premature activation of
proteolytic enzymes in the pancreas rather than in the duodenum,
causes acute pancreatitis. Any number of other factors including
endotoxins, exotoxins, viral infections, ischemia, anoxia, and
direct trauma may activate the proenzymes. In addition, any
internal or external blockage of pancreatic ducts can also cause an
accumulation of pancreatic juices in the pancreas resulting
cellular damage.
[0016] Anatomy, physiology, and diseases of the pancreas are
reviewed, inter alia, in Guyton A C (1991) Textbook of Medical
Physiology, W B Saunders Co, Philadelphia Pa.; Isselbacher K J et
al (1994) Harrison's Principles of Internal Medicine, McGraw-Hill,
New York City; Johnson K E (1991) Histology and Cell Biology,
Harwal Publishing, Media Pa.; and The Merck Manual of Diagnosis and
Therapy (1992) Merck Research Laboratories, Rahway N.J.
[0017] Metalloprotease
[0018] The metalloproteases may be one of the older classes of
proteinases and are found in bacteria, fungi as well as in higher
organisms. They differ widely in their sequences and their
structures but the great majority of enzymes contain a zinc atom
which is catalytically active. In some cases, zinc may be replaced
by another metal such as cobalt or nickel without loss of the
activity. Bacterial thermolysin has been well characterized and its
crystallographic structure indicates that zinc is bound by two
histidines and one glutamic acid. Many enzymes contain the sequence
HEXXH; which provides two histidine ligands for the zinc whereas
the third ligand is either a glutamic acid (thermolysin,
neprilysin, alanyl aminopeptidase) or a histidine (astacin). Other
families exhibit a distinct mode of binding of the Zn atom. The
catalytic mechanism leads to the formation of a non covalent
tetrahedral intermediate after the attack of a zinc-bound water
molecule on the carbonyl group of the scissile bond. This
intermediate is further decomposed by transfer of the glutamic acid
proton to the leaving group.
[0019] Metalloproteases contain a catalytic zinc metal center which
participates in the hydrolysis of the peptide backbone (reviewed in
Power and Harper, in Protease Inhibitors, A. J. Barrett and G.
Salversen (eds.) Elsevier, Amsterdam, 1986, p. 219). The active
zinc center differentiates some of these proteases from calpains
and trypsins whose activities are dependent upon the presence of
calcium. Examples of metalloproteases include carboxypeptidase A,
carboxypeptidase B, and thermolysin.
[0020] Metalloproteases have been isolated from a number of
procaryotic and eucaryotic sources, e.g. Bacillus subtilis (McConn
et al., 1964, J. Biol. Chem. 239:3706); Bacillus megaterium;
Serratia (Miyata et al., 1971, Agr. Biol. Chem. 35:460);
Clostridium bifermentans (MacFarlane et al., 1992, App. Environ.
Microbiol. 58:1195-1200), Legionella pneumophila (Moffat et al.,
1994, Infection and Immunity 62:751-3). In particular, acidic
metalloproteases have been isolated from broad-banded copperhead
venoms (Johnson and Ownby, 1993, Int. J. Biochem. 25:267-278),
rattlesnake venoms (Chlou et al., 1992, Biochem. Biophys. Res.
Commun. 187:389-396) and articular cartilage (Treadwell et al.,
1986, Arch. Biochem. Biophys. 251:715-723). Neutral
metalloproteases; specifically those having optimal activity at
neutral pH have, for example, been isolated from Aspergillus sojae
(Sekine, 1973, Agric. Biol. Chem. 37:1945-1952). Neutral
metalloproteases obtained from Aspergillus have been classified
into two groups, npI and npII (Sekine, 1972, Agric. Biol. Chem.
36:207-216). So far, success in obtaining amino acid sequence
information from these fungal neutral metalloproteases has been
limited. An npII metalloprotease isolated from Aspergillus oryzae
has been cloned based on amino acid sequence presented in the
literature (Tatsumi et al., 1991, Mol. Gen. Genet. 228:97-103).
However, to date, no npI fungal metalloprotease has been cloned or
sequenced. Alkaline metalloproteases, for example, have been
isolated from Pseudomonas aeruginosa (Baumann et al., 1993, EMBO J.
12:3357-3364) and the insect pathogen Xenorhabdus luminescens
(Schmidt et al., 1998, Appl. Environ. Microbiol. 54:2793-2797).
[0021] Metalloproteases have been devided into several distinct
families based primarily on activity and sturcture: 1) water
nucleophile; water bound by single zinc ion ligated to two His
(within the motif HEXXH) and Glu, His or Asp; 2) water nucleophile;
water bound by single zinc ion ligated to His, Glu (within the
motif HXXE) and His; 3) water nucleophile; water bound by single
zinc ion ligated to His, Asp and His; 4) Water nucleophile; water
bound by single zinc ion ligated to two His (within the motif
HXXEH) and Glu and 5) water nucleophile; water bound by two zinc
ions ligated by Lys, Asp, Asp, Asp, Glu.
[0022] Examples of members of the metalloproteinase family include,
but are not limited to, membrane alanyl aminopeptidase (Homo
sapiens), germinal peptidyl-dipeptidase A (Homo sapiens), thimet
oligopeptidase (Rattus norvegicus), oligopeptidase F (Lactococcus
lactis), mycolysin (Streptomyces cacaoi), immune inhibitor A
(Bacillus thuringiensis), snapalysin (Streptomyces lividas),
leishmanolysin (Leishmania major), microbial collagenase (Vibrio
alginolyticus), microbial collagenase, class I (Clostridium
perfringens), collagenase 1 (Homo sapiens), serralysin (Serratia
marcescens), fragilysin (Bacteroides fragilis), gametolysin
(Chlamydomonas reinhardtii), astacin (Astacus fluviatilis),
adamalysin (Crotalus adamanteus), ADAM 10 (Bos taurus), neprilysin
(Homo sapiens), carboxypeptidase A (Homo sapiens), carboxypeptidase
E (Bos taurus), gamma-D-glutamyl-(L)-meso-diaminopimelate peptidase
I (Bacillus sphaericus), vanY D-Ala-D-Ala carboxypeptidase
(Enterococcus faecium), endolysin (bacteriophage A118), pitrilysin
(Escherichia coli), mitochondrial processing peptidase
(Saccharomyces cerevisiae), leucyl aminopeptidase (Bos taurus),
aminopeptidase I (Saccharomyces cerevisiae), membrane dipeptidase
(Homo sapiens), glutamate carboxypeptidase (Pseudomonas sp.), Gly-X
carboxypeptidase (Saccharomyces cerevisiae), O-sialoglycoprotein
endopeptidase (Pasteurella haemolytica), beta-lytic
metalloendopeptidase (Achromobacter lyticus), methionyl
aminopeptidase I (Escherichia coli), X-Pro aminopeptidase
(Escherichia coli), X-His dipeptidase (Escherichia coli),
IgA1-specific metalloendopeptidase (Streptococcus sanguis),
tentoxilysin (Clostridium tetani), leucyl aminopeptidase (Vibrio
proteolyticus), aminopeptidase (Streptomyces griseus), IAP
aminopeptidase (Escherichia coli), aminopeptidase T (Thermus
aquaticus), hyicolysin (Staphylococcus hyicus), carboxypeptidase
Taq (Thermus aquaticus), anthrax lethal factor (Bacillus
anthracis), penicillolysin (Penicillium citrinum), fungalysin
(Aspergillus fumigatus), lysostaphin (Staphylococcus simulans),
beta-aspartyl dipeptidase (Escherichia coli), carboxypeptidase Ss1
(Sulfolobus solfataricus), FtsH endopeptidase (Escherichia coli),
glutamyl aminopeptidase (Lactococcus lactis), cytophagalysin
(Cytophaga sp.), metalloendopeptidase (vaccinia virus), VanX
D-Ala-D-Ala dipeptidase (Enterococcus faecium), Ste24p
endopeptidase (Saccharomyces cerevisiae), dipeptidyl-peptidase III
(Rattus norvegicus), S2P protease (Homo sapiens), sporulation
factor SpoIVFB (Bacillus subtilis), and HYBD endopeptidase
(Escherichia coli).
[0023] Metalloproteases have been found to have a number of uses.
For example, there is strong evidence that a metalloprotease is
involved in the in vivo proteolytic processing of the
vasoconstrictor, endothelin-1. Rat metalloprotease has been found
to be involved in peptide hormone processing. One important
subfamily of the metalloproteases are the matrix
metalloproteases.
[0024] A number of diseases are thought to be mediated by excess or
undesired metalloprotease activity or by an imbalance in the ratio
of the various members of the protease family of proteins. These
include: a) osteoarthritis (Woessner, et al., J. Biol. Chem.
259(6), 3633, 1984; Phadke, et al., J. Rheumatol. 10, 852, 1983),
b) rheumatoid arthritis (Mullins, et al., Biochim. Biophys. Acta
695, 117, 1983; Woolley, et al., Arthritis Rheum. 20, 1231, 1977;
Gravallese, et al., Arthritis Rheum. 34, 1076, 1991), c) septic
arthritis (Williams, et al., Arthritis Rheum. 33, 533, 1990), d)
tumor metastasis (Reich, et al., Cancer Res. 48, 3307, 1988, and
Matrisian, et al., Proc. Nat'l. Acad. Sci., USA 83, 9413, 1986), e)
periodontal diseases (Overall, et al., J. Periodontal Res. 22, 81,
1987), f) corneal ulceration (Burns, et al., Invest. Opthalmol.
Vis. Sci.; 30, 1569, 1989), g) proteinuria (Baricos, et al.,
Biochem. J. 254, 609, 1988), h) coronary thrombosis from
atherosclerotic plaque rupture (Henney; et al., Proc. Nat'l. Acad.
Sci., USA 88, 8154-8158, 1991), i) aneurysmal aortic disease (Vine,
et al., Clin. Sci, 81, 233, 1991), j) birth control (Woessner, et
al., Steroids 54, 491, 1989), k) dystrophobic epidermolysis bullosa
(Kronberger, et al., J. Invest. Dermatol. 79, 208, 1982), and l)
degenerative cartilage loss following traumatic joint injury, m)
conditions leading to inflammatory responses, osteopenias mediated
by MMP activity, n) tempero mandibular joint disease, o)
demyelating diseases of the nervous system (Chaotry, et al., J.
Neurochem. 50, 688, 1988).
[0025] Aspartic Protease
[0026] Aspartic proteases have been divided into several distinct
families based primarily on activity and structure. These include
1) water nucleophile; water bound by two Asp from monomer or dimer;
all endopeptidases, from eukaryote organisms, viruses or virus-like
organisms and 2) endopeptidases that are water nucleophile and are
water bound by Asp and Asn.
[0027] Most of aspartic proteases belong to the pepsin family. The
pepsin family includes digestive enzymes such as pepsin and
chymosin as well as lysosomal cathepsins D and processing enzymes
such as renin, and certain fungal proteases (penicillopepsin,
rhizopuspepsin, endothiapepsin). A second family comprises viral
proteases such as the protease from the AIDS virus (HIV) also
called retropepsin. Crystallographic studies have shown that these
enzymes are bibbed molecules with the active site located between
two homologous lobes. Each lobe contributes one aspartate residue
of the catalytically active diad of aspartates. These two aspartyl
residues are in close geometric proximity in the active molecule
and one aspartate is ionized whereas the second one is unionized at
the optimum pH range of 2-3. Retropepsins, are monomeric, i.e carry
only one catalytic aspartate and then dimerization is required to
form an active enzyme.
[0028] In contrast to serine and cysteine proteases, catalysis by
aspartic protease do not involve a covalent intermediate though a
tetrahedral intermediate exists. The nucleophilic attack is
achieved by two simultaneous proton transfer: one from a water
molecule to the diad of the two carboxyl groups and a second one
from the diad to the carbonyl oxygen of the substrate with the
concurrent CO--NH bond cleavage. This general acid-base catalysis,
which may be called a: "push-pull" mechanism leads to the formation
of a non covalent neutral tetrahedral intermediate.
[0029] Examples of the aspartic protease family of proteins
include, but are not limited to, pepsin A (Homo sapiens), HIV1
retropepsin (human immunodeficiency virus type 1), endopeptidase
(cauliflower mosaic virus), bacilliform virus putative protease
(rice tungro bacilliform virus), aspergillopepsin II (Aspergillus
niger), thermopsin (Sulfolobus acidocaldarius), nodavirus
endopeptidase (flock house virus), pseudomonapepsin, (Pseudomonas
sp. 101), signal peptidase II (Escherichia coli), polyprotein
peptidase (human spumaretrovirus), copia transposon (Drosophila
melanogaster), SIRE-1 peptidase (Glycine max), retrotransposon bs1
endopeptidase (Zea mays), retrotransposon peptidase (Drosophila
buzzatii), Tas retrotransposon peptidase (Ascaris lumbricoides),
Pao retrotransposon peptidase (Bombyx mori), putative proteinase of
Skippy retrotransposon (Fusarium oxysporum); tetravirus
endopeptidase (Nudaurelia capensis omega virus), presenilin 1 (Homo
sapiens).
[0030] Proteases and Cancer
[0031] 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 gelatinase's 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.
[0032] 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 intrapenrtoneally 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.
[0033] 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.
[0034] ATP-Dependent Proteases (Mitochondrial Lon Protease Homolog
1 Precursor)
[0035] The present invention provides a novel human ATP-dependent
protease. ATP-dependent proteases, such as Lon proteases, require
ATP hydrolysis for function and play critical roles in numerous
important biological processes, such as organism development, gene
transcription, intracellular proteolysis and protein biogenesis,
prevention of non-specific or excessive proteolysis (Goldberg,
Semin Cell Biol 1990 December; 1 (6):423-32), and intercellular
signaling. Therefore, novel human ATP-dependent proteases are
useful for modulating/regulating any of these important biological
processes, particularly for diagnosing, preventing and/or treating
defects in proteolysis, gene transcription, intercellular
signalling, and numerous human developmental disorders.
[0036] Many ATP-dependent proteases are involved in modulation of
proteolysis, insertion of proteins into membranes, and disassembly
or oligomerization of protein complexes (Suzuki et al., Trends
Biochem Sci 1997 April;22(4): 118-23). Proteolysis is critical for
maintaining the stability of important metabolic enzymes and for
effectively removing terminally damaged polypeptides (Porankiewicz
et al., Mol Microbiol 1999 May;32(3) 449-58). ATP-dependent
proteases may be found in mitochondria and chloroplasts, as well as
in the cytoplasm.
[0037] In E. Coli, Lon ATP-dependent proteases together with Clp
ATP-dependent proteases, account for 70-80% of the energy-dependent
degradation of proteins. Lon and Clp both interact directly with
substrates to cause degradation (Maurizi et al., Experientia 1992
Feb. 15;48(2):1178-201). Proteolysis in Escherichia coli, such as
by Lon proteases, eliminates abnormal and misfolded proteins from
the cell and also reduces the time and amounts of availability of
key regulatory proteins (Gottesman et al., Annu Rev Genet
1996;30:465-506).
[0038] Lon-type proteases catalyze the ATP-dependent degradation of
mitochondrial matrix proteins. In yeast, mitochondrial Lon-type
proteases has been found to be involved in a variety of critical
mitochondrial functions, including mitochondrial protein turnover,
assembly of mitochondrial enzyme complexes, and maintenance of
mitochondrial DNA integrity. Furthermore, Lon-type proteases are
essential for respiratory function (Barakat et al., Plant Mol Biol
1998 May;37(1):141-54.).
[0039] The improtance of Lon proteases in development is further
illustrated in Myxococcus xanthus, in which disruption of a lon
gene (specifically, the lonD gene), encoding a Lon protease, has
been shown to block development at an early stage. The
lonD-disrupted strains of Myxococcus xanthus could not form
fruiting bodies nor myxospores (Tojo et al., J Bacteriol 1993
July;175(14):4545-9).
[0040] The bsgA gene of Myxococcus xanthus encodes another
ATP-dependent protease that is critical for the regulation of early
gene expression during fruiting body formation and sporulation in
Myxococcus xanthus. Myxococcus xanthus strains with mutated bsgA
genes are unable to initiate a required cell-cell interaction,
thereby leading to an inability to transcribe normal levels of many
developmentally induced genes (Gill et al., J Bacteriol 1993
July;175(14):4538-44).
[0041] Novel Lon proteases may also be useful as markers during
spermatogenesis, and during mitochondrial and germ cell development
(Meinhardt et al., Hum Reprod Update 1999
March-April;5(2):108-19).
[0042] For a further review of ATP-dependent proteases, including
Lon proteases, see Schmidt et al., Curr Opin Chem Biol 1999
October;3(5):584-91; Etlinger et al., Revis Biol Celular
1989;20:197-216; and Langer et al., Experientia 1996 Dec.
15;52(12):1069-76. Barakat et al., Plant Mol Biol 1998
May;37(1):141-54, Suzuki et al., Science. 1994 Apr.
8;264(5156):273-6, Teichmann et al., J. Biol. Chem. 1996 Apr.
26;271(17):10137-42, van Dijl et al., Proc Natl Acad Sci USA. 1998
Sep. 1;95(18):10584-9, Van Dyck et al., J. Biol. Chem. 1994 Jan.
7;269(1):238-42, Rep et al., Science. 1996 Oct. 4;274(5284):103-6,
Campbell et al., Mol Biol Cell. 1994 August;5(8):899-905, Witte et
al., EMBO J. 1988 May;7(5):1439-47, Wang et al., Proc Natl Acad Sci
USA. 1993 Dec. 1;90(23):11247-51, Leonhardt et al., Mol Cell Biol.
1993 October;13(10)6304-13, Fu et al, Biochemistry. 1998 Feb.
17;37(7):1905-9.
[0043] Protease proteins, particularly members of the ATP-dependent
protease 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 ATP-dependent protease subfamily.
SUMMARY OF THE INVENTION
[0044] 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 ATP-dependent 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 retinoblastomas (eye),
melanotic melanomas (skin), endometrium adenocarcinomas (uterus),
adenocarcinomas (ovary), schizophrenic brain, kidney and human
heart.
DESCRIPTION OF THE FIGURE SHEETS
[0045] FIG. 1 provides the nucleotide sequence of a cDNA molecule
sequence that encodes the protease protein of the present
invention. (SEQ ID NO:1) In addition, structure and functional
information is provided, such as ATG start, stop and tissue
distribution, where available, that allows one to readily determine
specific uses of inventions based on this molecular sequence.
Experimental data as provided in FIG. 1 indicates expression in
humans in retinoblastomas (eye), melanotic melanomas (skin),
endometrium adenocarcinomas (uterus), adenocarcinomas (ovary),
schizophrenic brain, kidney and human heart. 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.
[0046] FIG. 3 provides genomic sequences that span the gene
encoding the protease protein of the present invention. (SEQ ID
NO:3) In addition structure and functional information, such as
intron/exon structure, promoter location, etc., is provided where
available, allowing one to readily determine specific uses of
inventions based on this molecular sequence. As illustrated in FIG.
3, an insertion/deletion SNP variant ("indel") was identified at
position 12469.
DETAILED DESCRIPTION OF THE INVENTION
[0047] General Description
[0048] 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 ATP-dependent 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 ATP-dependent 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.
[0049] 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 ATP-dependent protease subfamily and the
expression pattern observed. Experimental data as provided in FIG.
1 indicates expression in humans in retinoblastomas (eye),
melanotic melanomas (skin), endometrium adenocarcinomas (uterus),
adenocarcinomas (ovary), schizophrenic brain, kidney and human
heart. 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 ATP-dependent protease family or subfamily of protease
proteins.
[0050] Specific Embodiments
[0051] Peptide Molecules
[0052] 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
ATP-dependent 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.
[0053] 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.
[0054] 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).
[0055] 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.
[0056] 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. Ins 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.
[0057] 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 retinoblastomas (eye), melanotic
melanomas (skin), endometrium adenocarcinomas (uterus),
adenocarcinomas (ovary), schizophrenic brain, kidney and human
heart. 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.
[0058] 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.
[0059] The present invention further provides proteins that consist
essentially of the ammo 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.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] To determine the percent identity of two amino acid
sequences or two nucleic acid sequences, the sequences are aligned
for optimal comparison purposes (e.g., gaps can be introduced in
one or both of a first and a second amino acid or nucleic acid
sequence for optimal alignment and non-homologous sequences can be
disregarded for comparison purposes). In a preferred embodiment, at
least 30%, 40%, 50%, 60%, 70% 80%, or 90% or more of the length of
a reference sequence is aligned for comparison purposes. The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position (as used herein
amino acid or nucleic acid "identity" is equivalent to amino acid
or nucleic acid "homology"). The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences, taking into account the number of gaps, and the
length of each gap, which need to be introduced for optimal
alignment of the two sequences.
[0067] The comparison of sequences and determination of percent
identity and similarity between two sequences can be accomplished
using a mathematical algorithm. (Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds.; M Stockton Press, New York, 1991). In a
preferred embodiment, the percent identity between two amino acid
sequences is determined using the Needleman and Wunsch (J. Mol.
Biol. (48):444-453 (1970)) algorithm which has been incorporated
into the GAP program in the GCG software package (available at
http://www.gcg.com), using either a Blossom 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment,
the percent identity between two nucleotide sequences is determined
using the GAP program in the GCG software package (Devereux, J., et
al., Nucleic Acids Res. 12(1):387 (1984)) (available at
http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight
of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or
6. In another embodiment; the percent identity between two amino
acid or nucleotide sequences is-determined using the algorithm of
E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been
incorporated into the ALIGN program (version 2.0), using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4.
[0068] 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.
[0069] Full-length pre-processed forms, as well as mature processed
forms, of proteins that comprise one of the peptides of the present
invention can readily be identified as having complete sequence
identity to one of the protease peptides of the-present invention
as well as being encoded by the same genetic locus as the protease
peptide provided herein. As indicated by the data presented in FIG.
3, the map position was determined to be on chromosome 16 by ePCR,
and confirmed with radiation hybrid mapping.
[0070] Allelic variants of a protease peptide can readily be
identified as being a human protein having a high degree
(significant) of sequence homology/identity to at least a portion
of the protease peptide as well as being encoded by the same
genetic locus as the protease peptide provided herein. Genetic
locus can readily be determined based on the genomic information
provided in FIG. 3, such as the genomic sequence mapped to the
reference human. As indicated by the data presented in FIG. 3, the
map position was determined to be on chromosome 16 by ePCR, and
confirmed with radiation hybrid mapping. 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.
[0071] FIG. 3 provides information on a SNP that has been found in
the gene encoding the protease protein of the present invention.
Specifically, a thymine indel at position 12469 was identified.
[0072] 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.
[0073] Orthologs of a protease peptide can readily be identified as
having some degree of significant sequence homology/identity to at
least a portion of the protease peptide as well as being encoded by
a gene from another organism. Preferred orthologs will be isolated
from mammals, preferably primates, for the development of human
therapeutic targets and agents. Such orthologs will be encoded by a
nucleic acid sequence that will hybridize to a protease peptide
encoding nucleic acid molecule under moderate to stringent
conditions, as more fully described below, depending on the degree
of relatedness of the two organisms yielding the proteins. As
indicated by the data presented in FIG. 3, the map position was
determined to be on chromosome 16 by ePCR, and confirmed with
radiation hybrid mapping.
[0074] FIG. 3 provides information on a SNP that has been found in
the gene encoding the protease protein of the present invention.
Specifically, a thymine indel at position 12469 was identified.
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).
[0075] 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.
[0076] 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.
[0077] 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)).
[0078] 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.
[0079] 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.
[0080] 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).
[0081] 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.
[0082] Such modifications are well known to those of skill in the
art and have been described in great detail in the scientific
literature. Several particularly common modifications,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation, for
instance, are described in most basic texts, such as
Proteins--Structure and Molecular Properties, 2nd Ed., T. E.
Creighton, W. H. Freeman and Company, New York (1993). Many
detailed reviews are available on this subject, such as by Wold,
F., Posttranslational Covalent Modification of Proteins, B. C.
Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al.
(Meth. Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N.Y.
Acad. Sci. 663:48-62 (1992))
[0083] 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.
[0084] Protein/Peptide Uses
[0085] 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.
[0086] 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.
[0087] 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 numerous cancers, including retinoblastomas (eye), melanotic
melanomas (skin), endometrium adenocarcinomas (uterus), and
adenocarcinomas (ovary). Furthermore, expression has also been
observed in the schizophrenic brain and kidney. These expression
patterns have been determined by a virtual northern blot analysis.
In addition, PCR-based tissue screening panel indicates expression
in human heart. A large percentage of pharmaceutical agents are
being developed that modulate the activity of protease proteins,
particularly members of the ATP-dependent protease 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 retinoblastomas (eye), melanotic
melanomas (skin), endometrium adenocarcinomas (uterus),
adenocarcinomas (ovary), schizophrenic brain, kidney and human
heart. Such uses can readily be determined using the information
provided herein, that which is known in the art, and routine
experimentation.
[0088] 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 ATP-dependent protease
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
numerous cancers, including retinoblastomas (eye), melanotic
melanomas (skin), endometrium adenocarcinomas (uterus), and
adenocarcinomas (ovary). Furthermore, expression has also been
observed in the schizophrenic brain and kidney. These expression
patterns have been determined by a virtual northern blot analysis.
In addition, PCR-based tissue screening panel indicates expression
in human heart.
[0089] 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 Figure indicates expression in humans in
retinoblastomas (eye), melanotic melanomas (skin), endometrium
adenocarcinomas (uterus), adenocarcinomas (ovary), schizophrenic
brain, kidney and human heart. In an alternate embodiment,
cell-based assays involve recombinant host cells expressing the
protease protein.
[0090] 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.
[0091] 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.
[0092] 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).
[0093] 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.
[0094] 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.
[0095] 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 numerous cancers,
including retinoblastomas (eye), melanotic melanomas (skin),
endometrium adenocarcinomas (uterus), and adenocarcinomas (ovary).
Furthermore, expression has also been observed in the schizophrenic
brain and kidney. These expression patterns have been determined by
a virtual northern blot analysis. In addition, PCR-based tissue,
screening panel indicates expression in human heart. 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.
[0096] 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.
[0097] 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.
[0098] 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.
[0099] 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.
[0100] 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 retinoblastomas (eye),
melanotic melanomas (skin), endometrium adenocarcinomas (uterus),
adenocarcinomas (ovary), schizophrenic brain, kidney and human
heart. 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.
[0101] 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.
[0102] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for a protease
protein is fused to a gene encoding the DNA binding domain of a
known transcription factor (e.g., GAL-4). In the other construct, a
DNA sequence, from a library of DNA sequences, that encodes an
unidentified protein ("prey" or "sample") is fused to a gene that
codes for the activation domain of the known transcription factor.
If the "bait" and the "prey" proteins are able to interact, in
vivo, forming a protease-dependent complex, the DNA-binding and
activation domains of the transcription factor are brought into
close proximity. This proximity allows transcription of a reporter
gene (e.g., LacZ) which is operably linked to a transcriptional
regulatory site responsive to the transcription factor. Expression
of the reporter gene can be detected and cell colonies containing
the functional transcription factor can be isolated and used to
obtain the cloned gene which encodes the protein which interacts
with the protease protein.
[0103] 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.
[0104] 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 retinoblastomas (eye), melanotic melanomas (skin),
endometrium adenocarcinomas (uterus), adenocarcinomas (ovary),
schizophrenic brain, kidney and human heart. 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.
[0105] 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.
[0106] 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 or 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.
[0107] 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 maker 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.
[0108] 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.
[0109] 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 retinoblastomas (eye), melanotic
melanomas (skin), endometrium adenocarcinomas (uterus),
adenocarcinomas (ovary), schizophrenic brain, kidney and human
heart. Accordingly, methods for treatment include the use of the
protease protein or fragments.
[0110] Antibodies
[0111] 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.
[0112] 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.
[0113] 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).
[0114] 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.
[0115] 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.
[0116] 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).
[0117] Detection on an antibody of the present invention can be
facilitated by coupling (i.e., physically linking) the antibody to
a detectable substance. Examples of detectable substances include
various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, and radioactive
materials. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0118] Antibody Uses
[0119] 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
numerous cancers, including retinoblastomas (eye), melanotic
melanomas (skin), endometrium adenocarcinomas (uterus), and
adenocarcinomas (ovary). Furthermore, expression has also been
observed in the schizophrenic brain and kidney. These expression
patterns have been determined by a virtual northern blot analysis.
In addition, PCR-based tissue screening panel indicates expression
in human heart. 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.
[0120] 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 retinoblastomas (eye),
melanotic melanomas (skin), endometrium adenocarcinomas (uterus),
adenocarcinomas (ovary), schizophrenic brain, kidney and human
heart. 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.
[0121] 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 retinoblastomas (eye), melanotic melanomas
(skin), endometrium adenocarcinomas (uterus), adenocarcinomas
(ovary), schizophrenic brain, kidney and human heart. 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.
[0122] 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.
[0123] The antibodies are also useful for tissue typing.
Experimental data as provided in FIG. 1 indicates expression in
humans in retinoblastomas (eye), melanotic melanomas (skin),
endometrium adenocarcinomas (uterus), adenocarcinomas (ovary),
schizophrenic brain, kidney and human heart. 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.
[0124] 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.
[0125] 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.
[0126] Nucleic Acid Molecules
[0127] 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.
[0128] 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.
[0129] 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.
[0130] 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.
[0131] 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.
[0132] 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.
[0133] 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.
[0134] 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.
[0135] 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.
[0136] 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.
[0137] 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).
[0138] 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.
[0139] The present invention further provides non-coding fragments
of the nucleic acid molecules provided in FIGS. 1 and 3. Preferred
noncoding 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.
[0140] 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.
[0141] 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.
[0142] 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.
[0143] FIG. 3 provides information on a SNP that has been found in
the gene encoding the protease protein of the present invention.
Specifically, a thymine indel at position 12469 was identified.
[0144] 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.
[0145] Nucleic Acid Molecule Uses
[0146] The nucleic acid molecules of the present invention are
useful for probes, primers, chemical intermediates, and in
biological assays. The nucleic acid molecules are useful as a
hybridization probe for messenger RNA, transcript/cDNA and genomic
DNA to isolate full-length cDNA and genomic clones encoding the
peptide described in FIG. 2 and to isolate cDNA and genomic clones
that correspond to variants (alleles, orthologs, etc.) producing
the same or related peptides shown in FIG. 2. As illustrated in
FIG. 3, an insertion/deletion SNP variant ("indel") was identified
at position 12469.
[0147] 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.
[0148] 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.
[0149] 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 gen me,
to alter in situ expression of a gene and/or gene product. For
example, an endogenous coding can be replaced via homologous
recombination with all or part of the coding region containing one
or more specifically introduced mutations.
[0150] The nucleic acid molecules are also useful for expressing
antigenic portions of the proteins.
[0151] The nucleic acid molecules are also useful as probes for
determining the chromosomal positions of the nucleic acid molecules
by means of in situ hybridization methods. As indicated by the data
presented in FIG. 3, the map position was determined to be on
chromosome 16 by ePCR, and confirmed with radiation hybrid
mapping.
[0152] The nucleic acid molecules are also useful in making vectors
containing the gene regulatory regions of the nucleic acid
molecules of the present invention.
[0153] 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.
[0154] The nucleic acid molecules are also useful for making
vectors that express part, or all, of the peptides.
[0155] The nucleic acid molecules are also useful for constructing
host cells expressing a part, or all, of the nucleic acid molecules
and peptides.
[0156] The nucleic acid molecules are also useful for constructing
transgenic animals expressing all, or a part, of the nucleic acid
molecules and peptides.
[0157] 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 numerous cancers, including retinoblastomas
(eye), melanotic melanomas (skin), endometrium adenocarcinomas
(uterus), and adenocarcinomas (ovary). Furthermore, expression has
also been observed in the schizophrenic brain and kidney. These
expression patterns have been determined by a virtual northern blot
analysis. In addition, PCR-based tissue screening panel indicates
expression in human heart. 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.
[0158] 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.
[0159] Probes can be used as a part of a diagnostic test 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 numerous cancers,
including retinoblastomas (eye), melanotic melanomas (skin),
endometrium adenocarcinomas (uterus), and adenocarcinomas, (ovary).
Furthermore, expression has also been observed in the schizophrenic
brain and kidney. These expression patterns have been determined by
a virtual northern blot analysis. In addition, PCR-based tissue
screening panel indicates expression in human heart.
[0160] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate protease nucleic acid
expression.
[0161] 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
retinoblastomas (eye), melanotic melanomas (skin), endometrium
adenocarcinomas (uterus), adenocarcinomas (ovary), schizophrenic
brain, kidney and human heart. 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.
[0162] 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.
[0163] 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.
[0164] 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
numerous cancers, including retinoblastomas (eye), melanotic
melanomas (skin), endometrium adenocarcinomas (uterus), and
adenocarcinomas (ovary). Furthermore, expression has also been
observed in the schizophrenic brain and kidney. These expression
patterns have been determined by a virtual northern blot analysis.
In addition, PCR-based tissue screening panel indicates expression
in human heart. Modulation includes both up-regulation (i.e.
activation or agonization) or down-regulation (suppression or
antagonization) or nucleic acid expression.
[0165] 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 retinoblastomas (eye),
melanotic melanomas (skin), endometrium adenocarcinomas (uterus),
adenocarcinomas (ovary), schizophrenic brain, kidney and human
heart.
[0166] 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.
[0167] 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.
[0168] 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 a SNP that has been found in the gene
encoding the protease protein of the present invention.
Specifically, a thymine indel at position 12469 was identified. As
indicated by the data presented in FIG. 3, the map position was
determined to be on chromosome 16 by ePCR, and confirmed with
radiation hybrid mapping. 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.
[0169] Alternatively, mutations in a protease gene can be directly
identified, for example, by alterations in restriction enzyme
digestion patterns determined by gel electrophoresis.
[0170] 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.
[0171] 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)).
[0172] 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.
[0173] 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 FIG. 3 provides information a SNP that has
been found in the gene encoding the protease protein of the present
invention. Specifically, a thymine indel at position 12469 was
identified.
[0174] 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.
[0175] 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 a SNP that has been found in the
gene encoding the protease protein of the present invention.
Specifically, a thymine indel at position 12469 was identified.
[0176] 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.
[0177] 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.
[0178] 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
numerous cancers, including retinoblastomas (eye), melanotic
melanomas (skin), endometrium adenocarcinomas (uterus), and
adenocarcinomas (ovary). Furthermore, expression has also been
observed in the schizophrenic brain and kidney. These expression
patterns have been determined by a virtual northern blot analysis.
In addition, PCR-based tissue screening panel indicates expression
in human heart. 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.
[0179] Nucleic Acid Arrays
[0180] 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).
[0181] 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.
[0182] 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.
[0183] 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.
[0184] 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.
[0185] 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.
[0186] 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 a SNP that has been found
in the gene encoding the protease protein of the present invention.
Specifically, a thymine indel at position 12469 was identified.
[0187] 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).
[0188] 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.
[0189] In another embodiment of the present invention, kits are
provided which contain the necessary reagents to carry out the
assays of the present invention.
[0190] 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.
[0191] 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.
[0192] Vectors/Host Cells
[0193] 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.
[0194] 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.
[0195] 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).
[0196] 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.
[0197] 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.
[0198] 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.
[0199] 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).
[0200] 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).
[0201] 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.
[0202] 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.
[0203] 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.
[0204] As described herein, it may be desirable to express the
peptide as a fusion protein. Accordingly, the invention provides
fusion vectors that allow for the production of the peptides.
Fusion vectors can increase the expression of a recombinant
protein, increase the solubility of the recombinant protein, and
aid in the purification of the protein by acting for example as a
ligand for affinity purification. A proteolytic cleavage site may
be introduced at the junction of the fusion moiety so that the
desired peptide can ultimately be separated from the fusion moiety.
Proteolytic enzymes include, but are not limited to, factor Xa,
thrombin, and enteroprotease. Typical fusion expression vectors
include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New
England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway,
N.J.) which fuse glutathione S-transferase (GST), maltose E binding
protein, or protein A, respectively, to the target recombinant
protein. Examples of suitable inducible non-fusion E. coli
expression-vectors include pTrc (Amann et al., Gene 69:301-315
(1988)) and pET 11d (Studier et al., Gene Expression Technology:
Methods in Enzymology 185:60-89 (1990)).
[0205] 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)).
[0206] The nucleic acid molecules can also be expressed by
expression vectors that are operative in yeast. Examples of vectors
for expression in yeast e.g., S. cerevisiae include pYepSec1
(Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kujan et al,
Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123
(1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).
[0207] 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)).
[0208] 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)).
[0209] 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.
[0210] 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).
[0211] 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.
[0212] 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, NY, 1989).
[0213] 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.
[0214] 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.
[0215] Vectors generally include selectable markers that enable the
selection of the subpopulation eof 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.
[0216] 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.
[0217] 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.
[0218] 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.
[0219] 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.
[0220] Uses of Vectors and Host Cells
[0221] 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.
[0222] 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.
[0223] 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.
[0224] 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.
[0225] 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.
[0226] 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.
[0227] 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.
[0228] 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.
[0229] 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.
[0230] 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.
[0231] 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 2594 DNA Homo sapiens 1 gtgcgaaagg ctgccagcat gtcatcagtg
agccccatcc agatccccag tcgcctcccg 60 ctgctgctca cccacgaggg
cgtcctgctg cccggctcca ccatgcgcac cagcgtggac 120 tcggcccaca
acctgcagct ggtgcggagc cgccttctga agggcacgtc gctgcaaagc 180
accatcctgg gcgtcatccc caacacgcct gaccccgcca gcgacgcgca ggacctgccg
240 ccgctgcaca ggattggcac agctgcactg gccgttcagg ttgtgggcag
taactggccc 300 aagccccact acactctgtt gattacaggc ctatgccgtt
tccagattgt acaggtctta 360 aaagagaagc catatcccat tgctgaagtg
gagcagttgg accgacttga ggagtttccc 420 aacacctgta aaatgaggga
ggagctagga gaactatcag agcagtttta caaatatgca 480 gtacaattgg
ttgaaatgtt ggatatgtct gtccctgcag ttgctaaatt gagacgtctt 540
ttagatagtc ttccaaggga agctttacca gacatcttga catcaattat ccgaacaagc
600 aacaaagaga aactccagat tttagatgct gtgagcctag aggagcggtt
caagatgact 660 ataccactgc ttgtcagaca aattgaaggc ctgaaattgc
ttcaaaaaac cagaaaaccc 720 aagcaagatg atgataagag ggttatagca
atacgcccta ttaggagaat tacacatatc 780 tcaggtactt tagaagatga
agatgaagat gaagataatg atgacattgt catgctagag 840 aaaaaaatac
gaacatctag tatgccagag caggcccata aagtctgtgt caaagagata 900
aagagactca aaaaaatgcc tcagtcaatg ccagaatatg ctctgactag aaattatttg
960 gaacttatgg tagaacttcc ttggaacaaa agtacaactg accgcctgga
cattagggca 1020 gcccggattc ttctggataa tgaccattac gccatggaaa
aattgaagaa aagagtactg 1080 gaatacttgg ctgtcagaca gctcaaaaat
aacctgaagg gcccaatcct atgctttgtt 1140 ggccctcctg gagttggtaa
aacaagtgtg ggaagatcag tggccaagac tctaggtcga 1200 gagttccaca
ggattgcact tggaggagta tgtgatcagt ctgacattcg aggacacagg 1260
cgcacctatg ttggcagcat gcctggtcgc atcatcaacg gcttgaagac tgtgggagtg
1320 aacaacccag tgttcctatt agatgaggtt gacaaactgg gaaaaagtct
acagggtgat 1380 ccagcagcag ctctgcttga ggtgttggat cctgaacaaa
accataactt cacagatcat 1440 tatctaaatg tggcctttga cctttctcaa
gttcttttta tagctactgc caacaccact 1500 gctaccattc cagctgcctt
gttggacaga atggagatca ttcaggttcc aggttataca 1560 caggaggaga
agatagagat tgcccatagg cacttgatcc ccaagcagct ggaacaacat 1620
gggctgactc cacagcagat tcagataccc caggtcacca ctcttgacat catcaccagg
1680 tataccagag aggcaggggt tcgttctctg gatagaaaac ttggggccat
ttgccgagct 1740 gtggccgtga aggtggcaga aggacagcat aaggaagcca
agttggaccg ttctgatgtg 1800 actgagagag aaggttgcag agaacacatc
ttagaagatg aaaaacctga atctatcagt 1860 gacactactg acttggctct
accacctgaa atgccgattt tgattgattt ccatgctctg 1920 aaagacatcc
ttgggccccc gatgtatgaa atggaggtat ctcagcgttt gagtcagcca 1980
ggagtagcaa taggtttggc ttggactccc ttaggtggag aaatcatgtt cgtggaggcg
2040 agtcgaatgg atggcgaggg ccagttaact ctgaccggcc agctcgggga
cgtgatgaag 2100 gagtccgccc acctcgctat cagctggctc cgcagcaacg
caaagaagta ccagctgacc 2160 aatgcttttg gaagttttga tcttcttgac
aacacagaca tccatctgca cttcccagct 2220 ggagctgtca caaaagatgg
accatctgct ggagttacca tagtaacctg tctcgcctca 2280 ctttttagtg
ggcggctggt acgttcagat gtagccatga ctggagaaat tacactgaga 2340
ggtcttgttc ttccagtggg tggaattaaa gacaaagtgc tggcggcaca cagagcggga
2400 ctgaagcaag tcattattcc tcggagaaat gaaaaagacc ttgagggaat
cccaggcaac 2460 gtacgacagg atttaagttt tgtcacagca agctgcctgg
atgaggttct taatgcagct 2520 tttgatggtg gctttactgt caagaccaga
cctggtctgt taaatagcaa actgtaggtc 2580 caaatctcaa tttt 2594 2 852
PRT Homo sapiens 2 Met Ser Ser Val Ser Pro Ile Gln Ile Pro Ser Arg
Leu Pro Leu Leu 1 5 10 15 Leu Thr His Glu Gly Val Leu Leu Pro Gly
Ser Thr Met Arg Thr Ser 20 25 30 Val Asp Ser Ala His Asn Leu Gln
Leu Val Arg Ser Arg Leu Leu Lys 35 40 45 Gly Thr Ser Leu Gln Ser
Thr Ile Leu Gly Val Ile Pro Asn Thr Pro 50 55 60 Asp Pro Ala Ser
Asp Ala Gln Asp Leu Pro Pro Leu His Arg Ile Gly 65 70 75 80 Thr Ala
Ala Leu Ala Val Gln Val Val Gly Ser Asn Trp Pro Lys Pro 85 90 95
His Tyr Thr Leu Leu Ile Thr Gly Leu Cys Arg Phe Gln Ile Val Gln 100
105 110 Val Leu Lys Glu Lys Pro Tyr Pro Ile Ala Glu Val Glu Gln Leu
Asp 115 120 125 Arg Leu Glu Glu Phe Pro Asn Thr Cys Lys Met Arg Glu
Glu Leu Gly 130 135 140 Glu Leu Ser Glu Gln Phe Tyr Lys Tyr Ala Val
Gln Leu Val Glu Met 145 150 155 160 Leu Asp Met Ser Val Pro Ala Val
Ala Lys Leu Arg Arg Leu Leu Asp 165 170 175 Ser Leu Pro Arg Glu Ala
Leu Pro Asp Ile Leu Thr Ser Ile Ile Arg 180 185 190 Thr Ser Asn Lys
Glu Lys Leu Gln Ile Leu Asp Ala Val Ser Leu Glu 195 200 205 Glu Arg
Phe Lys Met Thr Ile Pro Leu Leu Val Arg Gln Ile Glu Gly 210 215 220
Leu Lys Leu Leu Gln Lys Thr Arg Lys Pro Lys Gln Asp Asp Asp Lys 225
230 235 240 Arg Val Ile Ala Ile Arg Pro Ile Arg Arg Ile Thr His Ile
Ser Gly 245 250 255 Thr Leu Glu Asp Glu Asp Glu Asp Glu Asp Asn Asp
Asp Ile Val Met 260 265 270 Leu Glu Lys Lys Ile Arg Thr Ser Ser Met
Pro Glu Gln Ala His Lys 275 280 285 Val Cys Val Lys Glu Ile Lys Arg
Leu Lys Lys Met Pro Gln Ser Met 290 295 300 Pro Glu Tyr Ala Leu Thr
Arg Asn Tyr Leu Glu Leu Met Val Glu Leu 305 310 315 320 Pro Trp Asn
Lys Ser Thr Thr Asp Arg Leu Asp Ile Arg Ala Ala Arg 325 330 335 Ile
Leu Leu Asp Asn Asp His Tyr Ala Met Glu Lys Leu Lys Lys Arg 340 345
350 Val Leu Glu Tyr Leu Ala Val Arg Gln Leu Lys Asn Asn Leu Lys Gly
355 360 365 Pro Ile Leu Cys Phe Val Gly Pro Pro Gly Val Gly Lys Thr
Ser Val 370 375 380 Gly Arg Ser Val Ala Lys Thr Leu Gly Arg Glu Phe
His Arg Ile Ala 385 390 395 400 Leu Gly Gly Val Cys Asp Gln Ser Asp
Ile Arg Gly His Arg Arg Thr 405 410 415 Tyr Val Gly Ser Met Pro Gly
Arg Ile Ile Asn Gly Leu Lys Thr Val 420 425 430 Gly Val Asn Asn Pro
Val Phe Leu Leu Asp Glu Val Asp Lys Leu Gly 435 440 445 Lys Ser Leu
Gln Gly Asp Pro Ala Ala Ala Leu Leu Glu Val Leu Asp 450 455 460 Pro
Glu Gln Asn His Asn Phe Thr Asp His Tyr Leu Asn Val Ala Phe 465 470
475 480 Asp Leu Ser Gln Val Leu Phe Ile Ala Thr Ala Asn Thr Thr Ala
Thr 485 490 495 Ile Pro Ala Ala Leu Leu Asp Arg Met Glu Ile Ile Gln
Val Pro Gly 500 505 510 Tyr Thr Gln Glu Glu Lys Ile Glu Ile Ala His
Arg His Leu Ile Pro 515 520 525 Lys Gln Leu Glu Gln His Gly Leu Thr
Pro Gln Gln Ile Gln Ile Pro 530 535 540 Gln Val Thr Thr Leu Asp Ile
Ile Thr Arg Tyr Thr Arg Glu Ala Gly 545 550 555 560 Val Arg Ser Leu
Asp Arg Lys Leu Gly Ala Ile Cys Arg Ala Val Ala 565 570 575 Val Lys
Val Ala Glu Gly Gln His Lys Glu Ala Lys Leu Asp Arg Ser 580 585 590
Asp Val Thr Glu Arg Glu Gly Cys Arg Glu His Ile Leu Glu Asp Glu 595
600 605 Lys Pro Glu Ser Ile Ser Asp Thr Thr Asp Leu Ala Leu Pro Pro
Glu 610 615 620 Met Pro Ile Leu Ile Asp Phe His Ala Leu Lys Asp Ile
Leu Gly Pro 625 630 635 640 Pro Met Tyr Glu Met Glu Val Ser Gln Arg
Leu Ser Gln Pro Gly Val 645 650 655 Ala Ile Gly Leu Ala Trp Thr Pro
Leu Gly Gly Glu Ile Met Phe Val 660 665 670 Glu Ala Ser Arg Met Asp
Gly Glu Gly Gln Leu Thr Leu Thr Gly Gln 675 680 685 Leu Gly Asp Val
Met Lys Glu Ser Ala His Leu Ala Ile Ser Trp Leu 690 695 700 Arg Ser
Asn Ala Lys Lys Tyr Gln Leu Thr Asn Ala Phe Gly Ser Phe 705 710 715
720 Asp Leu Leu Asp Asn Thr Asp Ile His Leu His Phe Pro Ala Gly Ala
725 730 735 Val Thr Lys Asp Gly Pro Ser Ala Gly Val Thr Ile Val Thr
Cys Leu 740 745 750 Ala Ser Leu Phe Ser Gly Arg Leu Val Arg Ser Asp
Val Ala Met Thr 755 760 765 Gly Glu Ile Thr Leu Arg Gly Leu Val Leu
Pro Val Gly Gly Ile Lys 770 775 780 Asp Lys Val Leu Ala Ala His Arg
Ala Gly Leu Lys Gln Val Ile Ile 785 790 795 800 Pro Arg Arg Asn Glu
Lys Asp Leu Glu Gly Ile Pro Gly Asn Val Arg 805 810 815 Gln Asp Leu
Ser Phe Val Thr Ala Ser Cys Leu Asp Glu Val Leu Asn 820 825 830 Ala
Ala Phe Asp Gly Gly Phe Thr Val Lys Thr Arg Pro Gly Leu Leu 835 840
845 Asn Ser Lys Leu 850 3 112132 DNA Homo sapiens misc_feature
(1)...(112132) n = A,T,C or G 3 atcattaaaa agtcaggaaa caacaggtgc
tggagaggat gtggagaaat aggaacactt 60 ttacactgtt ggtgggactg
taaactagtt caaccattgt ggaagacagt gtggcaattc 120 ctcaaggatc
tggaactaga aataccattt gacccagcca tcccattgct gggtatatac 180
ccaaaggatt ataaatcatg ctgctataaa gacacacaca cacgtatgct tactgcggca
240 ctattcgcaa tagcaaagac ttggaaccaa cccaaatgtc catcaatgat
agactggatt 300 aagaaaatgt ggcacatata caccatggaa tactatgcag
ccataaaaaa ggatgagttc 360 atgtcctttg tagggacatg gatgatgctg
gaaaccatca ttctgagcaa actatcgcaa 420 agaccgaaaa caaaacactg
caagttctca ctcataggtg gcaactgaac aatgagaaca 480 cttggacaca
gggtggggaa catcacactc aggggcctgt cgttgggtgg tggggagtgg 540
gggggaaggg ataccattag gagatatacc taatgtaaat gacgagttag tgagtgcagc
600 aaaccaacat ggcacatgta tacatatgta acaaacctgt acgttgtgca
catgtaccct 660 agaacttaaa ctataataaa aaataaaatt aaattaaaaa
catgaaaaaa aataaaagta 720 tcaaggttgt aaaaaaaaaa aaaattggac
gggcgcagtg gctcaggcct gtaatcccag 780 cacttttggg aggccaaggc
gggcagatca ctgaggtcag gagattgaga ccatcctggc 840 taacatggcg
aaaccccgtc tctactaaaa atacaaaaaa ttagccgggc agtggttgcg 900
ggtgcctgta gtccccagct actcgggagg ctgaagcagg agaatggcat gaacccggga
960 ggcggagctt gcagtgagcc gagatctcgc cactacactc cagcctgggt
gacagagcga 1020 gactccgtct caaaaaaaaa aaaaaaaaaa aaaaattgag
gacttgccac agattagaga 1080 acacctagga gatttcataa caaaacacct
aggagatttc acaacaggat cctggatatt 1140 ggatcctgga ccagatccaa
tgaaggacat tagtgggaaa actggcaaaa tttgggtaag 1200 gcctataggt
taaacgataa taatgttaat ttcctggttt tgatcattga actatgatta 1260
tgtaagatga taacagacga aactgggtga aaggtatata ggaactctgc tgtagttttg
1320 tacatctaaa atcaattcgg gccgggcacg ttggctcacg cctgtaatcc
cagcactttg 1380 ggaggccgag gtggacggat cgcttgaggt caggagttaa
agaccagcct ggccaacatg 1440 gtgaaatccc ctccctacta aaaatacaac
aattagctgg gtgtggtggc gggcatctgt 1500 aatcccagct actcgggagg
ctgaggcagg agaatcgctt gaacccggga ggcagaggct 1560 gcaagccgtg
ggtatcgcgc cattgcactc cagcctccgc gacagagcga gaatctgtct 1620
cagaataaat aaataaataa ataaataaat aattagttcg aatcaaaagt taaaaacact
1680 tcaagtatat gtaaaaaatc gaagaaaacg ttaaaaacac ttcaagtata
tacaattcaa 1740 ataagatcat ccttccaaat atactctgta agtgaggcga
aggtcgctgc acgcttgagt 1800 gcacgtcttt ccgcataggt aggacgctca
agtcttaccg ggaggctctc ctagagagca 1860 gcgcgaagcc atggcttttg
ggcccgggga cggaccgtag cgcgtagccg gaagcggagg 1920 cgtggaggcg
ggtctgaggt ttggtgactg cggggcaggc cgggggcagc tgtctgtctg 1980
gctctttttg acagccccca gtgcgaaagg ctgccagcat gtcatcagtg agccccatcc
2040 agatccccag tcgcctcccg ctgctgctca cccacgaggg cgtcctgctg
cccggctcca 2100 ccatgcgcac cagcgtggac tcggcccgca acctgcagct
ggtgcggagc cgccttctga 2160 agggcacgtc gctgcaaagc accatcctgg
gcgtcatccc caacacgcct gaccccgcca 2220 gcgacgcgca ggacctgccg
ccgctgcaca ggtaggcctg gctgcccccg cggcggcggc 2280 gggcggcgcg
gcctcctccg gggacctggg cccaggccac ggcctgcctt gagcgcgagg 2340
ctcagttcgg ggcggccttc gcggctcggt tccgcctctc tggtgctatc acttgcaaaa
2400 tggggatgtc agatacctgc cccatgacca tgaatgagat cgttcatgaa
gtagtgcctg 2460 acacctggtg aaactacgca gttccctacc gttctggata
atttaatttg aatcctcttc 2520 cccctctccg caattcctcg ccctcggtct
tcagcctcct aggccagtgc ttttaacttt 2580 ccaggccctt tctttctccc
cggtgatctc tgccttcact tgccttcgct tttcaccttt 2640 ctccccactg
ccctttactc ctatccgcct ccccttttct gtcacccatc atttttgtcc 2700
gctgaggcat tctctgctcc gtgagtttta acttttcctg tttcattcct aaactgcact
2760 atttgtgggt gcctttcttc tatactccct gccacccttc tccttctccc
cctaatcctt 2820 ctgtttccct ttgtaaaggg cctttactgc tcacattttc
gctggtcccc ctttctggaa 2880 ctttcctagc ttctcacctc tgctccttca
ctcataacat ttcttaggcc ccaggcttac 2940 tactatattg cccagtaccc
tcgccctatt ggtgtgactt tgggtgagag ctttaacctc 3000 tatttctttt
attctgcaat ttggaaactg acagcatcca tctcttaggc aagttatgaa 3060
gaataaattg aataatgtgt atattccact ttgcaccatg catgatggat gactttgctg
3120 tccagtactg tgtagtgcat gtggctcgtc aaattgagat gatagaattg
ccagttgtcc 3180 tggtttgctg cgattgtctg ttttagcatt gaaagtccta
tgttttagcc cctccgtccc 3240 agggaaacca ggaggttggt caccctaaat
gtgctgtaag tgtacaatac acgccagatt 3300 ttgaaaaaac tttttgatta
atacatttta tatggattaa atgttggaaa ggtaatattt 3360 tgagtacttg
gggttaataa aatgttaaga tttctgctgt ttttacttta taatgtggcc 3420
actaaaattt tatatgtggt ccacattata tttttattgg acaatgctgg tatatcgtat
3480 gctctcaaca agtatcttca aactcacctg ccaagcaccc gcctcctatt
cctaactcta 3540 ctggaggtgt tgtgttttca gtttagagct tctcctttcc
tggcagttat cccttatttt 3600 taaattaggg gttcctgact ctgaatggat
ttccggaggg ttggacatgt cttatttttc 3660 ctcaaaatct tgtgactatg
tacatttttt taggagaatc ctttgctttc ttcagattct 3720 caaaggagac
tggtacctcc ccccaccccc gttaaaagaa agcaaaacaa agcaacaaag 3780
accaacaaac cttccacagc agcccagtat tcatttatat tgtaaaagcc ttgattttct
3840 caagcatgga aaatattttg gctcccatct gacctgcttt ggttattgcc
tgagtggaat 3900 tggtcacatt ccaagtttca gtactctttg ataaattgta
ttggattcta gtttcccaac 3960 atacgactct gctccttctg cttacttttc
ccaaattatt ttgccttctg tgcccaggca 4020 cacttagttc cctgtctagg
caagagtggt cattattaga cttcattttc tttctactgt 4080 gcatatgtat
tgattagcca tgggcacatt gtgaacttga aaagtcgatt tagtcacatt 4140
ttaagtttca ctatttgttg gtattattct ggcaagattt tggaaggttt ttattattta
4200 ttcatttgtg tattttttga gacagagtct cattctgtct cctccgctga
agtgcagtgg 4260 cgtgatcgta gcccaccgca accttgattg aactcctggg
ctcaagtgat cgtcctgcct 4320 cagcttctgg agtggctggg actataggcg
tgcaccacta cacccagcta atttttaaat 4380 tttttgtaga aatggggtct
cactatgttg cttaggctgg tctcaaactc ctggactcaa 4440 gctatcccct
gctttggcct ctggagtagc tgggactata ggcaagcgcc accataccct 4500
tcaggttttt aatttatttt atgaaaatcc ctccaaagca acaatcctca attctcctgc
4560 ttgaaagtaa tcactaataa tcaggtactg tgtgatctga tccttgatgt
tcatattatt 4620 gcctttaact gagtagcaat gttaaaattt aatcatttaa
attagaaaac atatattgaa 4680 aagtcttcat agaagtccgg cattataaga
actcatcaga ccatctagtt atcctagaag 4740 tattgtttgc tacttaaaaa
gcctatgtgg aaagattgta ccatattcct tggtaatagt 4800 ttccaatgtc
tttttttctc taatagggcc tttaaaacac tctacttaaa aaaaaaaaaa 4860
aaaaaaggct ttaacaatac caatactgag taatccatag cattagcctg tttccacgca
4920 caagtctgtc cttccccagt tacctgcttt tctgtatggt agcccagagg
ccagaagagg 4980 ggctctgttc ctttctcttg tttcctttgc gctatccagg
tgacgctggc acagccttca 5040 aagagcagca gaagtaattt gctcccagcg
ttctttgcca catagagtgg cagggttaaa 5100 tgatttaaaa tttaatcatt
taaattagaa aacatagatt gaaaagtctt catagaattc 5160 cagcattaaa
agaactcatc agaccatcta gttatcctag aagtattgtt tgctacttca 5220
aaagcctatg tggaaagatt gtacgatatt ccttggtaat agtttcgaat gtcttttttt
5280 ctctaatatg gcttttaaag cactctactt aaaaaaaaaa aaagctttaa
taataccaat 5340 accgagttat ccacagtatt agtctgtttc catgcacaaa
tctgtccttc cccagttacc 5400 tgcttttctg tatggtagcc cagaggtgag
atgaggggct ctgttcctgt ctcttgtttc 5460 ctttacacca tccaggtgac
actggctgca gccttcaagg agcagcagaa gtaatttgct 5520 cccagcgttc
tttgccacac agagtggcag gattagatgt tgacttacct ctgccacttc 5580
cttggtggtt ttgagtagta cagtcccttt ctgcacgtta gtgtgcaggc atgttgcctg
5640 caggagcctt tttaaaggag gagctttgga cttgtcctgc agtatagaac
ttggctggca 5700 tgctgaccca gggcaccctg catttttctg cttagtagaa
ctgcattttt agtgcttcct 5760 gagtgaccca ttgttttctt agtgaaaagg
ggtcataatt tagtactacc tgtacaatat 5820 cctttcaagc atttcaagat
ggtcatccag ctttcttcca aatttacact tttcagggta 5880 catggcttca
tttcctcata gtgccgactt ctcagtctcc ctcaccaggc tggtgtcaaa 5940
cttgtgagct caagtgatcc tcctgcctct gtctcccaaa gtgttaggat tacaggcgtg
6000 agccaccatg cctggcctat gtttataatt cttgtaggta gaagtggtac
ctattgtcca 6060 ttgtaatgag aaaaaagtaa aatttgtctt aaaatataat
taaggaactc aatttattaa 6120 atttaaattt atcctttaaa ttttaaattt
aaatttattt cttaaattta tttctattac 6180 attttcttgt aaccatgtac
acctaagttg ttctacttta atttttttga gacagggtct 6240 cactctgtca
cccatgctgg tgcagtggtg ccatctcagc tcactgcaac ctttgcctcc 6300
caggttcaag tgatcctctc acctcagcct cctgagtgtc tgggattaca ggcatgtgcc
6360 acaatgccta gctatttttt tttttttttt ttggtggaga cggggttttg
ccatgttgcg 6420 caaactggtt tcgaactcct gagcccaagt gatccacttg
cctcggcctc ccaaagtgct 6480 gggattatag gtgtgagcca ccatgccatg
ttctaccttt ttgaatctca tttactcact 6540 tgtaataagg aaataatact
accttcttca tggggtgaag ggaggtataa aatgaagtat 6600 acatatgaaa
gccttttgaa actgcaaagc attctaaacc tatatccaaa tgggtagttt 6660
taaatgtaga ttttcacaaa aggggattaa agagaggagt ggggaggccc catattattc
6720 caacacgggc tgaactgaac taacatcatt gcaggaaggt cttggaagat
taaagattcc 6780 aagaaaaatt aagggctttg agtaaaaaaa ttttttaaaa
gtggctgggc ctggtggcac 6840 gtgcctgtac tcccatctac tcatgatgct
gaggcggagg attacttgag cccaggtgat 6900 cgaagctgca gtgagctata
atcgtaccac tgcactccag cctgggtgac agagcaagat 6960 tctgtctata
ggaaaaaaaa aaaaaaaaaa agcaagtgct gggcatatag gctggaatta 7020
gatatttaca taatatcctc atcttggaaa actttttcca gtagtgctgc ttttagattt
7080
tcccactact gcagttgatg gttcttaaat atgtttggaa ctcttatatt atttaggtca
7140 gtttccaaat tacacaaatt gtaaccattg tagtcagacc tcacttgaat
gaaaacaata 7200 ttttacaaac tctgagggta gattcgagtt aggatttgga
ttaaaacatt atcttaaaac 7260 ctctgagggt agattcgagt taggagtttc
aaaacttctt tgaacaatat cataattagg 7320 atgtagattt acagagctac
tagctaaagg gaaggacacc agtcattggg atgtataagt 7380 ttggatctgt
tgcaaaatta aaatgctgcc ttttgagcat gcctaataat gcacatacaa 7440
tagaagagcc agaattttta gaaaaatgac tgacttgata tacaaccttt tgtatatcat
7500 agaaggaaaa tattagttga gtattttgtt tatttacctg tttgtatata
taaaacctgg 7560 ggcccaatat acaatagatt ctttttcact atgcttttca
cccacagtgt ctcaccaggt 7620 actctgtttc tagccatcta taatttcata
gatgtttttc tttaaaaggg atgtattcta 7680 ggctgggcga ggtgggtctt
gcctgtaatc ctagcacttt gggaggccaa gatgggagga 7740 ttgcttgagg
ccagtagttg gagatcagcc tggtcaacat catgagatcc catctctgtt 7800
aaaaaaagaa aaaaaaattt ttttaaaggg ataatttcta gtcaactata agtgatttta
7860 agtaaaaagc aattaaggca tgtatacatc tgtacctttt gtaggcatag
tataaattca 7920 gcttaatctc ttcagtttgg aacatcttcc tttcacagca
aaaatattgt atttgcttta 7980 taagaaaacc ccttttggcc aggtgtggtg
gctcacgccc gtaatcctag cactttggga 8040 ggctgaggtg ggtggattac
cttaggtcag gagttcgaga ccagcctggc caacatagtg 8100 aaaccctgtc
tctactaaaa atacaaaaat tagctgggcg tggtggtgtg tgccctgtaa 8160
tcccagctag ttgggaggct gaggcacgag aatcccttga acccaggagg cagagtgcaa
8220 taagccgaga tcacgccatt gtacgtcagg ctgggcgaca gggtgagact
ccctctaaaa 8280 aacaaacaaa aaaaccacag tggctcacac ctgtaatccc
agcactttgg gaggccaagg 8340 tgggcgaatc atgaggtcaa gagatcgaga
tcatcctggc caacatggtg aaacctcatc 8400 tctacaaaaa atacaaaaaa
ttagctgggc gtggtggtgt gtgcctgtag tcccagctac 8460 ttgggaggct
gaggcaggag aatcacttga atctgggaga cggaggttgc agtgagccaa 8520
gattaggcta ctgcgctcca gcctggtgac aaagtgagac tccgtctcaa aaaaaaaaaa
8580 caaaaaacaa aaaacaactc tttagcatca ccttttagca atgacatagc
ccaaataatt 8640 aaatttgtct cctgatcgga gatttggatt tgtctcatct
ctctttctgg ttcctccttg 8700 gtttctactt tgtaaaccct ttaggccggg
gatccagttt cttgtctgtg gatgttttat 8760 atacaaacag gactgtgagc
tctttcagca ttgtacaaac agtgatgaat atcatctgca 8820 attaattatg
tttaagttat tctctaatca gtttagaggt ggctcacttc ctcaggcaat 8880
ctgagtgggc tttcaggaag tgggaaatat tatctactat tgattgaaga aaagcagcca
8940 caacacaaat aagtcaaaat aatagctaat tgctaaataa tttcaagttt
tttatgtatg 9000 tgattttttt ccctcaccaa tttatcttct cagttgtttg
gcttattatt taaatcagtt 9060 tttattgtaa acatggtaat gactgaaagg
taagaaaagg atagacgtag ttcagaataa 9120 actgagtggc agaaagaagc
caaaggctat gtgtaatcta cggaatgagt aatttataag 9180 gaagtaatca
agaattcact gtgtatagaa gtaagcaagt tcactcacat agtcacatac 9240
tgtattacat gatttattat ctttgagatg ggcaggtgtg gtgttcttct attaccgctt
9300 tcctagggtg ttgagagttc tagtccttct attttctttt ctggaattac
cacttttcct 9360 atggctgaag ggagaaaata ttatttattt tgggatctgg
aattgtcttc tcaatgttga 9420 tttttgtatt ttatataact gacttagttt
ggatgaggct tcctttctgt gaattaaatt 9480 tatatgtgac ttgatcagag
ttgtatttgc tgatgaggag ctgagacttg aagccttttc 9540 acctattgtt
aggtaaaatg attaccactt agaactaggt tgagaccttt tgagatgtgg 9600
gtctttcttt agctctcctc agtctatggc agtgtgtgga ctgtaatatt tagccctcac
9660 acttagaaat tcagtgttaa gggcatatat ataagttccc agtatgtgat
ggcagcttgt 9720 gataaggtgg gtatgtggaa gtttcataga ctgattatgt
aagaaaactg acttgatgtt 9780 agtagcacaa ctggtgttgg aacggagatt
tcttagattg gtttatgcta tttatattta 9840 aatgtattta aattgataat
atttatcctg gtataagatt gccttattct tagttgacaa 9900 tgttaattta
agatatgtaa ttctcagctg cttttctctt acatttttac gcttgaataa 9960
tccaagtgtt tacaaattcc tacctaattt tttaaaagag gtgcagatta tagtgagatg
10020 gtctgctttg ccatatagct gagggtagtg gcagaagagg ccacatactg
gatgctaagt 10080 taaatagaga aaaaatttat ttacacttca gatgtctttt
gcttaatgaa tgtatcagaa 10140 aagccaacac tttctgaagt gagtttctgt
tctaccgtat tgaatgtttg taataccgat 10200 gttttgtgtg tttttcagga
ttggcacagc tgcactggcc gttcaggttg tgggcagtaa 10260 ctggcccaag
ccccactaca ctctgttgat tacaggccta tgccgtttcc agattgtaca 10320
ggtcttaaaa gagaagccat atcccattgc tgaagtggag cagttggacc gacttgagga
10380 gtttcccaac acctgtaaaa tgagggagga gctaggagaa ctatcagagc
agttttacaa 10440 atatgcagta caagtaagtt gcttttattt tttcttaaaa
cccatttttc tttggttctt 10500 ttgctttcct aagatatggt gaatctgttg
gatagtgaag ttttaggaca gtatacattt 10560 aaatgagtta gtaacattat
atattaattc tgatttactc ttatctgggg ttgtacctaa 10620 atcattccag
gacatattgg cctacccttt ctaaagtttt ccaaatgtta tttctacagc 10680
tttccttcta acttctactg tctctaaact agataattat taaacctaaa tatttaaagc
10740 taaaaaacga aatactgcac agaagctgtc tgtcactaaa atatctaggc
accatttata 10800 taaattacaa tatattactt caaaagtcaa gatcacattg
tctagcagta actatggtag 10860 atcaagcctg tggtgggctg atttcaagta
tggttaaaac cttgattaac tagaatgctg 10920 ggaaggaagc acattttaga
tatgcattaa atatttgact ctttaattct agttcttttt 10980 ggttaactct
agatagaaca gaaagctcct attcccaccc cattttgttt caaaccttaa 11040
tgaaacataa aattataaag tatagtcttc tacttttcta ttagtttaat ccagtgacta
11100 taactagatc tatgaggatc agataatgtt taaaagtcac aattataaat
actactgatc 11160 attgaaatat gtgtggggca agtgttcata gccagtggta
tttgtatctg atgtggcatt 11220 tgaagagcca tacttacagt gtaatgaaca
ataacagaaa aatagtaaat ttgagggcca 11280 ggtgcgctgg tgcacacctg
taatcccagc actttgggag gctgaggtgg gtggattgct 11340 tgagcccagt
agttcgagat cagcctaggc agcatggtga gatcccgtct ctacaaaatg 11400
tacaaaaatt agccgagtgt gatggtgcgt gcctgtagtc ccagctactg gggaggctga
11460 ggtgggagga ttacttgaac ctagtaggtg gaagttgcag tgagccaaga
ttgcatcact 11520 gcattccagc ctgggcaaca gagcgagacc ctgactcaaa
aaaaaaaaaa gaaaaataga 11580 aaatttgaat ctgtaatttc tatatgggct
gaaagaaagc actttgagga aagaaatttc 11640 agtttgaaaa ctggaataag
tgaatatact gcttaggaat aaaggagatt gagagaaata 11700 gaatttcttt
ttcttttcag cagtgatgtt ccctgggtct ttgtgcctct attggacata 11760
gatagcttca tagcctcttt tgctttgctt ttacttcttt gtactttgaa tctagaggaa
11820 ctttttaaac ttgtaaagat tttgcagtga cattaaagga atttttagaa
ataaatagat 11880 caccacacat cttactgtca tcatgcatca aatttaattt
ttgttcgtct tctgggctca 11940 gttcatattc aattatatgt tttgtttttg
tatccatgtc tgatgttcat attaagtact 12000 tttgttaatt tcattgagtt
aatgtatact aattttataa tttctctttt tagacattaa 12060 agttatttcc
aattattctc tttcatcccc ttctgcatct acttctactt ctgcatctct 12120
tcaatgaact tcttcaatag catcctgtct cctagttctt ctgtcttgaa ccttttctct
12180 tcactgagcc tttctaaaag aagtctgggg catcccattc ccttgagtaa
aagactttaa 12240 tggctatagg atggacacca aatttcttag tataacatta
agaccgtttg caacttgtct 12300 tgggcctatc tgtcttgcgt caactctagt
tatcacctca ctgacaccct agttctagct 12360 ctactgaatg taaaacagct
tcacattgag ttattttatg tctctatgat tctgccttca 12420 gttctctgct
gggagtgctc ttccatctct gatttttttt tttttttttg aaatggagtc 12480
ttgccctgtt gcccaggctg gagtgcagtg gtgcaatttc ggctcactgc agcctccgcc
12540 tcccgggttc aagcgattct cctgcttcag cctcccaagt agctggcatt
acaggcatgc 12600 gccaccacgc ccggctaact ttttgtgtct ttagtagaga
tgaggtttca ccatgttggc 12660 caggctggtc tcgaactcct gacctcatga
tccaaccgcc accacgcccg gcctccatct 12720 ctgaatttta aaattgaatc
tatgctttcc caacagctgt aggctgttag cgctcatctc 12780 tgtgtgcctt
cacagtctgt catacatgtc atttaacata atgcttatca cattgtattg 12840
aaatgtatct tataggtatt ttttctctac caaacttgaa ttcacttttc tcctttagcc
12900 atcctgtact gagcagtgtt ttgggtctgg caaatagttt gtactcagta
aatgtttgga 12960 aaatgagttt taactgtttt attttcgtgg ggtgaattcc
tagtagcaag ggtattcaaa 13020 ttttattatc tacttcttcc acctgaacag
cttcatcgta attatacttt aattcccttc 13080 attctaggca ggtaatggat
aagttccaaa attacgatgt tgttggagag gtttgaatat 13140 tactagcaca
tgaaatctga tttgaactga ctaaatgaag gtttagtaca tcattatgaa 13200
ttagtgtgaa ctaagttttg ctatgttaac ttctctgaaa tctcagtcgc ataatgtgag
13260 tgtctttctg gctcatgctt catgcctgag actagtgggg gttgtgtctg
cctattaaag 13320 tcactcggac ccaggtggat tggagattca tctaaagaca
tgcttccctt atctctaagg 13380 caggaaaagg aaatggggcg catctctcat
tggcttgtaa tgcttctgcc cagaaggagc 13440 tgtcacttcc actacgtttc
atggatcaat ttaagactca tagacacacc tattagtata 13500 ttcgaaggaa
gttagaaaga gcagtgccca gaagaaaagg ggagtttgtc agtagcccta 13560
atgactatca cagttactga aagtgtgcct tgggcataat ctatcttaac tcccagatat
13620 acgctgacag ttgtttttct aaaagtcatt cacagtgctc agattctagt
tagtccaaat 13680 tgatatggtt tggctgtgtc cccacccaaa tatcaccttg
agttgtaata attcccatgt 13740 gtcagggggc ggtgccaggt gtagataatt
gaattatggg ggcggttccc ccatactgtt 13800 ctcttggtgg tgaataagtc
tcacaagatc agatggttat ataaatgata gttcccctgc 13860 acacgctgtc
ttgcctgcta ccatgtaaga caggcctttg cttctccttt gccttcctcc 13920
atgattgtga ggcctcccca gccatgtgga actgtgagtc cattaaacct ctgtctttta
13980 taaattaccc agtctctggt atgtctttat tagcagtgtg agaacagact
aatacaaaat 14040 gttatactaa atattaatat ttcatcctct gattggccgt
gataatagca tcaactatgc 14100 taaatttcta ataatacaca tatttctaat
aatatgcatc taatagggtt tatattgtga 14160 ttatgtaaga gaatattctt
gttcttaaga acaagggtcc ttaatctgtc acaggattag 14220 agatttaaag
aataaggatc tcgattctgc agcttatcct caaatgttca ttaattatgt 14280
gtgagtgtgg agagagagaa agcaaacatg gcaaaatgcc acttttcagt tggtgaattc
14340 aattggtgaa tctggaagaa ggatgtacag gagttattgt atgattcttg
caactttttt 14400 gtacatttga atttttttca atagaaagtt aaaaataatc
atggcacagg tttacaaaac 14460 ccttgtaaac attagtgtta actactttta
agccattatt gcttttcatt ctgattgatg 14520 ttttgaaagt acttttcttt
tcctctgagg cctgtaaaat acgtggacta tattaatcag 14580 tgatctttca
aaaacaaaga ctgaggccca aacattaaac ctagatggaa atctgatttt 14640
taaaaattca caaataatgc cagatttcat ttaaaagact ttttttcccc cttctagttg
14700 gttgaaatgt tggatatgtc tgtccctgca gttgctaaat tgagacgtct
tttagatagt 14760 cttccaaggg aagctttacc agacatcttg acatcaatta
tccgaacaag caacaaagag 14820 aaactccagg tacagtgttc ccttttgaac
gccaggttgc tttgtcactt tttattgaga 14880 actagatagt gagtagttaa
gttttgacct tcaagaaaaa gatattggag acccaaagta 14940 attgaaatgc
ttttacattt aaactgactt tcaaatgtga ttgttttata tttttgttga 15000
cacaagcagc tcttttattt tatatttttg ttgacacaag cagctctttt atttgcataa
15060 tcagtaatgg tagtcaattt acagaaaaag ttaaagcaaa gaatcataaa
aaggtaaata 15120 tttgactggg tgctcacgcc tgtagtccca gcactttggg
aggctgagat gggtggatcg 15180 cttgagatca ggagttcgag accagcctgg
ccaacatggt aaaaccccat ctctactaaa 15240 aatacaaaat tagctgggcg
tggtggtgcg cgcctataat cccagctact cgagaggctg 15300 aggcaggaga
atcgcttgaa cctgggaggc agaggctgca gtgagccaag attgcaccac 15360
tgcactccag cctgggcaac agagactctg cctctaaata aataaataaa taaatattta
15420 atttaactta aatatgtaga cattctttga ttcactattt ttaaacgtgg
agccatggcc 15480 cttcccttat gtgtggacct gctttcttag aatcttcatc
atgtttctta tataaatcac 15540 acctatgatg cattacttat aattttaaat
ttatatttat ttaaagtgaa atgaatttta 15600 aagacacttg aaaagtaatc
caagtataga atcctacatt tacatgactt aatccccaaa 15660 ctgtaatact
ttaagttttc ttgcacactt atttttaaga tatttttaaa gcagtatttt 15720
taatgaatca tcctagaata tttgtttgtt ttcagtgaaa cagctctttc atatgttatc
15780 agtttattta atacttaaat ccaactgtta taatagcaaa tacaactaac
acaaacaggt 15840 tggttataca caggaattca attaatccag tgggagtaga
agagttacag gactgccaga 15900 gagccccctg gctgtgggcg gcagcagtgt
gttttactgc gggaacagag agcggcctgt 15960 gctccgacaa atcactagtg
agagttggtt gagtgcttct gttctcttgt gtatgtaaac 16020 atttaatatt
ttgaacctat aatttgttta gatctaatat gaaaacacat tctgggcttc 16080
aagagagtaa ttcccagaaa gagttgacgt caactgtgtg tctggttttt tcatcttaaa
16140 aacacacagc ttcggccggg cgcagtggcc cacgcctgta atcccaacac
tttgggaggc 16200 cgaggtggga agatcacgag gtcaggagat cgagaccatc
ctggctaaca gagtgaaacc 16260 ctgtctctac taaaaataca aaaaattagc
cgggcatggt gtcgggtgcc tgtagtccca 16320 gttactctgg aggctgaggc
aggagaatga cgtgaaccca ggagggggag cttgcactga 16380 gccaagatct
cgccactgca ctccaacctg gggacagagc aagattccgt ctcaaaaaaa 16440
aaaagaaaaa aaaaaaccac acagcttcat tttaaagtga aaaaccaaga tcctgttttt
16500 tctttctttt ttaaggattc tgatattcat ctcaaacaac cttgctgatt
aatatagttc 16560 atttggttgt cttagccata gtgtagcttt gaatactgtt
aataattttt ttttaacttg 16620 gcaatttaaa ccatggctct gactgtctgt
ttttggattg tgtgtttctg agagagatcc 16680 tattgattga ctcacatttc
cttagatttt agatgctgtg agcctagagg agcggttcaa 16740 gatgactata
ccactgcttg tcagacaaat tgaaggcctg aaattgcttc aaaaaaccag 16800
aaaacccaag caagatgatg ataagagggt aaatatttat tttaacccat ttcagttttg
16860 aaaaaaaaat aaggagaata aagagaggaa caaagaagaa aagtttattg
tctcctacca 16920 ctcgcactac tgataaaatt taggtgtttc cctctcatcc
ttttctttgc ctggattttt 16980 ttttaaagca tgtaagcatt tttctcactt
tgttttggtt atcatccaaa aggataattt 17040 actgagccat ttcccctttt
gtgttgtttc caatgttttg tgtattgtaa acactaacaa 17100 ataactatga
tgggtgtctt tgagtataac atttttttac tgcatgtaat actaagaaac 17160
taatacaaaa ctctttctta aaaggactat atgttgtgtc aaaatttggc tgttttcaac
17220 ttataataag tttccatttt tatttagtca aactcttgat ctttttttgt
tttctaagct 17280 taagtcctct aaccttcagt ggcttgataa atattcactt
tcctttcagt ttaattttag 17340 ttgatttttt aaaaagtatt taattcttta
acccatatat tattttgaag acagcagttg 17400 tatttttccc tcaaatagct
ttttgtttga ctcaacacca ctaattaaat aatccttccc 17460 atccccatta
tcatctatta catttatatg tatgatggga tctgtttgaa gtctaccttg 17520
atctgctgat tttactattt ttatgtctgg acagagttta tattaggaag atatatttga
17580 tgtggacagg atgtgaaaat ggcatttctc tgaaggtgtt gagatgcagc
gctctgactt 17640 aagttgaggc gttgagaatt atgttagcaa tttgacgttc
atcagcgcag aagtcttgtc 17700 atcaaagaga atacattgta gagaaagcgg
agcagaaggg aagaactcct ccccggtggg 17760 actagagaag gggcagtcaa
gtaggctgag gagagagata ggaacagtga tgatcatgct 17820 ggcgattagt
actccaggac accatgctgt ttaaaacatg cagaaagctg gattatttct 17880
ggcttgagat caggtcaggg actcaattac tcattttgta tagagagaca aatccactgg
17940 gagttgcaga aaactgcaac ttactctcag taaagtttgc catcacttaa
aatgaaagtt 18000 tttcaaaagt gctccagaaa ataagcaaga gacagttatt
taaaaagtag gaattaggat 18060 aatatttgga gttaacctaa aactctctcc
tttttgttcc cctaagagtt gaaaagcact 18120 gttttagcag tcaggaagga
aaaatgcatt aaaaagtgct tttgtcttaa caatgaaatc 18180 actgatatgc
ttataaaaat ctcactttta aaaaatatat aatatgttca gttttttatt 18240
tataatattt tatctgctga tgacttatgt aagaataaaa gcatatattt agtacttgtg
18300 tttttataaa attaaatttt tatttactgc tttatgtttt aaacattttt
atatttgaat 18360 gtattaaata gataaatttt ccaggttaaa aaataagttc
tgggctgaat gcagtggctc 18420 atgcctgtaa tcccagcact ttgggaggcc
aaggaaggag aattgcttga ggccaggagt 18480 tcaagaccag gctgggcaac
atagtgagac ctcatcttta caaaaaaaat ttaaaaaatt 18540 agccagcatg
ctggtgtgtg tctgtagtcc cagctattta ggaagctgag gtggaaggat 18600
tacttgagcc agggaggttg aggctgcagt aagcagtgtt catgccattg cacttcagcc
18660 tggattacaa agcttgacct tgtctcaaaa aataaaatgt tctgggggct
tttaaattaa 18720 atgctagtat ataattttgc tccagtagtg gttgtttatt
catgaatttc aaggagcata 18780 taaggtagtt ttaacatatg atagagagat
catagagaat acaaaggcca tttgactttg 18840 cacagaatat gttttttaga
tttgaaagaa caattttggc aggatgggaa cagatgccga 18900 aggctcactg
aagtaattga tgaggtaggg gatctggtgg ttatagccac ttgctggaga 18960
agcagaactt cacaagaaag gaagtaaata gtgcgatagt taactagaag aaactagagg
19020 taagaaaaaa atattttgaa agcaggaaag ctttgaagac aaaatagagc
cagtggtgga 19080 aaggttgaag atgctaggaa gaaattttgt aatgtaggag
ataaaatgga atttttttca 19140 gtcaccaaat ggtaagaagt aatgtatttc
aagaaaatag tggctgcaat agtagctcaa 19200 agaaaggtaa ttcctagatg
gtttaattat ttctagtatc cagttccttg aaatttgttt 19260 tctcatgcaa
gtattattgt aagcatatac caaagaatca tgtctacctt acgttggtct 19320
acttctgcaa ttctgctgcc tctctgtata caactgcctt ttgattatca ttctgaactt
19380 cacttcctaa agatagagac tgtagtcata aaaatattta ttcagcacca
gtcataatct 19440 tatgtgtacc tgggtacttc gtttccaatt tattttgaca
tacggtttta cttttctgct 19500 ttctatgtta ggttatagca atacgcccta
ttaggagaat tacacatatc tcaggtactt 19560 tagaagatga agatgaagat
gaagataatg atgacattgt catgctagag aaaaaaatac 19620 gaacatctag
tatgccagag caggcccata aagtctgtgt caaagagata aagaggtaaa 19680
ttataaaagg catttgttca ttattgtttt cattcttggt actcctgatt aacaccactt
19740 tcactactct tttctccaat actgaggata cataatacaa atcttccacc
tgcagtgtgc 19800 tgtcaggcaa tataactctt gcagctgcct ttttgttgtc
tgaaagaaca gaccatgctt 19860 ctttgtttat acgtaatgtt tgttcagtta
gcatcatatt cttcacatgt gacttttctt 19920 ctctagatta taaactctca
agggcaagga ctgtccattt ctctttgtac aagacaaagt 19980 acagggaaac
cttgataaca gaataggata tatgggttga ttacattttc tggatatccc 20040
cagtgttaaa ctgaaagcca tttttccttt gcatactttt aactttataa ctcttattac
20100 attttctttt attagtgaat tgtagtgagc ctgcttgaat gcttagtgac
ttaatatttg 20160 actttctgag gcttacagtt aagaacatta gtaattgtag
ttgatgggta ttttatattg 20220 cctctgacat tagttaatat atgtagaaca
tttattatgt gcagaacact ttgctaagca 20280 ttgcatatat tatggaagta
gcatttgtta ttaaatatat gatattagct tgcttttatg 20340 agcagacctc
actcatctct gatacaaaaa aaaatgtatt gtattatgca tagttaggca 20400
cttacatctt attgtgataa gtaaaccaat ggatatatgt cacttgacta tccctgtgag
20460 cttaaaaggg acacacacta gtaaggccat atttccaggt tagaattaga
tataatgttt 20520 tctcctgcag tttgcaggta tctgccttat tttgttttgt
aagtacctta agtacttaga 20580 aaatatgaga atactttgta gagaaagcag
agcagaaggg aagaacccct ccctggtggg 20640 actccagaag gggcagttaa
gtaggctggg gagagagata ggagtggtga tcattacatt 20700 acaaaacaaa
ataaacgttt tattatctgg atactttaaa actttttcag atttgtttaa 20760
acatgcatga tatatctaac caagaaagag agctgtgttt gatttttctg ttatggaatt
20820 tttctgtgtt cttgaacatg tttgctgtgt attctttctc cacagactca
aaaaaatgcc 20880 tcagtcaatg ccagaatatg ctctgactag aaattatttg
gaacttatgg tagaacttcc 20940 ttggaacaaa agtacaactg gtaagccaaa
aaataacacc tgttttgcag tctaattgtc 21000 actcagaaag ctcatgcaat
ttttcatttc aaatttactc cactgattgt cgtactgtta 21060 aattattttt
gttttcaatt tttttgaaac cattttattg aagtgtgatt gtcgtacaaa 21120
aagctgtata taattaatga atacatctca gtgagtttca gaataagtat acacccatga
21180 aaccatcaca atcttcatag ccataaacat atccgtcacc tccaaagttt
cctcctacct 21240 cttttgtgat tattattatc atcattatta ttggcttttt
tcttttggtg ctggtggtaa 21300 gaacattgaa cataaggtct aatgttaaat
taacaatatt gttagcgata ggcacttttc 21360 tttatagtag atctctagaa
cttatttatc ttgcataagt gaaactttgt tccctttaac 21420 catcacctcc
catttccttc tcctctcatc ctgtggcaac tactagtcta ctctccattt 21480
ctatgagttt cactatttta gattccacat gcattaaata ggtgaaatca tacagtactt
21540 gtctttctgt gtctggctta tttcacttag catgatgccc tctaacctag
aggtccatcc 21600 atgttgtcac agatggcaag atttccttct tttttaaggt
gcataatatt ccattgtgtg 21660 tctataccac attttcttta ttcacttatg
tgtcagtaga catttcagtt atttccgtat 21720 cttggctatt gtaagtaata
ctgcagtgaa tacggaagtg cagataactc tttgagatcc 21780 tgatttcagt
tcctttggct gtttacccag aggtggcatt gctggatcat atgtaagttg 21840
tatttgaact tttttagtaa cttccatact gttttcataa tggctgttat cgggggacct
21900 gccccaataa tcatgtaggt tcttttctat tttcctaagc attggctggc
ttgagaaata 21960 aagagacaga gtacaaaaga gagaaatttt aaagctgggt
gtctggggga gacatcacac 22020 gttggtagga tccgtgatgc cccacaagcc
acaaaaacca gcaagttttt attagggatt 22080 ttcaaaaggg gagggagtgt
gcgaataggt gtgggtgaca gacatcaagt acttaacagg 22140
gtaatagaat atcacaaggc aaatggaggc agggcgagat cacaggacca cagctccgag
22200 gcgaaattaa aattgctaat gaagtttcgg gcaccattgt cactgataac
atcttatcag 22260 gagacggggt tttgagataa cggatctgac caaaatttat
tagatgggaa tttcctcttc 22320 ctaataagcc tgggagcgct atgggagact
ggagtctatc tcacctctgc aatctcgacc 22380 ataagagaca ggtacgcccc
gggggggcca gttcagagac ctacccctag gtgcgcattc 22440 tgtttctcag
ggacattcca tgctgagaaa aaagaattca gcgatatttc ttccatttgc 22500
ttttgaaaga agagaaatat ggctctgttc tgcccggctc accagcggtc agagtttaag
22560 gttatctctc ttattccctg aacaattgct gttatcctgt tctttttcca
cggtgctcag 22620 atttcatatt gcacaaacac acatgctgta caatttgtgc
agttaacgca attatcacat 22680 agtcctgagg ccacatacat cctccttggc
tgacaggatt aagagattaa agtaaagaca 22740 ggcataggaa atcacaagag
tattgattga ggaagtgata agtgtccatg aaatctttac 22800 gatttatgtt
tagagattgc agtaaagaca ggcataagaa attacaaaag tattaatttg 22860
gggaactaat aaatgtccat aaaatcttca caatccacgt tcttctgcca tggcttcagc
22920 cggtccctcc gtttggggtc cctgacttcc cgcaacacgc tgtaccaatt
tacattccga 22980 acaacagtgt acaagggtgc ccttttctcc atatcctcac
cttcactgat gatggttttt 23040 ttgtttgttt gtttgttttt ttaaataatg
gccatcctaa caggcataaa gtgctttctc 23100 attgtggttt tgatttgcat
ttccctgatg attagtcatg ataagcacct atttgatttt 23160 ttgccgttaa
gtttcatgag ttccttgtgt attttggata ttaacccctt atcagaaata 23220
tggtttgcac atattttctg ctgttacata ggttgccttc tcattttgct gaactttttt
23280 tattctgtac agaagctttt cagtttgata taatttcact tgttcatttt
tgcttttgtt 23340 gccttgactt tggtgtcaat atccaaaaat accatgccca
gaccaatgtc aaggagcttt 23400 taaaatatat tttgttctag gagttttaca
gtttcaggcc ttacatttaa gtctttaatc 23460 cattttgaat taatgtttgt
acatggtgtc atataagggt tcaagtgcat tcttctgcct 23520 gtgggtatct
ggttttccca caacattttc ttgaagagac tgccctttcc ctattgtata 23580
ttcttggtgc ccttgttgaa aattggttga ccttctaggt aactttatag gtttatttct
23640 gggccctcta ttctattcca ttggtccgtg tgtctgtttt tgtgccagaa
tcatactctc 23700 tgattactgt agcttcgtaa tataacttga agtcagaaag
tctggtgcct ccacgtttgt 23760 tcttgctcaa gattggtttg gctattcagg
gtcttttgta atttcttatt aattttagga 23820 tttttaaatc tatttttgtg
aaaaatgtca ttggaatttt aatagggatt acattgaact 23880 tgtaaattgc
tttgagtggt atagacattt taacaacatt cttctagtct acgaacatgt 23940
aatatctttc catttatttg tgtctgactt atttcatcag tgttttataa tttttagtgt
24000 acagacattt tacctccttg gttaagtttg tacttaagta tttcattctt
tctgaaacta 24060 ttgtaaatga gattgtttcc ttaatttcta tttatttatt
tatttttttg acaggagttt 24120 cactcttgtc gcccaggctg gagtgcagtg
gcatgatctt ggctcactgc aacctctgcc 24180 tcccaagttc aagcgattct
cctgcctcag cctcacgagt agccttaaat acaggcacct 24240 gccatgacac
ccggctaatt ttttgtattt ttagcagaga cggggtttca ccatgttgga 24300
caggctagtc tcgaactctt gacctcaagt gatccacctg cctcggcctc ccaaagtgct
24360 gggattacaa acgtgagcca ctgcgtctgg cccttaattt ctctttggag
aaaggttttt 24420 tttttttttg agctttattg aagtgtaatt gacgtacagt
aaacttcaca aatgtagtat 24480 gtacattttg atgagttttg acttacatat
acatctgtaa taccatcacc ataattaaga 24540 taatgagcat aaccctcacc
tccaaaagtt tcttcatgct ctttgataat cccttccttc 24600 ttccccgccc
ctttcctcct tgcctcctaa tccccaagca accactaaag attaatctgt 24660
attttctaaa atttcatata aatggaatca tagagtatga gccctttttt ctggcttctt
24720 taattcagca tgattatttt gaggttcatc catgttgctg tatataacag
taatttgttt 24780 ctttttattg ctggagttgt attctgttgt atggatatac
catcatttgt ttatcaattc 24840 atctgttgat agacatttgg gttgttttca
gttttttggc tattaaaaat aaagctgtct 24900 gggcacagtg gctcatacct
gtaatcctag cactttgaga gaccaaagtg gacagatcat 24960 ttgagcccag
gagtttgaga ccagcatgag taacacagga agaccccaac tctatttaaa 25020
aaaataaaat aataaatgaa ataaaaatat ttaataaaat atcaaaaaat aaagctactg
25080 tgaactgtgg tagtaaattt atttttaaat ttatgtaatg tttgcatgtc
gtgacaaaat 25140 actgcctttt agttgaaagg aaacatttct tggtactctg
agatgccatg tgtgtcagca 25200 ctagagatgt gtagcagcca tgtatccatc
atgaaaataa ttccattgtt tagcattgca 25260 catagcacaa agaactgaag
atgaataaat tatggtataa aaggagtcat gttaagctcc 25320 taaaccatta
ctacacagga ttatgtctag ataattgtga gtgtggttat aaaaccatga 25380
aaatgccatt catatatata tttttgagat ggagtctcgc tctgtcaccc agtctggagt
25440 gcagtggtgt gatcttgact cactgcagcc tccgcctcct gggttcaagc
aattctcctg 25500 cctcagcctc tcaagtagct gggattacag gcgcttgcaa
ccacacccaa ctcatttttg 25560 tatttttagt agagacaggg tttcactaca
ttggccaggc tggtctcgaa cttctggcct 25620 caagtgatct gcctgctttg
tcctccaaaa gtgctgggat tacagacctg agccactgtg 25680 tccagcctaa
atatctttgt ttgtttgttt gttcgttttt tgagatggtg tcttgccctg 25740
tcggccaggc tgtagtgcag tggtgtgatc tcagctcact gcaacccctg cctcctgtgt
25800 tcaagtgact ctcctgccct agtctactga gtagcaggga ttacaggcgc
ctgccaccat 25860 gcccagctaa tttttgtgtt tttagtagag atggggtttc
accatgttgg ccaggctggt 25920 ctcgaactcc tgacctcaag tgatcctccc
acctcggcct cccaaagtgt tgggattaca 25980 ggtgtgagcc accgagcctg
gccccccatt cataatttct gaaagagaag tttacctacc 26040 aagtagagat
ctcagatagt aaccgaaaac aaaaaggaaa gcagagagga aagagttgta 26100
ggaaatatgt ttgcagattt tcccagctta gaggagtcag tagataccat ttcaatcttc
26160 taattataaa taaggaaatt tatattgaaa tttgaaaaat tttttacatg
taatcacatg 26220 ttattcaaaa caggaagcat gctttctgaa tcattaaaga
gaataattag aaaaatatat 26280 cctgtataga aaagatagaa aataatttat
acagcatgga aatcaccttt acttaaaaga 26340 ttgaaagaac ttttaaaatt
gtctttactt ggcatatttc ttgcaagaaa tttcttcaca 26400 gtgttttcag
tcttttctaa attatcttga cttttattct taccttactg aatgtgttaa 26460
tcatgaatgg ataacgcatt ataacaagta cctttttagg tacaagatga tattttgatg
26520 gaaacttact cttcttgaac atgatgacat tgatgaccta acactgaacc
atgtttgcat 26580 aactaaaata aatcccactg ggacttagta tattattctt
tatagatttg atttactagc 26640 attttaatat ttacagctat ataaaaagat
ttgtctgagg ttttctttta tgtttactgt 26700 ggtaggtttt agtgtcaggg
ctagcactgt gaaacaattg agaaactctc tatctttcac 26760 ttcttcatat
attcattggt tgggttctgg agccaggaaa gggggaagaa attttagttg 26820
ttcttctcct acttcactca cctaggactc tgactaaaat caatagtact ataattaaat
26880 tatatagttt actgcttagc taggtttttt gggggactag cttgggaacc
aaattaccat 26940 ctcaggccat ttttttcctt tatgaaatat ccttagcaaa
ttctaaataa ttaattaaaa 27000 gatatgtatt aattaattaa aagatttctg
tgtatttctc tctcccatct tcttctttca 27060 ctgccagcat gatcaggtgg
ctgtgtatta taccctggca gccacccagc tagtgaattc 27120 attttggctt
ctgttacctg gtgtttaatc tgagtatttt aaatgctaaa tcttattagt 27180
aaacctgttg aaagcttggc tctagaaaca aagcctaact catacacttc tggtgagact
27240 ttgatacaac tttctgtgtg gcaattaggc aattctttac atcatctgtt
tttttttttt 27300 tttttgaccc agcacttctg ttcatagaag ataagctgaa
agaaatcatt gcagatatat 27360 gggaagattt agttccagtg atgcacagtt
gaagcatctt ttataaatgt aaagatgtgt 27420 aaacaacttg aatgctcagc
agtagggaat tagttaaatg aatatagata atttagtaat 27480 ggaacattaa
gtaaccatag aatgttactg ataaatatat gtgtgacagt gaaagttgtc 27540
tgtcatatat taagtgaaaa aaacatttta caaaacttaa aggccccata aaatcccatt
27600 ttgaaaaata ggtttgtaaa tgcacgcaca cagcctggaa ttacacatac
tgaagtaaag 27660 gtagtggtga tctcttgggg gcatgagatt atgggtaact
gttttcttct tttctgttag 27720 tgttatcagg ttttctggaa tgaacatatg
ttactactga aataaggaaa aaaatcaccc 27780 ttttttttaa aaaacaaatg
ccagcacaca tacaatatgt agaaattaag aagtaatgca 27840 taactagaaa
atcattccaa ataaaatgat atgaacattg agtttttaat tgtgtagtgc 27900
ctactatctc tggggacact aagtcttaag cagagaaacc aaaccaaatg cagatctcct
27960 agaatcctca tctagaaaga tccaagtctg ttcttatcac atctattttc
aaaaaaaata 28020 ttttgccctc gtcatgcttg aaaggagttc tttaacttaa
aaattttatg tgttctaatt 28080 atttctgttg ggttatttga cagaccgcct
ggacattagg gcagcccgga ttcttctgga 28140 taatgaccat tacgccatgg
aaaaattgaa gaaaagagta ctggaatact tggctgtcag 28200 acagctcaaa
aataacctga agggcccaat cctatgcttt gttggccctc ctggagttgg 28260
taaaacaagt gtgggaagat cagtggccaa gactctaggt cgagagttcc acaggattgc
28320 acttggagga gtatgtgatc agtctgacat tcgaggacac aggtagaaca
cttctctcag 28380 tttaatctct gattcctctt tctttttaat tgactagagc
tccctaaaag cttaggcata 28440 gcatacatct attttcctta aagggctatg
tgtggtacct tgaatgaaaa ggacatttac 28500 aagaagtatc agctagccta
gagcctctaa gcgtaatgat aaacccaaac taaccttgat 28560 ttgtatgaca
gtggatacta ctctgtgcct caactttcct ggaatctcat ttgaatgtaa 28620
ttataagtta tttatgattg gatattatta tgtctttaca ctcttttcaa cccagtagca
28680 tgccataaat aatgatccct aactctcaga gttaaaaaaa gtaactgcaa
tagggagggc 28740 caataggagg aggtgagaag tctttgataa caaacttgtt
ctgattgcag tctaaacttc 28800 ctcttatgaa ggttggtttg tattatgaat
atgagtaata aggataaatg ttagcataat 28860 tattaaggct tattcttgca
ttttggactc actttctata aaaaaacaat aaactgtaag 28920 aactgtccct
ctaggctggg cacagtggct catgcctgta atcctaacac tttgggaggc 28980
tgaggtgggt ggattgtttg agcctaacag tttgagacca gccggggcaa catagggaaa
29040 cacttttgtc tctacaaaat ttatatttaa attttttaat tttaaatttt
aatttttgtc 29100 tccacaaaaa ttaaaaaatt atgcaggcac agtggcatgc
acctgtggtc ccagctactc 29160 aggaggctga gatgggagaa tcatttaggc
cnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 29220 nnnnnnnnnn nnnnnnnnnn
naaccaagcg gcaaataagg aaactttgtc ttacacaagt 29280 aaatttactt
cttcatttac attaaatttg gttccacaaa aatataaaat taagctaggc 29340
acaatggcag gccttgtgtt cccagctctt agaaggtcta aatggagtat cattacgtct
29400 tgaaagttcc agtttgcagt aacccatatt gtcccctcgc acgccagcct
ggagacagag 29460 acattatctc aaacaaacaa acaaacaaac aacaacaaaa
ctgtttctga ttaatctgac 29520 attattagaa tcagatttgc atgttgcatt
cattgttctc actggtctct ttgttgatct 29580 gatggaaatt gccttgggaa
agcatgaatt tacatttcgt ggtttaaggg attcatagca 29640 attgtaagtt
gtgagaaaac atacctatag tgtatgtgtt aaagaacatg tttaaatgta 29700
ggaaccatga actgcttata aaagaatatg atgctttttt aatatcttgt tttctatttg
29760 ccttattcaa agggatccct atccatagac agggatggga aactgtttca
gaaacttttc 29820 tataagaaat ggttattttt attctctttt atttgctcac
ttaaaattct tacgcattta 29880 aaaagtatca ttactggcct tgtgtagtag
ctcatgcctg taatcccagc actttgggag 29940 gccaaggcag gcagttgctt
gagctcagga gttcaagaac agcctgggca acttggtgac 30000 accccatctc
taaaaaaata ataataataa attttaaaaa agactcatca caagatttta 30060
gtaaataaac aatgaggcgt gcagatcaga gtagagaatt gatttgggtg atttcttctg
30120 gcaatttcaa aagatatttt tgttgcctag acttcttatt cttgcatgta
ccactagagg 30180 ctatagtttg ctttcgtaaa ggaattggca tttctcttgg
accaaactca aagaagctgc 30240 gtctagggcc taaatcttct aattttagct
acagagtaag tatttgatgg catttagaga 30300 gtgagttcgt ggaattaatg
ctatgtgaaa ttgacatcat aagcacgtga catgtaggta 30360 atttgttctt
atttcttttc acattggtat tgattatttg ataaggcttg gaaagcactt 30420
attcaatacc tgacacacag tgagcattca ctaaaaatta gctttaacca ttatttaaat
30480 tctattaata aattctcagg aggacaaatt tagatttaca agcttcagta
tgagttttta 30540 taaatttcaa tctgattttt taattgcctt ctaaaatatt
tatcctattc tcagcattat 30600 tacttaattt atacggcaga attatgggaa
aatgcatttt tctgttgcct actaatggac 30660 agtgtatagt gtcatggttc
tcaccactta caaacatcac tggattaaaa taaatctcta 30720 ttttaaatcc
ttactgacat ataaaatttg ttcttttttt caagtgaata tgcttttgtg 30780
tatgtgactg tattaagaaa attgagtctg aagaaaataa gaattgactt tatgggtctt
30840 ttgtaaaagg aggttgtgtt acaatcacca ttgcctaaaa tatttgtaaa
tataaccttt 30900 ttagaaacgt atatatggag gctgtgattg ttgccgagta
aaaagtataa ggatttgttt 30960 tgtgaatcat tctattcagc ctgattttag
atacaccttg ctggtaagtg ttacttagcc 31020 atcagtgtac cagatgtttg
attaactact atagcaacct gcccttgtgc tgttggggac 31080 atattaccca
tctaccccgt gaattattaa agcctggtga aaaattttat ttcaaaccct 31140
gtttggaagc acgtggagag tagtggggtt cagttgttga ggaaagggtg agggcagagc
31200 atgcacttag gtcagttatg aattgaaggt gaataggagg aggagagaaa
gaacaaccga 31260 caattccagc acaaccatgg gtgtgcctgg gggaacatgt
ggttccatgt gacagttgag 31320 gcatttggga gacaacccag gtcttgacgt
ttgagtaccg gtcacatgct cacagttaga 31380 gttcatgaaa agttttgttt
ttcctcagcc tttgagtagg caccactgtt ccgcagcctt 31440 agaatagcca
aggaaaaaga aagccaggga aaaagaaagc tgctttgtta ttgtccttgc 31500
ttatcctctc gattttgcca ctcactctcc ctgttttccc atgtgtggaa cactttcctt
31560 ttgctaaaag tacctgcgta tgagaagaag gatgccgata agttggggat
tgattttaaa 31620 aacaagcaaa gatatgtttt ttatggttaa atgataatga
ggtgggagat ggggaagcaa 31680 aagagaggct tgccttaata tttaatctta
aacttggaaa ataatagtga tctgactaaa 31740 cattgcctca tttttgtctg
tattgttttg agtagcttaa aggaagaata atgtttatgc 31800 tacgtattaa
ctcattcagt ttttcagtct tttcgatatt tctcatttgg atttatctcc 31860
attgtgattt ttctgtccac tttgtaagcc acaaaatact cattcccttc tatcagtttt
31920 aacaacttaa atttttatat ttaagtatta catttaaata atttaagtca
attcacacaa 31980 atataaggta actaacttct tttaagatga agttttatga
aataatgttt gcataattgt 32040 ttttcatttg ttctttggta aaaagaaata
atatattatt gttatgatat atcttaaatc 32100 actgtggata ttaactccta
gaaatacttt accagctgtt tacttagata ataaaattat 32160 attattgcaa
gaaatccttg tctcaacttt caaacaagat gagaagaaaa atgaacttgt 32220
gatttccaca ttgatacatt ttcatatgca acctgaaatg gtaaagttat aaataaacta
32280 tttcattatt agtttctaca agggaaaaat aactgaagca gcaagcttct
aatgtatttt 32340 tttagcatag tgtaccagat atattatggt ttgcccacta
tcctttcaac ttacatttgc 32400 atgtagctct tctttgcctc tccaaaactt
aggtttattt taaggcctca acccaaggct 32460 tcctccatta atgtaagtgc
agtcagttat gatttcactc ttctctaaac tgaccaccta 32520 ttgtgctcct
ttatcgaata cgggcctctg gcatttctac catacaactg tggagatgaa 32580
acataaatac gtttataaaa agtacaagct ttctcaggca ggggatttat cgtctatctc
32640 ctttatgtac cccatgatgc ttatttaaca tggtgctaaa tgtggtgagc
gctctctggg 32700 tgttttgtga attcatgtaa gattaaaaca taatattttg
gaagttatgc aaccctttag 32760 acgagtacac ccatacaaat tagtctataa
aaagatttag gaatgactac cagaagaata 32820 attgcatttg tttagacatg
ctattataca ttaaaatccc agtttcttaa agactgtttt 32880 tctttttgag
atcattagga tcttttttaa actgattcct ttttccagtt tgagatacac 32940
acacacaccc acacacccac ccacacccac acccacacat ccacacaccc ttggtagaaa
33000 atgtgaaaaa taaggggaaa aaatcctcat gtttttctac cgtacaaaga
taatcactgt 33060 taacatttgt tttgttctgc cagacttatc attggatttt
aagtaacaga attgtaatcc 33120 tgtcattttc acttaacatt gtaacactta
aactcttttc tattccaaat tctttgtaaa 33180 ttttatttta acagtttgca
ttatagcctg cgggagccga gccctttaat tgaataggta 33240 ggaagagtgg
atggtgaaat gcctatattt ttctctcttg tctgctataa aagacatttg 33300
caaaagttgc ttccatgagg cagaaattga aatgggactc aaattcaggt gtactgaatt
33360 ctgctcttgt gctttttcca ggaaaccaga agtaaacttt aagtagctgt
tgctaataat 33420 gatgagcatc actggaaagc tcactgtgtg ccagggaccg
tgctgtgtgc tttgcctgtg 33480 ttctctcatg atccttatat taatataacc
caccaggttg acactatttt ccccatctta 33540 taggtgagga aactgaggct
taggtcaagt aatttgccca aaatagtatt cagaggcttg 33600 tactgtgtta
cctttagagt gctgatggaa agatgctttg agtgctggca cggtggatct 33660
ggtggggaac aatcttacag ctctatatct agcctctact ctgtggtaag accccgtctc
33720 tgtcataaaa gtgctcactg gctctataga ggaggttatt atacccatga
ataaaaacta 33780 ggttgtaagt aaccatcaga tgagttatgg ggccagtaag
tgctgtagac attgcattat 33840 tagagcgatc cctttgtgag aggtagtcag
aaaaagtttc ttagaattgt tgggatttac 33900 gtagcaggaa gaggagtatt
aagggcagga aggcaccata tttttaagaa aggtaaaaat 33960 ttttaagggg
cgtaatagta tcttgattgt ggttgaagca agaaagtaat ggcagcaagt 34020
tgggaagatg aatgggagct ggattgtgaa aagcctcgaa ctccagacaa aggaatttga
34080 accttattct gtaggctctg ggaagcaatg gaaagtgtaa gaggaattgc
ttatatacag 34140 tgtgagtaga atctaggatt ccaatttttt tagaaagggt
gcctacctag aatattattt 34200 tctctctgtg acttcaggtg tagaattgtc
agtacttgtt tttgaagttt actcatcaaa 34260 aaaggaaagg caaataaata
actgcagcaa aaaatgaccc attagagcct ttgagattct 34320 ttaaaaaaat
tcccttccct accactctta aaaatcagag taatggcaaa tctgtaagtt 34380
ctctagaaaa ataattggaa agaatttata aattctgagt ctcgtctttc ctgtatctga
34440 ttctgaaatc ttgaatgtgc taattcctta tattaacagg acaatgttta
ttgcctttgc 34500 ttccctgtgc cttagtcacc tttcccggat gaaaggcatt
cccatgatat ttttaaggct 34560 tgcttgcctt ttcaaagttc actctgttta
ttctgtccta ctttatacca gtcatgtggc 34620 agaaatcagg cctgctctgt
gaatcggctt tgtgcagatc atgaggtaac tgtggctgtt 34680 ccacttgtca
ttgatcattt tcttctcggc agtcaggctt ttatgccttt tcagagacag 34740
catttgcttt gcacaacata gacagcaggg ttataattaa aattagtaaa ttgctgcttt
34800 aagttttgct ggctttgtaa aaaagacacc ttttttggtt tgataaactt
atgtgttttt 34860 atttcatgcc acactctaca tctgtcataa ttatgtgggt
gattcttgtc caaatacaat 34920 aaagcaggct ctcacatttt aacgttcaac
aaaatacctg gctggctgaa cgtggttatt 34980 gccaattagt gcatatggga
tgaatacagt tttgttcaaa aggacagaat aatggaattc 35040 tgatataaat
actgttgacc ccagatcctt atactataat taatagatta tttcctctga 35100
aaataaaaga gattggagtt tttctttttt gttgttgttt ttggtctgca ttctgagtgg
35160 ctgtttgaac tgattttaat ttccttcatg aagatgatga tgttttagct
ggcccagggg 35220 cagccatttc agtgtgcata aaggtggttg cgttgggtag
ggggatgctc agaaaaatca 35280 tggaaagcat gggaattcat agggtacttt
ggacattttg gaatcttgaa gagtaagaac 35340 cgtaactggt gacttaagtg
tcgtgtttct tcatttcacc aaatggcaaa atgtgataca 35400 gttcttccaa
tatcatgggc aacttgtagc cagaattaag tagaagataa gattagaatt 35460
gaatataata acttttgatt tatcatagtg ccttttaaat acatagtacc tctttgctat
35520 attatagtga tagctaaatg atcttttcac attcctaagt tttgatttct
gaatggcgtc 35580 gctcctgcct cctgacatct cacactgtga atgtgctact
tgctttctct aggcgcacct 35640 atgttggcag catgcctggt cgcatcatca
acggcttgaa gactgtggga gtgaacaacc 35700 cagtgttcct attagatgag
gttgacaaac tgggaaaaag tctacagggt gatccagcag 35760 cagctctgct
tgaggtaaga tttggaaaat tccctgtctg tcttcatact ggaagagtat 35820
ggaggagggt tgataatcat attcaagtga tatacacagt ggtgtagctt tagttatggg
35880 aaaaacagtt tgataccggc tgaggtctga gcaatttggc acttaaatta
aaatgttttt 35940 gagatttctt tcactaagtc cccttttttt ttattttcct
tttgtatttt aatcagatag 36000 tttaacaaag ttttgtgcac acttattatc
tagaggccaa caattctaca cagttatggc 36060 aaaaaaaaca gcaagcaagt
ctccttctcc ctggggtccc ccatgccttc ttctgcactt 36120 tgacctcttc
agcttttagt tgattaaccc tattttcaaa atagcatggc tatcttgcac 36180
ttcctgattt tttttttttt tagtttttgt cattttctat agatgccccc caacaggagg
36240 tgaagatttt accttttttc ttccgttgtc cccactgtat catttttata
ccttagatct 36300 cgcaataaga atttttttct tgtttttttg ttgttttttt
cttgtgaata ctaatacatc 36360 catattagta tttacattat tatgattatg
taaatgcttt tcacagcagg agccacatgg 36420 taaactgtga tcacttttcc
tgttcctatt tttgtttttc tctacttttt aagaatattt 36480 tcagagttag
ctgtcttgtt tcttttgttt actttttcac caatcgtcta attctgtcaa 36540
gaccttcaga cactttaggt gttctatcca ttttatcttc ttaagcgtcc ggtctgaact
36600 ggttgttttt gacatccggt tttatggctt ccttcctagg ttctcccttc
acctctcacc 36660 atgttggatt tcctgtctcc tgtattccat ttcttgctct
ttcttggtcc attccctcat 36720 ttttgtggtg ttaactccct gatagtttcc
tgagaaagct tgcatgagtg gtaaatgttt 36780 tagactttgc atatctgaaa
atgtctttat gtttccctca tacttgatta gtaatttgag 36840 taaagaattc
tggttggaaa taatttttct atagaattgt actttgcctc cattttactt 36900
cactttccca tttccagtgt tgctgttggt aaaactgatt ccattcagtt cctatccttg
36960 cagacctgct ttaccctgaa aactttcagg ttcttccctt tatcctggga
ttctgaaatt 37020 tcctaataat ctgccttggc atgggtttct tttcatgcat
ttttgctcat tctttctttg 37080 aattcttcct gttctttggt tctaaaattt
ttcttaaatt cttttattga tgacttttcc 37140 cctttatttt ttggaactcc
catgacttgg atattatgtt tcagacttat cttttctctc 37200
ctattagtct ccacttttat gttttgctct actttctgtg cagactttct cagatttatc
37260 ttttaaaaac cctctgaatt tattatttca aaaactttct ctgcatgttc
ttttatagta 37320 tcctgttctt gttacatagt tgtaatatat cttatctcca
tgagaaagat acttatagat 37380 atattttaaa attttacttc tctgaccact
tggtatatta aaaagaaaaa gaaaaaaatt 37440 acttctcttt aagctgcttt
tatctgttta ttatatattt cttttagtct cttttatatt 37500 agagtctttc
attagatatc tggacatttt tgtttgtgtg tttatattta atagtaaggg 37560
acaaaaaggc tgattggagg ctatgagcat aggagtgggg cttatcaaca gtgagttcca
37620 caatagagtc agctggctgt gctgtttggt tgaggaatct tctactcaat
agctttaagt 37680 cttccttctt aggatggtca gattcctcag agaagacttc
ctgtctcttg ccttgagaat 37740 gaaggcctgg ctgccatcat tctgggaacc
aagcagggga agaatgattg gggtcggggg 37800 tatcactgca ttcagcatcc
gtgtatatgc attcacctga gctcttgttt tcagcatagt 37860 atatgttctt
atcagctgtg cccagggtcc cctgtgcaga gaaccactgt tttatgttct 37920
taagaaaata aacttccagt gttttgctgg ggtgggggag gggatctggg atctgactgc
37980 ttcctaaatt tatttcagcc agtcctcctt attttagcac atcagcccct
cctccctttt 38040 acccttgctt aaaatattat taatgcaaat tgatttgtaa
aattgaggaa aacttacttt 38100 gtgaaagttt ttattttttt cttgtttatt
tctgtgcttt gagctgcctc gtgcttcctg 38160 ttttttttct gtttttgtga
tcttagaaca ggatggcctg ggacatgtgt cttattaagc 38220 aggagaccat
acattctggt ttgcttggca cattcccagt ttatgcctaa tattaattgc 38280
actctttttt agtctcagaa gtgggttttg tttggacgat aaaaaagtac agttacctta
38340 cttaaaagcc ctggtatttg gaggtaaggg tttgatttgg ttcagttttg
ctacttttta 38400 ttgtaagatc attaccttct ggctccataa ctggttcttt
ttactatgaa gagtaaaata 38460 gtgaacatta tttaagattt tagtagtttc
ttatataata tctttagact ttcagtttaa 38520 tttatattgg gacatttttt
caggttatct gacagattct cccattagac acttacagtt 38580 atcctgttga
aaataatttt agagtattcc cctgacactt aaattttttc aacaactgtt 38640
ttgaagcaag ttcaccaaag acagctttac aagtagtagt agatgattaa gtcccctgtt
38700 tatttgttca gttgataaac aatatgtttt aggtcttcac ctatatatac
tttgtaatga 38760 ttcaataata tttgttaaat tgatctttga taacaagcag
ctagcataat gatattttct 38820 tgtctgatgt agaccttggt actcactttt
ttggcagtcg atttattagc attcaaaaaa 38880 aaggtatgaa aacctcaaat
gatatctcag agtaaatgcc ccctgggccc acgtactaat 38940 cactgtagtt
tagttatgaa tagcattggt tccttacaga ctgtaaatgc tataaaatga 39000
agcaagacat acatatggag gaactgagta tcttggtagc tgacagcctc ttcctccctg
39060 cttgcccaag tcctgggtaa aaacctcaga cctcacagat tgttgaaaca
attaaataac 39120 agtacatatt aaagcactct ataaatggta aagtactgta
cagatgttaa tttaatatcc 39180 actgatattt cttctgtgtc cattttgaaa
gccacttgct gcttccattg ccagtaggtt 39240 cacttaaatt taaaaaaaga
acaaactcaa ttacacaaca cgttacattt aaagtgaata 39300 ttcctgagag
tttggagacc caagtatagt tttattatct ttctacatag aaaacctgct 39360
tttaaaaaat gatatctaga tattatttgt aaaatgtata agattatttt atgtttaagc
39420 taattatatt attaaggtaa tatagcccag atgtgaagaa tgtaatagta
gatgtaaata 39480 tacactagag tgcttactct gaataaagaa taaacttttt
ctgctgtgta ttcttctttt 39540 tatttatgta ggatatgccc gtttccttga
cctaccatgt aattgttgct tatgtaaaac 39600 agaatgtatt tcaagttatt
acttaatatt gtccaaaaaa ggagaattca aaatttagat 39660 gatctctttt
gaaaatttat tggaagacta taaaaatagg tccaactact taattaataa 39720
atggtggtag gcagtagaat ttgggcaagt ctataactga gtagcactaa aatattagat
39780 ataaggaaag taagggcttg tatgtaatta atagacttga aagaaaatta
cagaattatt 39840 ttcttaccag atatatgtta tatttataac tggcacatgt
ccagacttta ttgttaaata 39900 tgaatgcata tctcaaatac atttttgtgt
gagtgggcaa ataaaatgca tggatacaat 39960 aattaattgt ctttataggc
aataatattt acagttcgaa aaacatatat tccccaaaat 40020 agagaagtca
ctagtctaga tatagtaaac ttcctttaaa actgaagttc ttacttaatt 40080
cgaattagat ccagttagta attagaccaa tagtatattt actacttaga tacagtagac
40140 atgatctttt gatttgagct atacaattat tgtcaaagaa tgtcagaaga
gagggactta 40200 gacatcatct aatccagctt catgctctta aggataaaaa
gcttaaggcc taagatatta 40260 ttttaatttc ttatttcact acatgctata
ttaatgatat aatttccaaa tatcgaatgg 40320 agttaaaaaa tgccttaaat
aaggcatacc ttgttttatt gtgttgtgct tcattgtact 40380 tcacagactg
tgttttttta acaaattaaa tgtttatggn nnnnnnnnnn nnnnnnnnnn 40440
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnngggcac ccgtgtatcc
40500 ccagcccctc ggaagcttga gccaataaca ataccttgac ccggggaggc
agagtttgcg 40560 gtcaccggag gggggggggg ggcgtcgcaa cctgggttac
aaaccaatac tctttctccc 40620 gtccccgaca aaaaaaagaa agaaagtgtt
tatggcaacc ccgtgtcaag caagtctgtt 40680 gacaccattt ttccaacatc
ttacttcatg tctgtatgtc acattttggt agttattgca 40740 atatttttaa
ctttttcatt attatatcct attatgatga tctgttatca gtgatctttg 40800
gtattgctat tgtgattgtt ttggggcacc acaaactgca cccatataag acagcaaact
40860 taatcaataa atgttgagta tgtactaact gctcaactgg ccaggcattc
ccctttctct 40920 ctccctctcc tctggctcct attccctgag acacagcaat
attgaaatta ggccaagtaa 40980 taaccctgca gtggcttcta agtgttgaag
tgaaaggaag agtcacacat ctcattgtaa 41040 atcgaaagct aaaaataatt
aagcttagtg aggaaggcat gttgaaagct aggcctcttg 41100 tgccagatag
ccaagttgtg agttcagagg aaaaattctc aaaggaaatt agaaatgcta 41160
ttccagtgaa cacaccaatg ataagaaagt gaaatggcct tattgctgat atgaagaaag
41220 ttttagtggt ctggataaaa gattaagcca actacaacat tcccttaagc
cgaaacctag 41280 tccagagcaa ggccctaagg ctcttcagtt ctatgaaagc
tgagagaggt gagaaagctg 41340 cagaagaaaa atttgaagct aacagaagtt
ggttcatgag atttaaggca agaagccatt 41400 tctacaacat aaagtgcaaa
gggaagcagc aagtactgat gtattgtaga agctgcatca 41460 tgttatctat
ccagaacatc tagctaacat cattgataaa ggtggctaca ctaaaaaaca 41520
gattttctat gtagatgaaa cagccttatt ttgtattgga agaagtgtca tttaggactt
41580 tcatggctag agaagtcagt acctggcttc aaagcttcaa agggcaggct
aactcttgtt 41640 aggggctaat gcagctggtg actttaagat gaagccagtg
ctcattgacc attctgaaaa 41700 ccctaaggcc cttaagaatg atgcaaaatc
tactctgcct ttgttctgta aatggaacaa 41760 caaagcctag gtgacaatgc
atctgtttat agcatggttt tactaagtac tttaagccca 41820 ctgttgaaac
ttaccgttca aaaaaaatag attcttttga aaatattact gctcgttgtc 41880
aatgcttctg gtcacccaag agctgtgatg gagatgtaca aggagattaa tactgttttc
41940 attccttata aaacaacatc cattctgcag cccatggatc aaggagttat
tttaactttc 42000 aagtcttatt atttaagaaa cacatttttt aaggctattg
ctcccataga ttatgattcg 42060 tcccatgcat cagggcgaag tacattgaaa
acccctagaa aagattcacc attctagatg 42120 ccattaagaa cattcatgat
tcacgggagg aggtcaaaat atcaacatga acaggagttc 42180 aggaagagtt
gattccagcc ctcatggatg actttgaggg gttcagactt cagtggagga 42240
agttaccgca gttgtggtag aaatagcaag agaactagaa ttagaaccca aagatgtgac
42300 tgaaatactg caatctcatg gtaaaacttg aacagatgag gagttgcttc
ttacagatga 42360 gcaaagaaag cgggtttctt gaaatggaat ctagtcctgg
tgaggatgct atgaaccttg 42420 ttgaaatgac aaccttgatg ttgtgaacct
tgttgaaatt ctaaacaaga tttagaatat 42480 tacataaaca tagttgataa
aggcagcaac agggtttgaa aggattgact tcaattttga 42540 aagaaattct
acggtgggca aaatgctatc gaatagcaat gcaggctata agaaattgtt 42600
tcatgaaagg aagagtcaat agatgaagca aattttactg ttgccttatt ttaagaaatc
42660 gccacagcca ccctaacttt cagcagccac cacctgatca gtcatcaacc
attaatattg 42720 agacaagaca ctccaccagc aaaatgacaa caactaacac
tgaagactca ggtgattagc 42780 attttatagc aagaaagtat ttgttaatta
aggcatgtac attgtttttt agacataatg 42840 ctattgcaca cttaatagac
tatagtatat tgtgtaaaca taacttttat atgcactggg 42900 aaacaaaaaa
aaacatacat gtgactcact ctgttgcaaa atttgcttta ttgcagtggt 42960
ctggaactga acccacagtg tctctgaggt atacctgtat tgaggagggg ttgcaaattt
43020 tagcacatag gcaaatttgc aaatatggaa taataaggat caactgtaat
tactgcttta 43080 tgccattatc ttttaaatca gataagaaaa agttacgtca
acaatatatt tacactgcct 43140 tttatgtttg caatgtaatc acttctgcca
gtgcgctcta tttctttgtg tggatactgt 43200 ctagtgtcct taaacttcag
tctttcatat ttcttgtctc atctcctggt gacatattct 43260 cagtttttgt
ttttctggga atgtcttaat ttctccttca tttttgaagt aattttgttg 43320
gtatagaatt tgggttgaca attgtttgct ttcagccctt tcgcatgtcc tctcaccact
43380 ttctggtctc tgtggtttct gctgtgaagc cagctgttaa gcttgtggcg
gatctcttat 43440 gcctaatgag ggcagcattt ttctctcata gttttcagta
ttctctcttt gtctttcatt 43500 tctgacagat tgactgtgtt tatgtgtgat
cctctgagtt tacttagttc tttttgagct 43560 tcttggatgt gtaggtaaat
gtttttcatc aaatttgaga agtatgtggc cagtatttct 43620 tcaaatattc
tttatgcccc tttctttttc ctctccttct gaaactcgta ttatggtgtg 43680
ttggtaatct ttgtggagtc ccgtaggtct ctaaagtgct gttcactttt tttaaagcct
43740 tttttctttc tattcttcag acaggatcat ctcagttgac ctgtcttcaa
gttcattgat 43800 tctttcttct gccagctgaa attgtcattc agcccctcta
gtgaattttt cattcaaatt 43860 actgtagttt tcaactccaa aatttctatt
ttaaaatttt tattatttat ctttgtttat 43920 attctctatt tgtcaagaca
tcattctcat actttcctgt aattgtttag acatgatttc 43980 ctttagtttt
tttaaatgtt agtaaatata acagaaaaag tcccattttt accactttta 44040
tgtgtacagt tcagtaatgt taagcacatt cgcattgttg tgcagccaat ctccagaact
44100 ttttcatctt gttaaagtga aggtgtatac tcattacaca gcaattccct
gtttctttct 44160 ccctccctca gtccctggca gctaccattc tcttttctgt
ttctatgagt gactactcta 44220 tatacctcat ataagtgcat catacggtac
ttatcttttt ataattgact gacttcactt 44280 agtttcctca aagttcatca
atgttggggc attagttttt taagcatatt tatagtagct 44340 gatttgtaat
cttttttttt ttttttttga gacggagtct caccatgttg cccaggctgg 44400
agtgcagtgg cgggatcttg gctcactgca agctccgcct cccaggttca caccattctc
44460 ccgcctcagc ctcccaagta gctgggacta caggtgcctg ccaccaggtc
tggctaattt 44520 tttgtatttt tagtagagat ggggtttcac catgttagcc
aggatggtct cgatctcctg 44580 accttgtgat ctgcccgcct tggcctccca
aagtgctgag attacagtcg tgagccaccg 44640 tgcctggccg ctgatttgta
atctttatct aataaatcca acatgtcttc cttagggatg 44700 gtttccattg
acttctcttt ttcttttttg agacagggtc tcgctctgtc acccagactg 44760
gagtgcagtg gcgcactcat ggctcatggc agccttgacc ttacccaggc tcaagtgacc
44820 cacccacctc agcctcccga gtagctggga ctacaggcac acaccagcat
gcctggccaa 44880 ttttttgtag agacagggtt tcgccatgtt gcccaggctg
gtctcgaact cctgagctca 44940 agcaatttgc tcaccttggc ctcccagagt
actgggatta caggcatgag ccactgaacc 45000 cagctgactt ctcttttttt
tttttactct ttagggccgt acttttgtat ttctttgtgt 45060 gtgtctcata
attttttttg ttgaaactga atatttagag tgttatattt atattaaata 45120
cagtcagata tataattgaa taatataacc ttaagggttt tttgtttgtg ctgttgttgt
45180 tgctgtttgt ttagtgactt tctggtttca ttctgtaaag tctgttttat
tcattaatgt 45240 gtgaccactg aagttgctca gtttgtttag tggtcagcta
gtgaccggac agagatttcc 45300 ttaagtacct ggacagtagc tctcccactc
cttgcccaag gggctcttat gtgtgtattg 45360 aagtgggcct ttcacacttt
ggcagatggt ttacaactct gccttagcct tcacttcctg 45420 cttttgcaga
gcctcagtgt ctgccaaaga tgagcttata gggccttctc aggtctttcc 45480
tggatatact tagagcctgc acattcacat gaaattttgg attctcaggc atatgtcaag
45540 gcttttcaaa gtccccatga atatctcatt tcccagtttt tccatttaag
ttttttggtc 45600 agcctcttgt tagtcccaac tagtttcatt gcctcaggca
gctgcagtgc taaaacagtt 45660 gccactggtt gtttttggca aatgtcctaa
ggataaaact gttctcacag agtgttctct 45720 gagttaagtc aaataaggat
atggagctct tctaaggaac tgccagagtc aaacagggac 45780 agttctctgg
ggatggggct tttgaaggat tgtaatcctt ttctaccccc taacaggatt 45840
gctaggctac tggttttcac agctactggg gttatgaggc tgttgatttt gctaccatga
45900 acttgagaga aagggatgag tgtaaagcaa gttaaaatat cacaaagctc
gttctgttta 45960 ttgagattca gctgtttttc ttgaataagc actcctcaaa
ttgttgcaag ttagtatgta 46020 gcattctgaa aaagttgatt ttgacaattt
ttgctagtgc tctcattgct tttctggagg 46080 agcagatttt cagagtttct
tactctacca ttatataata gaagtgcttc ctcccccatt 46140 tcattttgat
tctgtgcttg aatgatttca ctgcatgctt ctgatacttg tattttggtt 46200
tatcacttgt tcagatgaaa tatatcttca ggttacttca ttcaaagatt tgtgtgtgag
46260 ttgtattttg aatctcttct atatttgaga aggcttcttt gttgtctgca
ccagtagtaa 46320 tatatatgta aataaaataa gaatgtatta gtcttcttct
tttttttttt tttttttttt 46380 gagacggagt cttgccctgt cacccaggct
ggagtgcaat agtgcaatct tggctcactg 46440 caacctctgc ctcccaggtt
caagcgattc tcctgcctca gtctcctgag tagctgagat 46500 tacaggcacg
tgccaccacg cctgactaat tttttgtatc tttagtagag atgggctttc 46560
accatgttgg ttaggctggt ctcgaactcc tgacctcgtg atccatccgc ctcggcctcc
46620 caaagtgctg gtattacagg catgagccac cgcgcccagt cagaatgtat
tagaatgtat 46680 ttcttaagac tgccataaca aaataccaca gactgggtag
ctttgaagac caaacagaaa 46740 tttatttcct tatggttttg gaggctagaa
ttccaagacc aaggtgttta taggtttgat 46800 ttctcctaag gcctctctcc
ttggcttaca gacaaccgac ttgtggctgt gtcctcggga 46860 gacctgtgtg
catgcatccc tggggtctcc tctttcctct tataagggta ccaattgtat 46920
tagactaggg gcccactctt accttcattt aaccttaatt accttcttaa acaccctgtc
46980 tccaaataca gtcttcaccc tgactgccct tgagacagag cggagggggt
tagggattct 47040 gtcaattttg agggggcaca attcagtcca taacaaagga
catatataat agatacataa 47100 tatatatgta ccagtgtgcc catatcatgt
actttatgta aaacgaaatc agttttaaaa 47160 ggtaattata ttttcaatga
aagcactgtg ttctaattag ataattgttt ttacttcata 47220 atatgtctat
cctagcttat tatataaata aaagtgtcaa ctctgttatt ttcttgtggt 47280
tcataccttt gcctataccc tttttaatga tactttgcag gaatcttttt aaaccactca
47340 acccatttgt aatattaggc tctgtgaacc cggaaaattt gagacaggtc
tcagttaatt 47400 taggaagtat atttggccaa ggttgaggac gcgcgcccat
gacacagcct caggaggtcc 47460 tgacgacacg tgcccaaggt ggtcagagca
cagcttgatt ttatacattt tagggaagca 47520 tgagacgtca atcagcatat
gtaaggtgaa cattggtttg gtctggaaag gcaggacagc 47580 tctctggaga
gggcttccag gtcacaggta gataagagac aaacccttgt gttcttttga 47640
gtttctgatt agcctttcca aagggggcaa tcaggtttac ctcagtgagc agaggggtga
47700 ctttgaatag aatgggaggc aggtttgccc taagcgttcc cagcttgatt
tttccctcta 47760 gtctggtgat tttgggggcc aaatatattt tcttttcaca
gcacacatgg acagcaatgt 47820 gctgtaatta tagttaaggc agataagtga
ggacaccaca ggcagccttc gaccttatgg 47880 aacttcttct aagtgaagac
atcaattcca ttttggatat taaatattta caagctattt 47940 ttttctggta
tttataaata aaaaagataa atacaaatac taatattttc tacttgcact 48000
ttggtgggtc attttccact tttgtgacca ctggtctaaa tagataaaca aatgtcttca
48060 caaatgggta gtaggttcac aggtgttcat tttgttatta tgcatcatat
cttatatata 48120 ttacatatat ttgatgtatt caagattgta aaatatttta
aactagtgat aattttgctt 48180 gaaaattctg taggtgttat tctaatgaca
ttctcatttt tattgcacag gaggaggaat 48240 ctaaatcttt tcaatctata
gtgtcaaggt cttctagaat attttcgttt ctttaatccc 48300 tattttaatt
tactgagacc tcttctttag ttatattaac cagttatgaa ttgtatctct 48360
taatttttcc cgtatttatc ccctacatgt ctctaaagcc ctttttcttc tatgtcctga
48420 acacttttct caagtttgtc tttatcacag atttaatttc catagttgag
gatatagagg 48480 aaaagtaaac tcagtttctc ctactgcact ctcacaacac
agaacacctc tgaccaaatg 48540 cacgggtttt ttctccatat gccaagcaag
cagttcttca gcaaccgacc acagctgggt 48600 gtcctctaat tcaattctga
caaagtgtat cagatcctac gggttgagca ctgagtccca 48660 caagactgcc
tcccccttca gatgccagtc gtgagttgac ttccagaacg tgtgaccaac 48720
cagttataaa ttggagtacc cacaagcccc cctcctcagg tttgcttaat ttgctagagt
48780 agctcacaga actcagggaa acaatttact tgcatttact ggtttattaa
aagaatattt 48840 taaagaatac aaacaaacag cacaggagct tccatcccag
tgaagtcagg gtccaccagt 48900 cttcttgcac ctgggtgtgc tcaaattcac
cttcctggaa gcttcctgac ctcagtcctt 48960 tcgggttttt aatggaggcc
ttgtcacata ggcctgattg attaaatcac tggccattgg 49020 tgatcaactc
aactcttagc tcttctcccc tcccaagaga ttgggctggg gaactgacaa 49080
gtcctcagcc ctctaatcat gccttggtct ttcctgtgac cagcccacat cctgaagctg
49140 tggagggact gccagccacc agtcaatcac taacatacaa aatgatactt
atcactttgg 49200 tgattccaag gattttagga gttgcatgtc aggaaacaaa
gagatgaagg ccaaatatat 49260 attttacagt atcataatag tattaattgt
gtgtggcttt cagagctgat tttagttatg 49320 ttattttatc tttattttct
gttgtggaaa atttcaacca tagcaaaagc agagaagata 49380 gtataatgaa
ttctgtggac tcatcaccca gctttaatat cttgtttcat ctattgcttc 49440
ccattctccc ctacccaacc tctgattatt ttgaagcaga ttccagacat catcttttca
49500 taaatgtttc agtagctatc gacaaaagat atacactttt aaaaagcata
atcatactat 49560 atcacaccta aagatgacag ttacctagtc ttgtgtaatg
aactctatgt aatctattcc 49620 tggattgcct acagacatct atagttcttc
tcttgtcaga aattattatt gaagaataat 49680 tctcagtgta cattcctccc
acggttcatc ccattgtgac ttcacattcc taggaataat 49740 gcgtcatatc
acagctattt ccattcccag tcatactttg taggtaggaa ttatagtcct 49800
aggattgata cagaaaatct tttagttggg gagaataaag gagaaacagc cctaattatt
49860 tttgaaagtg gccctggatg tgggcagtag aatccctgct ctgaagttag
ggtaagaaga 49920 tgaggtttga tagctacaaa gctcttaatt gtaattttcg
tccttccatg gactcaccag 49980 tttgcctcgg agcttcatct gagtagtgat
taccagaaat tattttctgc cagaatattg 50040 atcagtattt ctgatgctgt
ttaaattcta tatgtctttt tatgcttttg aaaaccagaa 50100 agtatctgag
acaggtctca accagtttag aagtttattt tggcaacgtt ctccagagat 50160
gattgtgagg gcttcagtat ttaaagggga atgggcagat attggggaaa gaggaagaaa
50220 ttttaaaagg tatgagtaga caagagacaa acggttgcat tcttttgagt
ctttgatcag 50280 ccattcacct gtgagagggg agcagaggaa tagtcactga
cgcattcatc tagcttagtg 50340 aatctgcatt tctacataag ataaaataaa
tatagcgtac aggaagccat cagatatgca 50400 tttgtctcag gtgagcagag
ggatgacttt gagttctgtc ctttgtcctg tatgtgtaaa 50460 gaataagcta
tcaatttaca tggttggggt gaaattcaac agaactgtta caggttaaag 50520
atcttggggc ctacaaggaa tttctcagtg gggggattgt gagggagata tgtagctttt
50580 tttgtctttg tagctatctt atttggaaac aaaatgggag gcaggtttgt
gtgacgcagt 50640 tcccagcttg tctcttccct tttgcttagt gatttggggg
tcctgagatt tactttcctt 50700 tcacactctt cctgagtaaa agaggaaggc
aggcaaattg ggcacaaatt tagcctaagt 50760 ctgcctcctt acatattaat
attttaagtt tggcctaaag gtttcccctt acaaagtaaa 50820 ctgcagccta
actagctgtg taaacacact attcttaaca ccaatcacag attttcagca 50880
agtcacagga agtcagctgt taacaaactt taaataaagc aaacaccaag ctgtaagcaa
50940 tcccgctgtt tctgtacact ctttgttttc tgcatgtcgc tttccttttt
ctgtccataa 51000 atattatcaa accatatgcc agagtttctc tgaacctatt
ctgtttctgg gagctgccca 51060 atttgagact tgttctttgc tcaattaaac
tgttaattta tctagagttt ttcttttaac 51120 aagcatcact aattttttct
ccttataatc taggtattct gtcacactgt tttaaaaacc 51180 tccttcataa
ttcagaaaca ttgctttatt aattttccta ctttttaaaa acgctagtgt 51240
cttaaaattt taagagaaaa aaattacttg ttcaagtctg acagccattt ctaaaacata
51300 tccagcatat atgaattaca tatgcttaga gccattaaag aatagaattt
tttccggcca 51360 ggcatggtgg ctcatgcctg taatcccagc actttgggag
gccgaggtgg gcagatcacg 51420 aggtcaggag atcgagacca tcctggctaa
catggtgaaa ccccatctct actaaaaata 51480 caaaaaagta gccgtgcatg
gtggcgggcg cctgtagtcc cagctactcg ggaggctgag 51540 gcaggagaat
ggcgtgagcc cgggaggcgg agcttgcagt gagccgagat cgcgccactg 51600
cactctagcc tgggcgaaag aacgagactg tctcaaaaaa aaaaaagaat agattttttt
51660 ccttagctag tgttaaaaaa ttactcatga cgcttattaa aggtggtaag
gattacttta 51720 ttcaaggtgg gagactacgt ataagaaaca ctgcaatggg
gttttgcagt gacaggagga 51780 gagtgaatgg ggaatcagta gagggaaaca
ttctaagagg aagaattggg gttacggggg 51840 attctcacta gaaggacaca
acagaactct tgctgaaggg aggccagggt gaaaagatac 51900 tgggttagaa
gtgagaacag atacgtatgg gtatgggtca tttttgctaa cctgacttag 51960
caggattctt gctcaaattg gattttacaa agacagaggg aaggctgaca ttggcctagt
52020 tgagcagagg actcagagga gcctgactca agtttgcgtc aaaagaagag
cgtttttgtc 52080 actagatgat agttttaact attttccata cataaacatt
ttccgtacct aaacagtttg 52140 tttgttcatt tgtttgttag tttgtgttgg
attttcactc tgtcgcccac gctggagtgc 52200 agtggcgtga tctcagccca
cggcaacttc tgcctccaaa gttcaagcaa ttctcatgcc 52260
tcagcctccc gagtagctgg agctacaggc atgtgccacc ataccaggct aatttttgta
52320 ttttttttta gtagagacag agtttcacca tgttggctag gctggtctca
aacacctgac 52380 ctcaactgat ctgcctgctt cggcctccca aagtacttgg
attacaggtg tgagccaccg 52440 tgcccggcct gtgaacagtt tttagatgat
tagtagatag taagaccact cttaaccaat 52500 tcaatactga acataattag
ttttccttga ttacttgaaa gtacttgttt tttaatgata 52560 ttaaacatta
ttaagtcttg tgaaaatgtg aaattagagc tttctgggaa ttctagatag 52620
agtttccagt aataattaat gtttaacaaa attcagaatt atgtatgagg cctagaatta
52680 agactagctt ggggctgggc gtggtagcgc acgtctgtaa tccctgcact
ttgggaggcc 52740 aaggcaggtg gattgcttga ggccaggagt ttgagaccaa
tctggccaac atggtgaaac 52800 cccatctcta ctaaaattgc aaaaattagc
caggtggggg tggtacgcac ctgtaatccc 52860 agctactcag gaggcaaaga
ttgtagtgag ctggagacca tgccactgca cctcaacctt 52920 ggtgacaaaa
tgagactctg tctcaaacaa aacaaaacaa aacaaaacaa aaaactaact 52980
ttggatagtt ttgaaaataa gtaaaacttc agaaagaatc agaaggtagg aaaaactgct
53040 tatatagtta aattgtggtt ggtgagtata ttagtcattt tattgccttt
ttgaatatgt 53100 atggcaaccc tatttatagt aattgggcgt aagtgagagt
gttaatatgt ttaaggtttg 53160 gaacatgtag aagctgttgg tgccttatga
aagttctgca ccagcccctt agcaacaagt 53220 gcctgtgact tgaagctctt
taatgtacag ttgcacattt taagaatcca agttgactga 53280 taaattatct
aatgtatcta attcaaatat ttttaagagc tattgtaatc ccagtacttt 53340
gggagactga ggcaggcgga tcacttgagg tcaagaattt gagaccagcc tggccaacat
53400 ggtgaaaccc catctctact aaaaatacaa aagttagcca ggcatggtgg
cgcacacctg 53460 tagtcccagc tactcaggag gctgaggcag gagaatcgct
ggaacccggg aggcggaggt 53520 tgcagtgagc tgagattgtg ccactgcact
ccaacctggg caacagagta agactctgtc 53580 tcaagaaaaa aagagttatt
gatgttttgc ttattataag cagcaatgtt ttgtagtaag 53640 ccatttttaa
atagtgaatt ttttgctgta tcagaatata gtagcatagt aatttttact 53700
cttatttaac tcatagcaaa ggttactctt atttggaatt ctcctttcag ttaaataatt
53760 tataccagac tttctgaaaa tgtttgagga ggattatatg ggttcttatt
tactggttct 53820 ttgagaattt caaaatactt tacacatttg ctttatattc
ccatagcagt ttagataggg 53880 tgtgttacca agatggaaac tggttctgca
ggactggtaa cttatgatgg ccaaacaatg 53940 agtcattaat aaatagattt
ttgaacaaag cttgaaactg taatttctgc tgctttgtgc 54000 tattacattt
tcagaaattt tgacactgaa cgtattttat tttttaaaaa gtatgtagaa 54060
tgtagagaat gcaaataata atgctcagat gttagttttg tctgtttctt aaattcttct
54120 gagcagaaat accaaccttg ccagtacatc atgtgtgttt tcacttatat
acagccttct 54180 gttggcacta ctaaagtttt taaaatgttt tttgttctcc
cctaggtgtt ggatcctgaa 54240 caaaaccata acttcacaga tcattatcta
aatgtggcct ttgacctttc tcaagttctt 54300 tttatagcta ctgccaacac
cactgctacc attccagctg ccttgttgga cagaatggag 54360 atcattcagg
ttccaggtac ctgactctta aatcattatg atacatcttg cctttctgac 54420
cataacttta aaattagtta tgctatggag ttttgactaa aagaagttca tttgccaaca
54480 tacaatcttc agaagttctg aggaatgtat ataaatcagt ttctatgtag
cttcaaagtc 54540 tggaagagca aaacagcaaa cgttgacaac aacaatttca
gatttaatta gcatgaaaga 54600 atgataattt tatgacaaat aagacattct
tctttagtat aatttctaaa atggcaggct 54660 gtgtgtggtg gctcacacct
gtcatcccag cacttttggg aggctgaggc aggtggatca 54720 cttgaggtca
ggaattcgag accagcctgg ccaacgtggt gaaacaccat ctcaataaaa 54780
atacaaaaat tagcctggca tggtggcggg cgcctgtagt cccacctact cgggaggctg
54840 aggcgggaga attcccttga acctggggaa ggggaggttg cagtgagcct
cacgccactg 54900 cactccagcc tgggtgacag agtgaaactc catttcaaaa
aaaaaaaaaa aaaaagagta 54960 actgaacttt ctcataaaat ctggcctcac
ttttatatta aagtgcatgc cgcttttaaa 55020 ttcctcttga atctgtcaaa
tagttaaatt ttttaaatgt cttccctgtc actggagcgt 55080 gcaaaatgta
ttccttcagt tactaacact agataagtta tagcattttc accttatttt 55140
aattgctcag aattgttttt ccctggaaga gatcaaatat cactgagttt ttttttaatg
55200 tagagtagaa tctaaatgtc tttatttatt taattattta gagacagagt
ctagcttgtt 55260 gcccaggctg gagtgcagtg gcacgatctc ggctcactgc
agcctccgcc tccgaagttc 55320 aagtgagtct cgtgtgtcag cctcccaagt
agctgagatt acaggcactc gtgaccacgc 55380 ccaggtaatt tttgtatttt
tagtagagac catgttggcc agtctggcct cgaactcctg 55440 gcctcaagtg
atctgcctgc cttggcctcc aaaagtataa ggattacaga cgtgagccac 55500
catgtccagc ctaaatgtct tttacttatt ttttcttttt ttgagatgga gtctcactct
55560 gtcacccagg ctggaatgca gtggcacaat cttggctcac tgcaacctct
gcctcctggt 55620 tcaagcgatt cttgtgcctc agcctcctga gtagctggga
ctacaggtgt gcaccatcac 55680 acctggctaa tttttgcatt gttagtaggg
acagggtttc gccatattgg ccaggctggt 55740 cttgaactcc tgaccttagg
tgattcaccc gcctcagcct ccaaagtgct gggattacag 55800 gcgtgaaccg
ccacactcgg ccctaaatgt ctttagattc taaatgtaat ctaaatgtat 55860
ttttcatatt aatctgaaat atatttttac tactaagtga attataattg gatttctgtt
55920 tgtttttttt ttgagatgga gtctcactct gtcaccaggc tggagtgcag
tggcacgatc 55980 tcagctcact gcaacctcta tgtcccaggt tcaaacaatt
ctcttgcctc agcctcacaa 56040 gtagctggga ctacaggcgt gcaccaccac
gcccagctaa tttttgtatt tttagtagag 56100 atgggatttc accatgttgg
ccaggaaggt ctcaatgtct tgacctcatg atccacccac 56160 cttggcctcc
caatataact ggatttctta attatctgtg agcattgcag gttcctgtat 56220
ttagttttaa aatatggtag agtaaaaagt taattgtgtg tatttaaagt ctaaagtaaa
56280 taagtaatga attccctgga aactccaagt tatggcagaa aattcattag
atacactaaa 56340 gtaaagtgaa agaatcagga cagctgctgc agaggggagc
atatgatgcc accttcttcc 56400 tttggcagat ttagctgtcc gatcttctag
ctttcctggt gtttactaac ctctttccat 56460 tcaaaaggtg ccttatcaat
tcatattttt aatttttgct tgttaaatgg aaagggacat 56520 tagttggaat
tttgtcttac gggatttaga gacaaaggaa atctatattt attcaggcta 56580
ttaaataaga acattatgtg ttctaaatat actatatata gaaaaaatac atatatacat
56640 acataaatac atatgcacac atatataaat acatacacac acacacacac
atatatatat 56700 ataccatcat gtggaggaaa aaacctttta tatggacatc
ttaggttttc ttttgctgct 56760 acaatttatt ttatagtcat agttctggaa
acagtatctt tagagccctt cccttggaac 56820 ccactgctta tttaattgag
gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 56880 gtgtgtgttt
caagtataga tcaaattagg ctaaaaagat gcatttattc ttctatttga 56940
aatttcagag gatttgagga taaagagata attgtctcta agatttgagg tgttttcctc
57000 tttgggaaat atatcattta atcagaaaac tttcaagcac tgtgcttagt
aaatgcttgt 57060 tttgtttgtg aaaacgttgg aaattttaac aattattgac
ttagatcaaa tttctttttc 57120 tttttttttt tggaggcagt ctctgttgcc
caggctggag tgcagtggtg caatctcaac 57180 tcattgcaac ctccacctcc
ccagctgaag caattctcgt gcctcagcct ccagagtaac 57240 caggactaca
gacatgcgca accatgctca gctaattttt tgtgttttta gtagagacag 57300
ggtttcgcca tgttgcccag gctggtctca aactcttaag ttcaagtgat ccgcccgcct
57360 cagcctccca aagtgctagg attacaggtg tgagctaacg tgcctggcca
gaataaattt 57420 cttcattgta attatagtct catttgaaat aatacttaaa
tttgttctaa atctaagatc 57480 catttaatgc tacatttgat tcattaaaaa
agcatggcac tggctgggag cagtgactca 57540 tgcctataat cctcagcact
ttgggaggct gaggnnnnnn nnnnnnnnnn nnnnnnnnnn 57600 nnnnnnnnnn
nnnnnnnnnn nnnnctataa tctcagcact ttgggaggct gaggctggtg 57660
gatcacttga ggccaggagt ttgagaccag cctggccaac ttggcaaagc cctgtctact
57720 gaaaatacaa aaatcagcca gcgtggttgt gcatgcctgt aatcccagct
gctcgggagg 57780 gtgaggcagg agaatcactt gaacctgaga ggtggaggtt
gtagtgagcc gagatcacgc 57840 cactgcactg cagcctgggc gacagagcaa
gactctgtct ctaaaaaaaa aaacaaaaaa 57900 caaagcatgg cattatggga
gccatgtaaa taattacaaa acaagatctc ttcttttcca 57960 ggttatacac
aggaggagaa gatagagatt gcccataggc acttgatccc caagcagctg 58020
gaacaacatg ggctgactcc acagcagatt cagatacccc aggtcaccac tcttgacatc
58080 atcaccaggt tagttagcca tcctgaggct tcattaactc caggcaactt
ttgagtattt 58140 actgagttac caaacaggac atagagtatc aatatttgag
tttttcatct tttgagataa 58200 gccacagtct cctgaaaagg agattagttt
attggcatcc catagcatcc atttctcttt 58260 cttcaacaac ttccagcaag
tgttatcata actattgatt tacaccgttc tctacactag 58320 gcagaagttt
acagagaaac catttggaat attgttatag ctaaagctga aatttatgct 58380
ttgccacaat agcaatataa ggggttaatt tgatcattta aaaaccaaat acatggcaaa
58440 tatagagaca ctttttatgc ccaggatctt gaaagttgtt gaattctctt
aagaggtgat 58500 atgctacttt cagataatct gatttaagtt actcactttt
cttttcttct ctttggctga 58560 gagattttta aaatccttag aattttgatc
ttcagaatta acactggaac aatagagaag 58620 gtgccttccc aagtttacta
ccaaatgctt aagcctgtag caagcagtgt gtaaattatc 58680 tgaatagagt
attgcttagt ctaatttaca gattccctgt ttgaatggaa aatatactct 58740
gttgagaatt tatatccacc acagcctctt acagttttcc tagctcagta ttacagatcc
58800 attgcatcat ccagcaagtc atgtcaggct gccaagctct cctcttgcgg
cccttttcta 58860 gtaactactg tttttaagag atttgaagta tctctctatt
ttgaactttg acttagagtt 58920 tggccagact gtcttttgat ctatgccttc
ttatggatct atttagattt atatacaaag 58980 cagtaagact aagtcttacc
tgggggttcc ttttcttaat ttgtcttgtg atttatggtg 59040 tagataatgc
caggagaaat aaattaagtg acttatatgt ctgagtcttc caacaatatc 59100
attattccag ataacaccca tgatgccttt gggtaacttt caataagtca tttaacattt
59160 ttgatagctt ccccatctgt aaaatatgag ggatggagaa aaatccagag
tttatctgaa 59220 taataatgat tctgaagagt gatcattatt tatatttccc
agttgttacc tagagaactg 59280 tttctttttt tatgtatact tgttaactca
aaatatcaga tcttaaaagc tgtggacata 59340 aggaaatatc tggagcagtt
ttgttagttt tgatattgtt tttaaaaaca gcacaagtat 59400 gtactattcc
aggcacagtt tttggatatt tagtgagtta ccaaacttag gacatagagt 59460
atcaatattt gagtttttca tcttttgtga taagtcacag tcatagaccc taatgttcta
59520 gtctttctta tctccaagta taactcacct gcttgaatac ttccagtccc
agtatgctta 59580 attctagcga ataactacct tttcatgggt aattctaact
gtaacaaaga tattcttttt 59640 atttatttat ttatttttta agacaggttt
tcatgctgtt actcaggctg gagtgcagtg 59700 gcatgatctt gggtcactgg
agcctctgcc tcctaggctc aagccatctt gccatctcag 59760 ctcccaagta
gctgggacca caggtgcatg ccgggcgtgg tggtgtgtgc ctgtaatccc 59820
agctactcgg gaggctgagg caggggaatt gcttgaacca gggaggtgga ggttgcggtg
59880 agttgagatc gtgccactgc actccagcct gggcaacaga gtgagactcc
gtctcaaaaa 59940 aaaaaaaata gagatggggt tctcaccatc ttggccaggc
tggcctggaa ctcctgagct 60000 caagtgataa ttgttacaaa gatactcttt
ctattcactt ttctataatt ttcttcttct 60060 gccttatagg agcacctgga
atctaagtgt aattcctcct tgtacagccc ttctgacatt 60120 aagataaaat
actatcaggt gctgcacact aagtgttctc ttcttcaagc taaccattcc 60180
tctcctctgt accattcctc ttgatgtagt ttcaagactt ctcaccctcc tgattagtct
60240 tcttctgaaa gaatcctgta tatcaatgtg tcttttaaaa ttaaacaccc
agaattgaac 60300 acagtgtttc agatagagtc taaacagttc atggtatagg
aagcccatgc ttttcttatt 60360 ctgactatat tattttatga ctgtatctct
agattcttag ctttttaaag attattctct 60420 tccctttttc agtgaatttc
gctaagcttg gcatatccca ttttgtattt ataaagctga 60480 attttttaaa
gcccaaatgt agaagttgtt aagatgcctc cctgttttct cccttattga 60540
aattatacgt agttgcataa tataggcttt atatccttct atacctttga ctgaaatgag
60600 tattagagtg tttagctaag agctttttat ctgtcttttc tcagaacttt
taaaatctgc 60660 tttcctaaag tctacagtgt atgtctgact taatcaaatg
tatggctttg tcaaatccaa 60720 ttcttcagat aaaactgcat tctccacctg
atcctgtcca ttcaggtcca tccaaagctg 60780 agtggccaaa agtggtttca
ctatataatg gtctgtggaa tgacttaacg gagtttgatt 60840 ctaatgtaca
tgtgtttaaa gcagctctgc ttaaaccaca catagcatct ttttcacaaa 60900
gtcctcaaag tcagtgctgt catcacttag cataccttct tcctttagaa atcttcacaa
60960 tgaaaataca ctgaagaaag gtggttagca aagtgcctag tgaaaaccag
atttctgtct 61020 cagatttgtt tttgttttag ttccacaaag agcacaattt
ctcttattct ttcagtagta 61080 tttcaaatac aatgaattta tctagaattt
tcctaaattg acaaattttg tttaagaaaa 61140 ctcttcaaca aattaccgag
gagtaaatgg ttttttatat gctgccaagt ttactttggc 61200 aatgtaaatt
gaactagaac tagggttcat ttttaagtgt aggattataa ttcaagataa 61260
tctgtataaa ggaaattgtt gtagctgaaa atagatcaaa gtattgaaga aataacaata
61320 atgaggagtt ttaagtgtgg aaaagttagt actcaagaaa gggtaatgaa
cttttaaatg 61380 tacactgttt taccaaaaat gttaatcaca ttacctctct
atttttttaa gtggtatata 61440 gtcaaaaata aaatattttt gtttgatgac
aggtatacca gagaggcagg ggttcgttct 61500 ctggatagaa aacttggggc
catttgccga gctgtggccg tgaaggtggc agaaggacag 61560 cataaggaag
ccaagttgga ccgttctgat gtgactgaga gagaaggttg gtgaccttgt 61620
tctggcattc tcaggcctgg tggctaggag tgagtgacag aagaaggttg ggtatggagg
61680 ggaaggtgtt gggtagtcct tggagcagtg gcacacatga ctccactgtt
aaatgcatcc 61740 agtaagtaat accttaatgt ttcaacatat ttcatccaga
ggattgtctt ttacaaatag 61800 cacagtttta actggaataa taatatgaat
gctttgagga tataggaact gtattagggt 61860 tcactagagg gacaagacta
ataggataga tgtgtatatg aagaagagtt taaggagtat 61920 taactcacac
aatcacatgg tgaagtccca caataggcca tctgcaggcc gaggagcaag 61980
gaagccagtc caagttccaa aatctcaaaa gtagggaagc cgacagtaca gccttcagtc
62040 tgtggccgaa gccccaagag cccccagcaa accactggcg tacgttcaag
agtccaaaag 62100 ttgaagaact tcgagtccaa tattcgaggg caagaagcat
ccagcacggg agaaagctga 62160 aggccagaag attcagcaag tctgatcctt
ccagcttctt ttctctgctt tattctagcc 62220 atgctggaag ctgattagat
ggtgcccact cagattgagg gtgggtctgc ctctcctagt 62280 ccgctgactc
aaatgttaat ctcctttgac tatatcctca cagacacact ggaacaatac 62340
tttgcatcct tcaatccaaa gttgaaactc actattaacc atcacagtaa ctttctccag
62400 atgtataatg atggtgtacg ttatgtatgg gttctggtgt tatcttattt
ctttctgacc 62460 cagacagtta agtctttaaa taatttataa cataaaaagt
ttttacaaca taagacaatc 62520 catgctgttc aggtactgca aggacagacc
tttgtactct ggaatagctc catgtgtaat 62580 aatttttcac acattttctt
ttatggataa acaactaaat gtaatttaaa ttattcttta 62640 aaaaattatt
gtgaaggtgt tctattactg gaattaatca aatgtggatg ttcctttggt 62700
atctacttaa aatgttttaa ctggccaggc acagtggctc atgcctttga tcccagcact
62760 ttggaaggtt gaggcaggca gatgacttga ggtcaggagt ttgagaccag
cctagccaac 62820 acggtgaaac cccgtctcta ctaaaaatac aaaaattagc
caggcgtggt gttgggcgcc 62880 tgtagtcccc gctactctgg aggttgaggc
aggagaatcg cttgagccca aaagtcagag 62940 gttgcagtga gcaaaggtca
tgcccactgc actccatctg ggcaacggag cgagactcca 63000 tctcaaaaaa
ataaataagt aaataaaata aaatgtttta atttcttgcc ccaaaactgt 63060
aaggggtctc agttcatcat atcatgctgt tatgcagttt gccaaaactt gctttaacaa
63120 acatgagttg tagggaattg acaatttctt tcatagtaaa gagatttatt
agatttttct 63180 atcatttcca tagctgtttc cagaaaggag ttggatgact
gtgattaaag aaccataatt 63240 tatggtggac ccagttgaac agacacagcc
aaatgtcttt cttgtttttc catcagtcgc 63300 tgaacacagt gcattttaca
gcagtagcat cagagtcagc tttcacagaa tccttctgtg 63360 gccagtacag
tgcttcaccc ctgcctcccc acgcctggaa cctcactggt tcattttctc 63420
cagagagcga agctcctatc ttctgttgga ttggagggag gcagtgcctt cattatgtgg
63480 agtaggagta gaggtagtga gttctaattg tattttatcc agactttaaa
acttgtgctt 63540 tatttttatt atttttattt tattttactt tttgagatgg
agtctcgctc tgtcgtccag 63600 gctggactgc ggtggcacaa tcttggctca
ctgcaacctc cgtctccgag gttcaagtga 63660 ttctcctgcc tcagcctccc
cagtagctgg tactgtagac ggatgccacc acgcccggct 63720 aatttttgta
tttttagtag agacagggtt tcaccatgtt ggccaggctg gtcttcaact 63780
gctaacctca ggtgatctgc ccaccttagc ctgccaaagt gctgggatta caggtgtgag
63840 ccactgcgcc tggctttatt tttatttttt atttttactc tgccttggga
gaatctagaa 63900 aacttttgcc ttttgtccca ctcttcatcc atgctttcag
ggctaccttg aattctttag 63960 cttttgtaga cttttaggac ccacatcaac
ttgttgttct ctatctctag ccccacaaat 64020 gttgaggttt ctgctttctc
tagcctgtta agtgttggtt actttttgtc catgtacttt 64080 ttgtttccca
aaattttgtc agcatctctt gtcagctgat gtcctctttg tcattatttt 64140
tgttcttgtg ggtttatata ttttttattt cttaattgtc attttaatac tattcagaca
64200 ggaagtaaaa acgcatgctc agactaccat ttatagaaat ttgaatttaa
aaaaaatgtc 64260 ctaggtgagg gagtacctat caagggtgga aatcacttgt
gtagatgaca gtgacagtgg 64320 agaactgaag tctataaaag ttaagaccta
gatctagatg ctcctgaatt tccccttttt 64380 attcttaaca acacttcctt
tgtgctgtga tctcaagcaa ctgagcctag gtctttttat 64440 tcttgtctga
tataacagaa ggtagaggat gaaataaatg agtttattag gtaacacatt 64500
ttgaaaattg tgtttaagat ttagatgata tattttagaa cttctaataa attcagagga
64560 attcaatgtc aaaggaaact tttgtatagt tatacattgc ttaatgttta
tacatacatc 64620 catgtagcat acttctaata atatctttaa ttatactagt
tattttaaaa taacccacaa 64680 atactcaagg aattgttcag tttgtgaact
gtgtgagaac tacagttttt catggtaaca 64740 tttatttgtg tggtttttaa
aagtgatcac aggacatctc ctaaaagata atatagttaa 64800 gcagatttgc
ttagttaaga tattaccaag agcatctaga tgaataatta gaataaatac 64860
ttgtctcttg gagacgattt tgggtgtagt ctttactaga ggcataggta tggactccaa
64920 gttggctcta atattatgag atacccttga gtaaataaca gccattctct
agaccttagt 64980 agaatgatta ttaggtgtcc tgaattgttt atgacctcaa
ccaaaccaaa agaataattt 65040 ctacaaaaga gtctatgtta ggttttcata
gcaccaagtt caaatggagc ttagtaatga 65100 aaattttctc attaagaaat
gaattaatta aaattaagag cataaaataa gacagttgtt 65160 ttagaaactt
caagtaatac agtgtgggag ttatttttaa tgttaaaaat aaagctttcc 65220
taattcaagc acgagagaca gaaaaaaaat aataaggctg aacttggagt tactgccagg
65280 aagaaaagta attttaggcc acaagcttca aaacaggcag aaacctccag
tgtatcaaac 65340 aaactttctg gaataggccc agaagcactg atctgtgaac
agttgtcttt gtatttgtgg 65400 ggtcttaact ggcagttaaa gagactaaat
aatagcaggg agtttaaaaa gcaggtgaga 65460 tttagaattg atcgatctgt
gttagcggag gaacatttat ggtttcagtc acttacctat 65520 aaagtatgag
aattgtttct ttaaaagaat gctgcctctg tttttctgca tgttgttagt 65580
attttctgaa ttgccgtttt cctttctagg gtatttgttg ggttgagaga ttagttggat
65640 tacatgacta cagttttatt ctgctttttg cctgcctttt gccaagaaag
acacaaatgt 65700 cccatgtatt taattttgca cacttcagtg tttctaaaca
gggtaaatgt tcatttgttt 65760 aagtacccat gtatcatata ttcaatttat
atctagcaag atttttcctc aaaaattatc 65820 ctaagcaaag aaggatttat
attataatca gtccttataa agtttctcat aatacactgc 65880 attctcaatt
actttatttt tgaagaacat agtatttgag gaagttacat taaacagaaa 65940
gaacctgggt agatactagt ttctgattat tttcatagaa gtcacctgaa aaattggtta
66000 gaaaaaaaag acaaaattaa tacaaattta acagttattt gtgaaatatg
taaatgttgt 66060 gttattccat tttgctgtgc tacaaaggaa tacttgaggc
tgggtaattt ataaagaaaa 66120 gagatttgtt tgggtcagag ttctgcaggc
tctataacag gcacagtgct agcttataag 66180 gtgagacctt aggtagctta
taatcatgat ggaggacaat gggagagcag gcatgtcaca 66240 tggtgagaga
gggagcaagg aaagagccag ggacctttta acaaccagct gtcatgtgaa 66300
ctcattacca tggggaaggc accaagccat ttatcaggga tctgcccctg tgacccaaac
66360 atctcccagt aggtccctcc tccaacattg ggaaacaaag ctatagtaac
caaaacagca 66420 tggtactggt ataaaaatag acacatagat caatggaaca
gaatgcagaa actagaaata 66480 aagccacaaa tctacagcca actgatcttt
ggcaaagtag acaaaaacgt acactgggaa 66540 aggacaacct attcagtaaa
tggtgctgag aaaattggat agccatctgc agaaagaatg 66600 aaactgaacc
actctctctc ttattttata taaaaatcaa ctcgaggtta ggctaggtgg 66660
ctcacacctg taatctcagc actttgggag gctgaggtgg gtggatcact tgaggtcagg
66720 agtctgagac caacctggcc aaaatggtga aaccccgtct ctactaaaaa
tacaaaaatt 66780 agctgggcgt gctggtgcat gcctatagtc ccagctactc
gggaggctga gacaggagaa 66840 tcacttgaac ccaggaggcg gatggtgcag
tgagcccgag atcgcgccat tgcactccag 66900 tgtaggggta tcgcagcgag
actctgtctc aaaaaaaaaa aaaaaaaagt caactcaaga 66960 tagattaaag
actttaaatg taaaatccaa aactaaaaca tactagaaga aaatctagaa 67020
aaaattcttc tagacgttgc cataaacaaa gagttcatga ctaagacctc agaagcaaaa
67080 gcaacaaaac caaaagtaga cagatgagac ttaattaaac taaaaagctt
tttatacagc 67140 aaaagaaaca acagagtaaa cagacagctt gcagaataag
caaaaatatt tgcaaaatac 67200 atatgcaaaa gaccaatacc cagaatctac
aaggtaactc aagcaactca acaacaacaa 67260 aagaacccca aataacccca
ttaaaaagta ggcaaaggag atgaaagaca tttttcaaaa 67320
gaagacatac aagtggccag gaagcatttg aaaaaatgct caatatcact aatcatcaga
67380 gaaatgaaaa atctatgaga taccatctta taccagtcaa aatggctatt
tttagaaagt 67440 caaaagtaac agatgttggt gaggatgtgg agaaaaggga
gtgcttatat agtgctggga 67500 gaaatgtaaa ttagtaccac ctctatggaa
aacatatgga gagttctcaa agaacaaaaa 67560 atagaaccgt catttgatcc
agcaatccca ctactgggta tatacccaga ggaaaagaat 67620 tcattatgtc
aaaaagatac ctgcacacat atgttcgttt tatctgatat aaaaagtctg 67680
ttttatctgg tataaaaaga atggaatcat gccttttgca gcaatatgga tgaaactgaa
67740 ggctgtgaca ataactcaga aattcaaata ctgaatattc tcatttataa
gtggaagcca 67800 aataatgtgg acatatgaac atagagtgtg gaataataga
cacaagcatg agctatcatg 67860 cccagcctca aaaaatttaa tttccctctt
aattttgtca ttgacccaaa ggttgtccag 67920 gagcatgttg tttaatttac
atgtgtttgt atatttttga gagtttctct tcagattgat 67980 ttttagtttt
attccattgt gtgaagatac ttgatatgat tttgattttt ttttaaattt 68040
attgagactt gttttgtggc ctgacgtttg gtctgtcttg gagaatgtcc catgtgctaa
68100 tgagaaaaat gtatcttttg tggttgttgg gtagaatgtt ctgtaaatgt
ctgttaggtc 68160 catttggttt taagttcagt gtttctttgt tgactttgtc
tgtctcagtg ttgaagtccc 68220 acattttgta ttgctatctg tctcttttct
taggcctagt agtatttgtt ttattaatct 68280 ggtactccag ttttgggagt
atatacttag gattgttata tcttcttgtt gaattgatcc 68340 ctatgtcatt
atatactggc ctttaaaaaa aaaaaaacta ttgttgattt aaagtctgtt 68400
ttatctaata taagtatagt tactcttgct tgcttttggt ttccttttgc atggaacatt
68460 tttccacccc tttaccttca gtctgtgtgt ctttaacagt aaggcaaatt
tcttgtaagc 68520 agcatgtagt tgttgttttt taatccattg caccaattta
tatctttgaa gtggtgcatt 68580 caaggttaat actgatgcat gaggttttgt
tccagtcata atgttaattg ctatctagtt 68640 gctttgtaga tttttttttt
tcttttaagc aagagtcttg agtcttgctc tgtcacccag 68700 tctggagtgc
aatggcgcga tcttggctca ctacaacctc cacctcccaa gttcaagcga 68760
ttcccttgct tcagcctccc aagtagctgg aattacaggt gcatgccacc atgcctggct
68820 aatttttgta tttttagtac agacgggatt ttgtcacgtt ggccaggctg
gtctcgaact 68880 cctgacctca ggtgatcctc ccgccttggc ctcccaaagt
gctgggatta caggcgtgaa 68940 ccaccgcaac cagccagctt tgtagattct
ttgtttgttt tttgttcccg ctttgtggtc 69000 ttctggagtt ctgtcatgtt
gcccttttat ttctttcttt tccttatttg tataattgtt 69060 tcataaaact
tgtgagtttc atgtgttttt atgatagagt atcacctttt gttcccatgt 69120
ttagaacttc tttaaatatt tctcatagga ccaatcaagt ggtgatgaat tccctcattt
69180 gcttatctgg gaaacacttt atttctcctt catttgtgaa gcttacacta
gcaggataca 69240 aaattcgagt ttgaccattt tctttaagca ctttgaaaat
agaatccccg tctcttctgg 69300 cttctgaagt ttctgctgag aagtccactg
ttagtttgat gaagtttcct gtataagtga 69360 ctagacactt ttactgtatt
tagggatttt cccttcacat tgaccttaga cagcctgatg 69420 actagatgcc
atggtgagat ccttctcgca atgtatttgg ctggagtttg ttgagcgtct 69480
tgtatctgga tgtctagatc ctttgctaga ctagggaagg ttttctcaat tattttctca
69540 aataggtttt ctgaaatttt tgctttttct tctcctctag gaatacctat
gattcatagg 69600 ttccaatgtc ttatgtaatc ccttactttt cagaggctct
actcattttt taaaattctt 69660 ttttcttttt tttttttgtc tgactggatt
aattgaaaaa acctatctta aagttctgag 69720 gttctttctt ctgcttggtc
tagtctgttg ttgaagcttt caaatgtatt ttataattcc 69780 ttcaatgaat
tttttatttc caggagttct gtttggtttt ctttttaaaa tacctatctc 69840
tttggtaaat ttctcattca tttcctgaac tgattttctg acttctttgt attagttttc
69900 agatttctct tgtatcttgt tgagctactt tttttctttt aatttaattt
tattttgaaa 69960 cagggtctcg ctctgttgcc ttgtctggag tgcagtgatg
cagtcatagc tcattgtaag 70020 cccaagcagt cctctgcctc actgtcctaa
gtagctacaa attcaggcac ataccaccac 70080 acctagctta tttttttatt
ttttgtagag atggagggtt atactgtgtt gcccaggcta 70140 gtcttgaact
cctggcctta agtgatcctc cttcctcttg ccttggcttc ctaaactatt 70200
gggattgcag gcatgagtca ctgtgccctg cccctgacag cttcttcttt tttttttttt
70260 ctgagacaga gttttaccct gtcacccagg ctagagtgca gtggcacgat
ctcggctcac 70320 tgcagcctcc acctcctggg ttcaagtgat tcttgtgcct
cagcctcctg agtagctggg 70380 attacaagcg tgcgttacca tgcctggcta
atttttgtat ttttttagta gagatgcggt 70440 ttcaccttgt tggccaggca
ggtcttgaac tcctggcctc aagtgatcca tccaccttgg 70500 cttcctaaag
tgctaggatt acaggtgtga gccactgtat ccagcccctg atagcttctc 70560
taaatcagtg ttttgaattc tttatctggc attttgaaga tttgtttttt agttaggatc
70620 cattgctaga gaattactgt gtttctctgg gggtgtcata gcaccttttt
tttttcatat 70680 ttccaatatt actgtgctga ttcatttgta tctgggataa
cagttgcttc ttattatttt 70740 ttagtttact tttgttgggg caggactttc
tttcccttga ggatgtatct attatgtatg 70800 ttgagtaggg tcatttggct
ttgcttcagg gtgcattcag tgacatagac actgtatgat 70860 agccttggtt
ataaagtagt cttagtatgg tggctttctc aaatgccagt gacagtagta 70920
atgtacgggg tgggtgattg ggctcaaggc ctcctgccta gctggggtgg atgatggtgg
70980 cagcagaggt cgtgcaaaac ttgctttctt ccaaggcact atgcagttgt
atcaatagat 71040 gttgtaatgg gtggtgcagg ttgacttccc agctaggagg
tggtgcctgc agatgagcgt 71100 cagctgcaat agtggcagta gggtgattaa
cctttgtaat tcaagaatta ttcaggtatc 71160 tcaggtaccg agctgggccg
tgaaactctc aggggtcctg gtcttgtgct gtgcttccag 71220 ggtagattgt
ggggtgaagc caggcaggct ggaccagcca agctcatgtt tgagccccct 71280
gaatgggtac ttagggcctg ggataaaatt tccagaggct gcctcataca ttgtttcaag
71340 aattacttta tcttagataa tcttggtatc tggtagtgta agtcttccag
ctttgttctt 71400 cttcagaatt gggttggcta ttgtaggtcc ttcaaatatc
catgtaaatt ttaaagtcag 71460 tttgtcattt tctaccaaca agtaaataaa
taaaaactcc tggggcattt ttattatgat 71520 tccgttgaat ctgtaaatct
agttggggag aattgacaat ttgtattatc aagtcttcta 71580 attcatgacc
agcttcattt atttaagtct tcttacataa gttttttttc ttcagctttt 71640
aagttccagg gtacatgtgc aggatgtaca agtttattat gtaggtaaac atgtgccatg
71700 gtggtttgct gcacagataa tccatcaccc aggtattaag cccagcatcc
attagctatt 71760 cttcctgatg ctctccctcc cctcactccc acccacaaca
ggccccagtg tgtatttttc 71820 cctgccatgt gtccatgtgt tgtcattgtt
cagctcccac ttataagtga gaacatgcag 71880 tgtttggttt tctgatcctg
cattagtttg ttgaggataa tggcttctag tttcatccat 71940 gtccctgcag
aggacatgct ctcgttcctt tttatggctg catagtattt catggtgtac 72000
atgtaccaca ttttctttat ccagtctgtc attgatgcgc atttgggttg attccatgtc
72060 tttgctattg tgaatagtgc tgcaatgaat atatataaat cattctgttt
ctttggctat 72120 atacccagta gtgggattgc tggatcaaat ggtatttctg
cttctagatc tttgaggaat 72180 caccacactg tcttccacaa tggttgaact
aattaaactc ccaccaacag tgtaaaagca 72240 ttccttattc ttcacaacct
cgccagcatc tgttgtttct tgacttttta ataattgtca 72300 ttctgactgg
cgtgagatgg tatctcattg tagtttttat ttgcatttct ctaatgatca 72360
gtgatgttga gctctttgtc ctatgtttgt tggcaacata atgtcttctt ttgagaagtg
72420 tctgttcatg tcccttgccc actttttaat ggggttgttt ttttttttcc
ttgtaaattt 72480 gtgttcctgg tagactctag atactagact tttgtcgggt
ggatagattg aaaaattctt 72540 ttcccattct gtaggttgtc tgttcactct
gatgatactt tcttttgctg tgcagaagct 72600 ctttagttta attagatccc
atttgtcaat ttttgctttt gttgctattg cttttgtcat 72660 tttcttcatg
aaatctttgc ccgtgcctat gtcctgaatg gtattgccta gatttttttc 72720
taaggttttt atagttttgg gttttacatt taagtcttta attcatcttg agttattaaa
72780 taatttttgt ataaggtgta aggaaggggt ccagtttctg ttttctgcat
atggctagcc 72840 agttttccca gcaccattta ttaaatagag aatcctttct
tcattggtta ctagtacaaa 72900 aacagacaca tagaccaata gaatagaatg
gagaactcag aaataagacc acacatctac 72960 aaccatctga tcttcttaaa
taagtttttt aagagttttg atcattttct gtggcacact 73020 tttacataat
ttttctttag atatcttcct aggtatttga tctttatgtg tatattattg 73080
taaataacgt tcttaaaatt ttgttttcta attttttgtt ggtagtgtat gacaatgcaa
73140 tattggcctc ctgttcaaca aacttgccac attcacttat taatcataat
tgtttgtgga 73200 atcttttgga ttttctgcat ctaccatcct gtaatcacaa
atgcagatgt cagtttttac 73260 ttcttccttt ccaacgttat accttttatt
taatttcttc cctaatatgt tggctaggac 73320 ctcctgggaa atgctgaata
gaaataatga taatagacaa agtaagcagg ataaaagcct 73380 atgaagaaat
taccaactga cataggcttt gctttgtagc tttaggtcac ccctcatcac 73440
ctaatattat aaaatgacaa ttcggtagga ttctcagaaa ctgtccagtt tgaccctgat
73500 ttaattctca acattctcca gtaaacacta tgccttgcct gtttgacttt
gttaacagac 73560 atgtcagaca atcatgtggt gaagtgtgat tttacttgtt
tattcaacct gagatttgct 73620 gacagttcgt tctgtgttgc tgtaacagaa
taccacagac tgggtaattt taaatgagca 73680 gaaatgtatt ggttcacagt
tctggaggct gaagagtcca atgtcaaggt gccagcttct 73740 gacaggaacc
ttcttgctgc atcttcacat ggcagaaggg caaagaaaga gaagggggcc 73800
tgaactcact cttttataag gatatcagtc tcacccataa gggcagaatc ttcaggaacc
73860 taagagcaac ttgttacttc atggcctact gacctcttaa aagtctcact
acttaatatt 73920 gttacaatgg cagttaaatt tcaacatgaa ttttgaaggg
gacaaacatt taaaccatag 73980 cactgacttt cttgaatttg tatactcttt
tattggtttt ggaaagattt tggccattat 74040 cttttcaaat attcttccca
tttttttact cttccttctg ggattctgag aagagagccc 74100 ttcactgtct
cttatcctcc tttctatttt ttttttgttt gttaattttt ctctctcatt 74160
cagtttagat attttctgtt gccctgtatt ccagtttgtt attgctttct tctatttttt
74220 tgtggtctgc tattaagcct atgaagttct taattaccat attgtaattt
tttttttttt 74280 ttttttttac ttttagaatg gccactggat attttttttt
tctttcttta agacagagtc 74340 tcactctgtc acccaggcta aagtgcagtg
gcacgatttt ggcttactgc aacctttgcc 74400 tcctggattc aagcgattct
gatgtctcag cctcctgagt agctgggatt acaggcgtgt 74460 accaccatac
ccagctaatt ttgtattttt agtagagacg gggtttcacc gtgttggcca 74520
ggctggtctc gaactcctta ccttaggtga tctgccctcc tctgcctgcc aaagtgcaaa
74580 gtgctgggat tacaggcatg agccaccgcg cccagcccat tggattcttt
tttttttttt 74640 ttttttttga gacggagtct cgccctgttg ctcaggctgg
catgcagtgg cgtgaccttg 74700 gctaactgca accttcacct cccaggttca
agtgattctc ttgcttcagc ctcccgagta 74760 gctgggatta caggcgcccg
ccaccacacc cgaccaattt ttgtattttt agtagagacg 74820 gggtttcacc
atgttggcca ggctggtctt gaactcctga cctcaagtga tccacccacc 74880
ttggcctccc aaagtgctgg gattacaggc atgggccacc acacccggcc aggattcttt
74940 gtatatatat ggactccaat agattctcca ttgatatttt ctatcttttt
atctatttaa 75000 tccctccttt tccctatttt cttggacatg ctagtcatta
ttttgaaaat ctctacctta 75060 acactccatt atctgattca gttatgtttg
gtgtttgttt tgtttgtatt accttttttt 75120 cccccttgat ttctagtttt
ttgttctgtt ttttagcatt tcttgtattt ttttactgga 75180 tgccagacat
tggatgaaaa atacaagggc tgtaactatt atcctctgaa aagtgttaca 75240
ttttcttctg attggtaact acagtaccaa cctgtcactc tgtcctgtca aggctgagtt
75300 ttaggctttg tcaggactcg tcaatttcag tttgggtctt attactggga
tacagtcttt 75360 atttttatta tgtggtactc ccaggatgta gttcttattc
cttcgtgggt gacccttact 75420 tctagagcat gatctttctg agttctcaca
tgaaaatcca atcaggtctt tagcatcctg 75480 gcttctcctt tctcctgggt
ttctaaaaga ctcaccctga atacattcaa cttaggagtt 75540 agtcaacagc
ttgaggggga tttaagtgca gatttttgag atccttcttt ttggtttctt 75600
cctttattgg gattttgcca atgaagtccc agttgctttg acaacctcta attttcagaa
75660 ttacttttga ctaaatgttt tatgattcta aacataccat ctactctgtc
aattctgaat 75720 tatggtgata ctcaattcta cctcaaatcc caaagaaaag
agggggaaaa aacaacaaaa 75780 ctaagaagaa acattgcttt tgttttgtag
ctttaggctt ctacctatat aattgactat 75840 tataaaatct catttgagta
ggatctttag tagccaccta ctttgactgt gatttgattt 75900 ataaatccct
tcacaacatt cctcagtaaa caccatgctt tgcctgtttg acttggttaa 75960
cagacatgtc tttataaact tggctatcca ttttccagtc tgtaggaaaa gagaagctgt
76020 aagttggaga aaaggctagt ggttgggtgg tgagtcataa gcaataagat
ttgatgtcag 76080 tgatgacagg cctgtcctct tatgatagat tccttgagcc
ccctgctgac cacaaagctt 76140 tggctggcta gaccacaagt ctgtctccct
caatgacaat ttttgtagct caatatggat 76200 cctattttgt gtgagttgca
tttggagatt tattgtttat ctgctgtatt tgccttaggt 76260 gggacagtga
aatcaaccta atgtagtgga aggaagtagg tattacatcc ttaattcctt 76320
gatatacatc cttttattat gtggtactcc cgggatgtgg tttttcagat ttggagaaga
76380 atagttaaaa aaaaaaaatg cagaaaggat caaaagcact tgattctctc
gcagggacag 76440 cttcctgttt tggttgagga aggagctgca cttaaaataa
ctagcataaa gcatgcttag 76500 ggcttgcttt ccagacaacc tcaatttaaa
atgcatcaaa agccaggtgt ggtggctaac 76560 atctgtaatc ccagcacttt
gggaggctga agagggcaga tcacttgagg tcaggagttt 76620 gagaccagcc
tggccaacat ggtgaaaccc catctcttct aaaaatacaa aaattagctg 76680
ggcgtggtgg cacacacctg tagtcccagc tacttgggag gctgagatgg gaggatcatt
76740 tgaacctggg aggcggggat tgcagtgagc cgagatcaca ccacagcact
ctagcctggg 76800 caacagagca agactctgcc tcaaaaaaag aaagaaaata
aaattcatca aaataaaata 76860 tttgaatttt acagcactag ttcttttcat
tcattgactt tcattctccc actttaccac 76920 acctttaact attggcaaga
atgtggtgag tgggagaaag cgtatcctgc cacgtaagca 76980 agtataccta
gagccaaggg gtcagagtgt cacagaggag agccacatgc tgatgggctt 77040
gtgttcgttc ccactcactg actatgcaag cgcctcttct cttagccttt ctcaggatgc
77100 agttctccag ggaggaatca gccttctgtt gggctgcttt cagagctctt
tgttgtggct 77160 tcctgccatt gactttgcaa gccctaagca tgctttatgc
tagttatttt aagtgcagct 77220 ccttcctcaa ccaaaacagg aagctggctc
tgcaagagaa tcaagtgctt ttgatccttt 77280 cagctttttt tttttttgac
tattcttctc caaatctgaa acatatccat tctcgtctac 77340 ggccatgagt
gcatttatgt taacagaaaa tgctaaattt aatgtttaga aagtaacctc 77400
tgtggccaga catggtgact aatgcctgta atcctggcac tttgggaggc cgaggcaggc
77460 agatcacttg aggccaggag ttcgagacca gcctggccaa cacagtgaaa
ccctgtctct 77520 actaaaaata gaaaaaatta gttgggcatg gtggtgggtg
cctgtaatct cagctacttg 77580 ggagggtgag gcaggagaat cacttgagcc
caagaggtgg aggtcgcagt gagccaaaaa 77640 tcaagccact gcactctagc
ctggatgaca gagcaagact ctctcaaaaa aaataaaaag 77700 taacctctgt
gctttgtgta actttttgct aaattcctgt ctttgtcttc ttggaacagt 77760
cttctacttg ttacaggatc ttcctatctt ttggatttta tattagtttt aatataaaat
77820 taatatagtt ttatattata tagcccactg acatggctgt tagctgacct
cagttccttg 77880 ctgacttggc cagagccttc agtttcttat ctctggtaag
aggtaatgtg tctctcccta 77940 gggcaaggct gtgacagctg gcttctccca
gagggaatga tgtgtgagag aagcagggag 78000 agtaagaatc aagacaaaac
tgcagtcttt tatacccatc actattgcca tattctcttg 78060 gtcacacagc
ccaaccctgg tatgatatgg gaggcactaa ctccatgggg atgggatatc 78120
tgggcaccat cttgaaggct agctgacaca gattattttt tgtgcgtgtg cctgtaagaa
78180 ttttttggcc aggcgtggtg gctcacgcct ttaatcccag cactttggga
gggcgaggtg 78240 ggtgggtcac gaggtcagga gttcaagacc agcctggcca
agatggtgaa accccatctc 78300 tactgaaata caaaaattag ccaggcatgg
tggcaggggc ctgtaatctc aactactcgg 78360 gaggctgagg caggagaatc
gcttgaactt ggggggcgga ggttgcagtg agccgagatc 78420 acgccactgc
actctagcct gggcagcaga gtaagactct gtctcaaaaa aaaaaaaaaa 78480
aaaaagaatt tttctaagcc cgcattgaag tttatactgt agaatatcca tcaaacttga
78540 gctgatttct tatcaaagac ccaggttgca cagatagggg ttagaagttt
ggattcggtt 78600 ttgcattttc agtatttaaa gtcttgtttc atcttgttca
ttcttacctt tcctttgatt 78660 gtattagtag ctcaggacaa ataagaattt
ataattttcc aaggaactaa ggttgctgtt 78720 gaggaatatg ggtttcagag
acaagagttt aggcactggc tcattggtac taagcttcag 78780 gggtttgtag
tgttgttaga gctaattgga ttttacaaat aagccaagat tattaaaaaa 78840
aaaaaataga tctagagagt aacactttct gtgctaaatc cattgcattt gatgggatac
78900 taggcagtat gctatgtcca aacttctaaa atcaggcggt ggtctaacgt
tgaggtgaaa 78960 atatcatgtt gggtatatac tgccaatatc atgaagatat
actaaatatt attttctgag 79020 tctgacattt acactgattt actgatttat
ccctcatcaa tattggcctg gtttaagaga 79080 gacttgtttg cctgtacaga
ccgggaggaa gcttcaatga aggcaaaaat ctaactataa 79140 taggagccaa
acatttgtta tttgaattcc aattggggac aggaaaataa aatattatca 79200
aataattata aagtcatcat tctgttaaat gaatcatata ggaaaatgca ttgaccttaa
79260 aacagagtct ggctctgtta cccggactgg agtggagtgg cctggtttca
acttgctgca 79320 acctccacct cacgggctta agctgtcctc ccacctcagt
ccctagagta gctgggacca 79380 caggttttgc catgttgctc aggctgttct
caaactcctg agctcaagaa atccacctgt 79440 ctcagcctcc tgaagtgctg
ggattacagg cgtgagccac cgcgcccggc ctgcagtgac 79500 ctttggttgt
cattgttata cattatcaaa acaaactcaa gttacaagag tattaaagca 79560
atacttaatg gttttaaaaa aaatattaca aaaggtctct gcattttaac tactcatcta
79620 aataattgtc taggaatatt ttctgaatct ctaatacagg aaatgagatt
tattaataca 79680 taaaacccac tgaaaacagg ggtgcaaact ttcttgtctg
gtactaaaga tggattccta 79740 tgttttgggc ccttgtttat accagtttat
tcaatcagtg agtcagctag catttactga 79800 atagtcatat gcgttgctta
atgatgggga taatgttctg agaagtgcat ccctgggaaa 79860 ttttgtcatt
gtggaaacat catagagtgt acttacacaa acctagatgg tatagctttc 79920
tacacaccta ggctatatgg tatagcctgt taatcctagg ctataaactt ctacagcatg
79980 tgactatact gaatactgta ggcaattata acagagtggt atttgtatat
ctaaacaaca 80040 gatgaacaat aaagaaaaaa taaacaacaa ataaaagctg
gtacttctgt ataaaggcac 80100 ttaccatgaa tggagttgca ggactggaag
tagctctgcg tgagtcagca agtgagtggg 80160 agtgaatgtg aaagcctagg
acattactgt gtatatacta ctatagactt attaacactg 80220 tacacttagc
ctgtattttt taattttttt cttttttttt ttttacttct ttttcttttt 80280
ttgagacagg ctgtgttgct caggctggtc ttgaactctt gggctcaagt gatccttcta
80340 cctcatcctc ctaagtagct gggattacag gtgtgtgcca ccacacccag
ctttttaaaa 80400 cttttcaaat cttttataat aacactcagc ttaaaacaca
aatacactgt atagctatac 80460 aaaaaatatt tttaccccat ttatgcctag
tgctccatta ttggaacact aagcttgtgg 80520 gagttattta tatcctactg
ctcaaggtca ttgccaaggt ctgatttttc acaaaaaaaa 80580 attcacaact
tctggcataa atgggttaat atccttactg tatataagct tttttaaaaa 80640
ttgttttact ttttaaactt ctttgttaaa agcaaagaca cagacacaca ttagcccagt
80700 cctgaactag gtcaggatct tcagtttcac tgtcttccac ttccacatct
tggcccactg 80760 gaaggtcttc agaggcagta acatgcatgg ataacagtgc
cttctacctt ctgaaggacc 80820 tgcctgaggc tgttttacag ttaacttctt
ttttacagaa gggagtacac tctaaaataa 80880 tgatgaaaag catagtatag
tccaggcacg atagtgtgtg cctgtagtcc cagctactca 80940 ggaggctgag
gcaggaagat tgcttgaacc catgagttca agaccagtct gggcaacata 81000
gcgagactcc acctctaaaa atatatataa gaataaaaaa ttttttttaa atgaagcata
81060 gtaagtacat aaaccaataa catagtcact cactatgact atgaagtatt
atgtactgta 81120 tgtaattgta cgtgctgtgc atttatacag ctggcagcac
aataggtttg tgtacaccaa 81180 gcatcaccac aaagatttgg gtaatgcatt
ccattgccct aacggggcta caacatcact 81240 aggcaatagg aatctttcag
gtccgttgtt gtcttctggg acttctgtca tatatgtggt 81300 ctgcctttga
ccaaaatgtt gttatgcagt gcgtgactat acccactata tgttcaagtt 81360
ctaaattgga ttctgggaag ctgattaaag agaaaataat gtgtagtcta ttggaagagg
81420 tagataaaca atttttaagt gaaataattg ctaattttta acctctgtgg
aggcactgaa 81480 ctgatcattg aaagctctat tttacttact aaagatatgg
tagcttataa aaattactta 81540 tagtaaatgg acatgaaaag gtcatttgct
tacatctcta aattcatttt gatggaaaaa 81600 tagtggaaaa atgtttgcag
ataccctttt gtttgtttgt ttttttcata atagataatt 81660 gccactaaaa
ttgaagaatg gccaggtccg ttggctcatg cctgtaatcc cagcactttg 81720
ggaggccaag gcgggtggat tacttaagct caggagttca agattaacct ggccaacatg
81780 gcaaaacccc gtctctacta aaaatacaaa aaattagcca ggtgtggtgg
tgcacacgcc 81840 tgttgtccca gctacttggg tgactgaggc atgagaatca
catgagcctg ggaggcggag 81900 gttgcagtga gctgagattg tgccactgca
ctccagcctg ggcaacaggt gagactctgt 81960 ctccaaaaaa aaaaaaaaac
aactaaaatt gaaaaatacc tcacagtcat aacttccatc 82020 tgtatctcag
tggttattat gtagaaatgt tcagtaggta aacttgaaag aaaatgtatt 82080
tggtaatcgt aaggttgtgt tgccaccccc aaaataatga agaaaatacc aacagaaaga
82140 aaaaggattt attgctggcc tgaaggttct tctgggcatt tgatctacag
atttctccat 82200 tatagctagt tcctttaaaa aaataaaaaa cattgaaaat
atgcagaccc aaatgccttg 82260 gcagccctgg tcagtaactt gaatctcagt
tgcacttagc acaattcctc tggctgggaa 82320 gatgttgttt tggaaaagat
taacctgaaa tgacagcacg aattatacag ttggaaatac 82380
tcaggttttt ctgatttttt tcaaaagata ctttgctttt ccttttctgc cttaccatgg
82440 gaaggtcctt agatgcatca tatccttgtc agtttagcct tgtgacacat
atttctgcaa 82500 ttttgtgcaa taagaaagcc actcgaaatc tcagcatttc
atgtcacttt taaagtaggc 82560 tcagttaaaa caaaaccact tgattgtttg
tataaccaca accatatgtg tctttctctc 82620 catgcttaaa caaggtctga
aatcgtgtgt caaacagttg agatgtaaac atctcctcct 82680 cacacataac
ccctctgcca tgttgttatt tatatcccca gtaacacact tcttgtccct 82740
gacacaagta cagccgtctc cacattccat tttgctccta ctccatcagc ttgcaagaaa
82800 aattttaatc attcaaaaat aattgttaca taattacttt tcactgatta
aaaatatttg 82860 tttacttgac aaaattagca ttaaaaacag taattctttg
gcagattaat aagtattttg 82920 atgatttgtc atttttcaca gatgttgata
aaatttaaga attacatagc cgaaatttgg 82980 tctaattcaa caaaccacaa
ttgactcttt tggtaaggcc ctatgacgaa tggtatggga 83040 gagtggagtt
tatccaatct gactttcatt ttattgatac ggaaactggg gccccatttg 83100
ttcttttttt taattgctac ataatataca tatttatggg gtatagtgtg atgtttcagt
83160 acatgtatac attgtgtaaa aatcaaatca ggctgtttag catatctgtc
acctcatata 83220 tttatcattt ctttgtggta agtatattta aaattctcta
ttctagctat tttgaaatat 83280 acaatactgt taaccatagt cactgtgcaa
tagaacagtg gtccccaacc tttttggcac 83340 cagggaccaa tttcatggga
gacagttttt ccacggacct gtggggtggt ggtttcagga 83400 taaaactctt
ccacctcgga tcatcagcat tagattctca taaggagcac ccaccctaca 83460
tccctcacat gcacagttca taattcacaa tagagtttga gctcctatga gaatctaatg
83520 ccgctgctga tctgaccgga ggcggtgctc aggccgtaat gcttgcccac
ccgctgctca 83580 cctcctcctg acaggccatg gactggtact gaccagtcca
cagcctaggg tttggggacc 83640 cctgcagtag aacaccagaa cttattcctc
ctatttatct gcaattttgt acccattgac 83700 caatctctcc ccatccccac
tatctctccc cttgccagtc tcttgtaacc actgttctac 83760 tctctgtttc
tgtaagatca acttctttag attccacata taagtgagat catgcagtat 83820
ttgtcttttg gtgcctggct aatttcactt aatataatgt cctccaggtt caaccatgtt
83880 gccacatgtg acaggatttt attctttttg tggctgaata atattccatt
gtttatatat 83940 gtcacatttt ctttatccat tcatccgttg atggatgctt
acgttgattc catatattag 84000 ctattgtgaa tagtgctgca acaaacatgg
aagtgcagat acccctttga catattcatt 84060 tcctttggat aaatgcccat
ttgtgggatt gctggatcat atgatagttc aacttttaga 84120 ttttgagaaa
cctccatact gttttccata atggctgtac taatttacat tccagccacc 84180
agtgtgtaag agttctcctt tctccacatc cacaccaact acaggtggct tttctagact
84240 ggactttagg ttgggacaaa aagtgtcttt gagagtcagt agtcctaata
ctgtactgtg 84300 aatgctgtgg acttaggcag tttgtttaag cttgtttaaa
ctgggtctct ctttccttag 84360 atataaatgg agggttagac tggatcttta
agcttctgcc cagcatttaa tgttctgttt 84420 attgtggttc tagcctgtgc
ttcttgaatt cctgattctt cctgaattct gctaagcatc 84480 agaatgcagt
ctatacattc tcaacagctt cccaaagaca tgatattagt ataacagaaa 84540
cagtagtagt cctttcttgg aaaattatcc ccatttctgg accctatttt attgctggct
84600 gcaattaaca ggttcttgta tgtcccatcc ttccctcctc ctccctaacc
cacaggcatt 84660 aaaaacctgc tgtttgtgaa aatgaacact tctttgataa
tctggaagaa ggggttcctg 84720 ttaccagaaa atttagctct tgaactcctg
ggactgggct tgaaagcata gtactattat 84780 gcttcagatt aagcagggta
tagagaataa ggagtgatca caaaaattct gtcttgaata 84840 aagatgatga
tagatatccc agggccctct gtggttagat agtctccatt tctaccacat 84900
tctgaggaat tgtgggtgtt gcgcttttta tgtttctggc ctccctgcta cttgccattg
84960 gttggatcac tggccaagag ctaccgagaa ctaccatttt gcttcaagat
tttttcaaac 85020 agcaaggaac ttttttattt tttaacagag agctactgaa
gtttcctgag ttattacaac 85080 ccccttatcc ttcctcctta cttccccttt
caataattcc ctttcctccc tcttcccaca 85140 gcagttcttt ggctattggg
cctgttttca ttgaaatcat cttcctgtgg cagagggaaa 85200 atgaatagag
aagaacagtt gactgtgtcc aagtgatagc tgcttgctta ggaaaagcct 85260
ggtccttccc cagaggagtc tgtccctata ggacttccct ccataatagc tgtgcttcca
85320 tcagctctag aggatggctt agcccccttc gggggtacac cgcatttcac
tctcacttgg 85380 ctcacagcca tcaccacagt ccatgctgtg agtgcattgc
tggttctgcc cccgtgctgt 85440 gtgcatctct gctgctttaa tgctgggaaa
ctccgtggtt atgccccaac tatcttggca 85500 atgttctgaa tcagacatag
ataataccta ttaaaggtat taataggcca ataataccta 85560 gtaaagaaga
gctgggatat acctctgcat agattaaatc aactagaaaa cactagcccc 85620
ctcccatttt cagaccgatt ttatttcttt taagtgggaa aatagtcgaa gtgggatgaa
85680 gcagagctag cttattctac tcattttata tttctgtggc cttttcaacc
tctgtttaac 85740 agcactttat tacttagttt ttttgttttg ttttgttttt
ttgggatgga atctcacgtt 85800 gtcgcccagg ttggagtgca gtggcatgat
ctcggctcac tgcaacctcc acttcccggg 85860 ttcaagcgat tctcatgtgt
tagcctctca agtagctggg attacaggca cctgccacca 85920 ggtccggcta
atttttgtgt tttcattaga gatggggttt caccatgttg gccaggctgg 85980
tctcgaactc ctcacctcag gtgatctgcc cgcctcagcc tcccaaagtg ctgggattat
86040 aggtgtgaac caccacgccc agcctcactt tattactttt aagaatatgc
ttcaaaatag 86100 tttgtaaaga agattttaat agggagcact tatatgaaat
ataatagtga tatatagtat 86160 agcatagagc agagtcttca gtctttgtat
ctttttcttt ttttcttatg catatttaat 86220 gtatgtgatt cccaaccgtt
gtgtgattgt ggtcagagcc ctgtctgtgg gatgctgggt 86280 agaatgagat
tgtagagagc actttgtttt cttgtaattg aagggtttgg ggtgagaata 86340
tgtgagtcat agaaatctgt atagtaaata ttactctaaa aagggagcca tcaggatctg
86400 ggagaatttg ctaaaggaaa actaagaatg aaaaaaaggc caggtacagt
ggctcactcc 86460 tgtaatccca acactttgag aggccaaggc aggaggacct
gaggccagga gttcaagacc 86520 aacctggcca acatagtgaa accccgtctc
tactaaaaat acaaaaattg ggccgggcgc 86580 ggtggttcac acctgtaatc
ccagcacttt gagaggctgt ggcgggtgaa tcacgatatc 86640 aggagttcga
gactagcctg accaacatgg tgaaaccccg tctctactaa aaatacaaaa 86700
attgggccgg gcgcagtggc tcacacctgt aatcccagca ctttgagagg ccgtggcggg
86760 tggatcacga tatcaggagt tcgagactag cctgaccaac atggtgaaac
cccgtctcta 86820 ctaaaaatac aaaaattagc caggcatggt gacgtgtgcc
tgtaatctca gcttctcagg 86880 aggctgaggc aggagaatca cttgaaccca
ggaggtggaa gttgcagtga gccgagatca 86940 caccattgcc ctctagcctg
ggtgacacgg ggactccgtc tcaaaaaaaa aaaaaaaaaa 87000 aattggccag
gtgtggtggt acacacctgt aatcccagct acttgggagg ctgaggcatg 87060
agaatcgcat gaacacagac ggcagaggtt gcagtgagct gagatcacac cactacgctc
87120 cagcctctgt ctcaaaaaaa aaaggggggg aggggcggtg gggggagcgg
gagccagtat 87180 ataattcagt atctctcatc tatacatatt aaggcttttg
accattacca aattctccca 87240 gcagctctct gagagtactg taattctggt
tttgctgatt agaaaaccag atacaaagag 87300 gtaaagtcac cttgttctag
gccactaggt ggtaatctga gtcaggactg gagacaatga 87360 tttattttta
atatctcatg taatgttaat ctcataactc agggcataac tcttttacca 87420
ttttggacta tatcatttca ttcatatgat aaagacactg tagcttcccc ctcacctgca
87480 gcttcacttt ctgcagtttt agttacctgt ggtcaaccat cgtccaaaaa
tattaactgg 87540 aaaattctag aaataatcca ctcgtaagtt ttaaattgtg
cactattctg ggcagtgtga 87600 tgaaatgtcg agccatcctg ctctgtgtga
ccctggacag gaagcctctc tttgtccagc 87660 atatccatgc tgtatgactc
ccgccccttt agccactcag cagccatctc acttaccaga 87720 tcaactgtct
tggtttcagg gtgtttgtgt tcaagtaacc cttcctttac ttaataatgg 87780
acccaaagcc aagagcagtg atgctggcat tctgggttta ttttattagt attgttgtaa
87840 atctcttact ttgcttaatt tataaattaa acatgatcat aagtacatat
ctatagggaa 87900 aaaatggtat atatagggtt ctgaaccatc ctgcatttca
ggtatccacc gtgggtctgg 87960 aaatgtatcg cctgtggaga aggggtgact
actgtgtatg taaaaatcac cctgtgtgaa 88020 atgttatatc ctcccctttc
ctcagtttaa cgttgttttg aaagaatttt ctcacattac 88080 ttgaaaacac
ttaggaaacc atttttagtg actgtagtat tttaccagtt agatatgcca 88140
tggtttactt aaccatgttc ctaatgttgg gtacttatat tggatctaag ttttgctgtt
88200 atttgtagtg ctgcgatggg tgactgtgca caaacccttg cctgtacttt
tgtgtatttc 88260 cctaaggata gattgctgca aaaaagaacc actgagtgtg
agactgtaaa tatttggaag 88320 gctttcagtc tatttccata ttgctttcct
gaaagattga accagtttat acttctgtaa 88380 gcaacagtgt ttgagaagat
ctctttactt tttttaacat tgacctttgt catttcttaa 88440 actttactag
ttattttggt aaccggcttg tttttataat ttgaatttct ttgcttctca 88500
gtgaaataat agtttctttt ataggagtat taaccatttg ttaagaacca ctattttagt
88560 ccaaaagaaa ggtatataag aagaaaactg cacaattcca gtgggaagga
cttggggtca 88620 gggtccctga tatgttggaa ggttgaactt tttgttgttg
gtttttcccc ttgccttaaa 88680 aagtccatat tgcttgaatg ttgcaatctt
gggcaaggcc agcaattaat ccaagggatg 88740 atgccactgt cttctcctgg
tgctggtcct ttctgacaga gaacatggta ctagggctga 88800 gtgcttgaat
gcttgcacat aggacccaga aggtgcacat ataaccgggg gttcgttcct 88860
tgagtgatat ctttgtgaga tgacattttg cttgttggtt gtttgtttta taatgaggaa
88920 tcaaagtggg tattctagga agatccagtg tttccctact cacactttgc
attacacaca 88980 gtccaggggg tgactcagaa tccagtgctg tcctgcctct
cccagttggc tgacaccatt 89040 ttcttgactg gagccttagt tttctaggca
tatattctaa tgatggaaca ttttgaaatg 89100 cagattattt ttgaggttac
tgaatttttt aataacacag ctgctgtccc taaattgcca 89160 tcttttataa
ggtctagttg cattagaaat agctctccca accccactcc cccagtgctc 89220
agaacgctga accccgtact acacttggaa aaggattgga tgtcctaaag cattggttat
89280 gtaattgtgg gttggctttc acccactgag ctttacttcc tcctgtgatc
gtgaaataca 89340 agctggcaac agtaattaga tctcagaaaa gcttgtcaca
aagcaccaca gactagagaa 89400 acttgtaagc tctttttgca ctggctgaag
tttttgagta ccactacctt ccatctatag 89460 tgtagtaacc ttagacaggt
agtgcttttc ttctgtgcat taattttaat taagcaatga 89520 cacctacttt
cttttccact ctgagatctg catgtagcta aacttatcag gtgagtgctt 89580
tcccatcttt gatcattgat actgcttgga atataccgga aaaagagcag caagcagaaa
89640 atctcccatt tccacaagct gctgactaac tcagaattgc tagattttgt
gaagcaaatg 89700 aatgctataa aagaagtcag aaagatcagg gaagctgtcc
ctaggacttg gtcaggccaa 89760 accttgaaat atcaagtgat gttacagagg
tacaattatg agaatatata taactcaaga 89820 cttacatatg tgataaatag
tgcattgctc tttgccgtct ccaaaggatt ttcttttttt 89880 tttttttttg
agacggagtc tcactgtgtc gcccaggctg gagtgcagtg gcgcgatctc 89940
cgctcactgc aagctctgcc tcccgggttc acgccattct cctgcctcag cctcccgagt
90000 agctgggact acaggcaccc accaccacgc ccagctaatt ttttgtattt
ttagtagaga 90060 cggggtttca ctgtgttagc caggatggtc tcgatctcct
gacctcgtga tccacgcgcc 90120 tcggcctccc aaagtgctgg gattacaggc
gtgagccacc acgcctggcc aggattttat 90180 ttttaattct cacagcaatt
ctgcagagag aggtagtgag aggtttaatg ctttgttcaa 90240 cataatttgc
tgttaaatag ccattcattg gcagaaaatc tgaactgttg tgttttcctt 90300
cctgtgtcat tcatggtttc agtcctgaag aggagcccac tagagcccaa caggagagga
90360 gagtgggaga atccctcacc cagaagttca cagtggtatc atttagtgac
actcaggatg 90420 tctccagtta ttgttagaat ttaaagttag gttcatccct
gtgaggtcca agaaaatata 90480 aaaataaaat aagggtctac tagtattaaa
catactctgt aatcactttt gaaaggaaag 90540 gagttagtgg aaaaaatgga
agaaccatag cgaaactaaa ataaatatat gtagatatat 90600 tgctggacgt
ggtggctcac acctgtaatc ccaacactat gggaagctga ggcagccaga 90660
tcacttgagg tcaggagttc aagaccagcc tggtcaacat ggtgaaaccc cgtctctact
90720 aaaaatacaa acattaggcc aggctcagtg gctcacacct gtaatcccag
cagtttggga 90780 ggctgaggtg ggcggatcac ctgaggtcag gagttcgaga
ccagcctggc caacatgctg 90840 aaaccccatc tctactaaaa atgcaaaatt
tagctgggca tggtggcaca tgcctgtagt 90900 cccagctaca gggaggttga
gccaggagaa tcgcttgaac ccaggaggtg gaggttgcag 90960 tgagccatga
ttgtggcact acacgcccgc ctgggtgaca cagcgagact ccatctcaaa 91020
aaaaaaaaaa ttacatatat atacacatac acacacacac aaacattagc cgggcatggt
91080 gttgtgcacc agtaatccca gctactctgg aggctgaggc aggagaatcg
cttgaaccca 91140 ggaggcagag gttgcagtga gccgagattg caccactgca
ctgcagcttg ggtgacagag 91200 cgagactctg tctcaaaaaa tatagataga
tagacaatgt tagataactg cataattatt 91260 atatgtgtgt attaatatac
gaagcaatca ctttcagaag gaatagtgtg ttaaaaaaag 91320 gtaatgaaag
attttaaaac aaaacacttc atgagacaag aagttagaac aattacggca 91380
aactaaaaga aaaagctagg aatgagatcg aatacagcca agtatttcct gcagttttaa
91440 aacctctact ccccattttg ggtttctggc cacagattac gtaatatttt
tcgttacttg 91500 aactggaatt acaaagattg atacagaaga tggtccgata
agtcaattgg gtcctgctcc 91560 ttgtatgtct aggtccaaac caaaatgagt
caatatttgg acaagatatc agccatccag 91620 ggcttatagg caggtaaagg
agatggccca ttattacagg gatttcaaac caggctttgt 91680 attctcttac
cctggcactg ccaattatat ttatttattg gaaaatgata accttagagt 91740
taagctatat gcttataaaa gaggcactgc ttatatgggt tctatcatgt ccaggtttac
91800 attgcccgtt agaaaacagg acacctggct gggtgcagca actcatgcct
gtaatcccag 91860 cactttggga ggccaagcga gtgaggatcg cttgagccca
ggaggtcaag gcagcagtga 91920 gctgtgttca caccagtgca ctagacacca
tctcaaaaaa aaaaaaaagt gttgggggga 91980 gagagagaaa gagagagaga
gagagaagag gaggggaggg gaggggatac ctgatcagac 92040 tcctctgaag
agggaattga aaagtttgtc acaagccctg agttatgctg atataacaga 92100
gaattgttag atcagagaat ccaaagtaac ctactgcgct tagcccttca gtctttgtcc
92160 tagctatagg ccataaagtt gaatagtgcc gggaattgtt cttgacttaa
gaatataatg 92220 gtcaaaaagg acaggcaaag ttgtttccct tctggaactt
acactttaat gggggagata 92280 gacaataagc aagtaaaagt aattgaacaa
ggcaattgca aataccaccc tcggtgagct 92340 cttgaaacac aaattatttc
acctgcattc cacagataca caggtgaatg tttgccttga 92400 taaatgcata
aaagtgactg aacttttgag gtccactggg cttttgtttg atatttactg 92460
ctagtgaatt ttccagcctg caaatctctt agaacttcta aatacatttt tttttctttt
92520 aggttgcaga gaacacatct tagaagatga aaaacctgaa tctatcagtg
acactactga 92580 cttggctcta ccacctgaaa tgccgatttt gattgatttc
catgctctga aagacatcct 92640 tgggcccccg atgtatgaaa tggaggtgat
tcattctttt tatttctttt tgctccagtc 92700 aatgaaagga acactttatt
gaggccccag ggccgtaggg cctgggcagg aggctgccct 92760 ttggggaagg
aatagcctta ttcgaccttc tttttgggac gcaggttgtt ggtgtggccg 92820
cacttcttgc agcagttgac tgcatggggg cgcaggcgag cacagctctt gtggcacatc
92880 atcttcttgc agttgtattt ctgggcaagg tggcagaggg aaggctccgt
aatgccacct 92940 cacaggcaca gcatcaggcg cagggtggac tctttctgga
tgttgtagtc taagagtgtg 93000 tggccatcct tcagctgttt gccctcaaat
atcagacact gctggtcagg taagatgccc 93060 tacctgtctt gaattttggc
tttgacattc tcagtggcat cactgggctc gacctcaagg 93120 gtgatggtct
ggcctgtgag ggtcttcaca aagatccaca tctcagcgtc tgcagcttgg 93180
ccagtctcac tccattctca tttttttgtt ggtactcact ggtgtactca ggtggttgct
93240 taacagagaa gtaaaattgg atgtttccag aggctgaatt ttgccttaag
atggaaactt 93300 tatttctata tggtattgtg ttttagtgct tattgtgata
atatgacttg ccaggagcca 93360 gagatcccag ccatatcctc ttttagaacc
ccagtctcat tttattctct accattcagt 93420 tccattttaa ggacaatgcc
tctgactctt cttcttagaa aaattacata ttcttatgtg 93480 tactttaagg
agggatttct ttgtgctatc aagggcttgg gggaagaggc ggggaatcaa 93540
cctgatacag gtctgaaaac atgagcatag cttagcttca gactgtgcta gtgcagaccc
93600 agatgacatc tttcaggaac ctattgttcc attgttaata gttcctttag
ggttaaaccc 93660 acatgcaggt ctagccctat tttcatcttt ctctcctaac
tgtacctcac agcagaaggc 93720 ctgggtgcca agaccgagtt gaagcagctg
atggaaatag atgttagact ataactgcta 93780 agggcattgt gaaataattt
ataggtgctt agatgagctt tcataggttg gttactataa 93840 aaatgtttgt
attatactac tgaatttagc tttatcatca cctccttatc agtttaagga 93900
aaaaatattt tcagaaaata aatctgataa actatgtaga agataatctc tccatctaac
93960 atttgaaatc attaccagta gatatggttt tcctcaagtt cttacaactg
agcagatgag 94020 aaatagcccc caagcctgtc ttgtttatcc atttaaactc
taaactggtc attaaagcta 94080 atgagcctct ctacagagct ctcagttaca
agaatagaac ttgtttactc ttgacagtaa 94140 atctggactt gaacaataga
atcagaagca ttgttttgat tatttgaatt cttaagatat 94200 catggatttg
aattttgaag tgttgaaaga acttgagcaa aacattgttg attgagaaag 94260
tgaacaaaac ctgctttctc gttctgggag gatccagtga cattgtgagt gaagacgcaa
94320 acaggttttg actcctgcat ggccgatgac ctttttctgt aggcttacca
gaaaagtaca 94380 ttccaacagt tctttgagga tttaaactag agcagcaaat
aaagacaaaa gattaatgca 94440 tgtctctgtt gcatataccc ctctctccca
gccatttctg ctgatgttaa gtttggaagc 94500 attgctgaca ttcctggagc
attagcaaag aaagagccaa gagaacagaa atgagaaatt 94560 ttataaacac
tgcttaccag ttatccttgt tagcatggga gaaccttatt ttccttgtag 94620
catgtgagct ttaacatagt aacactttta ccaacatgag tctgcagaaa gactccagta
94680 gccattttgt cttttataga tagcatctta gaatggaaga tgtggtgtgt
cacatgcgtg 94740 cgtgcggaga gaccaccaaa caggctttgt gtgagcaaca
aggctgttat ttcacctggg 94800 tacaggtgag ctgagtccga aaagagagtc
agcaaaggga gataggggtg gggccgtttc 94860 ataggatttg ggtgggtagt
ggaaaattac agtcaaaggg ggttgttctc ttgctggcag 94920 gggcgggggt
cacaaggtgc tcagttgggg agcttctgag ccaggagaag gaatttcact 94980
aggttaatcg ctcagttaag gtgggacaga aacaaatcac aatggtggaa tgtcatcagt
95040 taaggcagga accaaccatt ttcacttctt ttgtgattct tcacttgctt
caggccatct 95100 ggatgtatac atgcaggtca caggggatat gatggcttag
cttgggctca gaggcctgac 95160 atcgtgtttt gagtgttggg aacattgtgt
tcattttttt catacttgaa agtgagaact 95220 caccctgtag ccgggtgtct
ctacctgtag tggtctgatg accaccagcc ccaaattact 95280 taaccacaca
gtctacctct gcttttgcat ctataaaatt aagatttatg gaacatttct 95340
ttcttgtccg tgagggctgt cactgtgcta ggagtgtaat tccattttac atacaaggga
95400 aaaagtttga agagattaaa tgaattgtac aaattcacgt aagtggcagt
tggtagagtt 95460 aggattcaga ctcagatcag cttattccaa gtccattatt
ctttctacct ttctacagta 95520 ccctgtcagg ccaaaataat tcctgccctt
gtctgctaga agagagtggc agtgatgtat 95580 gagagttttt taaaaaggca
tctgctctac atcagattct cattcatatt cttaccaact 95640 ctgttgctct
gttttggaat gggagaggct gggctcaact tgttgaccac tcccattttt 95700
gtatctcttg gctatcaggc actgtgtaag gccctccaca gtgatcattt aatcctcagt
95760 catggttgtc tttccaataa cagttgagga aacaggctta gagtatttaa
ataacttgag 95820 agaagacaca acttatgcca gaaatgagat ttggttctag
acctgaccaa ctccaaacct 95880 agtgctgttt attactctag aaaaacatca
caggcaacct gagcagggcc tctgttcatt 95940 gcagagagct cacaggtgga
cctgagcagg gcgtctgttc tttgcacctc acaagtggcc 96000 agtcttattt
ctctacttct ttgtgctttc ctaggcaaag aatctgaaga gagaggttat 96060
actaggaata ctggaataca tgttgaggtg ttcccaagat gttataagat acctttcatt
96120 tgtttgtttt tactttttga gatgaggtct cactctgtca cctaggctgg
attgcagtgg 96180 catgatcata gctcactgca acctccacct cctgggctcc
cacttcagcc tcctgagtag 96240 ctgggaccac aggcgtgtgc taccataccc
agctaatttt ctctgtattt ttttgtagag 96300 atggggtttc accatgttgt
cccagactgg tctcaaactt cctgagctca agccatccac 96360 ctgcctcagc
cttcccaaag tgctggaatt ataggcatga gccaccaaac ccagccgata 96420
cctttttttt gtctaaatgc ctgtattctc ccttagggta aattacagtc tagggtctgt
96480 ggtttcttct agaaagagtt tgattcattt aataaatacc tattaaggac
ctaacatgtg 96540 cttctggcaa cacagtagta aacaagcaag gtatgatgtc
tgccttcatg gatcccactt 96600 taatgcagga aaacaataga caagtaaaca
aataatcaca aattgaagtt gatgctatag 96660 agaaaacaaa cagggtggta
ctgagataga cagtaactac tctagctata tctgaggtct 96720 gttttagagg
tagaagtaga catgctgatg ggaaacattt ggggaatgaa ggaaacagtt 96780
atcaaaaggg acttacaggt ttctggccag agtgacaggg catgtgtagt agtgctgttt
96840 actgagatgg ggaagacttg gggagggaga tgaggagaga gtgttgcaaa
gaaaactgag 96900 agctcttttg aacacattac agttgaaata tccaggctgg
gcgcggtggc tcatgcctgt 96960 aatcccagca ctttgggagg ctgaggcagg
tggattgctt gagtctggga gttcaagacc 97020 agcctgggcg acacggcaaa
atcccttctc tacaaaaaat acaaaaatta gctgggtgtg 97080 gtggcttatg
cctgtagtca caactacttg ggaggctgag gtgggaggat cacttgagcc 97140
tgggagacgg aggttgcaat gagccaagat cacgccactg cattccagcc tgggtgacag
97200 aacaagaccc tgtctcaaaa aaataaaata aaagttagaa atatctgtga
ggcatagaag 97260 tagagacatt tggacattca gatctattgc tcagaggaaa
tacccaagat ggagatttta 97320 gaattattag aaaatagagg atatttagag
ccccagatat tgaggctttc acatcaccta 97380 agaaaaaagg atacattttt
aaaaagcagg tagtctagaa gcaagccctg aagaacagca 97440
ttatttaggg atcatataga gagaagagga gccaacaaag aagtcgggaa aaacagaaag
97500 ggactgggaa ggaacaagcc ttcagggaag aggaaaacca ggatgttgtg
ctgccataga 97560 gacagaagag gagagtattt caagaaagag gggacatcaa
aatgtgttta ctgtttgaga 97620 gatcaaaaga agatcaaggt cagaacaaat
gtgtattgga tttgatggca tgaaggttgt 97680 tggtgacctt gaaagagatt
tcacaaggaa ggagtggtgg ggatggtaga aattggagta 97740 tgttgaagag
agaatgggag gcgaggaagt agaattagtg tgtaggcagc tctttagaag 97800
tttggctgta aacaattgca gagaaatgag gcagctagaa gagaatatgg atgtcaaagg
97860 gagaatgttt tcaaaatagt agctgctgct gagagtaatc cagtagagag
cacagactga 97920 tgttgcagga cagagcagtg gtacgataga aacaaagtct
ccaggaaagt gagagggggt 97980 gggacccaaa gcaccagtga ggaaatggct
tttgttggga gaagggatac cttttgcagg 98040 atattatgta gaaagggaca
agaatattga gttatttata aggaaaagat tataatgatg 98100 gggctaacgt
gtgtgagctg cacaagagag gagtgaagtt agggcagagc tgctgtatga 98160
tgggaatgtg ctggagttca tggcttgagt acaggcgagc tagaaggata agaaatgatg
98220 gtcaggggtt tcagaggtag catggtttct gttggtgata agtacctgga
agagggtggc 98280 tgagttcagg aggcatttaa agaactgaga agccaggttc
tgggagagca tcatgccttc 98340 actgaagaca cccagggtga tagcaggggc
tggggcagaa aggaaggagc agagtttaga 98400 atcttcctga atgtcagaga
cagtgaagag agagtcagga tggtaaagcc agctgccata 98460 agcaggggct
cagaagggta gaagaataag gcctgaaagt tgcaaggcag cctcttactg 98520
actaaatttt aaacttagtc tctttgagct tgatgtcttc ctctgataaa tggtggtaag
98580 catgtgcacg ttatcacaga gttcaaattt ggtgagtcag tgtacccact
gcattgccca 98640 gtaatactaa aaaagaaaaa acaaatacta atttctgcaa
ctaccatact ccctaaaaac 98700 agagacctac ccccaatcac caaaaaatcc
ccattgtttt tctaatccaa attttgtaca 98760 tatttaataa ccttatacca
ccacttacta tttttttact ttcatcgaag atgaatctac 98820 aaaaatatat
taatgtcaaa aaatattact gacctagcaa actggcagtt gggaagtaag 98880
gtaagaaggc acacttttat taattaataa tatcttttgt attccctaaa cagattgaaa
98940 aatgatggat tagttcattc ttgcattcct ataaagaaat acctgaaacc
aggcacagtg 99000 gctcacgcct gtaaatccca gcgctttggg aggccaaggt
gggcggatcg cttgagttcg 99060 agaccaacct gggcagcaaa gtgagacctg
gtctctacaa aaaatacaaa atattacccg 99120 gaaggctgag gtgggatcca
cctgagccca gaaggttgag gctgcagtga gctgtgatca 99180 caccattgca
ctctagccta agtgacagag tgaaaactct gtctcaaaaa aaacaaagaa 99240
ccacctgaga ctgggtaatt tataaagaaa agaggtttaa ttggctcacg gttctgaagg
99300 ttctaaagga agcatagctc cagcattagg ccaggtgcat tggctcacac
ctgtaatccc 99360 agcactttgg gaggccaagg gcaggcggat catgaggtca
ggatttcgag accagcctgg 99420 ccaatatggt gaaaccctgt ctctactaaa
aatacaaaat tagctgggcg tggtggcgca 99480 cacctgtagt ctcagctact
cgagaggccg aggcagaaga atcacttgaa cccaggaggc 99540 ggaggttgca
atgagctgag atcgtgccac tgcactccag cttgggacac agagtgagac 99600
tccatctcaa aaataaataa ataaataaat aaataaatag ctccagcatc agcttctggg
99660 gaggcctcag gaaacttaca gccttggcag aaagtgaagg gggagccggc
atgtcatgtg 99720 gccagagcag gagcaagagt gcaggagggg aggtggccac
atgcttttaa acaacccacc 99780 tcccacaaga actcactcac tattgcgagg
acgacagtac caaggggatg gggctaaacc 99840 attcatgaga aatttccctc
cgtgatccag tcacctccca ccaggcccca cctccagcac 99900 tgaggattat
agttcaacat gagatttggt ggagacacag atccaaacca tatcaaatgg 99960
gttctaggaa cttagcctag atttcagatt taggaacagt atcataggtc accttttcaa
100020 aatacataaa gtttcctaca gaaacaatat caattaagtg catgttttaa
aaataaaaat 100080 aaaggttact acaaaaaaag tggggaggag caggagtggg
tgcaggtgtc cccaggaagc 100140 ctaggcatag ctcacactgc atgtgctatc
acggcgagac tcagaactgc cccgaatccg 100200 aggaggggcc atgcgagtag
gtgggcctag gcacctcctc agtcactggc tgtgcccttt 100260 cactctgtca
ctgggagaca gaatcctgag ttttctgctt cagggagcct gcatggaaag 100320
agtaggtcac tgccggaaat caggctagtt ttagcaaaag gaacggacat taggcacctc
100380 caaagggaca aaggaccaat atacctggtt ggggacagga ttctgtcatt
tgattattcc 100440 tgactcatgt tttcatgagg tagtccccca cctcatataa
aagcctcagt gttggcttct 100500 gaccatggtg tatgaaaagc ccttgtctaa
aggttactgc cctgagaaaa taataaagga 100560 agaagaggat agacatgaag
acactttaaa gcctcctgaa tagaatgcat ccagaagcga 100620 attccaggag
attctgtcat catgcttgcc tttcaagcaa acaaaattag ctgctagaac 100680
tgagaaagag tgtaaacacc aactaaatgc ctcaaagaat catggtagta aattacttct
100740 ccatgttgct ccatataaac ctgctgtgcc acctgttgaa ggcagcactg
atgctgcatg 100800 ttcagtctgg tccaaggccc caacaggaat ccgttgtgcc
aagaaaaggc cctactggaa 100860 ggattggaga gcagctggtt ctcagcaatg
caagcatcag gccaggctgg ggctgcttaa 100920 tgctgcttaa gagatgacag
tggtggaccc caacacctct ccaagggatg tagaatctgc 100980 ttttcccatt
tctgaatgct actgaaacaa atctacaact agaaaaatca aatattcatg 101040
aattcaagac ttgggatctc agtactaaga ctttaaagaa gttgccagat ggatcgcttc
101100 tgtggtgaca gccctggcag gagcattcaa gtgctctatg agctacaaaa
gaaaccagtt 101160 gatggtgtga acaccactac agagcaacct gcacaccaca
gcaatttgac agctcaggtt 101220 ctgtgtctca tgtggcaccg tgcttgtcct
tggaaagaag gcctacaaaa ttcttcatat 101280 ctccattcct tgacatctgc
tggcaaactc ccactcatat tttaagactc agcctctcct 101340 gtgacacctg
tgtcttctct ccaaacaggg agggacgctt gcctcttcag agctccccac 101400
actggagtat aactgctcct gtgtctgatg cccttagtct cagtgccagg aggtattcat
101460 gcttatgtcc ccatggcctg taacagagcc tgcatcagga tgcttggtaa
aggactgttg 101520 aatgaatgtc aaatatgggt ccctctgatg ggtctatacg
tgttgatcta ggattggaag 101580 ggtcacaaag agttgtgcat gcttacaatt
tcaatcaaat atcactattt ttagttaaga 101640 gggaagagta gtgtgaaatt
ggcaataatt agatactcca aatgttcttt aaaaactaat 101700 agcattgatg
tattaagaat gcaatcagcc gggcacagca gctcacacct gtaatcccag 101760
cactttggga ggctgaggca ggtggatcat gaggtcagga gttcgagacc agcctggcca
101820 agattgtgaa acccccgtct ctactaaaaa tacaaaaatt agccgggcat
ggtgacgcac 101880 acctgtagtc ccagctactt gggaggctga ggcaggagaa
ttgcttgaac ccaggaggtg 101940 gaggttgcag tgagcccaga tcgtgccatt
gcactccagc ctgggtgacg agcgaaactc 102000 agtctaaaaa aaaaaagaat
gcaatcatac attagaagac acattctgtt ttagattttt 102060 acttaaatat
tttaaatact tccttaatct gcatatttac cttattgata gatttcagaa 102120
gaaattgatc atttcatgga acaagattta ttagacacat aaggaaagtg aatcataaca
102180 actgtacagg tgggaaattg aacaacaaaa atgaccctga gatacccaca
ttctactttg 102240 gcatatagtg ggaaaaacat tctagacttc aagtctaggc
ctatcttggc taatgtaacc 102300 gatgacttca caaaccattt atgggactag
aagctgaaag gaaagtactg gtggataaac 102360 atcatattga aattatgttg
agtcacttat ttgctataaa acacaaattg ttttgtgtaa 102420 aggggttaag
atggctggaa aactgtctcc actcaagagc aagaaagcag catgtgtctt 102480
accctgtacc ttcattttta cttgtacttc ataatttctg agggagaaat acgtggaaac
102540 cagatgcttg atatagtttc agaacacgtc cttaaagaat atgactccaa
gtctaagaat 102600 tgtaggtcct ttgcttctta gataactact gttagccttg
atcacagaga ttccaggttt 102660 aataacttca gttctcccca ctgtgtatat
agatgttaag ttacacagat ttggcattat 102720 tcccattttc aggttaatat
cagaacactt gttatcaagt caggatagta attgtgagcc 102780 tagatgctct
aggtttggcc atacgtggtt atctacacca ccaactgttc caattaacaa 102840
tttaccagtt gcttctaccc aaagtaccaa gactccagca aatggggaat attggaaact
102900 ggcttggctt cttgaagcaa catggtaatc aataagaatc ttggctgggc
atggtggctc 102960 atgcctgcag tcccagcact ttaggaggcc aagatggaaa
gatgggaaga tcgctcaagc 103020 ccaggagttc aagaccagcc tgggcgacat
cgtgaaaccc catctctaca aaaaaataca 103080 aaaattagct gggtatggtc
gtgggtgcct gtagtcccag ctgctgggga gctgaggtgg 103140 gagatcacct
gagcccagga ggcagttgca gtgagccaag attgcaccac tgcactccag 103200
cctgggtgac agagtgagac tctgtctcaa aacaaacaaa acaacaatct ggctgggcgc
103260 ggtcgctaat gtctgtaatc ccaacacttt gggaggctga ggaggcagat
cacttgaggt 103320 caggaattcg agaccagcct ggccaacatg gtgaaacccg
tctctattaa aaatacaaaa 103380 attagccggg catggtggca cacacctgta
atcccagcta cttgggaggc tgaggcaaga 103440 gaattgcttg aaccaggagg
cagaggttgc agtgagctga gatcatgcct ctgcactcca 103500 gcctgagcta
cagagcgaga ctctgtctca aaaaaacaaa aaacaaaaac aagaagaatc 103560
ttactactgc ttcttcgggg atacttttgg tattattttg acaaatgaat tgtgaggatt
103620 caaatataag aaagggatta ttcttggtag agttaacaaa attgtaccaa
atgacttttt 103680 gtgttaaaca cgattcattc acccaaccct agaaaggagc
ctgaatgaag tctaatttgg 103740 gtgacagatt cccacacaaa ttagatgtat
gtcattcagg tatagagaat tgattttata 103800 ttagaaaaaa caaaccttgt
aaacagtttt ataaataact gtttcatgat tttccttaag 103860 tagtactgat
ctcttacata tagatcgttt gtgtctttcg cctcaagtta gtatagaaca 103920
gggcaagtgg caaagctcga ggaaagtgtg acctgaggta catgctgtca gcttgatgct
103980 ggagtttggc ctctcaaatc tctaacctgt taaatgaagt taattaggat
taattttttt 104040 taatgtatgt ttactactga aaataagtgc tcggccagac
gcagaggctc acgcctgtaa 104100 tcccagcact ttgggaggcc gaggctggca
gatcacctga agtcagggag tttgagacca 104160 gcctggccaa catggcgaaa
cactgtctct attaaaaata caaaaattag ctgggtgtgg 104220 tgatacatgc
ctgtaatccc agctactcgg agcctgaggc aggagaactg cttgaaccca 104280
ggaggcggag gttgcattga gccaagattg tgccattgca ctccagccca ggcgacagag
104340 tgagactcat gtctcaaaaa aaaaaaaaaa aaaaagagga aaagaagtgc
ccaatagctt 104400 caatggatgc cacataattt tggaataatt tttacaatca
ggaatttcat tgtccaagcc 104460 ccttagaaaa agaagcaacc cagccccata
cccagaaagt caagctgtat agtgctgttc 104520 cttagtgagg acggtcaact
ctcagtagaa aaatctcctg tttggattag tgcttagttg 104580 acctattgtg
ttcagttcct ctaacatgag taacttctat tggataggaa attttgaagc 104640
tcaaagggtg taatgagagt taacattact gattttccac tgttactttt tagtgttttc
104700 ataacttgga tgtgttaacc tatggcccat caactatgct cctagtctca
ggtgacaaca 104760 tgttcaattt aagatggcag gcagtacagt ggacctctct
catcccatgg gaaggaaccc 104820 aggatgttta ttatgtagta ttgtatagtc
tctgcagcag taatagagaa agttaaaggt 104880 aagcggtgga gaagtaaaat
ctagagtttc taatataacc cttctcactt ttcttttcaa 104940 aaaaaataag
agggtctcac catgttgccc acactggtct ctatcgaact cctgggctca 105000
agcgatcctg tcgtctcagc ctcccaaagt gctaggatta caggcatgag ccactctgca
105060 tggccaagct cactcttctt aaaggtctgc tagtaagagg gtttctactt
tttgaaacaa 105120 attcatgatt acctaaaatg aagctaggtt atgaagtata
tataaatatg cagcccaata 105180 ggctgggtgt ggtggctcac acctgtaatc
ccagcacttt gggaggctga ggcaggcaga 105240 tcacttgagg tcaggagttt
gagaccagtc tggccaacat ggtgagacca catctctaca 105300 aaaaatacaa
aaattagcgg gtgtggtggc ctgtgtgcgc ccatagtacc agccacttgg 105360
gaggcagagg caggagaatc acttgaagcc aggaggcaga gttttcagtg agctgaaatt
105420 gtgtcactgt acttcaagcc tgggcaatgg agtgagactg tctcaaaata
tatatatatt 105480 tgcagcccaa taaagatact tagataaaac tattgggttt
attccttgaa aactagggca 105540 tgtgtagcta gatctggctc ataaaaagca
aagttattta catatatttt aaggtaaaat 105600 tgcctctgat aaatgtcaaa
gaggaagttt aggtctttct tctggcagaa agccagagag 105660 taagtgctga
atgtgacgca gaatcatgtt aggtaacaag gactttgagg taagtggctg 105720
aagtcttctg tggagtcagc cgactcttgc aggattgtgt ggtatcagtc acctttagca
105780 tttgccaacc caactctgat cattcttctt ctttcaaggt atctcagcgt
ttgagtcagc 105840 caggagtagc aataggtttg gcttggactc ccttaggtgg
agaaatcatg ttcgtggagg 105900 cgagtcgaat ggatggcgag ggccagttaa
ctctgaccgg ccagctcggg gacgtgatga 105960 aggagtccgc ccacctcgct
atcagctggc tccgcagcaa cgcaaagaag taccagctga 106020 ccaatggtag
gagcctgcac ccggccaggc aggcgtgacc caggaggcgg taccttccat 106080
ggcggagact ggcatgagct cgagactgcc agttacacat ctagcaaagt acacaccgtt
106140 ttgaacccct gtggaaatcc tagttcccat ttcaggacta tttgactagt
gcctgaacta 106200 gaaactaatt caaaaggttt attttgtttt aatacgactt
agagtagaat ggaactgttc 106260 ttccacaccc tcacccaaat tgtactgtcc
accaatattt tgaagaattc atttacccaa 106320 aacattcatt tttgtttgtg
actttttttt taggagaaaa agaaaacagg tttaattttt 106380 ctacattaaa
gtcccttttt cctttttaaa gcttttggaa gttttgatct tcttgacaac 106440
acagacatcc atctgcactt cccagctgga gctgtcacaa aagatggacc atctgctgga
106500 gttaccatag taacctgtct cgcctcactt tttagtgggc ggctggtacg
ttcagatgta 106560 gccatgactg gagaaattac actgagaggt cttgttcttc
cagtaagtat gaaaaaacaa 106620 tttatatggt tattttttat ttaatttttg
aaaattaata ttatttttaa atacgggttt 106680 gccttctttc tatgaaaacc
ttggttttaa gtatatatta tatttttatg cctgtaacta 106740 attcatattt
taaaattttg atcaaataaa agaaaaactg acaatttttc acattttcct 106800
tttttttttt tttttttttt tgaaatagac aggtctcact ctgttgccca ggctggagtg
106860 cagtggtgtg actgtagctc actatagcca ccaagtcctg ggctcaagcg
atcctcctgt 106920 ctgtctcccg aatagctggg actataggag cacgccacca
tgctcagcta atttatttta 106980 ttttgcgtag agacagggtc tctctgtgtt
gtccaggctt gtctcaaact ccaggtctca 107040 tgcagtcctc tcatctccac
ctcccaaagt gctgggatta caggcgtgag ccaccacatt 107100 cagcccacgt
ttcccattct aagatttgct aagggaaaaa aatattagtg tggtcatcag 107160
aaatattggc agttacatga aaatttgagg ccttgttcta cttgacaaat tgttaaagat
107220 atagcacatg tgcaaaatgg gatagtagtt gtttttaagc tttaagccca
tttcttaaat 107280 ttgaagtttc tttgagacct cctgtccccc tgcagaaaac
tttgctagta tagaatggaa 107340 actctaataa agattaacca tatctaatga
ctacattttg aaaaggttct atacatgtgg 107400 ggtcttgagg ctccagatcc
taaactgctt ataaaaatag tgtgataaaa tgtacagaac 107460 ttgagagtat
ttaaagttgt tagttgagta ttagtctaca acagactaga ctacaatttt 107520
agtccacaac aagattttgg caggttcata gcaagatgag gaaaaaaaaa aagaaatagt
107580 ctttttttct tttttctatc gagatggagt ccggctctct tacccaggtt
ggagtacagt 107640 ggcacaatct tggctcactg caacctctgc ctcccaagtt
gaagtgattc tcctgcctca 107700 gtctctcaac tagctgggat tacaagcatg
cgccaccacg cccggataat tttttctatt 107760 tttagaacct ccatagaaca
aatgggtttt ctacttggtc ccctctcaga gcaaatcgta 107820 gcccaagtaa
aggcttctgc agcctcagga gagacagcca cagcggcctg gggtacacct 107880
tcagctccag accattacaa gaggcaggat ggaaagcagc agcacttgaa agaaaggcct
107940 gtgaaagctg gagaaaacct cctttgagaa cagaggacaa gacggggctt
tgggatttga 108000 aagtggtcaa agaattattc aggaaaaaac tatagtgaaa
aacaatttgt tgttagaact 108060 ccaacatcta aaaggagttc taacaaacag
gaaaatggaa tggaacaaat tatccaagaa 108120 ataactgaac atttcctaga
agttaaggca tcttgagatc gaaaggacca ttactaacca 108180 ggaaaaacat
ttcatcccct tgacttttca gattactgag gataaagcgg cctcagcact 108240
gacactggat gtgcagtacc ttcaaaacta tgagggaaaa tgggccaggc gtggcagctg
108300 acgtctgtaa tcccagcact ttgggaggct aaacaggagg atagctcaag
tccaggagtt 108360 caagaccagc ctgggaaata tatctctaca aaaattgttt
taaaaatagt aaggaggctg 108420 ggtgtggtgg ctcacgcctg taactccaac
actttgggag gccaaggtgg gcgtatcact 108480 tgaggttagg agtttgagac
cagcctggcc aacatggtga aaccctgtct ctactaaaaa 108540 tacaaaaaaa
ttatccggat gtggtggcgc atgcctgtaa tcccagctac tcaggaggct 108600
gaggcaggag aatcgcttga acctgggagg cagaaagttg cagtgagcca agattgtgcc
108660 actgcaactc tagcttgggt gacagagtaa gactgtctca aaaaaaaaaa
aaatagtaat 108720 gaaagctgtg agggaaaatg ttttacatct agtcttgtat
acatggcctt agtatcaatc 108780 aagtgtgaaa gtaaaatatt ttcaaacatg
caaggaatca gttcatctta cactcttttg 108840 aagaaggtac tttgaaggag
tacttcagca gcatgaacaa aaccttgaaa gaagatgcca 108900 gtggggcggg
aaggcctgga gcagccagcc agtcttaatt ggagcagatg caacacatta 108960
ccccaaagca agaatactcc atactcttca agttcctgtg ggccaggaat tcaggagagg
109020 ctgagctggg ttcttgtggc ccagggtctc tggccttaca gtctaggttc
cagccaggct 109080 gcagtcacat gaaggctgac aggctggaga aactgcttcc
atggtggttg actcatgtga 109140 ctggcaaatt ggtcccatct agtggcagga
ggccccagtt cctcacctga tggacttgcc 109200 cataggctgc ttgagtgacc
tcagacatta tgactggcca cctccagggc aggtgatcaa 109260 gagagattca
ggcagcagct ctcgtttttt gtgactcagc cgtggagatc atacagcatc 109320
actcccacca cactctgttt cttaccgagt cacaaagcct ggcccacatt caagcagggg
109380 gaccattgta gacatgtttg aaagccacca taggagccta gtttagggat
acattttctt 109440 cattaaccag catggaggtt ctggctttaa acctgtagag
agggaagtaa ccccagcaca 109500 cagctaagct ctgcaggagc ggcgctcatg
gtcagaatca cgtgctgctt tttcagatca 109560 acctaaagac tagacggttg
tgattacacc tgaatgccaa tttactttga cagcatttat 109620 aaaaacaatc
attgacagaa gaggaactca tacctatcaa caatttagaa tccccctcat 109680
cagagtcttt aatataacac caattgaaac attaaaaaaa ggttactact tatccttttt
109740 cctggctttc ctagctcatg ctataacaaa acggaagatg atttggatgt
tttaaaatag 109800 tagtggttaa attcagtgaa agaaagctgg gtcagggttt
ctttcagctt gagggtgatc 109860 attaacccta aaaacttttt tctctcctta
caggtgggtg gaattaaaga caaagtgctg 109920 gcggcacaca gagcgggact
gaagcaagtc attattcctc ggagaaatga aaaagacctt 109980 gagggaatcc
caggcaacgt acgacaggat ttaagttttg tcacagcaag ctgcctggat 110040
gaggttctta atgcagcttt tgatggtggc tttactgtca agaccagacc tggtctgtta
110100 aatagcaaac tgtaggtcca aatctcaatt ttttagaatt ttaagttatg
aagtgctcaa 110160 aggtactgac acagttgatt ttattcacac cattaggggt
atgcaagatg tccctgtttt 110220 ataaacataa tcacaacagt aataaacctc
aagtagtggc tagtgtttag tatagaaata 110280 taagatgttg atttagtaaa
ctgataaaaa tcgaattctt gtctttttag tgggatcctt 110340 actgtccctg
gaaagatata gcatagtggt tctcagcaca gtctccagaa cagaagcatc 110400
tgtagtacct ggtaacttgt tagaaatgta cattctcagg ctccacagca ggccgcctga
110460 atcaaatcct gggaggtggg gacagaaatc tgtgttttaa gaagccttcc
aggtaattct 110520 gctgcacact caagttcagg aaccaccggt atagaccatt
accttagtgg atttacctgt 110580 agagtttatt ggatcctgaa accaatcaat
tacttagaac taggcaaaga tgaaagtata 110640 gccaactatt cttggctata
tatatatatt caagtgggcc gggcgtgatg gctcacacct 110700 gtaattccag
cactttggga ggtcgaggta ggcagatcac cgagcccaag agttcaagac 110760
aatcctggcc aacggcgaaa ctctgtctct acaaaaaata tacaggcgtg ttagcatgtg
110820 cctgtaatcc cagcttcttg ggaagctgag gcacaagaat tgcctgaacc
caggaggtgg 110880 aggttgcagt gagctgggat cgcgccattg cactccagcc
tggctgacag agcgagactg 110940 tctctaaaaa aaaaagactc aagtggaccc
tacaatgaag cctacacatc ccaatagaag 111000 ccccttctta tgctgaggga
agcagccctc agaacatgat agcttgtatc cagcagagtg 111060 gcacgtgctg
gcacacctca cagaagcacc ctggccctgg atgcctgcaa cctcagaaga 111120
gtgcagctcc cagagggagg cagccatcca tctgggatgg tcctaagcat ggaatcctaa
111180 ctcctgattc cgtctcctat ttcttgcttg gctacgccag ttcccaaatc
tggtagatgt 111240 ccatgcccat gtgctcctgc tgggactcaa ttcaggctat
gtatgactat gaagtcaggc 111300 tcatctgctt actggctgtg tgaacttttt
gtatcttggt tttcttcatc catgaaatcc 111360 aagtaatact acctaattgt
tactgtggag attaagttca aatgcaatgt atagtaatat 111420 taagcaattt
ctagttatta ttctagccag taatggactt cagaatcttt tattacacaa 111480
tataagaata tgtatgtaaa gacattttgg aatttcctgg atgagaagga agtctgggct
111540 gggcatggtg gctcacgcct gtaaccctag cactttagga aatcgaggcg
agtggatcac 111600 ttaagctcag gagttcaagg ccagcctggg caacatggca
aaaccccatt tctacaaaaa 111660 atacaaaaat tagctgggca tggtggcacc
cgcctgtagt ccagctactt gaggctgaga 111720 tgggaggatg agggaggtcg
gggctgcagt gagccaagat cacgccactg cactccagca 111780 ccctgggcga
cagagtgaga ccctgtctca aaaaaaaaaa aaaaaaaaag attgggccaa 111840
aatactgtga taaaatagca ggcctgctga taaaagttta tctgaatgca ttgagaggaa
111900 aagtccagac ctaggactag ttatggcagt tggagagaaa gaacatcggg
atgtttgaaa 111960 atatgccatt gactatctta actactgtaa ttttatcatt
tccaacgtca tctaactggg 112020 gactagaaca aactgtgaat tcactttcag
caaccagagg gcgctaatcc acacccacat 112080 cgctctgccc tgttccaccc
agcaggggca acaaggatat aacttggggt tc 112132 4 884 PRT Homo sapien 4
Ser Asp Ser Pro Val Glu Leu Pro Ser Arg Leu Ala Val Leu Pro Phe 1 5
10 15 Arg Asn Lys Val Leu Leu Pro Gly Ala Ile Val Arg Ile Arg Cys
Thr 20 25 30 Asn Pro Ser Ser Val Lys Leu Val Glu Gln Glu Leu Trp
Gln Lys Glu 35 40 45 Glu Lys Gly Leu Ile Gly Val Leu Pro Val Arg
Asp Ser Glu
Ala Thr 50 55 60 Ala Val Gly Ser Leu Leu Ser Pro Gly Val Gly Ser
Asp Ser Gly Glu 65 70 75 80 Gly Gly Ser Lys Val Gly Gly Ser Ala Val
Glu Ser Ser Lys Gln Asp 85 90 95 Thr Lys Asn Gly Lys Glu Pro Ile
His Trp His Ser Lys Gly Val Ala 100 105 110 Ala Arg Ala Leu His Leu
Ser Arg Gly Val Glu Lys Pro Ser Gly Arg 115 120 125 Val Thr Tyr Ile
Val Val Leu Glu Gly Leu Cys Arg Phe Ser Val Gln 130 135 140 Glu Leu
Ser Ala Arg Gly Pro Tyr His Val Ala Arg Val Ser Arg Leu 145 150 155
160 Asp Met Thr Lys Thr Glu Leu Glu Gln Ala Glu Gln Asp Pro Asp Leu
165 170 175 Ile Ala Leu Ser Arg Gln Phe Lys Ala Thr Ala Met Glu Leu
Ile Ser 180 185 190 Val Leu Glu Gln Lys Gln Lys Thr Val Gly Arg Thr
Lys Val Leu Leu 195 200 205 Asp Thr Val Pro Val Tyr Arg Leu Ala Asp
Ile Phe Val Ala Ser Phe 210 215 220 Glu Ile Ser Phe Glu Glu Gln Leu
Ser Met Leu Asp Ser Val His Leu 225 230 235 240 Lys Val Arg Leu Ser
Lys Ala Thr Glu Leu Val Asp Arg His Leu Gln 245 250 255 Ser Ile Leu
Val Ala Glu Lys Ile Thr Gln Lys Val Glu Gly Gln Leu 260 265 270 Ser
Lys Ser Gln Lys Glu Phe Leu Leu Arg Gln Gln Met Arg Ala Ile 275 280
285 Lys Glu Glu Leu Gly Asp Asn Asp Asp Asp Glu Asp Asp Val Ala Ala
290 295 300 Leu Glu Arg Lys Met Gln Asn Ala Gly Met Pro Ala Asn Ile
Trp Lys 305 310 315 320 His Ala Gln Arg Glu Met Arg Arg Leu Arg Lys
Met Gln Pro Gln Gln 325 330 335 Pro Gly Tyr Ser Ser Ser Arg Ala Tyr
Leu Glu Leu Leu Ala Asp Leu 340 345 350 Pro Trp Gln Lys Val Ser Glu
Glu Arg Glu Leu Asp Leu Arg Val Ala 355 360 365 Lys Glu Ser Leu Asp
Gln Asp His Tyr Gly Leu Thr Lys Val Lys Gln 370 375 380 Arg Ile Ile
Glu Tyr Leu Ala Val Arg Lys Leu Lys Pro Asp Ala Arg 385 390 395 400
Gly Pro Val Leu Cys Phe Val Gly Pro Pro Gly Val Gly Lys Thr Ser 405
410 415 Leu Ala Ser Ser Ile Ala Lys Ala Leu Asn Arg Lys Phe Ile Arg
Ile 420 425 430 Ser Leu Gly Gly Val Lys Asp Glu Ala Asp Ile Arg Gly
His Arg Arg 435 440 445 Thr Tyr Ile Gly Ser Met Pro Gly Arg Leu Ile
Asp Gly Leu Lys Arg 450 455 460 Val Ser Val Ser Asn Pro Val Met Leu
Leu Asp Glu Ile Asp Lys Thr 465 470 475 480 Gly Ser Asp Val Arg Gly
Asp Pro Ala Ser Ala Leu Leu Glu Val Leu 485 490 495 Asp Pro Glu Gln
Asn Lys Ala Phe Asn Asp His Tyr Leu Asn Val Pro 500 505 510 Phe Asp
Leu Ser Lys Val Ile Phe Val Ala Thr Ala Asn Arg Met Gln 515 520 525
Pro Ile Pro Pro Pro Leu Leu Asp Arg Met Glu Ile Ile Glu Leu Pro 530
535 540 Gly Tyr Thr Pro Glu Glu Lys Leu Lys Ile Ala Met Lys His Leu
Ile 545 550 555 560 Pro Arg Val Leu Glu Gln His Gly Leu Ser Thr Thr
Asn Leu Gln Ile 565 570 575 Pro Glu Ala Met Val Lys Leu Val Ile Glu
Arg Tyr Thr Arg Glu Ala 580 585 590 Gly Val Arg Asn Leu Glu Arg Asn
Leu Ala Ala Leu Ala Arg Ala Ala 595 600 605 Ala Val Lys Val Ala Glu
Gln Val Lys Thr Leu Arg Leu Gly Lys Glu 610 615 620 Ile Gln Pro Ile
Thr Thr Thr Leu Leu Asp Ser Arg Leu Ala Asp Gly 625 630 635 640 Gly
Glu Val Glu Met Glu Val Ile Pro Met Glu His Asp Ile Ser Asn 645 650
655 Thr Tyr Glu Asn Pro Ser Pro Met Ile Val Asp Glu Ala Met Leu Glu
660 665 670 Lys Val Leu Gly Pro Pro Arg Phe Asp Asp Arg Glu Ala Ala
Asp Arg 675 680 685 Val Ala Ser Pro Gly Val Ser Val Gly Leu Val Trp
Thr Ser Val Gly 690 695 700 Gly Glu Val Gln Phe Val Glu Ala Thr Ala
Met Val Gly Lys Gly Asp 705 710 715 720 Leu His Leu Thr Gly Gln Leu
Gly Asp Val Ile Lys Glu Ser Ala Gln 725 730 735 Leu Ala Leu Thr Trp
Val Arg Ala Arg Ala Ala Asp Leu Asn Leu Ser 740 745 750 Pro Thr Ser
Asp Ile Asn Leu Leu Glu Ser Arg Asp Ile His Ile His 755 760 765 Phe
Pro Ala Gly Ala Val Pro Lys Asp Gly Pro Ser Ala Gly Val Thr 770 775
780 Leu Val Thr Ala Leu Val Ser Leu Phe Ser Asn Arg Lys Val Arg Ala
785 790 795 800 Asp Thr Ala Met Thr Gly Glu Met Thr Leu Arg Gly Leu
Val Leu Pro 805 810 815 Val Gly Gly Val Lys Asp Lys Val Leu Ala Ala
His Arg Tyr Gly Ile 820 825 830 Lys Arg Val Ile Leu Pro Glu Arg Asn
Leu Lys Asp Leu Ser Glu Val 835 840 845 Pro Leu Pro Ile Leu Ser Asp
Met Glu Ile Leu Leu Val Lys Arg Ile 850 855 860 Glu Glu Val Leu Asp
His Ala Phe Glu Gly Arg Cys Pro Leu Arg Ser 865 870 875 880 Arg Ser
Lys Leu
* * * * *
References