U.S. patent application number 10/389967 was filed with the patent office on 2003-09-04 for isolated human transporter proteins, nucleic acid molecules encoding human transporter 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., Merkulov, Gennady, Webster, Marion.
Application Number | 20030166153 10/389967 |
Document ID | / |
Family ID | 26941338 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030166153 |
Kind Code |
A1 |
Merkulov, Gennady ; et
al. |
September 4, 2003 |
Isolated human transporter proteins, nucleic acid molecules
encoding human transporter proteins, and uses thereof
Abstract
The present invention provides amino acid sequences of peptides
that are encoded by genes within the human genome, the transporter
peptides of the present invention. The present invention
specifically provides isolated peptide and nucleic acid molecules,
methods of identifying orthologs and paralogs of the transporter
peptides, and methods of identifying modulators of the transporter
peptides.
Inventors: |
Merkulov, Gennady;
(Baltimore, MD) ; Guegler, Karl; (Menlo Park,
CA) ; Webster, Marion; (San Francisco, CA) ;
Ketchum, Karen A.; (Germantown, MD) ; Di Francesco,
Valentina; (Rockville, MD) ; Beasley, Ellen M.;
(Darnestown, 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: |
26941338 |
Appl. No.: |
10/389967 |
Filed: |
March 18, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10389967 |
Mar 18, 2003 |
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09740041 |
Dec 20, 2000 |
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6562593 |
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60251035 |
Dec 5, 2000 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.5 |
Current CPC
Class: |
A61P 35/00 20180101;
C12Q 1/6876 20130101; A01K 2217/05 20130101; C07K 14/705 20130101;
A61K 38/00 20130101; C12Q 2600/156 20130101; A61K 31/00 20130101;
A61P 43/00 20180101 |
Class at
Publication: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.5 |
International
Class: |
C12P 021/02; C12N
005/06; C07K 014/47; C07H 021/04 |
Claims
That which is claimed is:
1. An isolated peptide consisting of an amino acid sequence
selected from the group consisting of: (a) an amino acid sequence
shown in SEQ ID NO:2; (b) an amino acid sequence of an allelic
variant of an amino acid sequence shown in SEQ ID NO:2, wherein
said allelic variant is encoded by a nucleic acid molecule that
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid
sequence of an ortholog of an amino acid sequence shown in SEQ ID
NO:2, wherein said ortholog is encoded by a nucleic acid molecule
that hybridizes under stringent conditions to the opposite strand
of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; and (d) a
fragment of an amino acid sequence shown in SEQ ID NO:2, wherein
said fragment comprises at least 10 contiguous amino acids.
2. An isolated peptide comprising an amino acid sequence selected
from the group consisting of: (a) an amino acid sequence shown in
SEQ ID NO:2; (b) an amino acid sequence of an allelic variant of an
amino acid sequence shown in SEQ ID NO:2, wherein said allelic
variant is encoded by a nucleic acid molecule that hybridizes under
stringent conditions to the opposite strand of a nucleic acid
molecule shown in SEQ ID NOS:1or 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 or3; (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 or3; (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 or3; (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
deternnning 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 transporter 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 transporter peptide having an arnino 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 transporter
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 transporter
proteins that are related to the differentation-associated
Na-dependent inorganic phosphate cotransporter (a type of
neurotransmitter transporter) subfamily, recombinant DNA molecules,
and protein production. The present invention specifically provides
novel peptides and proteins that effect ligand transport 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] Transporters
[0003] Transporter proteins regulate many different functions of a
cell, including cell proliferation, differentiation, and signaling
processes, by regulating the flow of molecules such as ions and
macromolecules, into and out of cells. Transporters are found in
the plasma membranes of virtually every cell in eukaryotic
organisms. Transporters mediate a variety of cellular functions
including regulation of membrane potentials and absorption and
secretion of molecules and ion across cell membranes. When present
in intracellular membranes of the Golgi apparatus and endocytic
vesicles, transporters, such as chloride channels, also regulate
organelle pH. For a review, see Greger, R. (1988) Annu. Rev.
Physiol. 50:111-122.
[0004] Transporters are generally classified by structure and the
type of mode of action. In addition, transporters are sometimes
classified by the molecule type that is transported, for example,
sugar transporters, chlorine channels, potassium channels, etc.
There may be many classes of channels for transporting a single
type of molecule (a detailed review of channel types can be found
at Alexander, S. P. H. and J. A. Peters: Receptor and transporter
nomenclature supplement. Trends Pharmacol. Sci., Elsevier, pp.
65-68 (1997) and http://www-biology.ucsd.edu/.about.msaier/-
transport/titlepage2.html.
[0005] The following general classification scheme is known in the
art and is followed in the present discoveries.
[0006] Channel-type transporters. Transmembrane channel proteins of
this class are ubiquitously found in the membranes of all types of
organisms from bacteria to higher eukaryotes. Transport systems of
this type catalyze facilitated diffusion (by an energy-independent
process) by passage through a transmembrane aqueous pore or channel
without evidence for a carrier-mediated mechanism. These channel
proteins usually consist largely of a-helical spanners, although
b-strands may also be present and may even comprise the channel.
However, outer membrane porin-type channel proteins are excluded
from this class and are instead included in class 9.
[0007] Carrier-type transporters. Transport systems are included in
this class if they utilize a carrier-mediated process to catalyze
uniport (a single species is transported by facilitated diffusion),
antiport (two or more species are transported in opposite
directions in a tightly coupled process, not coupled to a direct
form of energy other than chemiosmotic energy) and/or symport (two
or more species are transported together in the same direction in a
tightly coupled process, not coupled to a direct form of energy
other than chemiosmotic energy).
[0008] Pyrophosphate bond hydrolysis-driven active transporters.
Transport systems are included in this class if they hydrolyze
pyrophosphate or the terminal pyrophosphate bond in ATP or another
nucleoside triphosphate to drive the active uptake and/or extrusion
of a solute or solutes. The transport protein may or may not be
transiently phosphorylated, but the substrate is not
phosphorylated.
[0009] PEP-dependent, phosphoryl transfer-driven group
translocators. Transport systems of the bacterial
phosphoenolpyruvate:sugar phosphotransferase system are included in
this class. The product of the reaction, derived from extracellular
sugar, is a cytoplasmic sugar-phosphate.
[0010] Decarboxylation-driven active transporters. Transport
systems that drive solute (e.g., ion) uptake or extrusion by
decarboxylation of a cytoplasmic substrate are included in this
class.
[0011] Oxidoreduction-driven active transporters. Transport systems
that drive transport of a solute (e.g., an ion) energized by the
flow of electrons from a reduced substrate to an oxidized substrate
are included in this class.
[0012] Light-driven active transporters. Transport systems that
utilize light energy to drive transport of a solute (e.g., an ion)
are included in this class.
[0013] Mechanically-driven active transporters. Transport systems
are included in this class if they drive movement of a cell or
organelle by allowing the flow of ions (or other solutes) through
the membrane down their electrochemical gradients.
[0014] Outer-membrane porins (of b-structure). These proteins form
transmembrane pores or channels that usually allow the energy
independent passage of solutes across a membrane. The transmembrane
portions of these proteins consist exclusively of b-strands that
form a b-barrel. These porin-type proteins are found in the outer
membranes of Gram-negative bacteria, mitochondria and eukaryotic
plastids.
[0015] Methyltransferase-driven active transporters. A single
characterized protein currently falls into this category, the
Na+-transporting methyltetrahydromethanopterin:coenzyme M
methyltransferase.
[0016] Non-ribosome-synthesized channel-forming peptides or
peptide-like molecules. These molecules, usually chains of L- and
D-amino acids as well as other small molecular building blocks such
as lactate, form oligomeric transmembrane ion channels. Voltage may
induce channel formation by promoting assembly of the transmembrane
channel. These peptides are often made by bacteria and fungi as
agents of biological warfare.
[0017] Non-Proteinaceous Transport Complexes. Ion conducting
substances in biological membranes that do not consist of or are
not derived from proteins or peptides fall into this category.
[0018] Functionally characterized transporters for which sequence
data are lacking. Transporters of particular physiological
significance will be included in this category even though a family
assignment cannot be made.
[0019] Putative transporters in which no family member is an
established transporter. Putative transport protein families are
grouped under this number and will either be classified elsewhere
when the transport function of a member becomes established, or
will be eliminated from the TC classification system if the
proposed transport function is disproven. These families include a
member or members for which a transport function has been
suggested, but evidence for such a function is not yet
compelling.
[0020] Auxiliary transport proteins. Proteins that in some way
facilitate transport across one or more biological membranes but do
not themselves participate directly in transport are included in
this class. These proteins always function in conjunction with one
or more transport proteins. They may provide a function connected
with energy coupling to transport, play a structural role in
complex formation or serve a regulatory function.
[0021] Transporters of unknown classification. Transport protein
families of unknown classification are grouped under this number
and will be classified elsewhere when the transport process and
energy coupling mechanism are characterized. These families include
at least one member for which a transport function has been
established, but either the mode of transport or the energy
coupling mechanism is not known.
[0022] Ion channels
[0023] An important type of transporter is the ion channel. Ion
channels regulate many different cell proliferation,
differentiation, and signaling processes by regulating the flow of
ions into and out of cells. Ion channels are found in the plasma
membranes of virtually every cell in eukaryotic organisms. Ion
channels mediate a variety of cellular functions including
regulation of membrane potentials and absorption and secretion of
ion across epithelial membranes. When present in intracellular
membranes of the Golgi apparatus and endocytic vesicles, ion
channels, such as chloride channels, also regulate organelle pH.
For a review, see Greger, R. (1988) Annu. Rev. Physiol.
50:111-122.
[0024] Ion channels are generally classified by structure and the
type of mode of action. For example, extracellular ligand gated
channels (ELGs) are comprised of five polypeptide subunits, with
each subunit having 4 membrane spanning domains, and are activated
by the binding of an extracellular ligand to the channel. In
addition, channels are sometimes classified by the ion type that is
transported, for example, chlorine channels, potassium channels,
etc. There may be many classes of channels for transporting a
single type of ion (a detailed review of channel types can be found
at Alexander, S. P. H. and J. A. Peters (1997). Receptor and ion
channel nomenclature supplement. Trends Pharmacol. Sci., Elsevier,
pp. 65-68 and
http://www-biology.ucsd.edu/.about.msaier/transport/toc.htm- l.
[0025] There are many types of ion channels based on structure. For
example, many ion channels fall within one of the following groups:
extracellular ligand-gated channels (ELG), intracellular
ligand-gated channels (ILG), inward rectifying channels (INR),
intercellular (gap junction) channels, and voltage gated channels
(VIC). There are additionally recognized other channel families
based on ion-type transported, cellular location and drug
sensitivity. Detailed information on each of these, their activity,
ligand type, ion type, disease association, drugability, and other
information pertinent to the present invention, is well known in
the art.
[0026] Extracellular ligand-gated channels, ELGs, are generally
comprised of five polypeptide subunits, Unwin, N. (1993), Cell 72:
31-41; Unwin, N. (1995), Nature 373: 37-43; Hucho, F., et al.,
(1996) J. Neurochem. 66: 1781-1792;-Hucho, F., et al., (1996) Eur.
J. Biochem. 239: 539-557; Alexander, S. P. H. and J. A. Peters
(1997), Trends Pharmacol. Sci., Elsevier, pp. 4-6; 36-40; 42-44;
and Xue, H. (1998) J. Mol. Evol. 47: 323-333. Each subunit has 4
membrane spanning regions: this serves as a means of identifying
other members of the ELG family of proteins.. ELG bind a ligand and
in response modulate the flow of ions. Examples of ELG include most
members of the neurotransmitter-receptor family of proteins, e.g.,
GABAI receptors. Other members of this family of ion channels
include glycine receptors, ryandyne receptors, and ligand gated
calcium channels.
[0027] The Voltage-gated Ion Channel (VIC) Superfamily
[0028] Proteins of the VIC family are ion-selective channel
proteins found in a wide range of bacteria, archaea and eukaryotes
Hille, B. (1992), Chapter 9: Structure of channel proteins; Chapter
20: Evolution and diversity. In: Ionic Channels of Excitable
Membranes, 2nd Ed., Sinaur Assoc. Inc., Pubs., Sunderland, Mass.;
Sigworth, F. J. (1993), Quart. Rev. Biophys. 27: 1-40; Salkoff, L.
and T. Jegla (1995), Neuron 15: 489-492; Alexander, S. P. H. et
al., (1997), Trends Pharmacol. Sci., Elsevier, pp. 76-84; Jan, L.
Y. et al., (1997), Annu. Rev. Neurosci. 20: 91-123; Doyle, D. A, et
al., (1998) Science 280: 69-77; Terlau, H. and W. Stuhmer (1998),
Naturwissenschaften 85: 437-444. They are often homo- or
heterooligomeric structures with several dissimilar subunits (e.g.,
a1-a2-d-b Ca.sup.2+ channels, ab.sub.1b.sub.2 Na.sup.+ channels or
(a).sub.4-b K.sup.+ channels), but the channel and the primary
receptor is usually associated with the a (or a1) subunit.
Functionally characterized members are specific for K.sup.+,
Na.sup.+ or Ca.sup.2+. The K.sup.+ channels usually consist of
homotetrameric structures with each a-subunit possessing six
transmembrane spanners (TMSs). The a1 and a subunits of the
Ca.sup.2+ and Na.sup.+ channels, respectively, are about four times
as large and possess 4 units, each with 6 TMSs separated by a
hydrophilic loop, for a total of 24 TMSs. These large channel
proteins form heterotetra-unit structures equivalent to the
homotetrameric structures of most K.sup.+ channels. All four units
of the Ca.sup.2+ and Na.sup.+ channels are homologous to the single
unit in the homotetrameric K.sup.+ channels. Ion flux via the
eukaryotic channels is generally controlled by the transmembrane
electrical potential (hence the designation, voltage-sensitive)
although some are controlled by ligand or receptor binding.
[0029] Several putative K.sup.+-selective channel proteins of the
VIC. family have been identified in prokaryotes. The structure of
one of them, the KcsA K.sup.+ channel of Streptomyces lividans, has
been solved to 3.2 .ANG. resolution. The protein possesses four
identical subunits, each with two transmembrane helices, arranged
in the shape of an inverted teepee or cone. The cone cradles the
"selectivity filter" P domain in its outer end. The narrow
selectivity filter is only 12 .ANG. long, whereas the remainder of
the channel is wider and lined with hydrophobic residues. A large
water-filled cavity and helix dipoles stabilize K.sup.+ in the
pore. The selectivity filter has two bound K.sup.+ ions about 7.5
.ANG. apart from each other. Ion conduction is proposed to result
from a balance of electrostatic attractive and repulsive
forces.
[0030] In eukaryotes, each VIC family channel type has several
subtypes based on pharmacological and electrophysiological data.
Thus, there are five types of Ca.sup.2+ channels (L, N, P, Q and
T). There are at least ten types of K.sup.+channels, each
responding in different ways to different stimuli:
voltage-sensitive [Ka, Kv, Kvr, Kvs and Ksr], Ca.sup.2+-sensitive
[BK.sub.Ca, IK.sub.Ca and SK.sub.Ca] and receptor-coupled [K.sub.M
and KA.sub.Ch]. There are at least six types of Na.sup.+ channels
(I, II, III, .mu.l , H1 and PN3). Tetrameric channels from both
prokaryotic and eukaryotic organisms are known in which each
a-subunit possesses 2 TMSs rather than 6, and these two TMSs are
homologous to TMSs 5 and 6 of the six TMS unit found in the
voltage-sensitive channel proteins. KcsA of S. lividans is an
example of such a 2 TMS. channel protein. These channels may
include the K.sub.Na (Na.sup.+-activated) and K.sub.Vol (cell
volume-sensitive) K.sup.+ channels, as well as distantly related
channels such as the Tok1 K.sup.+ channel of yeast, the TWIK-1
inward rectifier K.sup.+ channel of the mouse and the TREK-1
K.sup.+ channel of the mouse. Because of insufficient sequence
similarity with proteins of the VIC family, inward rectifier
K.sup.+ IRK channels (ATP-regulated; G-protein-activated) which
possess a P domain and two flanking TMSs are placed in a distinct
family. However, substantial sequence similarity in the P region
suggests that they are homologous. The b, g and d subunits of VIC
family members, when present, frequently play regulatory roles in
channel activation/deactivation.
[0031] The Epithelial Na.sup.+ Channel (ENaC) Family
[0032] The ENaC family consists of over twenty-four sequenced
proteins (Canessa, C. M., et al., (1994), Nature 367: 463-467, Le,
T. and M. H. Saier, Jr. (1996), Mol. Membr. Biol. 13: 149-157;
Garty, H. and L. G. Palmer (1997), Physiol. Rev. 77: 359-396;
Waldmann, R., et al., (1997), Nature 386: 173-177; Darboux, I., et
al., (1998), J. Biol. Chem. 273: 9424-9429; Firsov, D., et al.,
(1998), EMBO J. 17: 344-352; Horisberger, J.-D. (1998). Curr. Opin.
Struc. Biol. 10: 443-449). All are from animals with no
recognizable homologues in other eukaryotes or bacteria. The
vertebrate ENaC proteins from epithelial cells cluster tightly
together on the phylogenetic tree: voltage-insensitive ENaC
homologues are also found in the brain. Eleven sequenced C. elegans
proteins, including the degenerins, are distantly related to the
vertebrate proteins as well as to each other. At least some of
these proteins form part of a mechano-transducing complex for touch
sensitivity. The homologous Helix aspersa (FMRF-amide)-activated
Na.sup.+ channel is the first peptide neurotransmitter-gated
ionotropic receptor to be sequenced.
[0033] Protein members of this family all exhibit the same apparent
topology, each with N- and C-termini on the inside of the cell, two
amphipathic transmembrane spanning segments, and a large
extracellular loop. The extracellular domains contain numerous
highly conserved cysteine residues. They are proposed to serve a
receptor function.
[0034] Mammalian ENaC is important for the maintenance of Na.sup.+
balance and the regulation of blood pressure. Three homologous ENaC
subunits, alpha, beta, and gamma, have been shown to assemble to
form the highly Na.sup.+ -selective channel. The stoichiometry of
the three subunits is alpha.sub.2, beta1, gamma1. in a
heterotetrameric architecture.
[0035] The Chloride Channel (ClC) Family
[0036] The ClC family is a large family consisting of dozens of
sequenced proteins derived from Gram-negative and Gram-positive
bacteria, cyanobacteria, archaea, yeast, plants and animals
(Steinmeyer, K., et al., (1991), Nature 354: 301-304; Uchida, S.,
et al., (1993), J. Biol. Chem. 268: 3821-3824; Huang, M. -E., et
al., (1994), J. Mol. Biol. 242: 595-598; Kawasaki, M., et al,
(1994), Neuron 12: 597-604; Fisher, W. E., et al., (1995),
Genomics. 29:598-606; and Foskett, J. K. (1998), Annu. Rev.
Physiol. 60:. 689-717). These proteins are essentially ubiquitous,
although they are not encoded within genomes of Haemophilus
influenzae, Mycoplasma genitalium, and Mycoplasma pneumoniae.
Sequenced proteins vary in size from 395 amino acyl residues (M.
jannaschii) to 988 residues (man). Several organisms contain
multiple ClC family paralogues. For example, Synechocystis has two
paralogues, one of 451 residues in length and the other of 899
residues. Arabidopsis thaliana has at least four sequenced
paralogues, (775-792 residues), humans also have at least five
paralogues (820-988 residues), and C. elegans also has at least
five (810-950 residues). There are nine known members in mammals,
and mutations in three of the corresponding genes cause human
diseases. E. coli, Methanococcus jannaschii and Saccharomyces
cerevisiae only have one ClC family member each. With the exception
of the larger Synechocystis paralogue, all bacterial proteins are
small (395-492 residues) while all eukaryotic proteins are larger
(687-988 residues). These proteins exhibit 10-12 putative
transmembrane a-helical spanners (TMSs) and appear to be present in
the membrane as homodimers. While one member of the family, Torpedo
ClC-O, has been reported to have two channels, one per subunit,
others are believed to have just one.
[0037] All functionally characterized members of the ClC family
transport chloride, some in a voltage-regulated process. These
channels serve a variety of physiological functions (cell volume
regulation; membrane potential stabilization; signal transduction;
transepithelial transport, etc.). Different homologues in humans
exhibit differing anion selectivities, i.e., ClC4 and ClC5 share a
NO .sub.3.sup.->Cl.sup.->- ;Br >I.sup.- conductance
sequence, while ClC3. has an I.sup.->Cl.sup.- selectivity. The
ClC4 and ClC5 channels and others exhibit outward rectifying
currents with currents only at voltages more positive than +20
mV.
[0038] Animal Inward Rectifier K.sup.+ Channel (IRK-C) Family
[0039] IRK channels possess the "minimal channel-forming structure"
with only a P domain, characteristic of the channel proteins of the
VIC family, and two flanking transmembrane spanners (Shuck, M. E.,
et al., (1994), J. Biol. Chem. 269: 24261-24270; Ashen, M. D., et
al., (1995), Am. J. Physiol. 268: H506-H511; Salkoff, L. and T.
Jegla (1995), Neuron 15: 489-492; Aguilar-Bryan, L., et al.,
(1998), Physiol. Rev. 78: 227-245; Ruknudin, A., et al., (1998), J.
Biol. Chem. 273: 14165-14171). They may exist in the membrane as
homo- or heterooligomers. They have a greater tendency to let
K.sup.+ flow into the cell than out. Voltage-dependence may be
regulated by external K.sup.+ , by internal Mg.sup.2+, by internal
ATP and/or by G-proteins. The P domains of IRK channels exhibit
limited sequence similarity to those of the VIC family, but this
sequence similarity is insufficient to establish homology. Inward
rectifiers play a role in setting cellular membrane potentials, and
the closing of these channels upon depolarization permits the
occurrence of long duration action potentials with a plateau phase.
Inward rectifiers lack the intrinsic voltage sensing helices found
in VIC family channels. In a few cases, those of Kir1.1a and
Kir6.2, for example, direct interaction with a member of the ABC
superfamily has been proposed to confer unique functional and
regulatory properties to the heteromeric complex, including
sensitivity to ATP. The SUR1 sulfonylurea receptor (spQO9428) is
the ABC protein that regulates the Kir6.2 channel in response to
ATP, and CFTR may regulate Kir1.1a. Mutations in SUR1 are the cause
of familial persistent hyperinsulinemic hypoglycemia in infancy
(PHHI), an autosomal recessive disorder characterized by
unregulated insulin secretion in the pancreas.
[0040] ATP-gated Cation Channel (ACC) Family
[0041] Members of the ACC family (also called P2X receptors)
respond to ATP, a functional neurotransmitter released by
exocytosis from many types of neurons (North, R. A. (1996), Curr.
Opin. Cell Biol. 8: 474-483; Soto, F., M. Garcia-Guzman and W.
Stuhmer (1997), J. Membr. Biol. 160: 91-100). They have been placed
into seven groups (P2X.sub.1- P2X.sub.7) based on their
pharmacological properties. These channels, which function at
neuron-neuron and neuron-smooth muscle junctions, may play roles in
the control of blood pressure and pain sensation. They may also
function in lymphocyte and platelet physiology. They are found only
in animals.
[0042] The proteins of the ACC family are quite similar in sequence
(>35% identity), but they possess 380-1000 amino acyl residues
per subunit with variability in length localized primarily to the
C-terminal domains. They possess two transmembrane spanners, one
about 30-50 residues from their N-termini, the other near residues
320-340. The extracellular receptor domains between these two
spanners (of about 270 residues) are well conserved with numerous
conserved glycyl and cysteyl residues. The hydrophilic C-termini
vary in length from 25 to 240 residues. They resemble the
topologically similar epithelial Na.sup.+ channel (ENaC) proteins
in possessing (a) N- and C-termini localized intracellularly, (b)
two putative transmembrane spanners, (c) a large extracellular loop
domain, and (d) many conserved extracellular cysteyl residues. ACC
family members are, however, not demonstrably homologous with them.
ACC channels are probably hetero- or homomultimers and transport
small monovalent cations (Me.sup.+). Some also transport Ca.sup.2+;
a few also transport small metabolites.
[0043] The Ryanodine-Inositol 1,4,5-triphosphate Receptor Ca.sup.2+
Channel (RIR-CaC) Family
[0044] Ryanodine (Ry)-sensitive and inositol 1,4,5-triphosphate
(IP3)-sensitive Ca.sup.2+-release channels function in the release
of Ca.sup.2+ from intracellular storage sites in animal cells and
thereby regulate various Ca.sup.2+-dependent physiological
processes (Hasan, G. et al., (1992) Development 116: 967-975;
Michikawa, T., et al., (1994), J. Biol. Chem. 269: 9184-9189;
Tunwell, R. E. A., (1996), Biochem. J. 318: 477-487; Lee, A. G.
(1996) Biomembranes, Vol. 6, Transmembrane Receptors and Channels
(A. G. Lee, ed.), JAI Press, Denver, Co., pp 291-326; Mikoshiba,
K., et al., (1996) J. Biochem. Biomem. 6: 273-289). Ry receptors
occur primarily in muscle cell sarcoplasmic reticular (SR)
membranes, and IP3 receptors occur primarily in brain cell
endoplasmic reticular (ER) membranes where they effect release of
Ca.sup.2+ into the cytoplasm upon activation (opening) of the
channel.
[0045] The Ry receptors are activated as a result of the activity
of dihydropyridine-sensitive Ca.sup.2+ channels. The latter are
members of the voltage-sensitive ion channel (VIC) family.
Dihydropyridine-sensitive channels are present in the T-tubular
systems of muscle tissues.
[0046] Ry receptors are homotetrameric complexes with each subunit
exhibiting a molecular size of over 500,000 daltons (about 5,000
amino acyl residues). They possess C-terminal domains with six
putative transmembrane a -helical spanners (TMSs). Putative
pore-forming sequences occur between the fifth and sixth TMSs as
suggested for members of the VIC family. The large N-terminal
hydrophilic domains and the small C-terminal hydrophilic domains
are localized to the cytoplasm. Low resolution 3-dimensional
structural data are available. Mammals possess at least three
isoforms that probably arose by gene duplication and divergence
before divergence of the mammalian species. Homologues are present
in humans and Caenorabditis elegans.
[0047] IP.sub.3 receptors resemble Ry receptors in many respects.
(1) They are homotetrameric complexes with each subunit exhibiting
a molecular size of over 300,000 daltons (about 2,700 amino acyl
residues). (2) They possess C-terminal channel domains that are
homologous to those of the Ry receptors. (3) The channel domains
possess six putative TMSs and a putative channel lining region
between TMSs 5 and 6. (4) Both the large N-terminal domains and the
smaller C-terminal tails face the cytoplasm. (5) They possess
covalently linked carbohydrate on extracytoplasmic loops of the
channel domains. (6) They have three currently recognized isoforms
(types 1, 2, and 3) in mammals which are subject to differential
regulation and have different tissue distributions.
[0048] IP.sub.3 receptors possess three domains: N-terminal
IP.sub.3-binding domains, central coupling or regulatory domains
and C-terminal channel domains. Channels are activated by IP.sub.3
binding, and like the Ry receptors, the activities of the IP.sub.3
receptor channels are regulated by phosphorylation of the
regulatory domains, catalyzed by various protein kinases. They
predominate in the endoplasmic reticular membranes of various cell
types in the brain but have also been found in the plasma membranes
of some nerve cells derived from a variety of tissues.
[0049] The channel domains of the Ry and IP.sub.3 receptors
comprise a coherent family that in spite of apparent structural
similarities, do not show appreciable sequence similarity of the
proteins of the VIC family. The Ry receptors and the IP.sub.3
receptors cluster separately on the RIR-CaC family tree. They both
have homologues in Drosophila. Based on the phylogenetic tree for
the family, the family probably evolved in the following sequence:
(1) A gene duplication event occurred that gave rise to Ry and
IP.sub.3 receptors in invertebrates. (2) Vertebrates evolved from
invertebrates. (3) The three isoforms of each receptor arose as a
result of two distinct gene duplication events. (4) These isoforms
were transmitted to mammals before divergence of the mammalian
species.
[0050] The Organellar Chloride Channel (O-ClC) Family
[0051] Proteins of the O-ClC family are voltage-sensitive chloride
channels found in intracellular membranes but not the plasma
membranes of animal cells (Landry, D, et al., (1993), J. Biol.
Chem. 268: 14948-14955; Valenzuela, Set al., (1997), J. Biol. Chem.
272: 12575-12582; and Duncan, R. R., et al., (1997), J. Biol. Chem.
272: 23880-23886).
[0052] They are found in human nuclear membranes, and the bovine
protein targets to the microsomes, but not the plasma membrane,
when expressed in Xenopus laevis oocytes. These proteins are
thought to function in the regulation of the membrane potential and
in transepithelial ion absorption and secretion in the kidney. They
possess two putative transmembrane a-helical spanners (TMSs) with
cytoplasmic N- and C-termini and a large luminal loop that may be
glycosylated. The bovine protein is 437 amino acyl residues in
length and has the two putative TMSs at positions 223-239 and
367-385. The human nuclear protein is much smaller (241. residues).
A C. elegans homologue is 260 residues long.
[0053] The Glutamate-gated Ion Channel (GIC) Family of
Neurotransmitter Receptors
[0054] Members of the GIC family are heteropentameric complexes in
which each of the 5 subunits is of 800-1000 amino acyl residues in
length (Nakanishi, N., et al, (1990), Neuron 5: 569-581; Unwin, N.
(1993), Cell 72: 31-41; Alexander, S. P. H. and J. A. Peters (1997)
Trends Pharmacol. Sci., Elsevier, pp. 36-40). These subunits may
span the membrane three or five times as putative a-helices with
the N-tertnini (the glutamate-binding domains) localized
extracellularly and the C-termini localized cytoplasmically. They
may be distantly related to the ligand-gated ion channels, and if
so, they may possess substantial b-structure in their transmembrane
regions. However, homology between these two families cannot be
established on the basis of sequence comparisons alone. The
subunits fall into six subfamilies: a, b, g, d, e and z.
[0055] The GIC channels are divided into three types: (1)
a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-, (2)
kainate- and (3) N-methyl-D-aspartate (NMDA)-selective glutamate
receptors. Subunits of the AMPA and kainate classes exhibit 35-40%
identity with each other while subunits of the NMDA receptors
exhibit 22-24% identity with the former subunits. They possess
large N-terminal, extracellular glutamate-binding domains that are
homologous to the periplasmic glutamine and glutamate receptors of
ABC-type uptake permeases of Gram-negative bacteria. All known
members of the GIC family are from animals. The different channel
(receptor) types exhibit distinct ion selectivities and conductance
properties. The NMDA-selective large conductance channels are
highly permeable to monovalent cations and Ca.sup.2+. The AMPA- and
kainate-selective-ion channels are permeable primarily to
monovalent cations with only low permeability to Ca.sup.2+.
[0056] The brain-specific Na.sup.+ -dependent inorganic phosphate
transporter (BNPI) belongs to a family of proteins that use the
inwardly directed Na+ gradient across the plasma membrane to
cotransport inorganic phosphate (Pi). Originally identified as a
sequence up-regulated by the exposure of cerebellar granule cells
to subtoxic concentrations of N-methyl-D-aspartate, BNPI mediates
the Na+-dependent accumulation of Pi in Xenopus oocytes. BNPI has
been implicated in adenosine 5'-triphosphate (ATP) production by
neurons and protection against excitotoxic injury. However, BNPI is
only expressed by glutamatergic neurons, militating against a
general metabolic. role in all neuronal populations. In
Caenorhabditis elegans, genetic screens for multiple behavioral
defects have identified mutants in the BNPI ortholog eat-4, and
recent studies indicate a specific role for eat-4 in glutamatergic
neurotransmission. The glutamatergic defect in eat-4 mutants
appears to be presynaptic, consistent with the localization of BNPI
to excitatory nerve terminals. The accumulation of cytoplasmic Pi
mediated by BNPI may activate the phosphate-activated glutaminase
responsible for biosynthesis of the bulk of glutamate released as a
neurotransmitter. However, the family of proteins including
BNPI/EAT-4 may have functions in addition to Pi transport.
[0057] BNPI shows sequence similarity to type I but not type II
Na+/Pi cotransporters. In contrast to the type II transporters that
exhibit robust Na+-dependent Pi uptake, the accumulation of Pi by
type I transporters is less striking. Rather, the type I
transporter NaPi-1 transports organic anions, including phenol red
and penicillin G, with substantially higher apparent affinity than
Pi. Human genetic studies have shown that mutations in another
protein closely related to BNPI and NaPi-1 account for disorders of
sialic acid storage. In these conditions, sialic acid accumulates
in lysosomes because of a defect in proton-driven export. Although
the sialin protein has not been demonstrated to mediate sialic acid
transport, these observations together with the report that NaPi-1.
accumulates organic anions with high apparent affinity suggest that
BNPI might also transport organic anions. Localization to
glutamatergic nerve terminals raises the possibility that it
transports glutamate. In addition, BNPI is localized to synaptic
vesicles in the brain and to intracellular membranes in transfected
cells, suggesting a role for BNPI in the transport of glutamate
into synaptic vesicles for regulated exocytotic release.
[0058] Glutamate transport into synaptic vesicles exhibits a number
of properties that distinguish it from glutamate uptake by other
transport systems. First, in contrast to plasma membrane glutamate
uptake, the accumulation of glutamate in synaptic vesicles does not
rely on a Na+electrochemical gradient. Consistent with this,
glutamate was transported by BNPI in the absence of Na+. Second,
vesicular glutamate transport has a substantially lower apparent
affinity (Km of .about.1 mM) than the plasma membrane excitatory
amino acid transporters (Km of .about.10 to 100 .mu.M). Glutamate
transport by BNPI is saturated with a Km of .about.2 mM, in the
same range as transport by synaptic vesicles. Third, plasma
membrane glutamate transporters recognize both aspartate and
glutamate as substrates, whereas vesicular glutamate transport does
not recognize aspartate. D-Glutamate partially inhibited the
transport of 3H-glutamate, and L-glutamine had no effect, also
consistent with prior work. Fourth, low micromolar concentrations
of the dye Evans blue inhibited the transport of glutamate into
both synaptic vesicles and membranes expressing BNPI.
[0059] For a review associated with the differentation-associated
Na-dependent inorganic phosphate cotransporter, see references
Bellocchio et al., Science, 289:957-960, 2000, Aihara et al., J.
Neurochem. 74: 2622-2625, 2000, Ni et al., J. Neurochem, 66:
2f227-2238, 1996, Takamori et al., Nature 407: 189-194, 2000.
[0060] Transporter proteins, particularly members of the
differentation-associated Na-dependent inorganic phosphate
cotransporter 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 transport proteins. The present invention advances the
state of the art by providing previously unidentified human
transport proteins.
SUMMARY OF THE INVENTION
[0061] The present invention is based in part on the identification
of amino acid sequences of human transporter peptides and proteins
that are related to the differentation-associated Na-dependent
inorganic phosphate cotransporter 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 transporter activity in cells and
tissues that express the transporter.
DESCRIPTION OF THE FIGURE SHEETS
[0062] FIG. 1 provides the nucleotide sequence of a cDNA molecule
or transcript sequence that encodes the transporter protein of the
present invention. 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 the pooled human
melanocyte, fetal heart, and pregnant uterus and human
leukocytes.
[0063] FIG. 2 provides the predicted amino acid sequence of the
transporter of the present invention. 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.
[0064] FIG. 3 provides genomic sequences that span the gene
encoding the transporter protein of the present invention. 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. 69 SNPs, including 14 indels, have been
identified in the gene encoding the transporter protein provided by
the present invention and are given in FIG. 3.
DETAILED DESCRIPTION OF THE INVENTION
[0065] General Description
[0066] 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 transporter protein or part of a transporter protein and
are related to the differentation-associated Na-dependent inorganic
phosphate cotransporter 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 transporter peptides and proteins that are related to the
differentation-associated Na-dependent inorganic phosphate
cotransporter subfamily, nucleic acid sequences in the form of
transcript sequences, cDNA sequences and/or genomic sequences that
encode these transporter 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 transporter of the
present invention.
[0067] 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
transporter proteins of the differentation-associated Na-dependent
inorganic phosphate cotransporter subfamily and the expression
pattern observed . Experimental data as provided in FIG. 1.
indicates expression in the pooled human melanocyte, fetal heart,
and pregnant uterus and human leukocytes. 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 differentation-associated
Na-dependent inorganic phosphate cotransporter family or subfamily
of transporter proteins.
[0068] Specific Embodiments
[0069] Peptide Molecules
[0070] The present invention provides nucleic acid sequences that
encode protein molecules that have been identified as being members
of the transporter family of proteins and are related to the
differentation-associated Na-dependent inorganic phosphate
cotransporter 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 transporter
peptides of the present invention, transporter peptides, or
peptides/proteins of the present invention.
[0071] The present invention provides isolated peptide and protein
molecules that consist of, consist essentially of, or comprising
the amino acid sequences of the transporter 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.
[0072] 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).
[0073] 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.
[0074] The language "substantially free of chemical precursors or
other chemicals" includes preparations of the peptide in which it
is separated from chemical precursors or other chemicals that are
involved in its synthesis. In one embodiment, the language
"substantially free of chemical precursors or other chemicals"
includes preparations of the transporter 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.
[0075] The isolated transporter 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 the pooled human melanocyte, fetal heart,
and pregnant uterus and human leukocytes. For example, a nucleic
acid molecule encoding the transporter 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.
[0076] 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.
[0077] The present invention fuirther provides proteins that
consist essentially of the amino acid sequences provided in FIG. 2
(SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA
nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the
genomic sequences provided in FIG. 3. (SEQ ID NO:3). A protein
consists essentially of an amino acid sequence when such an amino
acid sequence is present with only a few additional amino acid
residues, for example from about II to about 100 or so additional
residues, typically from 1 to about 20 additional residues in the
final protein.
[0078] 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 transporter 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.
[0079] The transporter peptides of the present invention can be
attached to heterologous sequences to form chimeric or fusion
proteins. Such chimeric and fusion proteins comprise a transporter
peptide operatively linked to a heterologous protein having an
amino acid sequence not substantially homologous to the transporter
peptide. "Operatively linked" indicates that the transporter
peptide and the heterologous protein are fused in-frame. The
heterologous protein can be fuised to the N-terminus or C-terminus
of the transporter peptide.
[0080] In some uses, the fuision protein does not affect the
activity of the transporter peptide per se. For example, the fusion
protein can include, but is not limited to, enzymatic fusion
proteins, for example beta-galactosidase fuisions, yeast two-hybrid
GAL fuisions, poly-His fusions, MYC-tagged, HI-tagged and Ig
fuisions. Such fusion proteins, particularly poly-His fusions, can
facilitate the purificafion of recombinant transporter 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.
[0081] 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 chimerie.gene sequence .(see Ausubel et al., Current
Protocols in Molecular Biology, 1992). Moreover, many expression
vectors are conunercially available that already encode a fusion
moiety (e.g., a GST protein). A transporter peptide-encoding
nucleic acid can be cloned into such an expression vector such that
the fuision moiety is linked in-frame to the transporter
peptide.
[0082] 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, alielic/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.
[0083] 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 transporter
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.
[0084] 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 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.
[0085] 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.,
Academiic 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.
[0086] 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 ofthe 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.
[0087] 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 transporter peptides of the present
invention as well as being encoded by the same genetic locus as the
transporter peptide provided herein.
[0088] Allelic variants of a transporter 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 transporter peptide as well as being encoded by the same
genetic locus as the transporter 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 12 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 transporter peptide encoding
nucleic acid molecule under stringent conditions as more fully
described below.
[0089] FIG. 3 provides information on SNPs that have been
identified in a gene encoding the transporter protein of the
present invention. 69 SNP variants were found, including 14 indels
(indicated by a "-") and 1 SNPs in exons.
[0090] Paralogs of a transporter peptide can readily be identified
as having some degree of significant sequence homology/identity to
at least a portion of the transporter 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 transporter peptide encoding
nucleic acid molecule under moderate to stringent conditions as
more fully described below.
[0091] Orthologs of a transporter peptide can readily be identified
as having some degree of significant sequence homology/identity to
at least a portion of the transporter 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
transporter 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.
[0092] Non-naturally occurring variants of the transporter 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 transporter peptide. For example, one class of substitutions
are conserved amino acid substitution. Such substitutions are those
that substitute a given amino acid in a transporter 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).
[0093] Variant transporter peptides can be fully functional or can
lack function in one or more activities, e.g. ability to bind
ligand, ability to transport ligand, ability to mediate signaling,
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.
[0094] 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.
[0095] 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
transporter 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)).
[0096] The present invention fuither provides fragments of the
transporter 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.
[0097] As used herein, a fragment comprises at least 8, 10, 12, 14,
16, or more contiguous amino acid residues from a transporter
peptide. Such fragments can be chosen based on the ability to
retain one or more of the biological activities of the transporter
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
transporter 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.
[0098] 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 transporter 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).
[0099] 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.
[0100] 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)).
[0101] Accordingly, the transporter 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
transporter peptide is fused with another compound, such as a
compound to increase the half-life of the transporter peptide (for
example, polyethylene glycol), or in which the additional amino
acids are fused to the mature transporter-peptide, such as a leader
or secretory sequence or a sequence for purification of the mature
transporter peptide or a pro-protein sequence.
[0102] Protein/Peptide Uses
[0103] 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
transporter-effector protein interaction or transporter-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.
[0104] 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.
[0105] Substantial chemical and structural homology exists between
the differentation-associated Na-dependent inorganic phosphate
cotransporter protein described herein and brain-specific
Na+-dependent inorganic phosphate transporter (BNPI) (see FIG. 1).
As discussed in the background, brain-specific Na+-dependent
inorganic phosphate transporter is known in the art to be involved
in transporting glutamate into native synaptic vesicles from the
brain and it is also a phosphate transporter, presumably at the
plasma membrane. Using fluorescence in situ hybridization, the BNPI
gene is to be located on th elong arm of 19q13, in close proximity
to the late-onset familial Alzheimer disease locus (Ni et al., J.
Neurochem, 66: 2f227-2238, 1996), Accordingly, the
differentation-associated Na-dependent inorganic phosphate
cotransporter protein, and the encoding gene, provided by the
present invention is useful for treating, preventing, and/or
diagnosing neurotransmitter related disease, brain diseases such as
Alzheimer and other disorders associated with this BNPI.
[0106] 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, transporters 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 transporter. Experimental data as provided in FIG. 1
indicates that transporter proteins of the present invention are
expressed in the pooled human melanocyte, fetal heart, and pregnant
uterus detected by a virtual northern blot. In addition, PCR-based
tissue screening panel indicates expression in human leukocytes. A
large percentage of pharmaceutical agents are being developed that
modulate the activity of transporter proteins, particularly members
of the differentation-associated Na-dependent inorganic phosphate
cotransporter 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 the pooled human
melanocyte, fetal heart, and pregnant uterus and human leukocytes.
Such uses can readily be determined using the information provided
herein, that known in the art and routine experimentation.
[0107] The proteins of the present invention (including variants
and fragments that may have been disclosed prior to the present
invention) are usefuil for biological assays related to
transporters that are related to members of the
differentation-associated Na-dependent inorganic phosphate
cotransporter subfamily. Such assays involve any of the known
transporter functions or activities or properties useful for
diagnosis and treatment of transporter-related conditions that are
specific for the subfamily of transporters that the one of the
present invention belongs to, particularly in cells and tissues
that express the transporter. Experimental data as provided in FIG.
1 indicates that transporter proteins of the present invention are
expressed in the pooled human melanocyte, fetal heart, and pregnant
uterus detected by a virtual northern blot. In addition, PCR-based
tissue screening panel indicates expression in human
leukocytes.
[0108] The proteins of the present invention are also usefiul in
drug screening assays, in cell-based or cell-free systems
((Hodgson, Bio/technology, Sept 10, 1992 (9);973-80). Cell-based
systems can be native, i.e., cells that normally express the
transporter, as a biopsy or expanded in cell culture. Experimental
data as provided in FIG. 1 indicates expression in the pooled human
melanocyte, fetal heart, and pregnant uterus and human leukocytes.
In an alternate embodiment, cell-based assays involve recombinant
host cells expressing the transporter protein.
[0109] The polypeptides can be used to identify compounds that
modulate transporter activity of the protein in its natural state
or an altered form that causes a specific disease or pathology
associated with the transporter. Both the transporters 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 transporter. These compounds can be
further screened against a functional transporter to determine the
effect of the compound on the transporter 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 transporter to a
desired degree.
[0110] Further, the proteins of the present invention can be used
to screen a compound for the ability to stimulate or inhibit
interaction between the transporter protein and a molecule that
normally interacts with the transporter protein, e.g. a substrate
or a component of the signal pathway that the transporter protein
normally interacts (for example, another transporter). Such assays
typically include the steps of combining the transporter protein
with a candidate compound under conditions that allow the
transporter 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 transporter protein and the target, such
as any of the associated effects of signal transduction such as
changes in membrane potential, protein phosphorylation, cAMP
turnover, and adenylate cyclase activation, etc.
[0111] 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).
[0112] One candidate compound is a soluble fragment of the receptor
that competes for ligand binding. Other candidate compounds include
mutant transporters or appropriate fragments containing mutations
that affect transporter function and thus compete for ligand.
Accordingly, a fragment that competes for ligand, for example with
a higher affinity, or a fragment that binds ligand but does not
allow release, is encompassed by the invention.
[0113] The invention further includes other end point assays to
identify compounds that modulate (stimulate or inhibit) transporter
activity. The assays typically involve an assay of events in the
signal transduction pathway that indicate transporter activity.
Thus, the transport of a ligand, change in cell membrane potential,
activation of a protein, a change in the expression of genes that
are up- or down-regulated in response to the transporter protein
dependent signal cascade can be assayed.
[0114] Any of the biological or biochemical functions mediated by
the transporter 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 transporter can be assayed.
Experimental data as provided in FIG. 1 indicates that transporter
proteins of the present invention are expressed in the pooled human
melanocyte, fetal heart, and pregnant uterus detected by a virtual
northern blot. In addition, PCR-based tissue screening panel
indicates expression in human leukocytes.
[0115] Binding and/or activating compounds can also be screened by
using chimeric transporter 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
ligand-binding region can be used that interacts with a different
ligand then that which is recognized by the native transporter.
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 transporter is derived.
[0116] The proteins of the present invention are also useful in
competition binding assays in methods designed to discover
compounds that interact with the transporter (e.g. binding partners
and/or ligands). Thus, a compound is exposed to a transporter
polypeptide under conditions that allow the compound to bind or to
otherwise interact with the polypeptide. Soluble transporter
polypeptide is also added to the mixture. If the test compound
interacts with the soluble transporter polypeptide, it decreases
the amount of complex formed or activity from the transporter
target. This type of assay is particularly useful in cases in which
compounds are sought that interact with specific regions of the
transporter. Thus, the soluble polypeptide that ,competes with the
target transporter region is designed to contain peptide sequences
corresponding to the region of interest.
[0117] To perform cell free drug screening assays, it is sometimes
desirable to immobilize either the transporter 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.
[0118] Techniques for immobilizing proteins on matrices can be used
in the drug screening assays. In one embodiment, a fiusion 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 transporter-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
transporter-binding protein and a candidate compound are incubat ed
in the transporter 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-immdbilized complexes, include immunodetection of complexes
using antibodies reactive with the transporter protein target
molecule, or which are reactive with transporter 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.
[0119] Agents that modulate one of the transporters 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.
[0120] Modulators of transporter protein activity identified
according to these drug screening assays can be used to treat a
subject with a disorder mediated by the transporter pathway, by
treating cells or tissues that express the transporter.
Experimental data as provided in FIG. 1 indicates expression in the
pooled human melanocyte, fetal heart, and pregnant uterus and human
leukocytes. These methods of treatment include the steps of
administering a modulator of transporter activity in a
pharmaceutical composition to a subject in need of such treatment,
the modulator being identified as described herein.
[0121] In yet another aspect of the invention, the transporter
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; 1019 and Brent
W094/10300), to identify other proteins, which bind to or interact
with the transporter and are involved in transporter activity. Such
transporter-binding proteins are also likely to be involved in the
propagation of signals by the transporter proteins or transporter
targets as, for example, downstream elements of a
transporter-mediated signaling pathway. Alternatively, such
transporter-binding proteins are likely to be transporter
inhibitors.
[0122] 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 transporter
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 transporter-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 transporter protein.
[0123] 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
transporter-modulating agent, an antisense transporter nucleic acid
molecule, a transporter-specific antibody, or a transporter-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.
[0124] The transporter 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 the
pooled human melanocyte, fetal heart, and pregnant uterus and human
leukocytes. The method involves contacting a biological sample with
a compound capable of interacting with the transporter 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.
[0125] 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.
[0126] The peptides of the present invention also provide targets
for diagnosing active protein activity, disease, or predisposition
to disease, in a patient having a variant peptide, particularly
activities and conditions that are known for other members of the
family of proteins to which the present one belongs. Thus, the
peptide can be isolated from a biological sample and assayed for
the presence of a genetic mutation that results in aberrant
peptide. This includes amino acid substitution, deletion,
insertion, rearrangement, (as the result of aberrant splicing
events), and inappropriate post-translational modification.
Analytic methods include altered electrophoretic mobility, altered
tryptic peptide digest, altered transporter activity in cell-based
or cell-free assay, alteration in ligand 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.
[0127] In vitro techniques for detection of peptide include enzyme
linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence using a detection
reagent, such as an antibody or protein binding agent.
Alternatively, the peptide can-be detected in vivo in a subject by
introducing into the subject a labeled anti-peptide antibody or
other types of detection agent. For example, the antibody can be
labeled with a radioactive marker whose presence and location in a
subject can be detected by standard imaging techniques.
Particularly useful are methods that detect the allelic variant of
a peptide expressed in a subject and methods which detect fragments
of a peptide in a sample.
[0128] The peptides are also useful in phannacogenomic 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. Phannacol. 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
transporter protein in which one or more of the transporter
funictions 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 ligand-binding regions
that are more or less active in ligand binding, and transporter
activation. Accordingly, ligand 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.
[0129] 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 the pooled human melanocyte, fetal heart,
and pregnant uterus and human leukocytes. Accordingly, methods for
treatment include the use of the transporter protein or
fragments.
[0130] Antibodies
[0131] 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.
[0132] 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.
[0133] 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).
[0134] 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.
[0135] Antibodies are preferably prepared from regions or discrete
fragments of the transporter 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 transporter/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.
[0136] 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).
[0137] 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.
[0138] Antibody Uses
[0139] 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 transporter
proteins of the present invention are expressed in the pooled human
melanocyte, fetal heart, and pregnant uterus detected by a virtual
northern blot. In addition, PCR-based tissue screening panel
indicates expression in human leukocytes. 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.
[0140] 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 the pooled human melanocyte,
fetal heart, and pregnant uterus and human leukocytes. 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.
[0141] 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 the pooled human melanocyte, fetal heart, and
pregnant uterus and human leukocytes. 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.
[0142] 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.
[0143] The antibodies are also useful for tissue typing.
Experimental data as provided in FIG. 1 indicates expression in the
pooled human melanocyte, fetal heart, and pregnant uterus and human
leukocytes. 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.
[0144] The antibodies are also useful for inhibiting protein
function, for example, blocking the binding of the transporter
peptide to a binding partner such as a ligand or protein binding
partner. 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.
[0145] 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.
[0146] Nucleic Acid Molecules
[0147] The present invention further provides isolated nucleic acid
molecules that encode a transporter 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 transporter
peptides of the present invention, an allelic variant thereof, or
an ortholog or paralog thereof.
[0148] 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 that 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.
[0149] 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.
[0150] 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.
[0151] 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.
[0152] The present invention fiurther 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.
[0153] The present invention flurther 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 comprise 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.
[0154] 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.
[0155] 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.
[0156] As mentioned above, the isolated nucleic acid molecules
include, but are not limited to, the sequence encoding the
transporter 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 purificafion. 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).
[0157] The invention fturther 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
transporter 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.
[0158] The present invention further provides non-coding fragments
of the nucleic acid molecules provided in FIGS. 1. and 3. Preferred
non-coding fragments include, but are not limited to, promoter
sequences, enhancer sequences, gene modulating sequences and gene
termination sequences. Such fragments are usefuil 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.
[0159] 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.
[0160] 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.
[0161] 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. As indicated by
the data presented in FIG. 3, the map position was determined to be
on chromosome 12 by ePCR, and confirmed with radiation hybrid
mapping.
[0162] FIG. 3 provides information on SNPs that have been
identified in a gene encoding the transporter protein of the
present invention. 69 SNP variants were found, including 14 indels
(indicated by a "-") and 1 SNPs in exons.
[0163] 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 6X sodium
chloride/sodium citrate (SSC) at about 45C., followed by one or
more washes in 0.2 X SSC, 0.1% SDS at 50-65C. Examples of moderate
to low stringency hybridization conditions are well known in the
art.
[0164] Nucleic Acid Molecule Uses
[0165] 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. 69 SNPs, including 14
indels, have been identified in the gene encoding the transporter
protein provided by the present invention and are given in FIG.
3.
[0166] 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.
[0167] 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.
[0168] The nucleic acid molecules are also useful for constructing
recombinant vectors. Such vectors include expression vectors that
express a portion of, or all of, the peptide sequences. Vectors
also include insertion vectors, used to integrate into another
nucleic acid molecule sequence, such as into the cellular genome,
to alter in situ expression of a gene and/or gene product. For
example, an endogenous coding sequence can-be replaced via
homologous recombination with all or part of the coding region
containing one or more specifically introduced mutations.
[0169] The nucleic acid molecules are also useful for expressing
antigenic portions of the proteins.
[0170] 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 12 by ePCR, and confinned with radiation hybrid
mapping.
[0171] The nucleic acid molecules are also useful in making vectors
containing the gene regulatory regions of the nucleic acid
molecules of the present invention.
[0172] 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.
[0173] The nucleic acid molecules are also useful for making
vectors that express part, or all, of the peptides.
[0174] The nucleic acid molecules are also useful for constructing
host cells expressing a part, or all, of the nucleic acid molecules
and peptides.
[0175] The nucleic acid molecules are also useful for constructing
transgenic animals expressing all, or a part, of the nucleic acid
molecules and peptides.
[0176] 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 transporter proteins of the present invention are
expressed in the pooled human melanocyte, fetal heart, and pregnant
uterus detected by a virtual northern blot. In addition, PCR-based
tissue screening panel indicates expression in human
leukocytes.
[0177] 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 transporter protein expression relative to normal
results.
[0178] In vitro techniques for detection of MRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detecting DNA include Southern hybridizations and in situ
hybridization.
[0179] Probes can be used as a part of a diagnostic test kit for
identifying cells or tissues that express a transporter protein,
such as by measuring a level of a transporter-encoding nucleic acid
in a sample of cells from a subject e.g., MRNA or genomic DNA, or
deteriining if a transporter gene has been mutated. Experimental
data as provided in FIG. 1 indicates that transporter proteins of
the present invention are expressed in the pooled human
mlelanocyte, fetal heart, and pregnant uterus detected by a virtual
northern blot. In addition, PCR-based tissue screening panel
indicates expression in human leukocytes.
[0180] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate transporter nucleic acid
expression.
[0181] 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 transporter gene, particularly
biological and pathological processes that are mediated by the
transporter in cells and tissues that express it. Experimental data
as provided in FIG. 1 indicates expression in the pooled human
melanocyte, fetal heart, and pregnant uterus and human leukocytes.
The method typically includes assaying the ability of the compound
to modulate the expression of the transporter nucleic acid and thus
identifying a compound that can be used to treat a disorder
characterized by undesired transporter nucleic acid expression. The
assays can be performed in cell-based and cell-free systems.
Cell-based assays include cells naturally expressing the
transporter nucleic acid or recombinant cells genetically
engineered to express specific nucleic acid sequences.
[0182] The assay for transporter 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 transporter 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.
[0183] Thus, modulators of transporter 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 transporter mRNA in the presence of the candidate
compound is compared to the level of expression of transporter 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.
[0184] 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 transporter nucleic
acid expression in cells and tissues that express the transporter.
Experimental data as provided in FIG. 1 indicates that transporter
proteins of the present invention are expressed in the pooled human
melanocyte, fetal heart, and pregnant uterus detected by a virtual
northern blot. In addition, PCR-based tissue screening panel
indicates expression in human leukocytes. Modulation includes both
up-regulation (i.e. activation or agonization) or down-regulation
(suppression or antagonization) or nucleic acid expression.
[0185] Altematively, a modulator for transporter 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 transporter nucleic acid expression in the
cells and tissues that express the protein. Experimental data as
provided in FIG. 1. indicates expression in the pooled human
melanocyte, fetal heart, and pregnant uterus and human
leukocytes.
[0186] The nucleic acid molecules are also useful for monitoring
the effectiveness of modulating compounds on the expression or
activity of the transporter 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.
[0187] The nucleic acid molecules are also useful in diagnostic
assays for qualitative changes in transporter nucleic acid
expression, and particularly in qualitative changes that lead to
pathology. The nucleic acid molecules can be used to
detect-mutations in transporter 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 transporter 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
transporter 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 transporter protein.
[0188] Individuals carrying mutations in the transporter gene can
be detected at the nucleic acid level by a variety of techniques.
FIG. 3 provides information on SNPs that have been identified in a
gene encoding the transporter protein of the present invention. 69
SNP variants were found, including 14 indels (indicated by a "-")
and 1 SNPs in exons. As indicated by the data presented in FIG. 3,
the map position was determined to be on chromosome 12 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.
[0189] Alternatively, mutations in a transporter gene can be
directly identified, for example, by alterations in restriction
enzyme digestion patterns determined by gel electrophoresis.
[0190] 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.
[0191] 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 transporter 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)).
[0192] 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 21 7:286-295 (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.
[0193] 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 transporter gene in an
individual in order to select an appropriate compound or dosage
regimen for treatment. FIG. 3 provides information on SNPs that
have been identified in a gene encoding the transporter protein of
the present invention. 69 SNP variants were found, including 14
indels (indicated by a and 1 SNPs in exons.
[0194] 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.
[0195] The nucleic acid molecules are thus useful as antisense
constructs to control transporter 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
transporter protein. An antisense RNA or DNA nucleic acid molecule
would hybridize to the mRNA and thus block translation of mRNA into
transporter protein.
[0196] Alternatively, a class of antisense molecules can be used to
inactivate mRNA in order to decrease expression of transporter
nucleic acid. Accordingly, these molecules can treat a disorder
characterized by abnormal or undesired transporter 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 transporter protein, such as
ligand binding.
[0197] The nucleic acid molecules also provide vectors for gene
therapy in patients containing cells that are aberrant in
transporter 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 transporter protein to treat the
individual.
[0198] The invention also encompasses kits for detecting the
presence of a transporter nucleic acid in a biological sample.
Experimental data as provided in FIG. 1. indicates that transporter
proteins of the present invention are expressed in the pooled human
melanocyte, fetal heart, and pregnant uterus detected by a virtual
northern blot. In addition, PCR-based tissue screening panel
indicates expression in human leukocytes. For example, the kit can
comprise reagents such as a labeled or labelable nucleic acid or
agent capable of detecting transporter nucleic acid in a biological
sample; means for determining the amount of transporter nucleic
acid in the sample; and means for comparing the amount of
transporter 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
transporter protein mRNA or DNA.
[0199] Nucleic Acid Arrays
[0200] 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).
[0201] As used herein "Arrays" or "Microarrays" refers to an array
of distinct polynucleotides or oligonucleotides synthesized on a
substrate, such as paper, nylon or other type of membrane, filter,
chip, glass slide, or any other suitable solid support.. In one
embodiment, the microarray is prepared and used according to the
methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT
application W095/11995 (Chee et al.), Lockhart, D. J. et al. (1996;
Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc.
Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated
herein in their entirety by reference. In other embodiments, such
arrays are produced by the methods described by Brown et al., U.S.
Pat. No. 5,807,522.
[0202] 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 that 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.
[0203] 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.
[0204] In another aspect, an oligonucleotide may be synthesized on
the surface of the substrate by using a chemical coupling procedure
and an ink jet application apparatus, as described in PCT
application W095/251116 (Baldeschweiler et al.) which is
incorporated herein in its entirety by reference. In another
aspect, a "gridded" array analogous to a dot (or slot) blot may be
used to arrange and link cDNA fragments or oligonucleotides to the
surface of a substrate using a vacuum system, thermal, UV,
mechanical or chemical bonding procedures. An array, such as those
described above, may be produced by hand or by using available
devices (slot blot or dot blot apparatus), materials (any suitable
solid support), and machines (including robotic instruments), and
may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or
any other number between two and one million which lends itself to
the efficient use of commercially available instrumentation.
[0205] 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.
[0206] Using such arrays, the present invention provides methods to
identify the expression of the transporter 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 transporter gene of the present
invention. FIG. 3 provides information on SNPs that have been
identified in a gene encoding the transporter protein of the
present invention. 69 SNP variants were found, including 14 indels
(indicated by a "-") and 1 SNPs in exons.
[0207] 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).
[0208] 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.
[0209] In another embodiment of the present invention, kits are
provided which contain the necessary reagents to carry out the
assays of the present invention.
[0210] 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.
[0211] 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 transporter 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.
[0212] Vectors/host cells
[0213] 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.
[0214] 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.
[0215] 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 procaryotic or
eukaryotic cells or in both (shuttle vectors).
[0216] 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.
[0217] 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 X, 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.
[0218] 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.
[0219] 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).
[0220] 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).
[0221] 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.
[0222] 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.
[0223] 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.
[0224] 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 enterotransporter. 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 1 Id (Studier et al., Gene Expression Technology:
Methods in Enzymology 185:60-89 (1990)).
[0225] 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)).
[0226] 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.).
[0227] 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)).
[0228] 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)).
[0229] 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.
[0230] 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).
[0231] 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.
[0232] The recombinant host cells are prepared by introducing the
vector constructs described herein into the cells by techniques
readily available to the person of ordinary skill in the art. These
include, but are not limited to, calcium phosphate transfection,
DEAE-dextran-mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection,
lipofection, and other techniques such as those found in Sambrook,
et al. (Molecular Cloning: A Laboratory Manual 2nd, ed., Cold
Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1989).
[0233] 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.
[0234] 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.
[0235] Vectors generally include selectable markers that enable the
selection of the subpopulation of cells that contain the
recombinant vector constructs. The marker can be contained in the
same vector that contains the nucleic acid molecules described
herein or may be on a separate vector. Markers include tetracycline
or ampicillin-resistance genes for prokaryotic host cells and
dihydrofolate reductase or neomycin resistance for eukaryotic host
cells. However, any marker that provides selection for a phenotypic
trait will be effective.
[0236] 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.
[0237] Where secretion of the peptide is desired, which is
difficultAto achieve with multi-transmembrane domain containing
proteins such as transporters, appropriate secretion signals are
incorporated into the vector. The signal sequence can be endogenous
to the peptides or heterologous to these peptides.
[0238] Where the peptide is not secreted into the medium, which is
typically the case with transporters, 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.
[0239] 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 pepfides may include an initial mnodified
methionine in some cases as a result of a host-mediated
process.
[0240] Uses of Vectors and Host Cells
[0241] The recombinant host cells expressing the peptides described
herein have a variety of uses. First, the cells are useful for
producing a transporter protein or peptide that can be further
purified to produce desired amounts of transporter protein or
fragments. Thus, host cells containing expression vectors are
useful for peptide production.
[0242] Host cells are also useful for conducting cell-based assays
involving the transporter protein or transporter protein fragments,
such as those described above as well as other formats known in the
art. Thus, a recombinant host cell expressing a native transporter
protein is useful for assaying compounds that stimulate or inhibit
transporter protein function.
[0243] Host cells are also useful for identifying transporter
protein mutants im 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 transporter protein (for example,
stimulating or inhibiting function) which may not be indicated by
their effect on the native transporter protein.
[0244] 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 oneuor more of the cells of the animal include a
transgene. A transgene is exogenous DNA that 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
usefuil for studying the function of a transporter protein and
identifying and evaluating modulators of transporter protein
activity. Other examples of transgenic animals include non-human
primates, sheep, dogs, cows, goats, chickens, and amphibians.
[0245] A transgenic animal can be produced by introducing nucleic
acid into the male pronuclei of a fertilized oocyte, e.g., by
microinjecfion, retroviral infection, and allowing the oocyte to
develop in a pseudopregnant female foster animal. Any of the
transporter protein nucleofide sequences can be introduced as a
transgene into the genome of a non-human animal, such as a
mouse.
[0246] 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
transporter protein to particular cells.
[0247] 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 arimals 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.
[0248] 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.
[0249] 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.
[0250] 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 ligand binding, transporter protein 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 transporter protein fimction,
including ligand interaction, the effect of specific mutant
transporter proteins on transporter protein function and ligand
interaction, and the effect of chimeric transporter proteins. It is
also possible to assess the effect of null mutations, that is
mutations that substantially or completely eliminate one or more
transporter protein functions.
[0251] 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 1811 DNA Homo sapiens 1 tcagaggtgc ccctcattca aaatgccttt
taaagcattt gataccttca aagaaaaaat 60 tctgaaacct gggaaggaag
gagtgaagaa cgccgtggga gattctttgg gaattttaca 120 aagaaaaatc
gatgggacaa ctgaggaaga agataacatt gagctgaatg aagaaggaag 180
gccggtgcag acgtccaggc caagcccccc actctgcgac tgccactgct gcggcctccc
240 caagcgttac atcattgcta tcatgagtgg gctgggattc tgcatttcct
ttgggatccg 300 gtgcaatctt ggagttgcca ttgtggaaat ggtcaacaat
agcaccgtat atgttgatgg 360 aaaaccggaa attcagacag cacagtttaa
ctgggatcca gaaacagtgg gccttatcca 420 tggatctttt ttctggggct
atattatgac acaaattcca ggtggtttca tttcaaacaa 480 gtttgctgct
aacagggtct ttggagctgc catcttctta acatcgactc tgaacatgtt 540
tattccctct gcagccagag tgcattacgg atgcgtcatg tgtgtcagaa ttctgcaagg
600 tttagtggag ggtgtgacct acccagcctg ccatgggatg tggagtaagt
gggcaccacc 660 tttggagaga agccgactgg ccacaacctc tttttgtggt
tcctatgcag gggcagtggt 720 tgccatgccc ctggctgggg tgttggtgca
gtacattgga tggtcctctg tcttttatat 780 ttatggcatg tttgggatta
tttggtacat gttttggctg ttgcaggcct atgagtgccc 840 agcagctcat
ccaacaatat ccaatgagga gaagacctat atagagacaa gcataggaga 900
gggggccaac gtggttagtc taagtaaatt tagtacccca tggaaaagat ttttcacatc
960 tttgccggtt tatgcaatca ttgtggcaaa tttttgcaga agctggacct
tttatttgct 1020 cctcataagt cagcctgctt attttgaaga ggtctttgga
tttgcaataa gtaaggtggg 1080 tctcttgtca gcagtcccac acatggttat
gacaatcgtt gtacctattg gaggacaatt 1140 ggctgattat ttaagaagca
gacaaatttt aaccacaact gctgtcagaa aaatcatgaa 1200 ctgtggaggt
tttggcatgg aggcaacctt actcctggtg gttggctttt cgcataccaa 1260
aggggtggct atctcctttc tggtacttgc tgtaggattt agtggcttcg ctatttcagg
1320 ttttaatgtc aaccacctgg acattgcccc acgctatgcc agcattctca
tggggatctc 1380 aaacggagtg ggaaccctct ctggaatggt ctgtcccctc
attgtcggtg caatgaccag 1440 gcacaagacc cgtgaagaat ggcagaatgt
gttcctcata gctgccctgg tgcattacag 1500 tggtgtgatc ttctatggga
tctttgcttc tggggagaaa caggagtggg ctgacccaga 1560 gaatctctct
gaggagaaat gtggaatcat tgaccaggac gaattagctg aggagataga 1620
actcaaccat gagagttttg cgagtcccaa aaagaagatg tcttatggag ccacctccca
1680 gaattgtgaa gtccagaaga aggaatggaa aggacagaga ggagcgaccc
ttgatgagga 1740 agagctgaca tcctaccaga atgaagagag aaacttctca
actatatcct aatgtctgag 1800 aggcacttct g 1811 2 589 PRT Homo sapiens
2 Met Pro Phe Lys Ala Phe Asp Thr Phe Lys Glu Lys Ile Leu Lys Pro 1
5 10 15 Gly Lys Glu Gly Val Lys Asn Ala Val Gly Asp Ser Leu Gly Ile
Leu 20 25 30 Gln Arg Lys Ile Asp Gly Thr Thr Glu Glu Glu Asp Asn
Ile Glu Leu 35 40 45 Asn Glu Glu Gly Arg Pro Val Gln Thr Ser Arg
Pro Ser Pro Pro Leu 50 55 60 Cys Asp Cys His Cys Cys Gly Leu Pro
Lys Arg Tyr Ile Ile Ala Ile 65 70 75 80 Met Ser Gly Leu Gly Phe Cys
Ile Ser Phe Gly Ile Arg Cys Asn Leu 85 90 95 Gly Val Ala Ile Val
Glu Met Val Asn Asn Ser Thr Val Tyr Val Asp 100 105 110 Gly Lys Pro
Glu Ile Gln Thr Ala Gln Phe Asn Trp Asp Pro Glu Thr 115 120 125 Val
Gly Leu Ile His Gly Ser Phe Phe Trp Gly Tyr Ile Met Thr Gln 130 135
140 Ile Pro Gly Gly Phe Ile Ser Asn Lys Phe Ala Ala Asn Arg Val Phe
145 150 155 160 Gly Ala Ala Ile Phe Leu Thr Ser Thr Leu Asn Met Phe
Ile Pro Ser 165 170 175 Ala Ala Arg Val His Tyr Gly Cys Val Met Cys
Val Arg Ile Leu Gln 180 185 190 Gly Leu Val Glu Gly Val Thr Tyr Pro
Ala Cys His Gly Met Trp Ser 195 200 205 Lys Trp Ala Pro Pro Leu Glu
Arg Ser Arg Leu Ala Thr Thr Ser Phe 210 215 220 Cys Gly Ser Tyr Ala
Gly Ala Val Val Ala Met Pro Leu Ala Gly Val 225 230 235 240 Leu Val
Gln Tyr Ile Gly Trp Ser Ser Val Phe Tyr Ile Tyr Gly Met 245 250 255
Phe Gly Ile Ile Trp Tyr Met Phe Trp Leu Leu Gln Ala Tyr Glu Cys 260
265 270 Pro Ala Ala His Pro Thr Ile Ser Asn Glu Glu Lys Thr Tyr Ile
Glu 275 280 285 Thr Ser Ile Gly Glu Gly Ala Asn Val Val Ser Leu Ser
Lys Phe Ser 290 295 300 Thr Pro Trp Lys Arg Phe Phe Thr Ser Leu Pro
Val Tyr Ala Ile Ile 305 310 315 320 Val Ala Asn Phe Cys Arg Ser Trp
Thr Phe Tyr Leu Leu Leu Ile Ser 325 330 335 Gln Pro Ala Tyr Phe Glu
Glu Val Phe Gly Phe Ala Ile Ser Lys Val 340 345 350 Gly Leu Leu Ser
Ala Val Pro His Met Val Met Thr Ile Val Val Pro 355 360 365 Ile Gly
Gly Gln Leu Ala Asp Tyr Leu Arg Ser Arg Gln Ile Leu Thr 370 375 380
Thr Thr Ala Val Arg Lys Ile Met Asn Cys Gly Gly Phe Gly Met Glu 385
390 395 400 Ala Thr Leu Leu Leu Val Val Gly Phe Ser His Thr Lys Gly
Val Ala 405 410 415 Ile Ser Phe Leu Val Leu Ala Val Gly Phe Ser Gly
Phe Ala Ile Ser 420 425 430 Gly Phe Asn Val Asn His Leu Asp Ile Ala
Pro Arg Tyr Ala Ser Ile 435 440 445 Leu Met Gly Ile Ser Asn Gly Val
Gly Thr Leu Ser Gly Met Val Cys 450 455 460 Pro Leu Ile Val Gly Ala
Met Thr Arg His Lys Thr Arg Glu Glu Trp 465 470 475 480 Gln Asn Val
Phe Leu Ile Ala Ala Leu Val His Tyr Ser Gly Val Ile 485 490 495 Phe
Tyr Gly Val Phe Ala Ser Gly Glu Lys Gln Glu Trp Ala Asp Pro 500 505
510 Glu Asn Leu Ser Glu Glu Lys Cys Gly Ile Ile Asp Gln Asp Glu Leu
515 520 525 Ala Glu Glu Ile Glu Leu Asn His Glu Ser Phe Ala Ser Pro
Lys Lys 530 535 540 Lys Met Ser Tyr Gly Ala Thr Ser Gln Asn Cys Glu
Val Gln Lys Lys 545 550 555 560 Glu Trp Lys Gly Gln Arg Gly Ala Thr
Leu Asp Glu Glu Glu Leu Thr 565 570 575 Ser Tyr Gln Asn Glu Glu Arg
Asn Phe Ser Thr Ile Ser 580 585 3 66804 DNA Homo sapiens 3
aacctctttt tgtctgagtt tcctgccagt aaaattgggg aaaataagaa gttatttacc
60 acagagtctt gctgggaaga ttgtggtgat acttaaagag tgcttaacac
agagccagga 120 ccctagaaag aactcaaaag atattagcaa tatttagcct
accaaggatt cagcacggac 180 ttagttgaac ttaattcaaa ttttggataa
tttggacagt ggcttgcaga ggatattgac 240 tggtcttgtg gaaatgactc
ctggggagcc tgagagccta tagcctatga tttgtcagtc 300 gcatgcagac
tggaggattg gaacacagga gcctcaaaga tgaagagttt tttttccacc 360
gcagcagcat ttacagaggc gtcatcctgc tgcccataaa tgtggccaca acttgcagcg
420 tttcagcccc agttcaacaa gtatttaggt aacgcccact ccctgccagg
ctctgctagg 480 gcagaggaca ggtgatttgg aggcacagag gagggacatc
tcaccttgcc catgcagttt 540 tctagaggat tgatatctta gcatgacctt
agaaccccta gaagttaccc agttgaaggg 600 gtgcagagag ttacccaggc
agagggcata gcttgagtaa agcccagagg caatagggag 660 cttgctgagt
tcagtgaaat gaggatgtgg aaagcagagt gacaagaaga aagacttagg 720
gtcccaggga aaggccttgt gtgccatgat aaagaattgt attgtaaata gtgctgcaat
780 aaacatacgt gtggatgtgt ctttgtagta gaatgattag aatacatgga
tacagagagg 840 ggaacatcac acaccggagc tggtcagggg ttggggggca
aggagaggga gagcattagg 900 acaaatacct aatgcatgtg gggcttaaaa
cctagatgat gggttgatag gtgcagcaaa 960 ccaccatggc acatgtatac
ctatgtaaca aacctgcatg ttctgcacat gtatcctgga 1020 acttaaagta
aaaaaaaaaa agtccatcta gagggagaaa aggggaaaaa acaaaaataa 1080
ttttatttat cctgaggaca atgaggagtc agtggagagt tctaagcagg ttctagatat
1140 cttccggctc agaaatcttc aattagatgg tcccaaatgg catctacgta
tcatactttg 1200 agagagcctg ctctgttgat taggagcaaa taaatgtcct
cctggatgta tgtggcctgg 1260 gttttgcatt tgggctactc aaatgcaagt
tcctcgtggg accacatcca tgctagtggc 1320 tggctgaaaa acggcttcat
gactctcatg aggggaataa aaggcatgga gtggtggctg 1380 tgagcctgtc
tgcagggcca gacctcagaa aagcaaaggg ctgtaaatgt ttcataaatt 1440
tctctctggg tgcctgctct ggctgagagc ccattcataa gcccaggcgg ctgaggggca
1500 ggtattgtgc cggttactat agcatcacct tggaaagtct cacttggtga
gagcggcagg 1560 cgagctgggg tggggcagga gggggacgcg gctggctgga
ggggctggag ctaggccacg 1620 gatactgctg ctggtctcag gactcctggt
ggtccggagc tcatgttagc gtccccagct 1680 gcagcccagg gagggagaga
ggctgcgctc agtctgagag tggctgcctg agacagctgc 1740 cacaggctgc
tgcagagcgt gcagcttttg caagggactg aattcccagc cagacacccc 1800
ttggactctt ttttggaggg gtggggagca gagagaggag ggagttgtct tatcttggaa
1860 gatccgagct gggtttcatc tcctttttga ttttgagtag ttccctccac
gagaactgac 1920 ttccaggtgt tcaccaaggg aaacaaggtg gttctcacac
tggaaatgag gaaggatgac 1980 agtttttgag actgactgtt aacggctcag
aggtgcccct cattcaaaat gccttttaaa 2040 gcatttgata ccttcaaaga
aaaaattctg aaacctggga aggaaggagt gaagaacgcc 2100 gtgggagatt
ctttgggaat tttacaaagg taaagtttga atgcgaactt tagttccttt 2160
ctgagtagct tcgtattgcc aatgtgtgag agacttggta tcacgttttt aaaaccacac
2220 tttaatgagg agaggatggg tcagattaga tccttctgga gccccttcta
gctccagtag 2280 tctatgcctg gaggaaaaac agatgcatga atagtattgg
gttgtattag gaaaagatca 2340 agacaaatat gctgtttata tagctggatt
agcactttct ggagatgatg atattgcata 2400 tggtatgttt ggcattgaat
tagaaaatat ttagggagat aatattttat gttaactcat 2460 tagtaatgac
aaatatgcct tgaactgaaa taatttttat gtttttcact gaatccacta 2520
taaatgaaaa ttaaatattt gcaattttta gcttatttaa taaaatacat aaagtggttc
2580 ctgattgtat agtttgcaaa gagaaggata gttacacatt aatttgaagg
aagtaactta 2640 aaaaatgtct ttgaagcaga aaatctcaca taattgcagt
gggaaaatgt taagtactat 2700 cactgaattg aatgagattt tagtccaaac
caaaaagtaa atatttttta aagtaaaata 2760 tattaatgga aggagagttt
gctataaatg attgaattaa tgtgacagtt taatttatga 2820 atttttatag
acatagtaaa tgccttctca aattatataa atgatttcat aagtggtcct 2880
tatgtgcaag gtaaaatgac tgctttatct ctctgatata aataaatgtg aaaaataact
2940 ttgatacact ttttatttgt ttggatgatt atttctaatc ctggtgagtg
aaaatgccat 3000 ctggtgtgtc cttttaactt ttctattatc tcttaaattt
aaaaactttt tcatttaaat 3060 gactatttcc aggcaatctg agattcatcc
catttcttgt gttttaaaac acatatgctc 3120 ctgtcagtgt taaattttcc
catggtatca ctgttaatat taactttcct aataagaaaa 3180 aagagttgga
caccttatta ttttagtaat tagaaacaaa aaagcttcaa tcagacctac 3240
actgaattag catgtctaga tgaaaaccta gctcagtgac agcagcataa accagccaaa
3300 tatagaaaaa attacaataa catttttttc agagtgtttt atccttccgt
tgagcactcc 3360 ccaggtaacg tcttattgtg ttggcgttca tttgattaga
aacgcaaaaa taatttttgc 3420 ataataagca cgatagctta attggcttat
tcaagtaatg acaaaggaat ctggcaaagt 3480 caagaataaa aaccataggc
cgggcgcagt ggctcacgcc tgtaatccca gcactttggg 3540 aggcggaagt
gggaggatcg cttgaggcca gaagttcgag actagcctgg ggaacataga 3600
gagaccatgt ctctacagaa atacaaaaaa ttagccagca tgatggtgca tgcctgtcat
3660 ctcagcttcc caagaagtgg gagtattgct tgagcccaga cattcaaggt
tgcagcgagc 3720 caagattgcg tctctgcact ccagctaggg tgacagagtc
agactctgtc tcaaaaaata 3780 aaaaaataaa ataaatttaa aaacctatga
cgttgggcca tagtcaccat tataaacagc 3840 aaactctgcc ttcatttata
aaatatttga tataaaaata cttaggaatt ttcttttcaa 3900 ccttaagttt
aattgctttt tgtgaaattt gattgctttt ttcaatagga attattgatc 3960
gaagagccgg ttttgctatg tttgattgga ggagctacat ggagatcttt ttgtttacaa
4020 aattgatttg cttagggata taacaaaatt ggcgattttc caaattgtgt
gacctcaacc 4080 agaaattggg ctatgtgtct aggactgttt gaatagtttc
ctcagaacaa tagaaaaaca 4140 gctagcacag tactagggac agagaatgca
ctaaacaaat gctagatatt gtcatggttg 4200 tcctaattgt agaatggctt
tagaaaaaat aaagccaagg tcaaatccct tttttcagtg 4260 atctatagag
agaaattatt ggcagaagaa acgaaaacag acattgcttg agcggtgatc 4320
caagttgatc ctcagttcta gtgaggaatt atcaagacca gctctgccac gtgtttggca
4380 ttaatcacag gtgtataagg taattgtatg taaatgaccc tgcccagagc
ctggcacata 4440 ctgggcattt ccctctcatt tcactgcttt tcacgtaaaa
ccagttgaca gaatcccatg 4500 taaaaaaatc acaaagaact gttttctgtt
ttgtaggagc ttttggaagc tagaagcccc 4560 tacattgtaa cttagaaggc
aatgtaaatc acagctgtct aataatgttt gaggctgagg 4620 tcatcatcta
aatggaattc ttgagatgct ttttaatcac agtgttcctc acagtcaggg 4680
gagtggcaat tgcacaggga agcatttgag agttcgcaca caggcttgat tacagtcagg
4740 catgattagc tttcctggaa aacagtcatt gataagaagc agctgagcaa
ttaatcagct 4800 aaaggtaaaa taatatttta gaagtgcagg aagaaagaag
atgcactcat ttatagttta 4860 gtattgaatt atatagatga catagaaagc
attaaacttg gaaactaatg tccagaaagt 4920 gacatgcaga tttgttcaat
ttaaattaca atttatgtgt cctttaattg ttcatgtcta 4980 aaaaacataa
cagtgacaaa acagtatctt tcagacactg taaactcatt taattctatt 5040
aaaatcccca tgaagagggg attactataa ttacaacttt tctttttttt gggatagggt
5100 ctcactctgt tgcctaggct ggagtgcagt gatgtgatca tagctcactg
cagcctcaaa 5160 ctcctggcct caagccatac tgcctccttg gcctcccaaa
gtgctaggat tacaggcatg 5220 agccacagca tctagcaata attttacaga
tgagaaaact gaggcacaga gaggttaagt 5280 agcttgccca aggtcacaca
gctataaatg gaagagctag gtttcaaacc agatgttcta 5340 tgcccatcat
tcttaatcac tacattatgt tacccctgta atcaagtgtc tttcctcttc 5400
ccactcactg tcttgatatt gggccactta tttaggttta gggaggtcta cttggactgc
5460 aatgtagcca gcaacttctg gatctgctgt caagtgtggg ctattctcct
aatcagttgc 5520 atctttattg aaggctttct ccaagggagg cttaagggga
gtctggtctc cttacaagta 5580 tgtctatctt ccctttaaat gaaactagtc
cctgcatcgt gtctgtcttc agcattcagg 5640 agtgtgccag atatgcactt
cctgctccat caacaaaggt gagtgtgtta aagcttgctc 5700 tgagatcagg
tgatcctggg ttccaactgc tgcaacatcc tttacttccc tgcctgcatg 5760
acctcaggca acttggctgc aatggggtga ctctaggaaa ccaagtcaga tcacatctca
5820 cccctgctca aaactacctc actcagagtt aaagccagtg ccctttcaat
ggccttcaag 5880 gacctctgtg atctaggact tttggaaggc tctctgagtt
catctgtgac attttcctgc 5940 ctcactctac tctggattca cgggcctcct
ggctcttatt agaactcccc cagattcact 6000 cctgtcccgg ctttcgccct
gtttcttttg cttaaatgct ttcctcccag atagcctgat 6060 ggctcattcc
ctcgctttct tcaagtatgt gctcaaagat ccccactttc ctggccattc 6120
tatttaaaca tgaagctcac ctgccctcct cctcctgccc tcttctctgt ccctctttcc
6180 tgctttactt cacctctgtc ttaggtaggt tccctaaaaa gcacagcctg
agacagggat 6240 ttgggtgagc ctagaatgtg atttaatgag ctcttcctga
aaaaactggg agggagtaaa 6300 acaagaaggg aaaggagagg ctgggtgtgg
tggctcacgc ctataatcct agcattttgg 6360 gagtccgagg caggcagatt
gcctgagctc aggagtttga gaccagcctg ggcaacatgg 6420 tgaaacctgt
ctctactaaa agacaaaaaa tgagccaggc atagaggcat gtgcctatag 6480
tcgtagctac tcaggaggct gaggcaggag aattgcttga atccgggagg cagaggttgc
6540 agtgagccga gatcacacca ctgcactcca gcctggacga cagagggaga
ctccatctcc 6600 aaaaaaaaca aaacaaaaaa aaacagaaag gagaaagagc
caagcaagga tgcatgctca 6660 caatgcccag tggccagatc caaaggggaa
ggctctggag cacaagcgat gtgctgagtc 6720 cttcctttgg ggcaagtggg
gcagcctttt atatctctgc ctcagtcagt catcagctct 6780 gggctgatgg
gggtgggtga ggggtttatt tggaggccac tgagcagtgg gaagttctcc 6840
agggttcctc atgccaggac tagaagccca ggcaaggagt caccatggtg gcaagggtca
6900 tgggtcctga tcctcaggag gaaccagaac tgtcacctca tcacgggagc
aggaagagat 6960 gtgtttggca ctgaggtggt ccactcggac atctcctgat
actgcctggg ctagttttat 7020 ttattttttt attttaattt ttaaaataat
agagatgggg gtctcaccat gttgattagg 7080 ctggtcttaa actcctgggc
tcaggagatc ttcctgcctt ggcctcccaa gtgctagaat 7140 tacaggcatg
agccaccgca ccctgcctag ttttaactgc aaatgggaaa atacagcaac 7200
cgtgacctgc tagcagctct ggaagtagaa gtgtgcttgc ccatcaaggg gaactgggga
7260 gtgtgctatg gtgtctatga cagcccaccc actgcaccgc tcagatcaac
ttgcttctca 7320 catgaagttc actccatcca ggtacagctt ctccaagact
ctatggttgt aattcctgag 7380 gagccttcca aagaagagtt attaagacag
actccaggcc ccactgtgat gactggtccc 7440 tcctctccac ccctttttga
tttccctcac ttctgtttgc ttgtctgatg ggttgcccca 7500 gatcttcatc
cctgagaggt ctaaatccct ggttaacata acctcaccag gtcatggttg 7560
ctgtatttgc ccactgacag ttaaaaacta gccaaggcag tatcaggaga tgtcccagca
7620 gatcatctgg gtgccaaaca tatttcttcc tgctcccatg atgaagcgac
agttctgatt 7680 cctcctgaag attaggatcc atgaccctta tcactgtagt
gacttcttac catgtcttct 7740 ggtcttggca cgaggacccc aaagtgactg
agcagcagtc gtaaccatat gttgactagg 7800 atttccattg tgttcctaaa
tggaagaatt cttccttgtg aatcgggatt tctagctcct 7860 cagagcctaa
gctgaagaga tgagatattc ctcaggtggg ttactgggaa tgatggcgag 7920
tggggccact ccttctctca tgccttgttt ctttgacctg tgtgttctgc ccactgggca
7980 cacagcacca tatcataact gttgggtttt ttgtttgttt gtttgggatg
gagtcccact 8040 ctgtcgccca ggctggatgc agcggcttga tctcagctca
ctgcaacctc tgcctcctgg 8100 gttcaagcaa ttctcctgcc tcagcctcct
gaatagtggg attacgggca cccaccacca 8160 tgcccggcta attttgtatt
tttagtagag atggggtttc ggtatgttgg tcaggctggt 8220 ttcaaacacc
tgacttcaaa tgatccaccc gccttggcct cccaaaatgc tggcgttaca 8280
ggtgcataac tgttgattta tggaatatac tgcatcctgg aagatagcac cttaccctcc
8340 cagggtttca tctccaagct gatgtcgcag ctgcatcttt aagaagcttc
ttcagaggcc 8400 aggtgccatg gctcacacct gtaatcccag cactttggga
ggccaacgca gatggatcat 8460 ttgaggtcag gagttggaga ccagcctggt
caacatggtg aaacatatat tttctactaa 8520 aaatacaaaa aattgccagg
cgtggtggtg ggcacctgta atcccaacta ctcaggaggc 8580 tgaggcagga
gaattgcttg attaaaccca agggggaaga ggttgcagtg agctgagata 8640
gagccactgc actccagcct gggtgataga acaagattcc atctcaaaaa aaaaaaaaaa
8700 aaaaaaagct tctttagctc tggcaggctg tcagcttctg gatggtgtgg
tatatggtgg 8760 ggcttgtgca tcccattgtc atgtgcccac tgctatgcca
tcttcaccat aaattgggtg 8820 ttttggtctg aaaaaatgtg atgtgagatc
ccatgttgag aaatcagaca ctgaatcctc 8880 agatagtgat gttggctgag
acttgtagtc tgaataggca aactcataca tggaatattt 8940 caaccccagt
caggatgaat tgctaccctt tccaggatgg aaggggtctg ttacaaacaa 9000
cttctgacca agagactggt ttgtcccctc aggaattgtg ccatctcagg ggctcagcat
9060 tagtcttgtt gctgaccgca gcaggagcta gctcagtcct ggtgagtggg
agctccgaca 9120 tagcctccat ccctgctgcc atggttactc tgttcataag
tgcactgctc tagcactggg 9180 tggctgagga cggaagctag ctgacatcaa
ctggccaagt cagcctgcct atcgtctgtt 9240 ttgtgcctct tccaagtggc
atgtgataat gtgcaatcag gagagctcat actaggcatc 9300 cactcataga
ttgatccaca tctcttcccc agatcttttt cccagtcctc caatattgct 9360
ttaaatgtcc cctgacctcc agtgaggtca ttcaccactg cctatgagtc catgtatatc
9420 cttacctttg aatgtttctc ttttcataca aaatgtgtgg ccaggagact
gctcaaaact 9480 ctgcctattg ggaggctttc cccctcactg tccttcaggg
ccatccctgg gtgggctgaa 9540 gtatagcagc agtccatttg caacatgctc
taacatacca aattgaccag ctctggacca 9600 aactaagttt tttttcctcc
tgtaatttgt tcacaggtgt gagctcatgg agaggcattg 9660 gtatagtaga
tataggtcag gtggaaaact ggcccctctg acatgggatt acttgtgtcc 9720
tgtggccctg ctggtgttag attccagatg gaccacttcc gtcttatgat ggacagttgg
9780 acttctcagg tctgatagaa cctagctcct gatgggtcgt tttggctgca
tggtctcttg 9840 atattccatg gtcagatgct gtgtctccac caggactcag
taacatgcca tgagatgttt 9900 tttgtctgtt gtatatttct ctactataga
tgacaaggcc ttgtcccaga atcctaaggc 9960 tctatgctat atttctccta
ttgagttttg ccagaaacct catatagtgt cttttcctat 10020 tcagatgact
ttagtaccaa gggtatgctg gagaatacag ccctagtggc aagatcaccc 10080
ttattgcagc ctagacttgc tacagaacat tttcttgttt ttggatttca ttaaaaacca
10140 gcagcctttt atgtcatgga ataagtgggt tagaacaata ttcccaagtg
ttgaatacac 10200 tgcctcaaaa acccaaagag gtctgccaac cattgtgctt
cttccttagt ggcaggaagt 10260 tcaaagcaca ataacttttc cttcactttg
aagggaatgt tctggaatgt ctcaaacact 10320 agactcctgt gaacttcgcc
cacgtgatgg acctgtgatc tttgtagcaa ttatttctgg 10380 agcacacatg
tcttactgag gcatccagag tacttgtcaa ttcttgctcc tgaggtctaa 10440
ataacataat gtcatctatt ggtcataata gatcaatgtg atgttctgca gcaggtccag
10500 aaggtctctc tgactatatt atgagagaga acaatccacg gatatatact
gtcattcatc 10560 tcatgtcatt gtgaatttct tagcctcctt tctaatggat
atggaaagaa tgcaccagat 10620 caagacaggc aagctatgta catgagttaa
gagtgtggta gttccctgtc atccaccgtg 10680 atcccttctt tttttttttt
tttttttttt cagggggtag gctggtgaat taccttggga 10740 tccaatgggg
gctaccaccc ctgcatcctt taaatctctg aaggtgcaat aatctctgtc 10800
attctgcata atataatact gtttttgatt tatcttctta cacagggatg acagttttaa
10860 ggacttccat ttgacttttt tctattacaa tagcttttat tctacaagtc
aaggaaccac 10920 ggaaagcgtt ttccaaatgc taggtgtctc tcttccaatt
atacatgtgg gaattataca 10980 tgggggaatg accactggat gggtccatgg
acatgaactc actatgagac gatcctgtgc 11040 caggactcca cttattacct
gacccttata agccccactc taatggagga agaatagtgc 11100 tataaatctc
tgagtatcaa catcaatttg cccctttatc caaaagtctg caaaagatag 11160
aggtatttct ctttcttcag tgtatgttta cccaaattaa tggtggtaga gtcctttggg
11220 gaaggactgt gggcatcatg actgcatttc cttctgtggt gttgcaggtt
ccttagtcac 11280 ggaaccttcg gtcttctcct tcagtctttg gattctggcc
tagaaactgg gcaagagagt 11340 gtgactttcc actggggtgg ccagcctcag
cctactgccc attcatcagc tctttatctt 11400 ttctggtttt atgtatatga
agcaataccc ttattggctg cccattttat ttttgttcct 11460 tggagcacca
tgttctgtga gtcatctctg agattcctat gggctgattc cctaactgta 11520
gttctgaatt ttctgccctt acctatgatg gttaagtgct cccaatcatc ccaattgcca
11580 ctggttctgg agcagcacct tctactgtga gcctcagcca gaagaggaca
gcagcttctg 11640 agcatcagtg gtgcctgtgg ccccatcacc actgcattcc
ttattatctt aagagcagga 11700 gtattcctca ggcctcctga gaaatatagt
tcgtttgtgg gttttctggt cttatataga 11760 aaatccattc ctgcatagtc
atttatttga ggcttttgat ctttctttat aatctgctgt 11820 aacagttccc
aacatttctc atttttaaag aaaataagtt aaagagagac cttttaattg 11880
atcaagagtg tgatcaacat taaagatata acaattatgg aattcttata ttccaaataa
11940 tagagatcaa aactttactt aaaggaatag aagatagcca atttaattat
cagtaattca 12000 tcgctatgac tggttcaaat tcagcaattt ttataccagg
cattaaaaaa tgaaataggc 12060 ttgtaaatta ggtttatata acaatgaagg
aaaagagagg atgtagacct ggaccaacca 12120 aaataaggac actcttgtgg
ccttaggcat tctctcctgg aatggataat tttttattct 12180 tttatttatt
tatttattta tttgagacag ggtctcactc tgtcacctag gctggagtgc 12240
agtggcacaa tcatacctca cggcagcctc aacctcccag gctcaagtga tcctcccacc
12300 tcagcctcct gagtagctga gactacagtt gcgagccacc atgcttggct
aatttttaaa 12360 atattctgta gagacgaagg tctcgctatg ttgcctagaa
tggtctcgaa ctcctgggct 12420 caagccatcc tcccacctca gcctgccaaa
ttgctgggat tacaggcgtg aacccctgtg 12480 cccagctttc aagttatttt
ttttaaaagt catggtggcc atatcctgta tctctgtgta 12540 taatgtaata
atgactagaa attagtacag aattatattt taaaagtcac caggctactc 12600
tggacatatc tattttgttt aagtttccaa gaaccgtatt agcagtttat caggatcatt
12660 tctcttaagg cctttgccgg gatgttagac cctgtgtcat gggaccatgc
cccctttatt 12720 agtttcctag ggctgctgta acaaagtacc acaaactagg
tagcttaaaa caacagaaac 12780 ttattctctc acaattctgg agaccagaag
tccaaaccca aggtgttggc agggccaagc 12840 tcctcctgaa ggctcttaag
gaggcctcat gcttgcctct tgctggctgc tggtagctgc 12900 tgggaatccc
aggcgtgcct tggcttgtgg atgcattgct ccaattgctg catttgttgt 12960
cacatggtct tctcccctgg tgtctgtgtc tatgatttca aattcccctc ttcttataag
13020 gacaccagtc atgaaatcaa tctattatga cctcatgtta acttgattac
atctgtgaag 13080 actccatttc caaataaggc tacattcaca ggtatcgggg
gttagaacat caacatatct 13140 attttggagg acagaattca atctacctcc
catattgatg aactctccct tatccaactt 13200 tattacccta ctccctccaa
atctagtaca ttcaggatcc attcccgggc atactttcct 13260 gcttcttgat
gtaaatgttc atcagattct acgactcctg ctcccagtat cttttcttag 13320
ctcaaaagtg tattttctca tctaaagttt atattctctc cttttacaac ttctcccaag
13380 tacttttaca acaatcaaat tttctaagtg cttcttaaag gttagtaagg
cctatagatt 13440 caatacctac agagtaaagc aaccatatta tatattttga
catagacaca ctacatatta 13500 acacatagaa ataggctcca cttctgcaag
gaaatatgtt gtatcattca aagttcttag 13560 ttgcaatcaa cagaatacac
tctagctaaa gtggaatgaa atttcgtaaa gaatgttaag 13620 aattgggctg
ggggcaatgg ctcatccctg taatcccagc actttgggag gccaaggcag 13680
ggagaggatc acctgaggtc tggagtttga gaccagcctg gccaacatgg tgaaatccca
13740 tctctactaa aactacaaaa attagccagg catggtggta cgtgcctgta
atcccagcta 13800 ctcaggaggc tgaggcagga gaactgcttg aacccaggag
gcagacgttg cagtgagccg 13860 aaatcccacc actgcactcc agcctgggca
acagagcaag actccatctc aaaaccataa 13920 attaataaaa aataaaagaa
tgttaggaat tgttcagact tcctggaagg atcaggtctg 13980 gatgctgtat
tctccaggaa aaagcagcag agaacatata ctgctagact gttctggata 14040
aaacacagct gccaccactg cctgcttcta agtgttgatt atattgatga cttgttccag
14100 aaattctgcc acagcagtca cagaggagcc agttgcctct gttgcatttg
aaaccatctg 14160 cactgccatt cccctgcatg ctgtatcctc ttcttgttct
gtcccgtatc taaatctcat 14220 tcaagtgctt tggatttagc agagtccacc
tctcatgcct gcattgtagc tgcaagagag 14280 cctaggaaaa gtaggtgttt
tttttgtttt tgtttttgtt gttttttatt ttgttttgtt 14340 tttgctgctc
cagcaagatt caaaatatca agaattcatt aagatattgg acagctataa 14400
atgatggttg tctgctacat atgtgtgcta ctagtctaat ttttattttt caacttttga
14460 tacagacatg ggtacaaaac atatttttct aatgtcttga ttttaactac
tagaaaagta 14520 acagtgcaag tataacgtta aatggcaact gagctcacta
tggaagtgac aatagggagt 14580 ggtggggact gtggtaaatt gagagccaat
tgtagccatg acagagtgag agcttgatta 14640 tttcaggtct tcagattttt
caaaatgaac aagaaatcca aagttttata tgtttgcttg 14700 tttctgcttt
tttgagctat ctcctgatat ttatttattt ttttatttat ttaatacaat 14760
ttttaaaagt agagatgggg gtcttactat gttgcccagg ctggtctcaa actcctggcc
14820 tcaagcaatc ctctcacctt ggcctcccaa agttccagga ttacaggtgt
gagccactgt 14880 gctgggcctt ggtttttaaa ctctgtcaat taatctaaat
ttatttttta ttttttattt 14940 tttatttttg agatggagtt ttgctcttgt
cacccaggct ggagtgcaaa ggcacaatct 15000 cagctcacta caacctctgc
ctcctgggtt caggcgattc tcctgcctca gccttctggg 15060 tagctgggat
tacaggcatg caccaccatg tccagctaat tttgtattta taatagagat 15120
ggagttttgc catgttggcc aggctggtct tgaactcctg acctcaagtg atctgcatgc
15180 cttggcctac caaagtgctg gggttacagg catgagccac cgtgcccagc
caattaatct 15240 aaattctaaa aaaaaaaaaa aaaaaaaaag caaagaccca
tacacacatt ataccagata 15300 aacaaaacat ggctatgggc cacatatggc
cattgggctt tcagcttgtc atctgtgact 15360 taggctttta aagccataga
gactatcttt ttttcctctt gttcatctaa tgatccctgc 15420 tgaggtaaga
agcagtgagt ctctgcttaa atggggggat aggaaagggt caaattacca 15480
ggaggaaaca aaaacagcat aggttaatac ctcaaaatct atgaagctgg gctgagtgct
15540 agggattttt ggttcctgac tttctgaaat tataatctac tggaagaggc
aaatattaat 15600 ttaaaaatga gagacataga tactggggga gcattgactg
ggctggcgtt ggccaggtgc 15660 actttattga gctccttttg aatgtggtgt
gctgaaatcc atgctgataa gatcctattt 15720 caaatctcaa actagctctg
gggatcgtat tttaaattct ccttcctttc tttaaaattt 15780 accatttatt
gattatttat caagtgccag gaattatgct aagcattttg taactcggtc 15840
tcatttaacg ttcacagtag tcccatcttc ctttcataaa tgagggaact caggttgagg
15900 gaagttaggt aatttgctca aggccacata cctaataaat accacagtca
gcattgaacc 15960 cagtactgtc tgtctccagg gcatgttctc tgaatcccac
tgcaatactc ctccagaacc 16020 tttaaaaaaa agtctctgta ggtaaagcac
tcgccattcg tcaggcgctt tctgattagt 16080 tcgtgtggca cactggtagc
aataggctgg atagcaaatc tcagttgtgt tctcccttca 16140 ccagctgcag
ctggatgatc cttgggcaag ttttttttgt ttgtttgttt tcttttcttt 16200
tgttttgttt tttaagtcag agttctcact ctgtcaccca ggctggagtg cagttcactg
16260 caaccgccac ctcccaggtt caagtgattc tcctgcttca gcctcctgag
tagctgggat 16320 tacaggtgct tgccagcaca cccggctaac ttttttgtat
ttttagtaga gatgggtttt 16380 caccatgttg gccaggctgg tcttgaactc
tgagctcagg tgatccacct gccttggcct 16440 tccaaattgt tgggattaca
gccgtgagcc accgtgccca gctgggcaag gttttaaata 16500 ttctgagtgt
ctcagtcttc tgagcgtctc agtcttctga gcagtaagat ggggatatct 16560
cctatttgtc aagactattt tgagaattaa gggagataat atatatttta tagaaacctc
16620 gtggagtccc tagagtgtag caagtagtca acgtccttca gttaattttc
ttcttccagt 16680 agaatagcaa ctcaaggatc gtgtaaaaga caacatgagc
taaatgggac cttttcagag 16740 ggcaaatttg aatgctgtat ttgtttgcta
gggctgccac aacaaaatac tacagaatgg 16800 gtggcttaac aaacagaaat
ttattttctc acagttctgg aagctagaag tccaagatca 16860 aggtttgatt
tctcctgagg cctttgtcct tggcttgcag atattgcctt cttgctatgt 16920
cctcagatgg ctttcctcta tgcatatgca tccctggtgt ctctgtgtgt ccaagtctct
16980 ttttatttat gtattttttt gaaacagggt ctcactctgt cacccagcct
ggagtgcagt 17040 ggcgagatca tagttcactg cagtgtccaa ctcctgggct
taagtgatcc tctcccctca 17100 gcctcccaag tagctgggac cacaggcatc
catgccacca cacctggctc aaatgtcctc 17160 ttcttataag gacattattc
atattagatg agggcccacc ctaagggcct catttaacca 17220 taattacgtc
cttaagcacc tcatcctaaa tatagccaca tttggtggta ctgggggtta 17280
agacttcaac acatgaattt tgggtcacac atttcagttc ataccaaata cagtgagcaa
17340 gtaaattgat ttaaaaatac tgttttatat atatatttaa ctttagatag
gctctctcta 17400 tattgcccat gctggtctcg aactcctggg ctcaagggat
cctcctgcct cagcgtccca 17460 aactgctagg attgcaggcg tgagccacca
cgcccagcca gtaaatggat ttttaaaata 17520 cgtaaaatta tctgcaagtt
ctctcacttt gtgctccaaa tgttgatctt attacctatg 17580 aaacaaaaca
aaacaaaacc ttttccgcaa ttagtgggaa catttgaatt gcaaagaaat 17640
agttctttaa gtgcctaagg actagttagc atatcttagg caattagacc cctggggctt
17700 ggatgtttgc tggacaactg tgcctgagaa cagagagcag gcacctccct
agtgtgcaga 17760 gggccagcag tctgcagacc gcggctgtct atatttggag
aaacaacaat gagaatgtca 17820 ctctagaaag aatgaagatt ctctgatcta
aaagaccaac tgcagtcaag cagggaagga 17880 aaacgaaatg ggataaatag
ctattatgga taattaaagt cctccaactc ctaagaaatg 17940 agttcgtttt
tcttctctta ttcttaaata actttctcgt ctcctcccct ttttataaag 18000
ccttttttct gggcaggatg aatagatcct taaccctgtc tgtaagtgct tcaagccagg
18060 agtgatgtct ggaattgatc caccaattcc attcagttgg acaaggattc
attgcttcca 18120 ggcacgatgc tgaacatgga gaataaagat gagttggaaa
tggtcctggg atcagggaga 18180 ccttcattca tatatggaca caaatcagtg
actttttttt tttttttttt tttttccgag 18240 acagagtctc gctctgtcac
ccaggctgga gtgcaatggc accatctcgg ctcactgcaa 18300 cctccgcctc
ctaggttcaa gagattctcc tgcctcagcc tcccgagtag ctgggattac 18360
aggtgccagc cactatgccc agctgatttt tgtattttta gtagagacgg ggtttcattc
18420 accatgttgg ttaggctggt ctcgaacccc taatttcagg tgatcctctc
gcctcagcct 18480 tccaaagtgc tcagattaca ggcatgagcc actgtgcctg
gcccaaatca gtggctattt 18540 acttagcacc tatgctgctg aatgaaaatg
actctaactc catgtgagaa gtgttctaac 18600 agaggaatgt ataaaatgcc
aaggaaacac cagggatggc agagacccta acgttcaggc 18660 aatgtctatt
catttattgg tgataatgtg ttagtctttg tagggtcggc tcatgtatct 18720
ctgtgagata aatatttatt gtacagaaga ggatatgtga gattcagaga ggccaggtta
18780 tttgccccca agtcacacag ctcgcgtatc agtggcagag ctggaaatca
aatccaggtt 18840 atctgactgc ccagaagcct ggtgtgttcc atgatacagg
gtgagggggt tctgtcttcc 18900 tctgtgagct aggctataca agaaatggcc
tgctatttga atgcttttaa aacaaatcaa 18960 atctggtcag gcatagtggt
tcacacctat aatcccaaca ctctgggaga ctgagatggg 19020 tggattgctt
gaggccagaa gttccacacc agcctggcca acacgctgaa accctgtctc 19080
tactaaaaat acaaaaatta gccgggcgtg gtggcctacg cctgtaatcc cagctactcg
19140 ggaggctgag gcacaagaat tgcttgaacc tgggaggcgg aagttgcagt
gagccaagat 19200 tgcgccactg cactccaacc tgggtgacag tgcaagactc
cgtctcaaaa aaataaataa 19260 aacaaaacaa atcaaatctg actctgagcc
ccctgcctgg gggaagttag atttctgttc 19320 attttgatgc tccccttttg
ccacagcaat attatgcaaa ggactcacaa acaactcagg 19380 aggtcctgct
aattattgat cctcatttgc tcctgagccc atgatccctt gaagtggtgg 19440
ctcagctgcc actttgggca aagaaaagtg agatcctgtg ctcagacccc tccccacagc
19500 tcctgatatc ccatctccaa ctggagagct gctgtgaggg gctggcttca
ggtcagccag 19560 ctgtaggtcc tgcttcttgt ggagcccaca gctccttctt
tcagggcttt ccctttgatc 19620 gttactttcc ccttctttct ccccatctcc
catactgtat gtcttccctc tggaaagtct 19680 cgggatgtct aagatgacac
tgtgcacaca gagggtgctt gtgttggttc aggtcttcca 19740 agaaagcaga
taccaagaca ggactcggca catacgagat atggtctcgc tctgttttcc 19800
aggctggagt gcagtggcac aatcacagct cactgcagcc tcaaactctt gagctcaagt
19860 gatcttcctg cctccgcctc ccaaagtact tggattacag gcatgagtta
ctacacctgg 19920 ccaagagatt tattgaggga aaatggggaa ggagctggag
gaggctgggg gagcattcaa 19980 actgctacct gtgtaggaga gagggaagga
agaaaagcta ggtgggaaga ctttcagact 20040 atattacaat actgggacat
tttggcatgg ccagtgcaga gtcctagagc cagtcgctgt 20100 cagaggagtc
ctgcctctgg caggaaagaa cggcctcaca tccctgcggt gctcagttct 20160
tggcagaata acagcctgtg agaaagaggc gctgtcccca cgccaaatgg gtggttgatt
20220 cagagcacag cagctggggc tgtctgcaat taagcagtgc aaagctccac
agcgctttca 20280 gttttcatta gccttcatct aaagcatctg catgtatata
gagagcgcta agcttatgac 20340 tggtgacact ttattaatag caatagtgat
agtacttacc acttattaat ataaagcact 20400 ttttacgtac caggcactgc
cgtgaatcat ttacatgcat caatcattga acaaccctat 20460 gagataccca
ttacgattag cccagtttag agatagggat tcttatgggc tgaattgtgt 20520
cttcatatgg atctgcccaa attcattatg gtgaaattct aaccctcagt acctcagaat
20580 atgagtatat ttggagatag ggtctttaaa aaggtaatta aggttaaatg
aggtccttac 20640 ggtaggccct aatcgaatat gactgatgtc cttatatgaa
gaaaaaattg ggacacacgg 20700 atacatagaa ggaagactat gtgaaggcac
agggagaaga gagccatctg caagccaaaa 20760 agaaaggcct cagaagaaac
caaggcctgc tgaaacctgg atctcagatt tctggctcta 20820 gaattgtagg
aaaatacatt tctgttgttt aggccaccta gtttgtggtg ctttgttaca 20880
gcatccctgg aagactagta gaaggtcaag taacttagcc aaagtcacag agctagcaca
20940 agggagagat agcactgggc atctctcagt ccagagtcca ttctcttccc
ctgctcttct 21000 gagtcatgat ggctgcgcaa ggactacaaa gtaacaggta
cagatgacaa agtgactcag 21060 gaagatcatt gagaaggagc atggcctggt
gtgctgggaa cacacaggaa agtggtccaa 21120 ggaacctaga cagcaaagga
gaagggtttc atatcttgcc tctacccact aagggctgtg 21180 tgaccttggc
caatttgttc ttgctttctg aactacagtt gtattttgtg tcaaatggga 21240
gtattagatt tcccatgtct cactgagctg tattaatgat caaataagag aattacatga
21300 aagtatctgt agaggagggc agagggagag aactgaattt gcctcataca
atattactgt 21360 ggttgttaca tattatcctt gttttagctg ctaggaatat
actattatag taatgtgtca 21420 atattagagc atcagttttc tttcttttct
tttctttttt ttgagatgga gtctcactct 21480 gttacccagg ctggagtgca
gcagtgcaat ctcagctcac tgtaacctct gcctccaagg 21540 ttcaagtgat
tctcatgcct cagcctccgg agtagctggg actacaggtg ctcgccacca 21600
tgcctggcta atttttgcat ttttagtaga gacggggttt tgccgtgttg gtcagtctgg
21660 tctcgaactc ctgacctcag gtgatctgcc cacctcagct tctcaaagtg
ctgggattac 21720 aggcgtgagc taccacgcca ggcctagagc atcagttttc
catcctactt aagttacacg 21780 tatttggttg ccagaaattc atggagacta
ctagggcagc ccattataaa gtcctatcat 21840 ccaactgcct ctcagagcta
atggcatcaa tgctaagtct agcatcatag actcattaag 21900 tgacggtgag
gattaacgta ataaaaatag ctggtatatg ttgcttttta ttatgtggca 21960
agttctgttc taaattacct aagtttgata actcatttat gacaatccta agaacaaccc
22020 tatgaagaag aaactattat aattcctagc ttacagatga agaaactgaa
gtccagggag 22080 tttaagtaat taggctaaag tcacacagct gagtaagtgg
gcgactcaac attcaaagta 22140 aggtacatga gctcctcagt tggacataga
ttggagaagt gaggcatcca agatggcttc 22200 aagatatata tatatatata
tttttttttt tttttttttt ttgagacgga gtctcactgt 22260 catgaagact
ggaatgcaat gccgctatat cagctcactg cagcctccgc ctcccagatt 22320
caagtgattc tcctgcctca gtctcccgag tagctgggac tacaggcgcg tgccaccacg
22380 gccagctaat ttttgtattt ttagtagaga cggagtttgc catgttggcc
aggctggtct 22440 cgaactcctg acctcaggtg atctacctgc cttggcctcc
caaagtgctg ggattacagg 22500 cgtgagccac cgcgcccagc ctgatggctt
caagattttt gctggagcaa ccaaagtagc 22560 aaaattgtca ttacttatga
tgagaataac ttcaggaatt aatttttttt taggggaagt 22620 cagtttggac
atgttaagtt taagctgcct tttaggtgtc caaggagatg tcagataagt 22680
ctagttataa agattgggag ctgttagcat atacatggta tctaaagccc agagcctgct
22740 tagatgtcca gagggcatag acagaaagca agagacccga gaatggagtc
ctaggcattc 22800 tagtgtatat aggttgaggt aagaaggaat cagctataag
agataaaaca gaagaattag 22860 gaggatgacc aagtgttttc ctggaaaaac
ataaaatggc caagaaagag aaagtggtca 22920 attgtatcaa atgctgctgc
taggttgatt aaatcagatg aggactgaaa atgacctttg 22980 gactgagcca
tgaggagggc attgataacc ttaagtaggg cagttttggg ggctcaggtt 23040
gggaatacct ggctggagtg ggtccaggag agaacaggag gagaggaatt gaagacagtc
23100 atttctttct taaaaaaagg aaaatgagaa ataggaggat aactgaaaga
gaaaatgtct 23160 tttattttag attctaatat gggaggaata aaagcttatt
tataggcaac aggaatgatc 23220 tattatacta gggaggagaa cataatgaat
gaagcgtggg ggtggggatt tctggagcaa 23280 tattcgtgag gggataaaag
gggacaagat ctagtgtcca gggaaagggg ctggacttag 23340 ctagaagcat
ggacaactgc atagacccca tcagtataaa tgcaggccgg caggtaggta 23400
ggtacattgg tagggaaatg gttaggttct tttccaattg ctttaatgtt ctggcacatt
23460 tactaagctt ctactctggg ctcaccggtt gaaattcaaa gctccttccc
ttgttctacc 23520 attgcttttc actttgattt caataaaacc cacatcatcc
agtaattata gctgcttgta 23580 tatgtgtctt tcttccccat cagcctaaga
gctggaagaa ggcagataat atgtcacgtt 23640 gtctattgct ccccaatact
tagcccagta cctgagacac agtaggcgct caatatatat 23700 ctgatgaact
gaattgaatc cagtgtattt gtttctctat acttgtgccg gaaatttgat 23760
ttccttgagt cataagaacc tgccaaggtg ccgggggcgg tggctcacgc ctgtaatccc
23820 agcactttgg gaggccaagg tgggcggatc acgaggtcag gagatcgaaa
ccatcctggc 23880 caacatgttg aaaccccgtc tctactaaaa atacaaaaat
tatctgggtg tggtggcgca 23940 tggctgtaat cccagctact caggaagttg
aggcaggaga attgcttgag ctagggagtc 24000 agagattgca gtgagccgag
aatcgtgcca ctgcactcca gcctggcaac agaccgagat 24060 tccgtcccca
aaaaaaaaaa aaaaagaacc tgccaaggtt atctttcata tgaacttgtg 24120
ggcaaatgac ttgtgtttta tccaaactat tgggttaacc attatattag ctatttatca
24180 ctgcatttaa tatttatgaa aacttgcaag ctttaattat ttttaaaaag
acttggacct 24240 taagtgggcc atgacagtat cctcagaaag atgacaataa
gtaagaggat acaacttcct 24300 ttataattga cagatagggt tccgtttgtc
caattacttt ttttttaaaa gaagagataa 24360 attcactgta atgaatgtgc
cataattgga atctatagag gtctaccatt tgaataaaag 24420 gtgctggatg
atcacctcct tagaggaacc atctaaggag aaaaggatat acaaccaaat 24480
gggtgtgcat tgtgatagaa aatgtccctc tccacctcca cttagtattt tattaagact
24540 tagaaaaatt aggccgggca cagtgcctca cacctataat
cccagcactt tgggaggctg 24600 aggcgggcgg atcatctgag tcgggagttt
gagaccagcc tgaccaacat ggagaaaccc 24660 cgtctctact gaaaatacaa
aaattagcct ggcatggtgg tgcagacctg taatcccagc 24720 tactcaggag
gctgatgtga gagaatcgct tgaacctggg aagcagaggt tgcggggagc 24780
cgagatcgtg ccattgcatt ccagcctggg caacgggcaa caaaagcaaa actccgtctc
24840 aaaaaaaaaa aaaaaaaaag acttagaaag gttaaggtca actgtatcag
ctgggtcgag 24900 caatgtgaac aaagtctgtc aatgctcttt cagcaggaaa
tgcagtatag catattgttt 24960 tagacataga ctctggactt gggcctctat
cctacctcaa atgacttagt ttcctcatct 25020 ataaaatgac atgatgacac
tgtctacctc atggggttgt tataaaattt aaatgattga 25080 ttgaatgttt
ataaaagtcc cacacaatac ccagaacatc agtagtttta gccactataa 25140
cttactttaa taataataat aatatttaat aataataata acttacttta ataataatag
25200 taatacctcc atagtattct actatgggtc ttcctttttg tttttcatct
gctggtacct 25260 tttttctttt tgcttagtat actttctttt tcctttaatc
ctggctttta ttttctgcct 25320 atcctttttc ccatgtagaa aaatcgatgg
gacaactgag gaagaagata acattgagct 25380 gaatgaagaa ggaaggccgg
tgcagacgtc caggccaagc cccccactct gcgactgcca 25440 ctgctgcggc
ctccccaagc gttacatcat tgctatcatg agtgggctgg gattctgcat 25500
ttcctttggg atccggtgca atcttggagt tgccattgtg gaaatggtca acaatagcac
25560 cgtatatgtt gatggaaaac cggaaattca ggttggtatc agtccatggt
ggaagacttt 25620 tctttttgag acagggtctc gctcggtctc ccaggctaga
gtacagtggc acgatcttgg 25680 cttactgcag ccccaacctg ccaggttgaa
attaacctcc catctcagca tcctcccatt 25740 tcagcatctc agataagtag
ctcctcccat ctcagcatct cagcatctca gcatctcagc 25800 atctcagatc
agtagctgag actacaatcc tgaggaaact gttgactgca gctgtgtcaa 25860
tactttgctc cttgagagaa agccctgcaa ttccttcagt gatatgacaa aaatggagag
25920 tggctacttg tgctgggcat tgtgcagaat gatggggata gaaaggtgaa
tgacctagac 25980 tgagccctgt cctcatggag acaagtaagt gatgacagtt
tgagggggta ggtgccacgt 26040 tggaggtaca caggattctt gggctcatag
gagagggcac agcccagact tccctattgt 26100 gaacaaattc ccaaagtgat
ggctggacca ggcaaagagg gtgtggtgtg gtgggaagaa 26160 gaatgtttga
agaaaaaggt actgtgaagg actgtaagaa agagacagag agagagagag 26220
agagagagaa cgtacacatg ctatgtaggt atattttagg aactgaaaca ggagctcatc
26280 atcttttctg tgtcatggac tcctggagat gactaatgaa cctttgccaa
agtaatgttt 26340 taagttctta aaataaaaca caaaggatga caaaagaagc
caattatatt aaaatataaa 26400 taccaaaaca tttaaaaatc acatttgtga
catagaaaca tatgggcttc tttagtagta 26460 catcagtgac aaaatctagt
attgggtcta acatttactc tgattttaag ttggaatgta 26520 tgccattgtt
ggaaatagtg gccatgactg taatacgatt tgaacatatt tgctatttcc 26580
acgtgggaca cagtcatagg tactagtcat atgacggtgg cttgttgcct acattcataa
26640 tggcagaaaa tgctaaattt tggttaagag tgaaaataaa gatgcatgtt
ttcttcccat 26700 ccaagttctc agatgcacag gattccatcc acagactcca
ggttgagaac tcccagtgat 26760 tgggtagagc acgttgaggt ggaggcagcg
aagtaaatag ggggctgatc atccatagcc 26820 tggtaggcat gtagcaaggg
gctgcaagca tggaatgatc acatctgtgc tccagattgt 26880 tcactgcccc
attgcagggg gccagattga ggtaagatag gaatggaggc cacagggcca 26940
gttcagaggc catcatagtt ataagcaagg atacttcgaa gtgacttaaa tagtattgtt
27000 ttaggaatca ctggaaacat aaaatctggt ttgctgctta aacgatagac
ctagagaagt 27060 actgaggtta tggggtaaaa gaaacaaaca aaaatgtctg
cccagtggac accccataaa 27120 tgcatgtttc atcgtactaa actcacacac
tgcaatgact catgcagaaa tccgttcatc 27180 tgcagagaga catttaatag
ttctctggtc cctccctcta tttgaagaaa catttagatc 27240 acagtttttt
gaactagtgt ctgggaaatc actgcactgc aggctgtgcc atgaagaagg 27300
cagtgcgaga cctggagccc atactgtgct gtgtcttatg agactttcca agagggagac
27360 gtggtaggca atattttctg gactgacttg atcatagaat gctctctttc
atgccatatc 27420 tattagcatc atctggcaca gtctcctgcc aggcactggt
ttgagaaaat ttgatttcaa 27480 tctgtcaaaa gaagtcttta gttggtctgc
aagctatttg tttttgcttt tttcaaacca 27540 agagattatt ctgccagagg
aaaacagcac catggagatc ctcctaacta gtctctattt 27600 gatgccacag
ccaaatctgt cctaaaagga tatcctgtct tttgtggggt gtgggggata 27660
gaggtagaag ggcatatcat gcgtttttaa aataaagaat gatgtatatt agcaaggttt
27720 cagatgtgta tcacatgcat tctttcagcc ttttgtgagc aagaccagct
aattaaaact 27780 tgtctgctga ggcccagatc aaaatgagat gctgttttgc
atttgtttgt tgcctgaaaa 27840 gatagacctt ggtcaataga gtctgctctg
aggcatatgg aaaagacatt ttgattaacc 27900 cgaggaacaa tgctagtgtg
cgctctctag tttctacggc tgtgccctct ggagtcttag 27960 agaaactgat
taaaatctga aatatggttt aaattttttt cctctggact caggagtagg 28020
aatttagtat cagtaactct agtacagctc taatttatag cagattattt ctcttgtccg
28080 cctagaacaa agcttagata tcaagtgagc atgttcaacc aaatgacaaa
tactttgcta 28140 attgtattaa gaaaggctct gaatggctgg tatgtttgtt
tggtttttct gttttaaggg 28200 aaaaactaga tatttggcac tgagatatct
ttaaatcttt atttcaaaag aaggagagaa 28260 ataagcagta tgaataggta
gatctttcaa atatgtggca tatgttctac aaggggtatg 28320 aagagtgatt
ttaactaaag cgtgaacact tttttttttt tttgaaacgg gatctctgtt 28380
gcccaggctt tagtgaagtg gtgtgatcat agttcaccgc agccttgacc tcctgggctt
28440 aagtgatcct cccacttcag tttccaagta gctgggtcca caggctcatg
ccaccattct 28500 tagctaatta aaaaaaattt tttttagaga tgggatcatg
ccatgttgcc caggctgatc 28560 tcaaacccct ggcctcaagg gatcctcctg
ccttggtctc ccaaagtgct gggacaagca 28620 tgcaccactg tgcctggccc
atattttaaa tttaatagtt atgagttaaa acatgtgaac 28680 tcttagaaaa
gtgtttggca tatagtaaga aaataaaatg accgaagttt gagaaacttg 28740
tgattttgtt ttctcattac tctcaggaaa agtccaaagt tcttcccatg gattgtgggc
28800 cctgtaggat tcagagcatg ggctttggaa ctggccagac ctggttttaa
tgagctctgg 28860 gaccttgaat aagttgccct tgtgtcctgg tcagagattg
ctggttgcga agaaatgtgc 28920 agtgaaactg gctcgagtta aaaggggatt
attggggccc ggcatggtgg ctcatgcctg 28980 taatcccagc aatttgggag
gccaaggtgg gtggatcacc tgaggtcagg agttctagac 29040 tagcctggcc
aacatggtga aaccccatct ctactaaaaa atacaaaaaa tttggccaga 29100
catggtggcg cacacctgta gtaccagcta cttgggaagc tgaggcaaga gaatcctggc
29160 agttggaggt tgtagtgagt cgagatgtgt gagactccat ctaaacaaac
aaacaaacaa 29220 acaaaaaatg gtagtgggga ttattgtagg gctgtaagag
gatctcgtga aagccaaggg 29280 cagaaagcag gtctgtggtg tatgtgtgca
gtctgcaccc aggacgcaga agccagcctg 29340 aggtggggct gaaacccagg
ctgtcctcca ccctgaggag ggaagggagt ctttatgtaa 29400 ttctttctga
ggccgcagga caggccctgc cagaagtgct gaatggagct ttccctcgtg 29460
ggaactagag aagcctttgc taaggtctcc agcttgcttg ccccacagag tctttcattg
29520 gcttttcttg gagtcagctc cgttttccct ggtccttcat ggactgcttt
ctttcctctt 29580 ccctggcttc tcactgccct ccacagtgga agtgccttga
gcctttgtct tgctaggaag 29640 ctgatttact tggccctgac tctgtgactc
cgtgggactt atttgggttc aagagtgcac 29700 tattgtctaa ctagaatctc
tgtgggtttg ggttgctgtc tctctctctc tctctgtgtg 29760 tgtgtgtgtg
tgagagagag agagagagag agaaagagaa agagacagag acacagagag 29820
agggagaggc tgactggctg agcctagcct atggctttgc tgtcttaaac attttttttt
29880 tttttttttt ttgagacaga atcttcctct gttgcccagg ctggagtgcg
gtgacatgat 29940 ctcagctcac tgcgacctcc acctccccgg ttcaagcgat
tctactcctt aggctatcaa 30000 gtagctggga ttacaggtgc atgccacaac
gcccagctaa ttttcgtatt ttaaaaatag 30060 agacgaggtt tcaccatgtt
ggccaggctg gtcttgaact cctgacttca ggtgatctgt 30120 ccaccccggc
ctcccaaagt gctgggatta caggcgtgag ccaccacacc tgactggctt 30180
ggctgtctct actcaggtgt ccagtcagct gtggtagtca gtcggggaga atcccatgtt
30240 gcgggggaag gtgcaatcct ctcagaagtg tgagcagaca ggaactgaca
tttctagaag 30300 ttccttgcta accctcattg cccttattgt gaaatgggaa
taaaaggact gctttgaaga 30360 tcaaataagc taacctatat taaataccta
tattagttcc ctaaggctgc cgtaacatat 30420 taccacaaac ttgatggctt
aaaacaatag aaatttattc tctcagagct gtggagaccg 30480 gaagtctaaa
tcaaggtgtt ggcagcacct catgccctct gaagactcta gcagagaatc 30540
tttccttgac tcttctagct tctagtggct gcagcagatc ctcggtgtgc gacaatgtca
30600 ctctcatgtc tgcctccatc ttcacgtgga catctttctg cgtgtctcct
cttttgtctc 30660 aaatctccat ctgtctttct cctataagga cacttgtcat
tgggtttagg gcccagctgg 30720 atagtccaga tatctcattt taagattctt
gacattttca catcagcaaa gacttgtttt 30780 ccagataagg tagcatttat
aggtcctggg gatttgatgt ggatatcttt tgggggccat 30840 tttttggcct
ttcacaatat ctgacacagt gtttggttta ttatagtgat ggtccatata 30900
cagggccatt tttttaaaaa tttataattt taaaaaattt tattgtgata agaatgctta
30960 acatgagagc tactgtttta ataaagtttt tagtgtacaa tacattatgg
ttgactctaa 31020 gtacaatgtt gaatagcaga tctctagagc gtgttcattt
tgcttgactg aaactttttc 31080 ccattaatta gtaactcctc atttccccct
cccccagcac ctgacaacca tcattctact 31140 cttcaagtct atgaatttga
ctattttagg tatgtcatgt aggtggaatc atgcagtatt 31200 tgtctttctg
tgactggctc atttcactga gtgtaatgtc ctccaggttc atgccagttg 31260
ttacatcttg cagaattttc ttctttataa aagatgaata gtattccatt ggtgtgtata
31320 ccacatttcc tttttttttt ttttgagatg gggtcttact ctgtcaccca
ggctggagtg 31380 cagtggcaca atcttggctc actgcaactt ccgcctccca
ggttcaagcg attctcctgc 31440 cccagcctcc tgagtagttg ggattacagg
catgtgccac catgccaggc taatttttat 31500 atttttagta gagacggggt
ttcaccacat tggccaggct ggtctcgaac tcctgacctc 31560 aagtgatcta
cccgccttgg gctcccaaag tgctgggatt acaggcatga gccactgcgc 31620
ccagccacat tttctttatt catctgtcaa cgggcattca ggttttttcc acgtcttggc
31680 tattgtgaat aatgcttcag tgaacatggg ggtactaata tctttttgga
tcatgatttc 31740 aactcttttg gataaatacc cagaagtggg attgctaagt
catacattcg ttctgttttt 31800 aagttttgga ggaacctctg tactgtttcc
atggtggctg cacccattcc caccaacagt 31860 atataagggc tttattttct
cttcatccgc accaacactt cttgtctttt gtttttgata 31920 atggtcatcc
taacaggtat aaagtgacgt cttatggtgg ttttgatttg catttccctg 31980
atggttagtg acattgaccg cctcttcatg tagatattgg ccatttattg gtcttctttg
32040 gagaaatgtc tattcaagtc tttagtccac tattatggtt ttaatgggtc
tcaaatgaca 32100 atgaaagtca gttctcagca gcctaggggc tcttcttcat
gtattatttc tttcagagat 32160 tgacagaagc actatttccc cagagagaaa
ggcatgagaa agggatgttg tgattgacaa 32220 ttagcagctg gttgaagtgg
gagttagaga aagggtctag ttctccctct gtcttggatc 32280 ctcaggtaat
tctgtggatc tgggcaaaga agtcttgtct ctccttagtg agaaaattaa 32340
gtctctccaa gcaatagaaa gaatatcgtg ttttggggtt aggcagatga gaggttttgt
32400 gtcccctttt ccttgcaaat agttgtatga ccttggacaa gtaaactaat
ctctctaagc 32460 cttagtttcc tcatttgcaa ttacctctag gtgttttaaa
gattaaagga ggaaatctgt 32520 agaaagcacc ttagtgaaat catattccac
ctctgctcaa attttccaat ggttttcatt 32580 tctctttgtt taaaagccag
agttccggtg atgtcttaaa gaacccttca tcattgtaac 32640 ctctcttgca
ttaacaccta ttctcttcct cctcattcat taccctccag ctgtactgac 32700
atactgcttt tcctctaaca cgcaagacac aaccctacct tgggtccttt gtacttgctg
32760 tttctctgcc tggaaagctc acatctcaaa tgaccatatg acttgctccc
ttcctttctt 32820 taggtcttta cttaaaactc atcttctcag tgaagacttc
cctggccgtt ctatctaaaa 32880 tttaccccac cacactgcca tccaacactt
catattccct tcccttcttt attttttcat 32940 cttattgctg gttaccatct
aactctgcct gtaattgttt atcacctgct atctccactg 33000 gcatcttcaa
aatggcagga gtcactacag ctgttcactg ctgtacccca gtgcatagaa 33060
ctatgcgtgt tacacaataa acacaaaata cagatttggt gagctgattt gaattaatga
33120 tagctagcta gttccttttt accattgagc ttcaactttc taatccgtaa
aatgagaaat 33180 agagagtata ggccaaagtg gcttggactg tgagctccta
gaaggcaaag acaatgcttg 33240 tttgagtctg tatttacact gtccagcacc
taacattgca ttcaggaagc acaggacgaa 33300 cgttgaacag atgggcggat
aaatatgtaa taacttgtga caggaaaata agataagcag 33360 tgatgaaaat
ttataaaaca tagtatgttg ataattagga accctcttac tccatatttg 33420
cattttgata tcaaaaagct ttacaaagcc attcatttat tcattcattt ggcgaataca
33480 cacttgtacc ttctatgttc ccaggcgttt gatttaggta ctaagactat
aagtcaaaca 33540 ggacatggct gctatcttag agtttcttgg tgccctgtgt
gggaaattga catgtggatg 33600 tccattcact gaagacagca ctgtgttggt
gccatggctg tgggaccaaa ggtctgtaag 33660 acaagcccaa gaaaaaggga
ccagttcaat tttcgggatt caggaagttt cctctgagga 33720 aggaccattt
acaatgagtc taaaaagaat gagttacttt acctgggaaa gaatatgagg 33780
aaagggattc cagccctaga gaatcacatt ttcaatggcc taagggttgt ggaaggttgt
33840 gttgttgcta ttgccatcag catagtatca gtaatggctg ctaacattta
ttgagtctac 33900 actgtgtgcc agtcactatc ctaatctgtt acatgcaaaa
tctctaagca gagagataac 33960 ctactagaaa tattcatgcc atttatcccc
acacatccta tggataggta gaatgggctt 34020 tattgtcctc attaagaaat
gagagactta agactctaat tctctttgtg ctatcacaaa 34080 actggcatct
gaataatgta gtaaataact tagtagcccc ccaaaacccc attttttgtt 34140
ttattcacaa gctattttat tttctcctta gcattcattg ctattttgtg ttttttctct
34200 ctgtgtatat acatatatac acacacatta tatatattat atatatatag
agagagacac 34260 acacacatta gatatatgta tttttagaga caggagcttg
ctctgtcact cccactggag 34320 tgcagtgtgt gtttgtagct taccttaacc
ttgaccaact cctgggttcc agggatcctc 34380 ccatctcaac ctcctgagta
gctaggacta caggcacaca ccaccacacc tggctagatt 34440 tgtattatta
ttattattat tattattatt actattgaga tggagtctct ctcagtcacc 34500
caggctggag tgtagtggtg tgatcttggc tcactacaac ctctgcctct tgggttcaag
34560 tgattctcct gcctcagcct cccaagtagc tgggattaca ggcgtctgcc
accacaccca 34620 gctgattttt atatttttag tagagatggg atttcaccat
gttggctagg ctggtctcaa 34680 actcctgacc tcaaatgatc cacccacctc
tatctcccaa agtgctggga ttacaggcgt 34740 gagccattgc acctggccta
gctggctaga tttttgattt tttgtagaga tggggtctcg 34800 ccacgttgcc
caggctggtc ttgagctcct ggcctcaagt aatcctcttg cctaggcctt 34860
ccaaagcatt gggattacag gtgtgagtca ccatgaccat taatataaat acatatatat
34920 ttaaatttgt acataatctc ttattacaag gtgaaatcta tgagagcagg
gacttttgtt 34980 tgtttgtttc attttttttt tttgagatgg agtctcactc
tgttgcccaa gctggaatgc 35040 agtggtgcaa tctcagctca ctgcaaattc
catctcccag gttcatgcca ttctcctgcc 35100 tcagcttcct gagtagctgg
gagtacaggt gcccgccacc acgcccggct aatttttttt 35160 gtatttttag
tagagatggg gtttcaccgt gttagccagg atggtctgga tctcctgacc 35220
tcgtgatcca cccgcctcag cctcccaaag tgctgggatt acaggtgtga gccaccgcac
35280 ccggccggtt ttgtttttta agatggggtt tcactctgtt gcccaggctg
gagtgcattg 35340 gcactatctt ggctcactgc agccttgacc tcctgggctc
aagccaggag gctcaagcca 35400 ggctgaggtc ccacctcagc ctcctaaata
actgggacta caggcacaca ccactacgcc 35460 tggcccagga cttttgcttg
ctgctatccc caagtatgta agatgccctc cataagtatg 35520 tgttaaataa
atgaaaaaag aaagacctca tgaggtaatt attgtgtagg ctcattggta 35580
aaaaatggtt gtcagccttt ttctaacaaa cacaactata tctgatttct catttccaga
35640 cagcacagtt taactgggat ccagaaacag tgggccttat ccatggatct
tttttctggg 35700 gctatattat gacacaaatt ccaggtggtt tcatttcaaa
caagtttgct gctaacaggt 35760 aagataaatt gatataacat gatacaaacc
aatgaaatgt ggctttgtac ctataaattc 35820 tgcatagctg gctctcaatt
tgggggtgca gaatgaaaaa caggagccat ctggatagat 35880 gcaattcaca
gatactgatc ccaaatgacc ctgatcttaa tttattttta tttttatttt 35940
tgagacggtg tctcactctg tcacccaggc tggagtgcag tggtgtgatc ttggctcact
36000 gcaacctctg ccccaccccc tcccccaccc cactcggcat tcaagcaact
ctggttcctc 36060 agcctcctga acagttggga ttaaaagtgt gcaccactac
acccagctaa cttttgtatt 36120 tttggtagag acgaggtttc accatgttgg
ccaggctgat ctcaaactct tgacctcaag 36180 tgatccaccc gccttagcct
cccaaagtac tgggattgca ggcgtgtgag acaccagcgc 36240 ccagtcaaga
gtttcttttt atttcgtttt tcatccaatt aaatttacct tgcaactctt 36300
caagtgatta tgtggtaaaa agaccaatca actctgagtc aggagaaatg gttcctgccc
36360 cctaactgga tcactgggtg acctctattg agtcactttc cttccctccc
tgggcctcag 36420 tttcttcatc tgtgaaatga aacattggac tagattgtat
ttcagttccc cttgaccagt 36480 gacattctgt aatcttaggt taatatcacc
cagtaccata aaggttttct cagatgagtg 36540 gtgggggctt gcctctagac
tgcaagatgt gtctctaatg tccttgagac tctgtagtgg 36600 gtgtttgagc
aattaaaagt acccaagaac agagtgagct gtctcaagaa gcagtgagtt 36660
ctctgtcacc ggaggtattc aagcagagga ggatggccac ttgggggaga tgttgtagaa
36720 tgtatcatgt attagaaaag gagtgaacta aatctctcca ggtcgcttcc
aattgtattt 36780 cccatatgac tttccataaa tggatttcat gaagtgcatt
ccatttttaa aaagtggttt 36840 tttttttcaa attctaaagc acaactcatt
agatagttgt gaaaacaata taatgattta 36900 ccaatgagca tttttaaaaa
agagagaaat agaagaaaag ataatcaaat agaataaaat 36960 agaaaatatc
tgaatgcatt gcacataagg gtaaatattg gtttttgaga cttgtttcag 37020
ttacatttgt atgtatgagt atggactggg ttgcaatata aaatatattt tgtttatggg
37080 ttaaggtaaa aaaattggaa gccactacca taagatctaa ataggaataa
gcatatattt 37140 atttaggttc ttgtctaatt tatgtctttt atttattgtt
agttatctat tgtattcttt 37200 ttaaaaagtg ataaaatatt ggttgctatg
gtttcctggg ttaccgctta cacctcagcc 37260 ttgaaaaaaa atcacacata
atctaatttc ccagcacata aaaagagtgg aaacatcatc 37320 aacataagtg
agaggggaag aaaatgctgc ttgctctctt ttcccagggc accctgagct 37380
ggccaggaaa tgggagctaa gacaggtaca caacctgtct tgtgcttggc tggtcccagg
37440 acatacaatg cttcttggat agtcagtgtt tctgactctg ggaagcagga
aacaacctca 37500 aacatacagt aacagtcaga aaagatcagt cggtggggaa
ccaggcagga tggtaggtct 37560 ctagcaagct tacctgaacc tggccaatct
ccaacttttc aggacatcat ccaggcagga 37620 catccctgtg ccaccaaaaa
tttgttcata gttggtccag gggccagagc ttgggaatca 37680 aagaagccca
agagtctagc ttggggtgca ctagacccta acacatctat ttctccaaat 37740
tacaggtgcc agccgccatg cctggctaat tttttgtatt tttagtagag atggggttca
37800 ccatgttggc caggctggtc tcaaactcct gacctcaggt gatccaccca
cctcagcctc 37860 ccaaagtgct gggattacag gtacgatctt tccacaggga
tttccacagg gatctttcat 37920 gaactgttag gtttgtttct ggtgcttagc
tgaagtagca catccatcag cagacctgcc 37980 gaataacaca atgctttggt
cccccagggt ctttggagct gccatcttct taacatcgac 38040 tctgaacatg
tttattccct ctgcagccag agtgcattac ggatgcgtca tgtgtgtcag 38100
aattctgcaa ggtttagtgg aggtaggaga tactttcctt acagtttttg atattgctag
38160 agacagcgca gtcctttaga aaattcacct tctgaagaaa atccccttta
ctcagttttt 38220 ttctatattt tcttcctttt cctgctgttt ccattctctg
gtaatggcta aaattgcaag 38280 aattttaatt aaaatgcctt gtgtgatttt
acatttatga acaataaagt acccttgcat 38340 aatgatctta gagataatct
aacctgaccc tcttcatttt aatagatagt gtaactgaag 38400 cccaaatcta
cagttcacat agcagaggct cattccacta aaacaattta agtggattca 38460
ttaataaatc tgtacatttt caagggtgta gtctgatgca gagatttaat tcaatgaagg
38520 aggcagcatg atatggagcc agaaggtaaa tattttggac tttacaggcc
ttacagtgtc 38580 tgttgcatct actcaaccct gctgttatag tgcaaaagca
gccagagaca atatggaaac 38640 aaatgggcat ggctacgttc caattaaaca
ttttacaaac tgaaatttga acttcatatg 38700 attgttgtgt gccattaaat
attactcttc ttttgatttt ttttctcacc attttaaaat 38760 gtaaaaaaga
ttcttagctt gtgggctata caaaaacaga tggtgaacca attggcccat 38820
agtttgccaa ccctcgatat acagcaatgt ttcccaaaca cagtcattca cctctgacct
38880 tcgccagttt gttatgccca tgtacaactt gtactattat ttgcctattg
ttttccccta 38940 gatcgactca tttaaaacaa aaaacaaaag atacctatta
ctctaagcaa taccatcttt 39000 gaaatcatgg gtttgatgtg ttagttacat
cttttccttt tttttttttt tttttttgag 39060 atagagtctc gctctgtagc
ccaggctgaa gtgcggtggc atgatctcgg cccgttgcaa 39120 catctgcctc
ccaggttcaa gcgattctcc tgcctcagcc tcctgagtag ctgggactac 39180
aggtgccagt taccacaccc ggctaatttt ttgtattttt agtagagatg gggttttacc
39240 atgttggcca ggctggtctt gaactcctga cctcaggtga tccgcccacc
tcagcctccc 39300 aaaatgctgg gattacaggt gttagccacc acacccagcc
actagttaca tctttttcaa 39360 agcatacata tatatagtag aattatatat
aaatttaatt atatatagat taattataac 39420 atatatacta gtgtatatat
gtatatataa tatatacata tatagtatat atataatata 39480 tatagtgtat
atatatactg tatcatatat agtgtatgta tataatatac atacactagt 39540
atatatatta taattaaaaa tgtaagttgt tatatcattt caaatccaac tctagtccca
39600 ctagagggac atatatgaca ctttgggatg tacccgtgta
gtggaaagaa cacgatatta 39660 gcatccatga agactaaatt ttagtcactt
aacagccctg agtctcaggt tctgtatctt 39720 gaaatgagtg gatggaccaa
ctgattgtgg aaggctcttc ctacactgat agtctatgat 39780 aatatgaaat
ataaatataa agaccttttc ccccatctcc taccatgctt acatgtgaag 39840
tgtatttgaa tttcagcatc tgtactgtga gtcaaaatag ctcaatcatg ctgtttagtg
39900 tctgttttag tccatttggg cttctacaga ataccataaa ctaggtaggt
tataaacaaa 39960 agaatttttt tttttttttt tgagacagag tctcactgtg
tcaccgaggc tggaggcagt 40020 ggtgtgatct cagctcactg caacctctgc
ctcccaggtt caagcgattc ttctgcttca 40080 gcctcctgca tagctgggat
aacaggcaca tgccactgca cccggctaat ttttgtattt 40140 ttggtagaga
taggattttg ccatgttggc caggctggtc tcgaactcct gacttaggtg 40200
atccgcccac ctcggcctcc caaactgttg ggattacaag cataagccac tgtgcctggc
40260 cttttttttt tttcagtctc gctctgttgc ccaggctgaa gtgcagtggt
gcaatctcag 40320 ctcactgcaa tctctgcctc ctgggttcag gcgattcttg
tgcctcagcc tcccaagtag 40380 ttgggattac aggcatgcac caccatgccc
aactagtttt tgtattttta gtagagatgg 40440 ggtttcatca cgttggctag
gctggtcttg aactcctggc ttcaagtgat ccacccacct 40500 cggcctctca
aagtgctggg actacaggcg tgagccaccg ctcctggcct agaaatgtat 40560
ttcttacagt tctggaggct gaggagtcaa agatcaaggt gctggcagat cggtgacttg
40620 ggagagctag cttcctggtt cataaacaac taccttctct ttgtctgccc
atggcagaac 40680 ggatgaggga gctctctgga gtttctttta caaggcacta
atctcattca tgagggctac 40740 acccttatta cttagtcact tcccaaaggt
ccatctccaa ataccatcac attgggaatt 40800 aggttttaac ataggaattt
ggtggggaca caaacattca acctacaaca gtgtctgtaa 40860 attgggcttt
tatattgtag cctgtgtgaa gaagcagcat ccatatttta aacacaagca 40920
gaaactacag tcaaatcaac taatctattt tcaactcttc tgccagggtg tgacctaccc
40980 agcctgccat gggatgtgga gtaagtgggc accacctttg gagagaagcc
gactggccac 41040 aacctctttt tgtggtgggt atattagaat cgtaacaaat
tttatttatg aatgcttttt 41100 ttgggttcat gcagtggctc acgcctgtaa
tctcagcact ttagggaggc cgaggcagga 41160 ggatccctgg agcccaggag
ttcgagatca gcctggacaa tatagtgaca cttcgtcttt 41220 aaaaaaaaaa
aaaaaaatta gccgagcatg gaggtgtgtg cctgggatcc tagctactag 41280
ggaggctgag gcaggaggac tgcttgagcc tgggaggttg aggctgcatt aagctatgat
41340 ggccacagca ctccagcctg agtgacagag tgagaccttg tatctaaaaa
gaaaaaagaa 41400 aaaagaaatg gaatgctttt ttggcttcaa gcaactgaaa
accctactaa gggccttaaa 41460 atgagtctat ttattttata taacagaatt
ctaaaggtga gtggtggcta gtgttggttc 41520 tgctgctcaa aaatccatcc
agggcctagg catgttctga ctttctactc tgctatcctc 41580 agacatagct
ttttatttac ttctgtgctt attccatctg tccctttcat caggaaaaca 41640
aaagctttcc caaagccccc taccaacctt ccactttaat ttctttggcc ctaactgtat
41700 catatgcttt actaaatgca gaggaggcta ggcaagcaga tgcctagctt
caccagcctc 41760 ttcaggagtg aaggggaagg gagaaagggt tggaagtggt
tgttggatta gccaacaaat 41820 gacatttgct aaggacaaaa gtggaaagat
gggatcatca agcatcccac gcctcttctt 41880 tttatatgaa actaaagttc
agtgacttgc ccaagatcat ggagctagaa caagacctga 41940 actgttgatc
tggaactttc cttacttcac gctcctacca tgtacacatt gtcatataga 42000
aatgtaaatt aatttttgtc attatatccc agataataag aagtagagac catccatctt
42060 atctgaaagt aaatgagtag cccccaagta gtatgtgact ttaattcctg
catctccaaa 42120 cttcaccttg ctgaggttgc catctccaag ctacccctgt
gggacaggcc tctctaggtg 42180 tggctgggtc cctaggaatc aatcaacaac
agaacaacaa cagcacatgc cgctgccatc 42240 aacacagtgg taaatgtgtc
gggggaaggg gcccatgaag gtaaaagtac cttagaccag 42300 ccaggcatgg
tggctcacac ctgtaatccc agcactttgg gaggctgagg tggaggattg 42360
cttgagccta ggagtttgag accaacctgg gcaacatggt gaaaccccat ctctaccaaa
42420 aatacaaaaa attagctggg tgcggtggct catgtctgtg gtcccagcta
ctcaggaggc 42480 caaggtggga ggatcgcttg agcccggagg tggaggttgc
agtgagccga gatcacacca 42540 ttgtactcca gcctgggtga cagaggaaga
ccccgtctca aaaaaaaaaa aaagtacctt 42600 agaccacaaa agtcacagtg
tggcctaggc agtgtgaatt acagcttagg tctgtctgat 42660 tttcaaacta
gcacactttt cctaagatat tcttctttgc taaagggaga aagatagctt 42720
tctatttatt tctgcatatg ttttaatttt cctcttcctg ctggcctttt acctccttga
42780 aataataata aagtaatcct gagaatgtgg tgtgaggtat tcaccgctat
gcctactttg 42840 tgcctcgttg ggaattgcat gctcagctga gatgtcttta
catattcagt gtctcttgtc 42900 cttagaaacc atctccatcc gctcatttgc
agtttaagca tctccatccc tactactgtg 42960 cttataccaa ctctagaaga
ggataagact caccccagct ggccttgtgg cttgttagat 43020 ccttgacctt
actttctttg gatggtttat ttgtaagacc tttcattttg atttgccagc 43080
aaaatgagca tgactagcag ccactcccca ttcttagtgt gtttttatag ccctaaaagg
43140 gctgatttaa gaaatggttt gactctcaag gaaagttacc tgatcaagga
cacaggcctc 43200 attacatgtc ccagctaagg tgtggccttg gtttcaaaga
acagccaaag gaaaatgtgg 43260 aagaaggaaa cccaggcttg gagtgtataa
attcttaatc tcaaaagata ttggagttag 43320 aagggattct agaaaacatc
cagtgatatg gtttggctct gtcgccaccc aaatctcatc 43380 ttgtagctcc
cataattccc atgtgttatg ggagggacct ggtgggaatt gattgaatca 43440
tgggggtggg tctttcccat gcttttctcg tggtagtgaa tgggtctcat gagatctgat
43500 ggttttaaaa acgggagttt ctctgcacaa gctctctctt tgcttgccgc
catccacgta 43560 agatgtgact tgctcttcta tgccttccgc catgattgtg
aggcctcccc cgccacgtgg 43620 aactgtgagt ccaattaaac ctctttcttt
tgtaaattgc tcacacttgg gtttgtcttt 43680 atcagcagca tgaaatcaga
ctaatacatc cagttacaac ccattgtttt atagttgagg 43740 aaactgaggc
tgagggagga aaaaagattt aaattcttac agctagtgag ggccgaaccg 43800
ggggctcttt ctcaccccca gttctgttct tccttctttg cataccattc aacaatcatc
43860 tgaggcccag gggactgagc tgcagtctgc tccccagggc agtctgggag
cagctggggg 43920 cagctgcagt aagggctgag tgccctgttg tttgctcaag
gggctgtgtc taataggaac 43980 tgacattgga gaatgtctaa aaggatgagg
aagatttttt ctgatagaaa agaagggtag 44040 tttaggtcac attgtgtatt
agtctgtttt cacataacta taaagaacca cctgagactg 44100 ggtaatttat
aaaagaaaga ggtttaatca actcacagtt ctgcatggct ggggaggcct 44160
caaggaactt acaatcacgg caggaggcaa aaggggaggc aaggcacatc ttacatggtg
44220 gcaggagaga gagagagaga gtgaagggga aggtgccaca cttttaaacc
atcagatctc 44280 atgagatctc actcactatc gtaagaacag cacgggggaa
atccgccccc atgacccagt 44340 cacctcccac caggttcttc cctcaacaca
tggggattac aatttgagat gcaatttggg 44400 tagggacaca gagccaagcc
atatcacatt gtaaagtttc cccaatgata gaatgctttt 44460 tactatgtaa
ggggaattat taggtgcttt tgagtgaagg aggcatgact gaatgattaa 44520
ataagagtaa gggctttggg gttccacaga cctgggctcc tgtcctgtga cttgtcactt
44580 ctacctgtgt gacctcaggc aatctgcctc ccctcctcca gcctggcttt
ctccttataa 44640 aatgggggtc atattggtac ttaccttgtc aggttgaagg
agagttaaac aaagtcatag 44700 gtacagtata cttagcatgg tactaggcac
ccagaaagca ctcagtgcat cttagttggt 44760 ggggttattc tctacctgcc
cctgtcccag gcattctttt gcattaccta aaccagactc 44820 acccacccca
cctcccaggg tatttggcct ggggacaaag gccaccctat ctccacgcac 44880
agcagaatga gacctgcagc ccattttcaa cacatgcctg gagtgctcac cttattggtt
44940 tgaggagccc tgagattgtt ttttgagtgt gttgtcattc tgtacatgat
aatagcggta 45000 atagctggca tttgtgtaac ccattatagc ttacaaagca
tcttcacata catagtttat 45060 ttgaatctca aaacaacccc ttgagatgga
tatttcattc ccatcttatc tctgaggaaa 45120 atgagtctct tgacttcctc
gggtgtcatg atgttcagat tccagatctc aggctgggcc 45180 tttcaccgag
ggtcaggctc accttggaaa gatgtgattt aatctatttc tctggaagat 45240
ccccaacctc ccatttccta aagatcttcc ttagcatcaa attctgggat atagaatttc
45300 ctttcaccac tcactttttc tgaagcaaga gttttttcat tcacagccca
gggggagttt 45360 cagagagtaa cttctccttt cagctaataa ctcccaataa
tgggaggtca cagggctcat 45420 ctttccctac cagacgtcca gaggatagca
gaggtcagct cactgcctct agtcacaatt 45480 atcttgtcta gacaagataa
acattcacac acaggtaagc atttgcaagg ttaagtttta 45540 caaagtaaga
aatacatgta aaaatgtacc cattcaggag ctgaatggag acagcagccc 45600
tcttgccatc tggaatttaa ttgttcaccc ctcacctttt tttttttttt tttttttgat
45660 acagtcactc tgtcacccag gctggagtgc agtggtgaga tcttggctca
ctgcaacctc 45720 cgcctcacgg gttcaagcaa ttcccgtgcc tcagccgccc
aagtagctgg gattacaggc 45780 acgcgccacc atgccaggct aattttttgt
atttttagta gagatggggt tttgctatgt 45840 tgaccaggct ggtcttgaac
tcctggcctc aagtgatctg tccacctcag cctcccaaag 45900 tgttaggatt
acaggtgtga gccaccgtgc ctggcaaccc tctccttttt ttttttaatc 45960
aagactttaa aaatcatgat cttttaaata attcaatgtc cctcatttaa agatctggat
46020 gagaatcctc ccagtcctcc taagcaaatt ttgtatgttc ctttgcttgc
tctttttagc 46080 ttccaatatt gcgcctggtt gaattttcaa aatttctctt
agattttttt catcttctga 46140 ttccattctc tcatgtaatt ccaaactgtg
atgctggagc aatctttgtc taaatcctgt 46200 gtggtctctg gatgaagtta
aagggcatct tggtgacctt cctctcctgg aagccctgtt 46260 ctgtggcaca
ctgggagttt gcctgtctct gcacggaggc agtctgattc ctgctcagtt 46320
tgattaattc ctgactttac catatgaatt ctaaatgagc tgaaaaggct tgcatgatga
46380 ttggtcagat tccctcaatc ttttcttgtt ccaggttcct atgcaggggc
agtggttgcc 46440 atgcccctgg ctggggtgtt ggtgcagtac attggatggt
cctctgtctt ttatatttat 46500 ggtgagtgat ttgacttcac aagttcacat
gtgactcata gagatggtat tttactgcat 46560 atgggtttgg ctcagagttc
attacatcaa aatagagatt actaaaacaa gtttattgta 46620 taaatggaat
actttatcta tgatttgatt aatatttata ttaaagttga cctaaaaaaa 46680
taagtagaac attgtctttc tttaaatacc agttaacaag aggaacgtca acaaaatact
46740 tacccctagc tgaacatact gccatttgga aatattgtaa agatcctttt
gtagttcata 46800 aatgtgataa ttgggtgttc acgtgcatgt atgagatgtc
tgagtccctc aaaccttgtt 46860 acaacattgg tacattaccc attttacctg
aaaaaaatat atatggtaaa aattgaaaaa 46920 tttagaaacg gaagaaaatg
agaccatata acccagcctt ttctttttta actgcaggca 46980 tgtttgggat
tatttggtac atgttttggc tgttgcaggc ctatgagtgc ccagcagctc 47040
atccaacaat atccaatgag gagaagacct atatagagac aagcatagga gagggggcca
47100 acgtggttag tctaagtgta agtataaaaa gtcagatgaa gacttacctt
ttttcataag 47160 tgattgtgtt gccttcttac agaaaaaatg tcaatatctt
tactaaaaat atcatggtat 47220 ttttactccc tagaaattta gtaccccatg
gaaaagattt ttcacatctt tgccggttta 47280 tgcaatcatt gtggcaaatt
tttgcagaag ctggaccttt tatttgctcc tcataagtca 47340 gcctgcttat
tttgaagagg tctttggatt tgcaataagt aaggtaaaca cacagatgct 47400
ccaaatattt ttgaacttta aatctcttga ttctacagag aataactttg tatgataaaa
47460 taattaaatt gctgatcata attcataaca gttctgtgac acctaatagc
ctggctgtca 47520 gacaagttat acattctatg catagtatgc atagctgttt
aatttcttct tagcaaggat 47580 cagagccgta ttaagctgct ttaaagattt
atgttgtacc caatcttaga gtgtttttga 47640 agctagctca aggacggcat
attaggcaag gataaaaaga tttgagggtg tgggttttct 47700 ttttttcctg
taagctactc agtgagtagc agtaagaacc ttaccattca ttttgcagaa 47760
caccccttct ccataatggt ggctatagca gtaacaatca ttgcttgcaa tgggttagaa
47820 agaacctctt tctgccaggc gtggtggctc acgcctataa tcccagcatt
ttgggaagcc 47880 aaggctggcg gatcacctga ggttaggacc agcctgacca
acatggcaaa accctgcctc 47940 tactaaaaat acaaaaatta gctgggcgta
gtgatgcaca cctgtgatcc tagttactca 48000 ggaggctgag acaggagaat
cacttgaacc caggaggcag aggttgcagt gaggcgagat 48060 tgcaccactg
cactccagcc tgggcaacag agcaagactc tgtctaaaaa aaaaaaagaa 48120
gaagaaaaaa taaacagaaa aaaaagaaag aacctctttc aatgctccca gacattatca
48180 tcaagccaat tgtgttttag ggaggaaggg tgtggatagt gaatcatcaa
ccatcatcat 48240 aagataaacc tctttcctac aagggaaaga acagcagccg
agcaaacaca aatgtctgcc 48300 tagctacaga tactgtcaga agtgaccatg
gaagagctgg cataatcatg aaatggtggc 48360 tgtcatcagt catcagtgct
cactgggtgc caagtgcttt atctcccatg tgccatgccc 48420 tctgtgatga
ataaaagtca tcgctgccct caaggagctt ccaatctggt agaggacaca 48480
gataggtcta aaatcattcg ctcattcatc atttatttat tatgaaattc aggcctaccc
48540 agctcccaca taattagatg cttaaatttg gtggtggtag gtaggggggc
tgtggagtgg 48600 aggtgggcaa gggaattagg gaggcccctc tctcagaaat
aatgacaaac tgcttactgt 48660 ttctttccct tccaggtggg tctcttgtca
gcagtcccac acatggttat gacaatcgtt 48720 gtacctattg gaggacaatt
ggctgattat ttaagaagca gacaaatttt aaccacaact 48780 gctgtcagaa
aaatcatgaa ctgtggaggt actgtggatt tcatagatgg cttaggcagc 48840
ttttgtagaa ttagggtaaa ctgaactgca gagcatatat taagaagtga catttagtca
48900 ttggagtgga tcttaaagac ctctaagtct gtccctcagc agacacttga
gtgttgtcca 48960 tcacagtgct gccaagaggt catccagctg ggacctttcc
atacatcctt ccacatttat 49020 tgtttgctta tgtagtttat tcccttctct
gcttaccttt ctacctatcc atatgttttg 49080 gtaagaaaca gaagaaaagt
agtctttcct cctagcctat gcttgtgcat gggacacaca 49140 cacacacaca
cacacacaca cacacacaca ccattttctt tcttgatttt atttagctcc 49200
tgctttatgt tttaattttg taaagacaaa gtgaatgtta ggtgatttcc caaaagaggt
49260 aggcgaaagt aattgtgaac ccctacaatg ttcatgagtg ctttttaaaa
aactcatctt 49320 ttttgtttag cttttaaaat taacatttat tgaatgcttt
ctgtgccaga cactaagcta 49380 aatcttctac atacattatt ttatttaatc
ttcataacca ccatgtggag caggtactat 49440 tactatatgc aatttgcaat
gaggaaacag aggtaaaata aagggacttg ctcaagtagc 49500 agatccctgc
aaggtatcag gtaggccgga gcctaccgcc aaagctctta gtttgcggct 49560
acccctctgg aggactagtc aggatgagcg agcaggaggt agaggatagc gccacctatg
49620 ggcaagagct cacaactgtg atattaagtt gaaagggacg gattgcgtat
gctctgacag 49680 atagctaggt ctggcacatt tagaagtgaa gactataccg
agggacacag gagcaggcat 49740 gatctgatcc catagcattt cgggaagaaa
gcctaagagt ctgttggcac ctgttctccc 49800 agttccttga ctgctggtcc
caggcaggga tgtgtgggcc tgaccttagc ttgaactttc 49860 ttgtagagga
ctgagggtta gcggatatag gcctgctatc tggtgggcag gaggtgaagc 49920
tctgggacat tgcattcaag tcctctccaa gagagctgta gcagctagaa taatgcccat
49980 gtcctaatcc tcagaagctg tgaatatgtt tccttacatg tcaaaaggga
ctttgcaggt 50040 gggattaaat tgaggttctt gagatgggag tttatcctgc
attatctagg tgggcccaat 50100 ataatcacaa taatccttat aaaaggagga
aggagggtca gagtcagaaa agaagatgtg 50160 atggtggagg caagagtcag
agtgatgcag ccacaaacca aggaatgcaa gcagacccta 50220 gaagctggag
aagacaagaa gagattccgc catagcacct ctagaaggaa tgcaactctg 50280
taggctgctg ccttgacttt agccctgtac cattttggat ttttggcctc cagaactgta
50340 caatagtgca gagagtattt tagaggtgac atctaatcat tggaatagat
cttaaagacc 50400 cctaagtcta tccctcagca gatacttgat atttgtgttg
ttttgagcca ctgagtttgt 50460 ggtaatttat tacagcagca aatgaaaact
aacacagcgg taggcagggt gcagtggctc 50520 actcctgcaa tcctagcact
ttgggaggtt gaggcgggca gaccacttga gctcaggagt 50580 tcgaaatcag
tcagggcaat agtgagaact tttctctatt aaaaaataaa acatttataa 50640
aatgaaaact aatacagtag ccaaagcctc acccttctaa tgataaaatt ctgctccagc
50700 tgaacagccc tcacccaagc cctgaacata tctttctgtc tctgactttg
cccactccct 50760 ttctctttcc ctgtgagttc tcaccttcac ctctcaatcc
agtcctctct atacatccag 50820 ctcaattctt ctcctcttat gtttccttaa
agccatgcca ttctccagtg atccctctga 50880 atatgtccac atggctagat
tggcaactca tcatgtggtg ccttattgca gctctctcag 50940 gaaaagattt
taggcagagg gaatagtatg tgcaatgacc ctggggcagg caggaatgtg 51000
gcctgtgtga gaatagaagg aaggggagtc agaatggctg agtgacggga gacgggatcg
51060 ggatgttttt ctagggtcag atcatggcag gccttgtcgg cgtatgcaga
gcttgggttt 51120 tatttgaagt acattgagat gcagatgatt taaagcacgg
aatggatatg atctcatttt 51180 tttttttttt tgagacagag tctcgctctg
ttacccaggc tggagtgcag tggtgcaatc 51240 tcagctcact gcaacctccg
cctcttgggt tcaagtgatt ctcctgcctc agcttcctga 51300 gtagctggga
ttacaggcat gggccaccat gcctggctaa tcttttgtat ttttgtagag 51360
acagggtttc actatattgg ccaggctggt ctcaaactcc tgacctcaag taatccgccc
51420 gcctcggcct tccaaagtgc tgggattaca ggcatgagcc acctcgcctg
gccttgctta 51480 tttattttta atctggggaa ttatgcaggg tacaagagta
aaagaaggga gaccaggtag 51540 gaggtgattt cagttgtcct gtctagagaa
gatggtggct tagacaaatg aggtggcaat 51600 ggagatggag agaggggggt
caatttctaa attctcagag ccaacagttc tcatctttaa 51660 attacataat
aatatttact tcagaggata gttatgagag ttaaatgata caacgtatga 51720
atgcacctag tgcggtgttc aacctataaa aagttctcaa caaatgttaa tgctgctttt
51780 tttctcctat gttcaagaca caaaaaacac agaagttttt caaagagttc
tttaacaaat 51840 atctgtgatt gtatttcctt tggacaaaaa aatgtacttc
taaactggca actttaaata 51900 agtttctgga ttttaaacac tatttgcaca
acctcttcta aacccagatg cattggatat 51960 tcttgagcat attttgtggg
aatgtcttgt tcctatttaa ttctgcccca gtacctctgc 52020 tgtttctcca
taattggtgg tgattatgtt atgttgtggt gatgagaact ttcaaagatg 52080
tttaattgct aacaaagtgc ctgttgagag gaaatagttt tttttctgca gaaactagaa
52140 ggcatatgtg gaatctttct gcctcatctc ccatctttaa aaaatacctc
ttcacatggc 52200 ttttcatgtt catatatata tatatttttt ttgtttgttt
gttttgtttt gttttgtttt 52260 tgagatggag tctcgctctg tcacccaggc
tggagtgcag tggcgtgatc tcagctcact 52320 gcaagttccg cctcccaggt
tcacaccatt ctcctgcctc agcctcccga gtagctggga 52380 ctacaggcac
ccgccaccac acccggctaa ttttttgtat tttttagtag aggcggggtt 52440
tcaccgtgtt agccagggta gtctcgatct cctgaccttg tgatccaccc acctcggcct
52500 cccaaagtgc tgggattaca ggcatgagcc accgtgcccg gccattcttt
tatattttga 52560 catagtagga ccagtgagtt atatatagaa aataaaattt
ttaaaaagac cataatggtc 52620 ccactttttc tgcttaaata cagagatgct
agagcagaga taactacatg aaaacaaagt 52680 tttgtgccat cagtgaagaa
tgcaggttga tttggaaatg atgaagcact ggtatgatct 52740 tccagagaat
tttggttggc tttttggttt cctactaaga aatatagaag gcatttctca 52800
tctgagaagg atcacacata tcttggagcc tgtcatcttt tatttccata gattttaata
52860 tgccattaaa atcatttaaa gcaaaacaga tcacttaaga catgatgttc
aattcattct 52920 gaatcagggt ctacgtctat gatgcttaaa gacagatgcc
aaattcttgt cctgccccct 52980 ctatagaaca tgcaaagtgt aactgaggtc
aaaaattcta ttctggctga atcagttgca 53040 agtgtgaact tcagattatt
ttaatatgaa ataaaatatt tcttaggcct ttaagtccta 53100 gttttgtttt
tcttgtcaac tctaaatagg ttcaatttta aggatctcct gattacccct 53160
aaagttgaaa ttttatcctt aagctcctga aacatgcagc cctgtctcta gtattttaac
53220 tgtcagtaga aaccatttag gctcttaaat gctttttttt ccactggcaa
tctgctattt 53280 ggccaaaatt ttttttctta cagatgaact gatgtatcat
ttgtaagttt tattctttat 53340 acaatgtcat cattctaatt ctttggggga
attgactttc tgcatgcttc tgttcagagt 53400 gtaaaaataa aagaagtttc
agccagatgc cttgttattt aggataggca cttctaagac 53460 acatatagtt
agtatatgaa acactagcta tttttcccta tgtgtagtct taaatgttga 53520
aacaaaatta agaacaagta gcaatgatat aaagcctata gttttaaaag taagacttcc
53580 ctaattacat ttcatcctct ttagaagcca tttaaaacaa ttattagttc
ttgcccttct 53640 ttatagtagt gttgaagaaa taggttcaaa aaggtaaata
ttaataactt aaccatcatt 53700 tacggtaagt acttcagctt gtgaatctta
ttttcttctt tctgggtccc atttcctttc 53760 ctttgcatta attcattaaa
cgttatgtat gtatgtatgt atgtatgtat gtatgtatgt 53820 atgtatgtat
gtatttagag acagagtctc actctgttgc ccaggctgga gtgcagtggt 53880
gcaatcttgg ctcactgcaa cctccacctc ccggtttcaa gtgattctcc cgcctcagcc
53940 tcctgagtag ctgggattac aggcacatgc aaccatgcct ggctaacttt
catatgttta 54000 gtagagaagg ggttttgcca tgttgcccag gctggtcttg
aactcctgac gtcaggtgat 54060 ccgcctgcct cgtcctccca aagagctgga
attataggtg tgcaccacca tgcctggcca 54120 aacgttattt attgagtgca
tactacatgc tagacagact ctgtgttaaa tatacagttt 54180 tgtgggagag
gcagaaacac aaatgaaaag ttacaaagca atattgaaaa gttctataaa 54240
atgatgagaa ggtgatgtca gcttcattgg ttgagggtag ggagagggtt gttagggaag
54300 ctttctagag gaggcactat ttaatctgga ctttaaaaat agtaagattt
atccagaaaa 54360 agagaaaatg atgagagaag agtatcccag gtaaagaaac
aatgtgtgaa aatatgtaca 54420 ggcatgagat agtattgtgt ggttagaaaa
cagctaatag aggagtatgt ctgtggcaca 54480 gagggctatc cacagaatgg
gggcagtaag caaagagatg agggctggaa gaagatgaaa 54540 ctggaacagc
aggaggtatt cattatagaa cactatactc atgatatgga gctcatgaca 54600
aacacgttaa gcacaggagc aaataatgag gtgtgtggct tagaaagaca gtggtattga
54660 gaatgcatca gaggaggacg agttgggaag actaccaaag
tggcttattg tggctgagca 54720 tggtggctta ggcctgtaat cccagcactt
tgggaggcca aggcaggcag atcacctgag 54780 gtcagaagtt ggagaccagc
ctggccaaca tggggaaacc cggcctctac taaaaataca 54840 aaaattagac
tgggcgtggt ggctcacgcc tgtaatccca gcactttggg aggctgaggt 54900
gggtggatca cgaggtcagg agactgagac catcctggct aacacggtga aaccccatct
54960 ctactaaata tataaacaat tagctgggca tggtggtggg tgcctatagt
cccagctact 55020 caggaggctg aggcaggaga agggcacgaa cccgggaggc
agagcttgca gtgagccaag 55080 atcgcgctgc tgccctccag cctgggtgac
agagcaggac tccatctcaa aaaaaaaaaa 55140 aagttagccg ggcgtggtgg
tggactataa tcccagcgac gggggaggct gagtcaggag 55200 aaccacttgc
acccgggagg cagaggttgt aatgagctga gattgcacca ctgcactcca 55260
gtctgggtga cagagcacga ctccatctca aacaaaagaa gaaaaaaagg tggcttattg
55320 cagttttcct ggtaagaggt cacggggcct ggaactaaag cagtgacagg
ggaggggaaa 55380 gtggcagttg cactggacag atgtttccga ggccaaacct
gcagatttgt atatgaaagc 55440 tcaggcagga ggagaagtcc aaggtagttc
tgaagtttct gcatcggact tctggctatc 55500 atttgttgag ctgtgcccat
gtgccacact cagtacctca tataccaatt tcatttactt 55560 ttccgatacc
tcacaaggct gtggtactat ctccagcttt tggatgagga atctaagagg 55620
tgtagtaact tgttcaaggt cacaaaatta gtgattttga agtggaaagt gaacccatac
55680 cagtttgact ctaaagattg ggttctaaac acagaatatg gaagattaat
ttagaggaga 55740 agaaagcacg tggtggcgat ggtttggtga tggtttgctt
gtttgtttag gagtaaaaaa 55800 ataggggaag aggccagggg tggtggctca
tgcctgtaat cccagcactt tgggaggctg 55860 aagtgggcgg accacctgag
gtcaggagtg gccagcctgg ccaacatggt gaaaccagcc 55920 tggccaacat
ggtgaaaccc caactctact aaaaatacaa aattagctgg gcgtggtagc 55980
acatgcccat aatcccagct acttgggagg ctgaggcagg agaatcattt gaacttggga
56040 ggcagaagtt gcagtgagcc aagatcatgc cgttgcactc cagcctgggt
gataagagca 56100 agactctgtc tcaaagaaaa aataaataaa taaataaata
aaaatagggg atgagagaat 56160 tgatttgggc atgttgcctt tgaggtactg
tagaacaatt gtgtggagat gtctggaatc 56220 agcagacagt ctccaaatga
agcaccacta attgtctctt ccccctccta aggcactcta 56280 tatacttgga
aatgatattt atatcatttt tctgtctgtt gtcagctgaa cttttttttc 56340
gggtgagaag gaacttcttc ataatttcct cattcttttt attttttatt gtgctagact
56400 cacttattct gaatgaaagg aacagaaagt acttttgttc tgcaatattt
tctgtgcaaa 56460 attctcatgt attgtttgtt tttttttttt ttaagaggcc
tgagagcttg gtgaactttg 56520 aaatagaaaa attttgactt ttgctttaca
aggggtgaag tgctgttttt gtttgtttct 56580 ttgtttgttt ttgtttcaga
tatttgctac agttttctgg ttgcttttgg caataaatat 56640 tagagtgttg
tcattttact tttaagggaa aggccataac tagtcaaagg ggaatcatta 56700
ccacagttat atagtagagt tttagtattt aacaatggca gggacagcta cccatgaagc
56760 aactaataat taacatccct catctcagga gcatcattgg aacctattgg
gaccgtgtgg 56820 tgttcaaggt gcaccgcgat aatgttagaa agtttgtgaa
cacccaggga atattagcaa 56880 agtcatgtag tcatgaaagt cctgggtggc
attgtaagca ctgtaccaga atgtaggtct 56940 gtggaggaac agaaaaccaa
acactgcatt tccccactca taagtgggag atgaacaatg 57000 agaacacatg
gatacaggga ggggatcatc acacactggg gcctgctagg gggcaagggg 57060
agggacagca ttagggcaaa tacctaatgc atgtggggcc caaaacctag atgatgggtt
57120 gataggtgga gcaaaccatc atggcacatg tatacctatg taataaacct
gcacattctg 57180 cacatgtatc cctgaactta aatcccagaa cttcaagtaa
agttaaaaaa aaaaaaaaaa 57240 aaacttaaat tccagaactt aaagtaaaaa
aaaaacatag acacaaacaa aataaactta 57300 ggtctgtgga attataggtt
agttcttatt tgataaataa atgaacttgg gttgaccgat 57360 atgaaaatga
catttttttc ccttgctgtt tccatttgca ggttttggca tggaggcaac 57420
cttactcctg gtggttggct tttcgcatac caaaggggtg gctatctcct ttctggtact
57480 tgctgtagga tttagtggct tcgctatttc aggtaatgtg tcctttgggt
ttccagatct 57540 tgactataga ttcaacaagt cccaggaaga aggaaggaca
aggatattgt agcaccttct 57600 ttcagtagcc agtccattct cagagagcag
gaccaccgtc cagagaatgt gatctagtgg 57660 gggtgatttt gtaagatcac
tgagaactgg gcttgggagc tcagttaagg tggaattttt 57720 cctacttact
ttgttacggg aaaagacaca aagtgcagat gacccttctg agacacgagc 57780
agaggcccaa gcatatgtcc tgggtgaagt ggactttcat actttagcac catgtcaccc
57840 tacctgacag aggctcctgt gactttttca agcctcgccc tcttgctaga
gaactgcgag 57900 tgtcattaca gtcataggat cagaagtttt tttaagagtg
aaaaccttct ttagattttt 57960 gtctactcca ttgctttcat tttccaaaca
agaaaatgcg ggtccataga ggggaagtga 58020 ctttctgaac agggtaaaga
ataatgacaa tgatgatgtg agctagcgat gaccaagcac 58080 agattctgtg
ccagggaata ttccatgaga tctgcatata ttaagccctg tctctctcac 58140
aactaccctg ctgggtatca gtgctattac ggtccccatt ttacaggagc agaaaccagt
58200 ctactatatg tgagagaaag gccagagtgc aatcatatca gaagcttcct
atgcaaaact 58260 gggtcaaaga gtgaaattta gttgtttgtc tatctttaaa
acatcgtaat aagaatatgg 58320 ttactggccg ggtgcgctgg cttacgcgtg
taatcgcagc actttgggag accgagacga 58380 atggatcact tgagcccagg
agttcaagac cagcctgggc aacatggcaa aaccccatct 58440 ctacaaaaaa
tacaaaaagt tagctaagtg taatggcgca cacctgcagt cccagttagt 58500
caggaggttg aggtgagagg atggcttgag cctgggagtt ggaggttgcg gtgagctgag
58560 ttcgtgccac tgcattccag cctggatgac aaagcgagac cccttctcaa
gaaaaaaata 58620 aataaataaa ataaaaataa aaaatggtta cttaaagaaa
atttcacata tattgtatat 58680 atatcataac attgtgaagc aagtagtagt
atatcactat gctactgggt ttttcactat 58740 tttactaaag ctcagaaaaa
tttgatactt tcttaatatc acacagttag tggcaaagga 58800 aggatgacag
aacagttctg cctggcccaa aggccgtgct ccttccatta ttccaggttg 58860
ccttaaatat caaacagtgt tagtgtccca gaatagaaaa atatggaacc tctggtctaa
58920 actgccctaa gacaggggct tgtatctttc aaaataaata gagttgatga
ataaattaga 58980 aaataaagta aaagtctaaa ttaaaagtaa cttgcagcta
agtaatttgg tttagagatg 59040 catagacctg ggtttgaggc cctctttact
atttactatt tataaaataa aaaatttgct 59100 aaattatgaa aactctcaag
cttcagtttt ctcatctaga gattggagag atgaaacagc 59160 aacctcatag
ggttgttggg aggataaact tagataattc atgtatttcc ccgcacttct 59220
tgtgggctgg gcattattct tagcactggg gatattgcag tgaataaatg aaagtgtcca
59280 tccccataaa gtttacattc tagtggaaat acttattcaa ataaaaacct
tagctgtatt 59340 tatttgaagt ccttagcaca gtgccagatg cataacaaaa
ttaatgagtg ttcaccatta 59400 ttgttctatt agtacacaca ccagcccagt
gcctctcaaa gtgttatgtg aaatcaccat 59460 aagatatttc agaatgcaga
ttctgatttg gtagctctag ggtggagcct gagattctgc 59520 agttttagca
agttccccag agctgctgct gctgcagggc agtccacact ttgagtagca 59580
agggcagagc aatcacgatt tgcttccagt aggaagcgga ggaacgcctt cccttgataa
59640 ctttgtgatg caaaagagat ccatatcctg ttcccagaga tactgaaatg
ttcaagttca 59700 tattgcttcc tttcccccga ttgccaatta agtcacaatc
tgaaggagag aaacccaata 59760 ctccaaatca cataaactgc ttttttgttt
tccttttttt ttagacaggg tctcttgcct 59820 tgtgcagtgt ctcatgacta
taatcccagc actttgggag gccgaggcag atggatcacc 59880 tgagatccag
gagttcgaga ccaacctggc caacatggtg aaaccgcatg tctactaaaa 59940
atacaaaaac tagttggttg tggtggtatg tgcctgtagt cccagctact ggggaggctg
60000 aggttgcagt gagccaagat tgcaccactg cactccagcc tgggtgacaa
agagagattc 60060 tgtctcaaaa aaaaaaaaaa aatagacagg gtctcgctct
gacacacagg ctggtgtgca 60120 gtggcatgat cgcggatcat tgcagcctct
acctcccatg ctcaactgat tctcctgcct 60180 cagcctcctg agtagctggg
gctacaggca tgtgccacca cttccagata tatatatatt 60240 ttttcgagac
agggtctcac gatgttgccc aagctggtct cgaactcctg gcctcaagtg 60300
attctcctgc cttggcctct caaagtattg agattacagg catgagccac cacacctggc
60360 cttcttgcca ctttttaaac atgatttcat ttaatcctca ttgcaacctt
gatgagaaag 60420 gtattgctat attcacttta ttggtgggga aaccaaagtg
tggtttaact tgccgagtga 60480 agtggctggg agtgtggaat aaaggtctgt
tggtcccagc aatgacactg tgggagggat 60540 tgcagccaca ggggcaataa
ttcctcagaa tctactgtct gccaactttt aaaggaataa 60600 acatagatgt
cagggaagac tgactggcac aatttaggag ctgattatag acaagactgc 60660
tgagatagat gaagttaaaa ataggcaaga gatgagtgat gcctgttttg ggaaatgtcc
60720 tatacagaag atagattctc tcagtttatg tgtaattttt ttatctgcta
taaaaatcta 60780 tcaatatctc aatttctcag tgattttccc ccctccccaa
atgtcaggat tgtgcagcta 60840 gaaacctaaa tggcttttcc cacattatct
ttagctgaat gcagatgccc aggctttgta 60900 tcagagcata atactcaaca
atcatattaa ttgcttctta tctctggatt cttttctaat 60960 aaagtgttta
tcacattcaa atccatggta agattaatga acttgcagct gttttatatt 61020
ctgatcattt ggcacattga cctgaaagat aaggtatgtt tattattacc aaaaagtttt
61080 ctcaaaattt ctccctgaag ggaagtagga aagacaacca accagtgtgc
cagattagaa 61140 caaaaaaatg ttttaagtcc tattttcagt tttttttttt
gcacagaata gagaaataaa 61200 aagcaaagca aaggaagaca aaaagatgaa
taaagcctac aaccccttgc tataatttca 61260 gtagctgaag ctggtaatta
atttagcaac tatttattga gtgactacaa tgtgccaggc 61320 actttgctag
ttcaggggag atggtggtaa acaagacgga tggctaacca cctgtaaaga 61380
gcatgcatgt tggtttacac gtctatgcac catgtagtta acatacatta tttaacttaa
61440 ttcctacatc aattttataa gaatcattat cccgttatgt agatgaaact
aaggttcagg 61500 aagtttaaat ccttggtcta ggcttgcatc tcaactaagc
tgccagaact gaggtctgtc 61560 tgatttgaac atgcacccct gcaatatatt
gacaaagtca gatctcagct cgctgtaacc 61620 tccaactcct gggttcaagt
gattctcctg tctcagcctc ccaagtagct gggattacag 61680 gcatgtgcca
ccatgcctgg ctaatttttg tatttttagt agaggtgagg ttttgccatg 61740
ttggccaggc tggtcttgaa cttctgacct caggtgatcc acccgcctca gcctcccaaa
61800 gtgctgagat tataggcgtg agcaaccatg cccggccagc agcattatct
tttgatagaa 61860 gacctcaaag agagggagtt actttgcaat ggcagcagaa
ggtagcagta gtagtagtgg 61920 tagttagcat agctttgata tttgccaagg
gcttcacata cctatttccc ctgagtctct 61980 atcacagcac ctctgtgaag
tgaatagtaa tattatcctc atattggaga tgaagaaaca 62040 aaggccccca
aattacttgt ttacatagta gaaataagat tcaagtccag atttacagac 62100
tccaaatcaa gtaggtgtgt gaaagtgttt cataaattac agaaggttct cccaatgttt
62160 gtgcaaatgt ttcattaaaa agcacccttt tcattgtgtg aaaatgtggc
catgtggcca 62220 ataaagtagg cttacccttg gctgcctttt aagagtaagt
caggggtagg agtgggaata 62280 ttataaagca aggtttggtc tagtcatact
gtatgtgatt gtatgattat ttactctgaa 62340 taaatgtgat tcaggcttta
ggcttttcaa tattgtgcca aacaccgtat tttggaattc 62400 agaacctaca
aggtagagat gccataattc tctttataga gagagccctt gatagatatc 62460
cataatcaat tccagcattg tctaccagtg ctgctttgtg cagacacagc ctcttgaacc
62520 cagtcctctt ggtctggaaa ctagtcatat actagaggaa accaaacaga
ttggtaaagg 62580 ctggggcaac tgagtatttt ccaaagcata tttgaaattc
tgttcttgac tctgattttg 62640 aggttttggc ttcactgtag gttttaatgt
caaccacctg gacattgccc cacgctatgc 62700 cagcattctc atggggatct
caaacggagt gggaaccctc tctggaatgg tctgtcccct 62760 cattgtcggt
gcaatgacca ggcacaaggt aaaggtctcc tttgtggcta tgggttacaa 62820
tatcagagga ctggagctct acacaaactt gagatttcaa ggctctactg cagtctgtaa
62880 atgtgtatgt ccttgacctt gactgagtca gctgaacttc tttttttttt
cttccttctt 62940 ctgattttca aatcattgct tatcaatggc accaaggcta
gttgttgttt tgttctatgt 63000 tttctcaatt gaggaataat agtctgggga
gaggggatgg gccatagaaa ctgtttagag 63060 acccaaagaa gaaactgagg
cagtcaactt gggataaatg agttactgaa gattgttttc 63120 tcattctcag
tgattaaacc ttatagccta tttccatcca ttgcttagca tgtttcagca 63180
taaaaagatg agtgctattc tacttccttg ttaagaataa aataaacagg acattgataa
63240 cctacccagt tgttactgag cctttgtgaa tttagacaag ggtggatggt
agaggcagat 63300 ccatccagag ttcaaccaca gcccacatga tttctttatc
tttgtcactg aaacgtctca 63360 agatgctgct ttctgcaaat aagaattctt
tgataccatg ggattttttt cccccatcta 63420 ttttcttagt tggattgcct
attacaaata taacttcaga agtttttgca gcttcctgca 63480 gaagaaagtg
tgagataaat tttcttactt tttgacagaa aaggtaggat tttataggca 63540
gagaattcat gttttccatc tctgttcatg aaatgatagg attgataacc tgactattaa
63600 atccaagata tcttccccca accttagaca caaattccca ttattttttg
acatactttt 63660 ttttacactg aaaatattat aaagttcttg tcagtcaagg
gtgagaactt taatggctca 63720 aatattgtta tgtatccaac aacaagcaag
aaggagactt ctgatattta aaacggtggg 63780 ttcctaaaac aattttaatt
tagctgacta tgtgaaggga aaccccattt gagtattcaa 63840 aaagctatgc
aatggtgctg caggtattaa tatttgtata tgttgtttat tttaaaatgt 63900
attttcttgt aatcccagca ctttgggagg ccaaggcggg tggatcatga ggtcaggaga
63960 tcgagaccat cctggctaac acagtgaaac cccgcctcta ctaaaaatac
aaaaaattag 64020 ccaggcgtgg tggcgggcac ctgtagtccc agctactcag
aggctgaggc aggagaatgg 64080 tgtgaaccca ggaggcggag cttgcagtga
gccgagatcg cgccactgca ctctagcctg 64140 ggtgacagag cgagactcca
tctcaaaaaa aaaaaaaaga attttctaaa ttaaaaaaat 64200 acgtatttat
tgttttgtct aactttcata ttcattgttg tcttaacttt cattttttaa 64260
gtttttcttt taaatttggt ttgaatcccg gatggtgctt ctgacacacg tcctcccgcc
64320 caaggagcct ctagagcatc gccttccaaa tgggcaggtg ctttttcaca
gtggaggcct 64380 ccaggacata ctggtaatct ctagttttag ttaaaacatt
aattggcact ttatttcctt 64440 atttagaccc gtgaagaatg gcagaatgtg
ttcctcatag ctgccctggt gcattacagt 64500 ggtgtgatct tctatggggt
ctttgcttct ggggagaaac aggagtgggc tgacccagag 64560 aatctctctg
aggagaaatg tggaatcatt gaccaggacg aattagctga ggagatagaa 64620
ctcaaccatg agagttttgc gagtcccaaa aagaagatgt cttatggagc cacctcccag
64680 aattgtgaag tccagaagaa ggaatggaaa ggacagagag gagcgaccct
tgatgaggaa 64740 gagctgacat cctaccagaa tgaagagaga aacttctcaa
ctatatccta atgtctgaga 64800 ggcacttctg tcttctcctt actttagaaa
cagaaagtat ccatacctat tgcctttctt 64860 gtagcccagc ttgccagagg
tccaaatatt gggaggggag aagatctaac cagcaacagg 64920 gaaaagagaa
atattatctt tcaatgacat gtataggtaa ggagctgcgc tcagttgata 64980
acatagttga taatacatat tttttgaatt gacagttgac ccttctctca aagagctaaa
65040 cttattcaga aaggaatgac tagaagaaaa aggagacaat accatgttgt
tcaaagaaac 65100 attgaaggaa attgggatgt ttggccagaa ggaatgtaaa
cagtagtagt agctgccacc 65160 acatctctag ggtagccatg cagaggaggg
cttcatattc ccaataaacc ccacgttgtg 65220 gcaggtgctt tataaacact
cttatttaat ctccacacct ttatgacaca catttcttat 65280 ccccatttta
caaccaaggc atctaaagca acaagaaatg aacttgccca aggtcatctg 65340
ccagggtcag tgctgagact gttgaagctc tcaataggtg gcagttttag ggaagatttc
65400 cattcagtgt agggaagaca tttgtaataa tgaaaactga aaatggagta
attgtgagta 65460 actcaccact ttagcaggtg ttggggaagg gaaacatttg
ggttgatgag gcagagggga 65520 ttcaaatgtg tgagaggcta gattcaaaga
ccctcagtgt tctatgttat ctgaagagtc 65580 aaatggtttt gtgactccat
agtttttaaa gtaataaggg tcaaagacta catcagagat 65640 tcaaataggt
ttttaaagaa aagctaagca agagagccaa atttttagaa atctgatggt 65700
caaaatagct gaaagcagta aacaagagat tggctattaa atttcaactt tccataatat
65760 taagaatgta gctaaatgat gtcccaaact acttacaaac ttttaagaca
tttaataatt 65820 taagaagtag gttcatgtgt tttcttaggt aaagttcttc
tgaaagaatt ttctattttt 65880 aaaaaatgta tctctttagc cttttctgct
ggagattata ttaggaagtt tcatcagatt 65940 gtataaaatt atgattttgt
atcaaaagta ttcatgatga ctctatttgg aatgatattc 66000 agggaaatca
caataatata gcagtagtta tacagagaaa tactacaatg aaaacatttg 66060
gggcaattag acctacagtt actgttgaaa aattcacctt tgattgcata aggcaattac
66120 atggatactt ttagatatat ttaaaatttt aacattggca tctaaagtgt
tatttgaaaa 66180 taaaattatt ttcctgttca ttgattttaa acattttatt
cctactttca gaagaaaaat 66240 ataatacgga aaaaattata gatttacttg
tagcttatta ttgtaaagtg gttttttttt 66300 tttttttttt ttttctaatt
tctcccacat gtatttctgg tccccagtga tactagctga 66360 gttgtagtgt
attttataaa tggaataatc ttggggaaaa attgcgattc ttcattaaat 66420
aatattcttt atgtcactag catacaattt atgttagtag acatctttaa atctctttaa
66480 tgagtgaatc catgcaagcc ccataaaaca gttcctagca tgcagaaaat
gcccacgtaa 66540 atagctgtca tcatcattat cttttaacat tttgggggac
tttccagttg aaaagaaaac 66600 atgctatgtc atttttatcc attatccctg
gaacttattg tgaaagttgt gctgttttct 66660 aagtaaaata aaaaataaaa
aattagcaat ttatgatagc cagtgtttta ttttgtgtgt 66720 gtgttagtaa
agtcaaataa ttgtatttta aaaactcacg ataatcctta aggtagtatt 66780
gtatattgtg acacaaagtt gtat 66804 4 578 PRT Rattus norvegicus 4 Glu
Ser Val Lys Gln Arg Ile Leu Ala Pro Gly Lys Glu Gly Ile Lys 1 5 10
15 Asn Phe Ala Gly Lys Ser Leu Gly Gln Ile Tyr Arg Val Leu Glu Lys
20 25 30 Lys Gln Asp Asn Arg Glu Thr Ile Glu Leu Thr Glu Asp Gly
Lys Pro 35 40 45 Leu Glu Val Pro Glu Lys Lys Ala Pro Leu Cys Asp
Cys Thr Cys Phe 50 55 60 Gly Leu Pro Arg Arg Tyr Ile Ile Ala Ile
Met Ser Gly Leu Gly Phe 65 70 75 80 Cys Ile Ser Phe Gly Ile Arg Cys
Asn Leu Gly Val Ala Ile Val Asp 85 90 95 Met Val Asn Asn Ser Thr
Ile His Arg Gly Gly Lys Val Ile Lys Glu 100 105 110 Lys Ala Lys Phe
Asn Trp Asp Pro Glu Thr Val Gly Met Ile His Gly 115 120 125 Ser Phe
Phe Trp Gly Tyr Ile Ile Thr Gln Ile Pro Gly Gly Tyr Ile 130 135 140
Ala Ser Arg Leu Ala Ala Asn Arg Val Phe Gly Ala Ala Ile Leu Leu 145
150 155 160 Thr Ser Thr Leu Asn Met Leu Ile Pro Ser Ala Ala Arg Val
His Tyr 165 170 175 Gly Cys Val Ile Phe Val Arg Ile Leu Gln Gly Leu
Val Glu Gly Val 180 185 190 Thr Tyr Pro Ala Cys His Gly Ile Trp Ser
Lys Trp Ala Pro Pro Leu 195 200 205 Glu Arg Ser Arg Leu Ala Thr Thr
Ser Phe Cys Gly Ser Tyr Ala Gly 210 215 220 Ala Val Ile Ala Met Pro
Leu Ala Gly Ile Leu Val Gln Tyr Thr Gly 225 230 235 240 Trp Ser Ser
Val Phe Tyr Val Tyr Gly Ser Phe Gly Met Val Trp Tyr 245 250 255 Met
Phe Trp Leu Leu Val Ser Tyr Glu Ser Pro Ala Lys His Pro Thr 260 265
270 Ile Thr Asp Glu Glu Arg Arg Tyr Ile Glu Glu Ser Ile Gly Glu Ser
275 280 285 Ala Asn Leu Leu Gly Ala Met Glu Lys Phe Lys Thr Pro Trp
Arg Lys 290 295 300 Phe Phe Thr Ser Met Pro Val Tyr Ala Ile Ile Val
Ala Asn Phe Cys 305 310 315 320 Arg Ser Trp Thr Phe Tyr Leu Leu Leu
Ile Ser Gln Pro Ala Tyr Phe 325 330 335 Glu Glu Val Phe Gly Phe Glu
Ile Ser Lys Val Gly Met Leu Ser Ala 340 345 350 Val Pro His Leu Val
Met Thr Ile Ile Val Pro Ile Gly Gly Gln Ile 355 360 365 Ala Asp Phe
Leu Arg Ser Lys Gln Ile Leu Ser Thr Thr Thr Val Arg 370 375 380 Lys
Ile Met Asn Cys Gly Gly Phe Gly Met Glu Ala Thr Leu Leu Leu 385 390
395 400 Val Val Gly Tyr Ser His Thr Arg Gly Val Ala Ile Ser Phe Leu
Val 405 410 415 Leu Ala Val Gly Phe Ser Gly Phe Ala Ile Ser Gly Phe
Asn Val Asn 420 425 430 His Leu Asp Ile Ala Pro Arg Tyr Ala Ser Ile
Leu Met Gly Ile Ser 435 440 445 Asn Gly Val Gly Thr Leu Ser Gly Met
Val Cys Pro Ile Ile Val Gly 450 455 460 Ala Met Thr Lys Asn Lys Ser
Arg Glu Glu Trp Gln Tyr Val Phe Leu
465 470 475 480 Ile Ala Ala Leu Val His Tyr Gly Gly Val Ile Phe Tyr
Ala Leu Phe 485 490 495 Ala Ser Gly Glu Lys Gln Pro Trp Ala Asp Pro
Glu Glu Thr Ser Glu 500 505 510 Glu Lys Cys Gly Phe Ile His Glu Asp
Glu Leu Asp Glu Glu Thr Gly 515 520 525 Asp Ile Thr Gln Asn Tyr Ile
Asn Tyr Gly Thr Thr Lys Ser Tyr Gly 530 535 540 Ala Thr Ser Gln Glu
Asn Gly Gly Trp Pro Asn Gly Trp Glu Lys Lys 545 550 555 560 Glu Glu
Phe Val Gln Glu Ser Ala Gln Asp Ala Tyr Ser Tyr Lys Asp 565 570 575
Arg Asp
* * * * *
References