U.S. patent application number 11/504059 was filed with the patent office on 2006-12-28 for murine genomic polynucleotide sequence encoding a g-protein coupled receptor and methods of use therefor.
This patent application is currently assigned to Wyeth. Invention is credited to Mark H. Pausch.
Application Number | 20060294614 11/504059 |
Document ID | / |
Family ID | 23218416 |
Filed Date | 2006-12-28 |
United States Patent
Application |
20060294614 |
Kind Code |
A1 |
Pausch; Mark H. |
December 28, 2006 |
Murine genomic polynucleotide sequence encoding a G-protein coupled
receptor and methods of use therefor
Abstract
The present invention particularly relates to a newly identified
murine genomic polynucleotide that encodes an ortholog of the human
P2T receptor which is expressed at high levels in the central
nervous system, the use of such polynucleotides and polypeptides,
as well as the production of such polynucleotides and polypeptides.
The invention relates also to identifying compounds which may be
agonists, antagonists and/or inhibitors of P2T, and therefore
potentially useful in therapy.
Inventors: |
Pausch; Mark H.; (Rocky
Hill, NJ) |
Correspondence
Address: |
CHOATE, HALL & STEWART LLP
PATENT GROUP
TWO INTERNATIONAL PLACE
BOSTON
MA
02110
US
|
Assignee: |
Wyeth
Madison
NJ
|
Family ID: |
23218416 |
Appl. No.: |
11/504059 |
Filed: |
August 14, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10208483 |
Jul 30, 2002 |
7115724 |
|
|
11504059 |
Aug 14, 2006 |
|
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60314068 |
Aug 22, 2001 |
|
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Current U.S.
Class: |
800/18 ;
424/143.1; 435/320.1; 435/354; 435/6.16; 435/69.1; 435/7.2;
514/13.8; 514/17.7; 514/20.6; 514/44R; 530/350; 530/388.22;
536/23.5 |
Current CPC
Class: |
C07K 14/705 20130101;
G01N 2500/00 20130101; G01N 33/6893 20130101; A01K 2217/075
20130101; A01K 2217/05 20130101; C07K 14/723 20130101; C12Q
2600/136 20130101; C12Q 1/6883 20130101; C12Q 2600/158
20130101 |
Class at
Publication: |
800/018 ;
435/069.1; 435/354; 435/320.1; 530/350; 530/388.22; 536/023.5;
424/143.1; 514/044; 514/012; 435/006; 435/007.2 |
International
Class: |
A01K 67/027 20060101
A01K067/027; C12Q 1/68 20060101 C12Q001/68; G01N 33/567 20060101
G01N033/567; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; A61K 39/395 20060101 A61K039/395; C12N 5/06 20060101
C12N005/06; C07K 14/705 20060101 C07K014/705; C07K 16/28 20060101
C07K016/28; A61K 48/00 20060101 A61K048/00; A61K 38/17 20060101
A61K038/17 |
Claims
1-19. (canceled)
20. An antibody specific for a murine P2T receptor polypeptide
comprising the amino acid sequence of SEQ ID NO:2.
21. The antibody of claim 20, wherein the antibody is selected from
the group consisting of monoclonal, polyclonal, chimeric, humanized
and single chain.
22. The antibody of claim 21, wherein the antibody is
monoclonal.
23. The antibody of claim 21, wherein the antibody is
humanized.
24. An antibody specific for a polynucleotide comprising the
nucleic acid sequence of SEQ ID NO:1 or a degenerate variant
thereof.
25. The antibody of claim 24, wherein the antibody binds to a
nucleic acid sequence comprised within nucleotide 1 to about
nucleotide 45,167 of nucleic acid sequence SEQ ID NO:1 or the
complement of SEQ ID NO:1.
26. The antibody of claim 25, wherein the antibody is monoclonal,
polyclonal, chimeric, humanized and single chain.
27. The antibody of claim 26, wherein the antibody is
monoclonal.
28-69. (canceled)
Description
[0001] This application claims priority to prior application U.S.
Ser. No. 10/208,483, filed on Jul. 30, 2002 (the '483 application);
the present application is co-pending with and shares at least one
common inventor with the '483 application, and is a divisional of
the '483 application.
[0002] This application further claims priority to prior
application U.S. Ser. No. 60/314,068, filed on Aug. 22, 2001, (the
'068 application); the '068 application was co-pending with and
shared at least one common inventor with the '483 application. The
entire disclosure of the '483 and the '068 applications is hereby
incorporated by reference.
FIELD OF THE INVENTION
[0003] The present invention relates generally to the fields of
neuroscience, cell signaling and molecular biology. More
particularly, the invention relates to a newly identified murine
genomic polynucleotide that encodes an ortholog of the human P2T
receptor which is expressed at high levels in the central nervous
system, the use of such polynucleotides and polypeptides, as well
as the production of such polynucleotides and polypeptides. The
invention relates also to identifying compounds which may be
agonists, antagonists and/or inhibitors of P2T, and therefore
potentially useful in therapy.
BACKGROUND OF THE INVENTION
[0004] Platelets play a central role in blood clot formation
critical to the maintenance of normal homeostasis (Harder et al.,
1995). Pathological thrombus formation producing vascular occlusion
can result in stroke, myocardial infarction and unstable angina.
Platelet aggregation and shape change are induced by ADP released
from damaged vessels and red blood cells. ADP also is secreted by
platelets from dense granules on activation, potentiating the
aggregation response induced by other agents. Three types of
purinergic receptors mediate the platelet response to ADP: P2X, an
ADP-gated ion channel and two types of G-protein coupled receptors:
P2Y1, coupled to increases in intracellular calcium via Gq, and P2T
(P2Y.sub.ADP, P2Y.sub.AC, P2Y.sub.CYC, P2T.sub.AC), an ADP receptor
coupled to inhibition of adenylyl cyclase through Gi (Ralevic and
Burnstock, 1998; Kunapuli, 1998; Jantzen et al., 1999; Boeynaems et
al., 2000). Interestingly, the P2T receptor is irreversibly
inactivated by active metabolites of the anti-clotting drugs,
ticlopidine and clopidogrel (Savi et al., 2000); and patients with
a rare heritable clotting disorder lack P2T receptors (Catteneo et
al., 1992; Nurden et al., 1995).
[0005] In addition, the P2T receptor may have a role in the central
nervous system (CNS). ADP receptors with pharmacological properties
similar to the platelet P2T receptor have been identified in B10
brain endothelial cells and rat C6 glioma cells (Webb et al., 1996;
Boyer et al., 1993). Until recently however, the P2T
G-protein-coupled receptor had remained uncloned (Zhang et al.,
2001; Hollopeter et al., 2001).
[0006] In the CNS, nucleotides and related compounds are often
released in concert with other neurotransmitters and act as signal
transduction modulators. It has been reported that treatment of
astrocytes with P2T selective agonists produces an increase in
arachidonic acid release via a pertussis toxin sensitive
G-protein-dependent mechanism (Chen and Chen, 1998). ADP-induced
arachidonic acid release from astrocytes stimulates glycogenolysis,
suggesting that the ADP receptor may be involved in regulation of
glycogen metabolism in the CNS. Arachidonic acid acts directly on
glial glutamate transporters to inhibit uptake of glutamate
(Robinson and Dowd, 1997), further suggesting that glial ADP
receptors may regulate neurotransmitter re-uptake. However, the
exact role played by the P2T G-protein-coupled receptor in the CNS
is not well understood.
[0007] It is well established that many medically significant
biological processes are mediated by polypeptides participating in
cellular signal transduction pathways that involve G proteins and
second messengers, e.g., cAMP, IP.sub.3 and diacylglycerol
(Lefkowitz, 1991). Some examples of these polypeptides include
G-proteins and G-protein-coupled receptors themselves (e.g.,
G-protein-coupled receptor families I, II, III and IV),
G-protein-coupled receptors such as those for biogenic amine
transmitters (e.g., epinephrine, norepinephrine and dopamine)
(Kobilka et al., 1987(a); Kobilka et al., 1987(b); Bunzow et al.,
1988), effector polypeptides (e.g., phospholipase C, adenyl cyclase
and phosphodiesterase) and actuator polypeptides (e.g., polypeptide
kinase A and polypeptide kinase C) (Simon et al., 1991).
[0008] One particular pathway of cellular signal transduction is
the inositol phospholipid pathway. In this pathway, an
extracellular signal molecule (e.g., epinephrine) binds to a
G-protein-coupled receptor. The G-protein-coupled receptor (GPCR)
subsequently associates with a specific trimeric G-protein, wherein
the trimer is comprised of .alpha., .beta. and .gamma. polypeptide
subunits. In the GPCR/G-polypeptide associated state, there is an
exchange of GDP for GTP at the G-polypeptide .alpha.-subunit,
resulting in the dissociation of the .alpha.-subunit from the
.beta./.gamma. subunits. The GTP bound .alpha.-subunit is the
active state of the polypeptide. The active .alpha.-subunit further
activates phospholipase C, which catalyzes the cleavage of
PIP.sub.2 to IP.sub.3 and diacylglycerol (DAG). The IP.sub.3 and
DAG serve as second messengers in further signal amplification
(e.g., Ca.sup.2+ release and phosphorylation). Hydrolysis of GTP to
GDP, catalyzed by the G-protein itself, returns the G-protein to
its basal, inactive form. Thus, following GPCR binding a ligand
molecule, the ligand activates a G-protein. The G-protein serves a
dual role, as an intermediate that relays the signal from receptor
to effector, and as a clock that controls the duration of the
signal.
[0009] GPCRs are membrane bound polypeptides, comprising a gene
superfamily characterized as having seven putative transmembrane
domains. GPCRs can be intracellularly coupled by heterotrimeric
G-proteins to various intracellular enzymes, ion channels and
transporters (see, Johnson et al., 1989). Different G-protein
.alpha.-subunits preferentially stimulate particular effectors to
modulate various biological functions in a cell.
[0010] The G-protein-coupled receptors include a wide range of
biologically active receptors, such as hormone receptors, viral
receptors, growth factor receptors and neuroreceptors. Examples of
members of this family include, but are not limited to, dopamine,
calcitonin, adrenergic, endothelin, cAMP, adenosine, muscarinic
acetylcholine, serotonin, histamine, thrombin, kinin, follicle
stimulating hormone, opsins, endothelial differentiation gene-1,
rhodopsins, odorant, and cytomegalovirus receptors.
[0011] The seven transmembrane domains are believed to represent
transmembrane .alpha.-helices connected by extracellular or
cytoplasmic loops. GPCRs have been characterized as including these
seven conserved hydrophobic stretches of about 20 to 30 amino
acids, connecting at least eight divergent hydrophilic loops. Most
GPCRs (also known as 7TM receptors) have single conserved cysteine
residues in each of the first two extracellular loops which form
disulfide bonds that are believed to stabilize functional
polypeptide structure. The 7 transmembrane regions are designated
as TM1, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 has been implicated
in several GPCRs as having a ligand binding site, such as the TM3
aspartate residue. TM5 serines, a TM6 asparagine and TM6 or TM7
phenylalanines or tyrosines are also implicated in ligand binding
in certain receptor families.
[0012] Phosphorylation and lipidation (palmitylation or
farnesylation) of cysteine residues can influence signal
transduction of some GPCRs. Most GPCRs contain potential
phosphorylation sites within the third cytoplasmic loop and/or the
carboxy terminus. For several GPCRs, such as the
.beta.-adrenoreceptor, phosphorylation by polypeptide kinase A
and/or specific receptor kinases mediates receptor
desensitization.
[0013] Presently, more than 800 GPCRs from various eukaryotic
species have been cloned, 140 of which are human GPCRs for which
endogenous ligands are known (Stadel et al., 1997). In addition,
several hundred therapeutic agents targeting GPCRs such as
angiotensin receptors, calcitonin receptors, adrenoceptor
receptors, serotonin receptors, leukotriene receptors, oxytocin
receptors, prostaglandin receptors, dopamine receptors, histamine
receptors, muscarinic acetylcholine receptors, opioid receptors,
somatostatin receptors and vasopressin receptors have been
successfully introduced onto the market for various indications
(see Stadel et al., 1997). This indicates that these receptors have
an established, proven history as therapeutic targets. The search
for GPCR genes has also identified numerous genes whose products
are members of the GPCR family, but for which their natural ligands
are not known, commonly refered to as orphan receptors. In fact,
more than 100 of the 240 human GPCRs identified (i.e., about 45%)
are orphan receptors, and it is estimated that there are at least
400-1000 more GPCR genes that have yet to be identified (Stadel et
al., 1997).
[0014] Thus, there is clearly a need for the identification and
characterization of further GPCRs, their genes and their ligands,
which can play a role in preventing, ameliorating or correcting
dysfunctions or diseases, including, but not limited to, infections
such as bacterial, fungal, protozoan and viral infections,
particularly infections caused by HIV-1 or HIV-2; pain; cancers;
anorexia; bulimia; asthma; Parkinson's disease; acute heart
failure; hypotension; hypertension; platelet formation and
aggregation; stroke; urinary retention; osteoporosis; angina
pectoris; myocardial infarction; ulcers; asthma; allergies; benign
prostatic hypertrophy; and psychotic and neurological disorders,
including anxiety, schizophrenia, manic depression, delirium,
dementia, severe mental retardation and dyskinesias, such as
Huntington's disease or Gilles dela Tourett's syndrome.
SUMMARY OF THE INVENTION
[0015] The present invention relates to a newly identified murine
genomic polynucleotide that encodes an ortholog of the human P2T
receptor which is expressed at high levels in the central nervous
system, the use of such polynucleotides and polypeptides, as well
as the production of such polynucleotides and polypeptides. The
invention relates also to identifying compounds which may be
agonists, antagonists and/or inhibitors of P2T, and therefore
potentially useful in therapy.
[0016] In particular embodiments, the invention is directed to an
isolated murine genomic polynucleotide comprising the nucleic acid
sequence of SEQ ID NO:1. In other embodiments, the isolated
polynucleotide is selected from the group consisting of DNA, cDNA,
RNA, antisense RNA and pre-mRNA. In yet other embodiments, the
polynucleotide further comprises heterologous polynucleotides.
[0017] In another embodiment, the invention is directed to an
isolated murine genomic polynucleotide comprising a nucleic acid
sequence of SEQ ID NO:1 encoding a P2T receptor polypeptide
comprising an amino acid sequence of SEQ ID NO:2. In a preferred
embodiment, the polynucleotide encoding the polypeptide sequence of
SEQ ID NO:2 is active in glial cells or platelet cells. In another
embodiment, the polynucleotide is selected from the group
consisting of DNA, cDNA, RNA, antisense RNA and pre-mRNA and may
further comprise a polynucleotide encoding a heterologous
polypeptide.
[0018] In yet another embodiment, the invention is directed to an
isolated polynucleotide which hybridizes to a polynucleotide
comprising the nucleic acid sequence of SEQ ID NO:1 or the
complement of SEQ ID NO:1, under stringent hybridization
conditions. In a particular embodiment, the polynucleotide
hybridizes to the nucleic acid sequence comprised within nucleotide
1 to about nucleotide 45,167 of nucleic acid sequence SEQ ID NO:1
or the complement of SEQ ID NO:1. In another embodiment, the
polynucleotide hybridizes to the nucleic acid sequence comprised
within nucleotide 45,168 to about nucleotide 46,358 of nucleic acid
sequence SEQ ID NO:1 or the complement of SEQ ID NO:1.
[0019] In certain other embodiments, the invention provides a
recombinant expression vector comprising a polynucleotide encoding
a murine P2T receptor polypeptide, wherein the polynucleotide
comprises the nucleic acid sequence of SEQ ID NO:1 or a degenerate
variant thereof. In a particular embodiment, the polynucleotide is
selected from the group consisting of DNA, cDNA, RNA, pre-mRNA and
antisense RNA. In another embodiment, the vector DNA is selected
from the group consisting of plasmid, episomal, YAC and viral. In a
preferred embodiment, the vector is a yeast expression plasmid. In
another embodiment, the vector polynucleotide is operatively linked
to one or more regulatory elements selected from the group
consisting of a promoter, an enhancer, a splicing signal, a
termination signal, a ribosomal binding signal and a
polyadenylation signal.
[0020] In yet another embodiment, the invention is directed to a
genetically engineered host cell, transfected, transformed or
infected with a recombinant expression vector comprising a
polynucleotide encoding a murine P2T receptor polypeptide, wherein
the polynucleotide comprises the nucleic acid sequence of SEQ ID
NO:1 or a degenerate variant thereof. In one preferred embodiment,
the host cell is eukaryotic, wherein the eukaryotic cell is further
selected from the group consisting of yeast, mammal, insect and
plant. In yet another preferred embodiment, the host cell is
prokaryotic. In still another embodiment, the polynucleotide is
expressed to produce the encoded polypeptide, a biological
equivalent thereof, or a fragment thereof.
[0021] In one particular embodiment the invention is directed to an
antibody specific for a murine P2T receptor polypeptide comprising
the amino acid sequence of SEQ ID NO:2. In a preferred embodiment,
the antibody is selected from the group consisting of monoclonal,
polyclonal, chimeric, humanized and single chain. In a more
preferred embodiment, the antibody is monoclonal. In another
preferred embodiment, the antibody is humanized.
[0022] In another particular embodiment the invention provides an
antibody specific for a polynucleotide comprising the nucleic acid
sequence of SEQ ID NO:1 or a degenerate variant thereof. In a
preferred embodiment, the antibody binds to a nucleic acid sequence
comprised within nucleotide 1 to about nucleotide 45,167 of nucleic
acid sequence SEQ ID NO:1 or the complement of SEQ ID NO:1. In
another embodiment, the antibody binds to a nucleic acid sequence
comprised within nucleotide 45,168 to about nucleotide 46,358 of
nucleic acid sequence SEQ ID NO:1 or the complement of SEQ ID NO:1.
In another preferred embodiment, the antibody is selected from the
group consisting of monoclonal, polyclonal, chimeric, humanized and
single chain. In a more preferred embodiment, the antibody is
monoclonal.
[0023] In still another embodiment, the invention is directed to a
transgenic animal comprising a polynucleotide comprising the
nucleic acid sequence of SEQ ID NO:1. In one preferred embodiment,
the transgenic animal comprises a mutation which modulates P2T
receptor activity. In a more preferred embodiment, the P2T receptor
activity is in a glial cell. In another embodiment, the
polynucleotide has at least one mutation selected from the group
consisting of nucleotide deletion, nucleotide substitution and
nucleotide insertion. In one preferred embodiment, the transgenic
animal is heterozygous for the mutation. In yet another preferred
embodiment, the transgenic animal is homozygous for the mutation.
In still another embodiment, the animal is selected from the group
consisting of mouse, rat, rabbit and hamster.
[0024] In one particular embodiment, the invention is directed to
an RNA molecule which is antisense to a polynucleotide comprising
the nucleic acid sequence of SEQ ID NO:1 or a degenerate variant
thereof. In one preferred embodiment, the RNA is antisense to the
polynucleotide of SEQ ID NO:1 from about nucleotide 1 to about
nucleotide 45,167. In another preferred embodiment, the RNA is
antisense from about nucleotide 987 to about nucleotide 1,046 of
SEQ ID NO:1. In yet another preferred embodiment, the RNA is
antisense from about nucleotide 35,843 to about nucleotide 35,976
of SEQ ID NO:1. In still another preferred embodiment, the RNA is
antisense from about nucleotide 43,330 to about nucleotide 43,494
of SEQ ID NO:1. In yet another preferred embodiment, the RNA is
antisense from about nucleotide 45,168 to about nucleotide 46,358
of SEQ ID NO: 1.
[0025] The invention is directed in other embodiments to a method
for assaying the effects of test compounds on the activity of a P2T
receptor polypeptide comprising the steps of providing a transgenic
animal comprising a polynucleotide encoding a murine P2T receptor
polypeptide comprising the amino acid sequence of SEQ ID NO:2;
administering a test compound to the animal; and determining the
effects of the test compound on the activity of the P2T receptor
polypeptide in the presence and absence of the test compound. In
particular embodiments, the effects of the test compound on the
animal are phenotypic. In a preferred embodiment, the receptor is
expressed in glial cells or blood plasma cells of the animal. In
yet another embodiment, the polynucleotide has at least one
mutation selected from the group consisting of nucleotide deletion,
nucleotide substitution and nucleotide insertion.
[0026] In one particular embodiment, the invention is directed to a
method for assaying the effects of test compounds on the activity
of a P2T receptor polypeptide comprising the steps of providing
recombinant cells comprising a murine P2T receptor polypeptide
comprising an amino acid sequence of SEQ ID NO:2; contacting the
cells with a test compound; and determining the effects of the test
compound on the activity of the P2T receptor polypeptide in the
presence and absence of the test compound. In a preferred
embodiment, determining the effects of the test compound are
selected from the group consisting of measuring ADP levels,
measuring K.sup.+ levels, P2T kinase activity, measuring platelet
morphology, measuring platelet aggregation, measuring P2T
phosphorylation, measuring phosphatidyl inositol levels, measuring
GTPase activity, measuring GTP levels, measuring cAMP levels,
measuring GDP levels and measuring Ca.sup.2+ levels. In another
embodiment, the polynucleotide has at least one mutation selected
from the group consisting of nucleotide deletion, nucleotide
substitution and nucleotide insertion.
[0027] In still another embodiment, the invention is directed to a
method for assaying the effects of test compounds on the activity
of a P2T receptor polypeptide comprising the steps of providing
yeast cells comprising a polynucleotide encoding a murine P2T
receptor polypeptide comprising the amino acid sequence of SEQ ID
NO:2 and a reporter gene operatively linked to the polynucleotide;
contacting the cells with a test compound; and determining the
effects of the test compound on the activity of the P2T receptor
polypeptide by measuring expression levels of the reporter gene in
the presence and absence of the test compound. In a preferred
embodiment, the reporter gene is selected from the group consisting
of .beta.-galactosidase, HIS3, CAN1, CYH1, URA3, TRP1 and LYS2.
[0028] In yet another embodiment, the invention is directed to a
method of producing a murine P2T receptor polypeptide comprising an
amino acid sequence of SEQ ID NO:2 comprising transfecting,
infecting or transforming a recombinant host cell with an
expression vector comprising a polynucleotide comprising a nucleic
acid sequence of SEQ ID NO:1 or a degenerate variant thereof;
culturing the host cell under conditions sufficient for the
production of the polypeptide; and isolating the polypeptide from
the culture.
[0029] The invention further provides a method for the treatment of
a subject in need of enhanced P2T receptor activity comprising
administering to the subject a therapeutically effective amount of
an agonist to the P2T receptor polypeptide; and/or administering to
the subject a polynucleotide encoding a P2T receptor polypeptide
comprising an amino acid sequence of SEQ ID NO:2, in a form so as
to effect the production of the P2T polypeptide in vivo.
[0030] The invention is further directed to a method for the
treatment of a subject in need of enhanced P2T receptor activity in
the central nervous system comprising administering to the subject
a therapeutically effective amount of an agonist to the P2T
receptor polypeptide; and/or administering to the subject a
polynucleotide encoding a P2T receptor polypeptide comprising an
amino acid sequence of SEQ ID NO:2, in a form so as to effect the
production of the P2T receptor polypeptide in vivo.
[0031] In another embodiment, the invention is directed to a method
for the treatment of a subject in need of inhibiting P2T receptor
polypeptide activity comprising administering to the subject a
therapeutically effective amount of an antagonist to the P2T
receptor polypeptide; and/or administering to the subject a
polynucleotide that inhibits the expression of a polynucleotide
encoding a P2T receptor polypeptide comprising an amino acid
sequence of SEQ ID NO:2; and/or administering to the subject a
therapeutically effective amount of a polypeptide that competes
with a P2T receptor polypeptide for its ligand.
[0032] In yet another particular embodiment, the invention is
directed to a method for the treatment of a subject in need of
inhibiting P2T receptor polypeptide activity in the central nervous
system comprising administering to the subject a therapeutically
effective amount of an antagonist to the P2T receptor polypeptide;
and/or administering to the subject a polynucleotide that inhibits
the expression of a polynucleotide encoding a P2T receptor
polypeptide comprising an amino acid sequence of SEQ ID NO:2;
and/or administering to the subject a therapeutically effective
amount of a polypeptide that competes with a P2T receptor
polypeptide for its ligand.
[0033] In other embodiments the invention is directed to a method
for the diagnosis of a central nervous system disease or the
susceptibility to a central nervous system disease in a subject
related to the expression or activity of a P2T receptor polypeptide
in the subject comprising determining the presence or absence of a
mutation in a polynucleotide encoding a P2T receptor polypeptide
comprising the amino acid sequence of SEQ ID NO:2; and/or assaying
for the presence of P2T expression in a sampled derived from the
subject, wherein the P2T expressed is a polynucleotide encoding a
P2T polypeptide comprising the amino acid sequence of SEQ ID
NO:2.
[0034] In yet another embodiment, the invention provides a method
of inhibiting expression of the P2T gene in a cell comprising
providing the cell with a polynucleotide antisense to the nucleic
acid sequence of SEQ ID NO:1. In a preferred embodiment, the cell
is a glial cell or platelet cell.
[0035] In a further embodiment, the invention is directed to a
neural cell line stably expressing a P2T polypeptide comprising the
amino acid sequence of SEQ ID NO:2, a variant thereof or a fragment
thereof. In a preferred embodiment, the cell is a glial cell.
[0036] In one particular embodiment, the invention is directed to a
method for treating a subject for a nervous system disorder
comprising modulating the activity of the P2T receptor polypeptide
comprising the amino acid sequence of SEQ ID NO:2 and/or modulating
the expression of a polynucleotide encoding a P2T receptor
polypeptide comprising the amino acid sequence of SEQ ID NO:2.
[0037] In another embodiment, the invention provides a method for
producing a transgenic animal whose genome comprises a functional
disruption in a polynucleotide encoding a P2T receptor polypeptide,
the method comprising providing a polynucleotide encoding a P2T
receptor polypeptide having a functional disruption; introducing
the disrupted polynucleotide into embryonic stem cells; selecting
those embryonic stem cells that comprise the disrupted
polynucleotide; introducing the embryonic stem cell into a
blastocyst; transferring the blastocyst to a pseudopregnant animal;
and allowing the transferred blastocyst to develop into an animal
chimeric for the disruption. In a particular embodiment, the method
further comprises breeding the chimeric animal with a wild-type
animal to obtain animals heterozygous for the disruption. In
another embodiment, the method further comprises breeding the
heterozygous animal to generate animal homozygous for the
disruption. In a preferred embodiment, the animal has a central
nervous system disorder. In another preferred embodiment, the
animal has a blood plasma disorder.
[0038] The invention is directed in a further embodiment, to a
method for assaying the effects of test compounds on the binding
interaction of a P2T receptor polypeptide and a P2T substrate
polypeptide comprising the steps of providing yeast cells for a
yeast two-hybrid system comprising a P2T receptor polypeptide
having an amino acid sequence of SEQ ID NO:2 and a P2T substrate
polypeptide; contacting the cells with a test compound; and
determining the effect of the test compound on the binding
interaction of the P2T receptor polypeptide and the P2T substrate
polypeptide in the presence and absence of the test compound.
[0039] In a particular embodiment, the invention is directed to a
method for inhibiting the expression of a P2T polynucleotide in a
cell the method comprising providing the cell with a nucleic acid
molecule antisense to the polynucleotide of SEQ ID NO: 1.
[0040] In yet another embodiment, the invention is directed to a
method of providing a P2T protein to a mammal comprising
introducing into the mammal a homologously recombinant cell which
produces the P2T protein, the homologously recombinant cell being
generated by the method comprising providing a vertebrate cell, the
genomic DNA of which comprises an endogenous P2T gene; providing a
DNA construct comprising a targeting sequence of the nucleic acid
sequence of SEQ ID NO:1 from about nucleotide 1 to about nucleotide
45,167 of SEQ ID NO:1, which is homologous to a target site
upstream of the endogenous P2T gene, an exogenous regulatory
sequence, an exon and an unpaired splice-donor site at the 3' end
of the exon, wherein the exogenous regulatory sequence is
operatively linked to the exon and transfecting the cell with the
DNA construct, thereby generating a homologously recombinant cell
in which the splice-donor site is operatively linked to the second
exon of the endogenous gene and the exogenous regulatory sequence
controls transcription of the construct-derived exon, the
endogenous P2T gene and any sequence between the construct-derived
exon and the endogenous P2T gene, to produce an RNA transcript that
encodes a P2T protein.
[0041] In still another embodiment, the invention provides a method
for the treatment of a subject in need of enhanced P2T receptor
activity in platelet cells comprising administering to the subject
a therapeutically effective amount of an agonist to the P2T
receptor polypeptide; and/or administering to the subject a
polynucleotide encoding a P2T receptor polypeptide comprising an
amino acid sequence of SEQ ID NO:2, in a form so as to effect the
production of the P2T receptor polypeptide in vivo.
[0042] In another particular embodiment, the invention is directed
to a method for the treatment of a subject in need of inhibiting
P2T receptor activity in platelet cells comprising administering to
the subject a therapeutically effective amount of an antagonist to
the P2T receptor polypeptide; and/or administering to the subject a
polynucleotide that inhibits the expression of a polynucleotide
encoding a P2T receptor polypeptide comprising an amino acid
sequence of SEQ ID NO:2; and/or administering to the subject a
therapeutically effective amount of a polypeptide that competes
with a P2T receptor polypeptide for its ligand.
[0043] In still another embodiment, the invention is directed to a
method for the diagnosis of a blood platelet disease or the
susceptibility to a blood platelet disease in a subject related to
the expression or activity of a P2T receptor polypeptide in the
subject comprising determining the presence or absence of a
mutation in a polynucleotide encoding a P2T receptor polypeptide
comprising the amino acid sequence of SEQ ID NO:2; and/or assaying
for the presence of P2T expression in a sampled derived from the
subject, wherein the P2T expressed is a polynucleotide encoding a
P2T polypeptide comprising the amino acid sequence of SEQ ID
NO:2.
[0044] Other features and advantages of the invention will be
apparent from the following detailed description, from the
preferred embodiments thereof, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIG. 1 shows an amino acid sequence alignment of human (SEQ
ID NO:11), rat (SEQ ID NO:12) and mouse (SEQ ID NO:2) P2T
receptors. Non-conservative replacements are highlighted in black,
conserved amino acid replacements are highlighted in grey. Putative
transmembrane domains (TM1-7) are underlined. ClustalW was used to
perform the alignment.
[0046] FIG. 2 shows the pharmacological characterization of mouse
P2T receptor expressed in yeast cells. MPY578t5 cells containing
pMP344 were assayed for agonist-induced stimulation of
.beta.-galactosidase activity. Symbols: square, 2MeSATP; triangle,
2MeSADP; inverted triangle, 2CIATP; diamond, ADP; circle,
ATP.gamma.S; open square, ADP.beta.S.
[0047] FIG. 3 shows a northern blot analysis of mouse P2T receptor
mRNA expression. Hybridization of a mouse P2T probe to mouse
multiple tissue northern blot (Clontech).
[0048] FIG. 4 shows the murine P2Y12 agonist-induced release of
intracellular calcium. NIH3T3 cells expressing the mouse P2Y12 and
a chimeric G.alpha.q/i3 were assayed for agonist-induced release of
intracellular calcium as described in the Methods and Materials
section. Symbols: square, ADP; triangle, 2MeSADP; inverted
triangle, ADP.beta.S; diamond, ATP.gamma.S.
DETAILED DESCRIPTION OF THE INVENTION
[0049] In an effort to identify genes encoding orphan G-protein
coupled receptors (P2Ts), the present invention has identified a
genomic polynucleotide sequence encoding a murine ortholog of the
human P2T G-protein coupled receptor, hereinafter the P2T receptor.
The murine ortholog of the human P2T receptor was identified by
querying mouse genomic and cDNA databases using the human orphan
GPCR sequence, EBI-2 (U.S. Pat. No. 6,060,272). A yeast-based GPCR
expression technology (Pausch, 1997) was employed to demonstrate
that the murine polynucleotide sequence of the invention encodes a
GPCR with pharmacological properties equivalent to the human P2T
receptor. The murine mRNA was expressed at high levels in the
brain, particularly in glial cells and in various peripheral
tissues, including those that contain platelets. Results of the
present invention, using the yeast GPCR expression system (Pausch,
1997), are in excellent agreement with the three different measures
of human P2T pharmacological activity.
[0050] Thus, the present invention relates to a newly identified
murine genomic polynucleotide sequence that encodes a P2T receptor
which is expressed at high levels in the central nervous system,
the use of such polynucleotides and polypeptides, as well as the
production of such polynucleotides and polypeptides. The invention
relates also to identifying compounds which may be agonists,
antagonists and/or inhibitors of P2T, and therefore potentially
useful in therapy.
[0051] In certain embodiments the present invention relates to an
isolated genomic polynucleotide comprising a nucleic acid sequence
of SEQ ID NO:1, encoding a P2T receptor polypeptide or fragments
thereof. In other embodiments the invention relates to P2T receptor
polypeptides comprising an amino acid sequence of SEQ ID NO:2. In
yet other embodiments, the invention provides recombinant vectors
comprising a polynucleotide encoding a P2T receptor polypeptide. In
another embodiment, a vector comprising a polynucleotide encoding a
P2T receptor polypeptide is comprised within a host cell, wherein
the vector expresses the polynucleotide to produce the encoded
polypeptide or fragment thereof. In further embodiments, methods
for assaying test compounds for their ability to modulate the
activity of P2T receptor polypeptides, methods for producing P2T
receptor polypeptides, and methods for the diagnosis of a disease
or the susceptibility to a disease in a subject related to the
expression or activity of a P2T receptor are provided, as well as
methods for treating a subject in need of inhibiting or activating
P2T activity.
A. Isolated Polynucleotides that Encode the P2T Receptor
[0052] Isolated and purified P2T polynucleotides of the present
invention are contemplated for use in the production of P2T
receptor polypeptides. Thus, in one aspect, the present invention
provides isolated and purified polynucleotides that encode P2T
receptor polypeptides. In particular embodiments, an isolated
murine genomic polynucleotide comprises a nucleic acid sequence of
SEQ ID NO:1 and encodes a P2T receptor polypeptide comprising an
amino acid sequence of SEQ ID NO:2. In particular embodiments, a
polynucleotide of the present invention is a DNA molecule. In a
preferred embodiment, a polynucleotide of the present invention
encodes a P2T receptor polypeptide comprising an amino acid
sequence that has at least 95% identity to an amino acid sequence
of SEQ ID NO:2, or a fragment thereof.
[0053] In another embodiment of the invention, an isolated and
purified P2T polynucleotide is a genomic mouse cDNA sequence
comprising the nucleic acid sequence of SEQ ID NO:1. The nucleic
acid sequence of SEQ ID NO:1 comprises a 46,358 base pair
nucleotide sequence encoding the mouse P2T promoter and transcript
(see SEQ ID NO:1). The mouse P2T mRNA of SEQ ID NO:1 is encoded in
four exons (Exon I, SEQ ID NO:3; Exon II, SEQ ID NO:4; Exon III,
SEQ ID NO:5; and Exon IV, SEQ ID NO:6), three of which comprise the
5' untranslated region. The entire mouse P2T open reading frame is
encoded in a single uninterrupted exon (Exon IV, SEQ ID NO:6) from
nucleotide 45,167 to nucleotide 46,358 of SEQ ID NO:1.
[0054] In another aspect of the invention, an isolated and purified
polynucleotide comprises a nucleic acid sequence that has at least
95% identity to the nucleic acid sequence selected of SEQ ID NO:1,
a degenerate variant thereof, or a complement thereof.
[0055] As used herein, the term "polynucleotide" means a sequence
of nucleotides connected by phosphodiester linkages.
Polynucleotides are presented herein in the direction from the 5'
to the 3' direction. A polynucleotide of the present invention can
comprise from about 40 to about several hundred thousand base
pairs. Preferably, a polynucleotide comprises from about 10 to
about 3,000 base pairs. Preferred lengths of particular
polynucleotide are set forth hereinafter.
[0056] A polynucleotide of the present invention can be a
deoxyribonucleic acid (DNA) molecule, a ribonucleic acid (RNA)
molecule, or analogs of the DNA or RNA generated using nucleotide
analogs. The nucleic acid molecule can be single-stranded or
double-stranded, but preferably is double-stranded DNA. Where a
polynucleotide is a DNA molecule, that molecule can be a gene, a
cDNA molecule or a genomic DNA molecule. Nucleotide bases are
indicated herein by a single letter code: adenine (A), guanine (G),
thymine (T), cytosine (C), inosine (I) and uracil (U).
[0057] "Isolated" means altered "by the hand of man" from the
natural state. If an "isolated" composition or substance occurs in
nature, it has been changed or removed from its original
environment, or both. For example, a polynucleotide or a
polypeptide naturally present in a living animal is not "isolated,"
but the same polynucleotide or polypeptide separated from the
coexisting materials of its natural state is "isolated," as the
term is employed herein.
[0058] In certain embodiments, an "isolated" polynucleotide is free
of sequences which naturally flank the nucleic acid (i.e.,
sequences located at the 5' and 3' ends of the nucleic acid) in the
genomic DNA of the organism from which the nucleic acid is derived.
For example, in various embodiments, the isolated P2T nucleic acid
molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,
0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the
nucleic acid molecule in genomic DNA of the cell from which the
nucleic acid is derived (e.g., neuronal or red blood cells).
However, the P2T nucleic acid molecule can be fused to other
protein encoding or regulatory sequences and still be considered
isolated.
[0059] However, in other embodiments, an isolated polynucleotide
comprises a murine genomic polynucleotide sequence disclosed in SEQ
ID NO:1.
[0060] Polynucleotides of the present invention may be obtained,
using standard cloning and screening techniques, from a cDNA
library derived from mRNA from human cells or from genomic DNA.
Polynucleotides of the invention can also synthesized using well
known and commercially available techniques.
[0061] The invention further encompasses nucleic acid molecules
that differ from the nucleotide sequence shown in SEQ ID NO:1, (and
fragments thereof) due to degeneracy of the genetic code and thus
encode the same P2T receptor polypeptide as that encoded by the
nucleotide sequence shown in SEQ ID NO:1.
[0062] In another preferred embodiment, an isolated polynucleotide
of the invention comprises a nucleic acid molecule which is a
complement of the nucleotide sequence shown in SEQ ID NO:1, or a
fragment of this nucleotide sequence. A nucleic acid molecule which
is complementary to the nucleotide sequence shown in SEQ ID NO:1 is
one which is sufficiently complementary to the nucleotide sequence
SEQ ID NO:1, such that it can hybridize to the nucleotide sequence
shown in SEQ ID NO:1, thereby forming a stable duplex. Examples of
hybridization stringency conditions are detailed in Table 1.
[0063] Orthologues and allelic variants of the murine P2T receptor
polynucleotides can readily be identified using methods well known
in the art. Allelic variants and orthologues of P2T will comprise a
nucleotide sequence that is typically at least about 70-75%, more
typically at least about 80-85%, and most typically at least about
90-95% or more homologous to the nucleotide sequence shown in SEQ
ID NO:1, or a fragment of this nucleotide sequence. Such nucleic
acid molecules can readily be identified as being able to
hybridize, preferably under stringent conditions, to the nucleotide
sequence shown in SEQ ID NO:1, or a fragment of this nucleotide
sequence.
[0064] Moreover, the polynucleotide of the invention can comprise
only a fragment of the coding region of a P2T polynucleotide or
gene, such as a fragment of SEQ ID NO:1.
[0065] When the polynucleotides of the invention are used for the
recombinant production of P2T receptor polypeptides of the present
invention, the polynucleotide may include the coding sequence for
the mature polypeptide, by itself, or the coding sequence for the
mature polypeptide in reading frame with other coding sequences,
such as those encoding a leader or secretory sequence, a pre-, or
pro- or prepro-polypeptide sequence, or other fusion peptide
portions. For example, a marker sequence which facilitates
purification of the fused polypeptide can be encoded (see Gentz et
al., 1989, incorporated herein by reference). The polynucleotide
may also contain non-coding 5' and 3' sequences, such as
transcribed, non-translated sequences, splicing and polyadenylation
signals, ribosome binding sites and sequences that stabilize
mRNA.
[0066] In addition to the P2T nucleotide sequences shown in SEQ ID
NO:1, it will be appreciated by those skilled in the art that DNA
sequence polymorphisms that lead to changes in the amino acid
sequences of a P2T receptor polypeptide may exist within a
population (e.g., a mouse, rat or human population). Such genetic
polymorphism in the P2T gene or polynucleotide may exist among
individuals within a population due to natural allelic variation.
As used herein, the terms "gene" and "recombinant gene" refer to
polynucleotides comprising an open reading frame encoding a P2T
receptor polypeptide, preferably a mammalian P2T receptor
polypeptide. Such natural allelic variations can typically result
in 1-5% variance in the nucleotide sequence of the P2T
polynucleotide. Any and all such nucleotide variations and
resulting amino acid polymorphisms in a P2T polynucleotide that are
the result of natural allelic variation are intended to be within
the scope of the invention. Such allelic variation includes both
active allelic variants as well as non-active or reduced activity
allelic variants, the latter two types typically giving rise to a
pathological disorder.
[0067] Moreover, nucleic acid molecules encoding P2T receptor
polypeptides from other species, and thus which have a nucleotide
sequence which differs from the murine sequence of SEQ ID NO:1, are
intended to be within the scope of the invention. Polynucleotides
corresponding to natural allelic variants and non-murine
orthologues of the murine P2T cDNA of the invention can be isolated
based on their homology to the murine P2T polynucleotides disclosed
herein using the murine cDNA, or a fragment thereof, as a
hybridization probe according to standard hybridization techniques
under stringent hybridization conditions.
[0068] Thus, a polynucleotide encoding a polypeptide of the present
invention, including homologs and orthologs from species other than
murine, may be obtained by a process which comprises the steps of
screening an appropriate library under stringent hybridization
conditions with a labeled probe having the sequence of SEQ ID NO:1,
or a fragment thereof; and isolating full-length cDNA and genomic
clones containing the polynucleotide sequence (see, Table 1). Such
hybridization techniques are well known to the skilled artisan,
e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., chapters 9 and 11, and Ausubel et al., 1995, Current
Protocols in Molecular Biology, eds., John Wiley & Sons, Inc.,
sections 2.10 and 6.3-6.4, incorporated herein by reference. The
skilled artisan will appreciate that, in many cases, an isolated
cDNA sequence will be incomplete, in that the region coding for the
polypeptide is cut short at the 5' end of the cDNA. This is a
consequence of reverse transcriptase, an enzyme with inherently low
"processivity" (a measure of the ability of the enzyme to remain
attached to the template during the polymerization reaction),
failing to complete a DNA copy of the mRNA template during 1st
strand cDNA synthesis.
[0069] Thus, in certain embodiments, the polynucleotide sequence
information provided by the present invention allows for the
preparation of relatively short DNA (or RNA) oligonucleotide
sequences having the ability to specifically hybridize to gene
sequences of the selected polynucleotides disclosed herein. The
term "oligonucleotide" as used herein is defined as a molecule
comprised of two or more deoxyribonucleotides or ribonucleotides,
usually more than three (3), and typically more than ten (10) and
up to one hundred (100) or more (although preferably between twenty
and thirty). The exact size will depend on many factors, which in
turn depends on the ultimate function or use of the
oligonucleotide. Thus, in particular embodiments of the invention,
nucleic acid probes of an appropriate length are prepared based on
a consideration of a selected nucleotide sequence, e.g., a sequence
such as that shown in SEQ ID NO:1. The ability of such nucleic acid
probes to specifically hybridize to a polynucleotide encoding a P2T
receptor lends them particular utility in a variety of embodiments.
Most importantly, the probes can be used in a variety of assays for
detecting the presence of complementary sequences in a given
sample.
[0070] In certain embodiments, it is advantageous to use
oligonucleotide primers. These primers may be generated in any
manner, including chemical synthesis, DNA replication, reverse
transcription, or a combination thereof. The sequence of such
primers is designed using a polynucleotide of the present invention
for use in detecting, amplifying or mutating a defined segment of a
gene or polynucleotide that encodes a P2T receptor polypeptide from
mammalian cells using polymerase chain reaction (PCR)
technology.
[0071] In certain embodiments, it is advantageous to employ a
polynucleotide of the present invention in combination with an
appropriate label for detecting hybrid formation. A wide variety of
appropriate labels are known in the art, including radioactive,
enzymatic or other ligands, such as avidin/biotin, which are
capable of giving a detectable signal.
[0072] Polynucleotides which are identical or sufficiently
identical to a nucleotide sequence contained in SEQ ID NO:1 or a
fragment thereof, may be used as hybridization probes for cDNA and
genomic DNA or as primers for a nucleic acid amplification (PCR)
reaction, to isolate full-length cDNAs and genomic clones encoding
polypeptides of the present invention and to isolate cDNA and
genomic clones of other genes (including genes encoding homologs
and orthologs from species other than mouse) that have a high
sequence similarity to of SEQ ID NO:1 or a fragment thereof.
Typically these nucleotide sequences are from at least about 70%
identical to at least about 95% identical to that of the reference
polynucleotide sequence. The probes or primers will generally
comprise at least 15 nucleotides, preferably, at least 30
nucleotides and may have at least 50 nucleotides. Particularly
preferred probes will have between 30 and 50 nucleotides.
[0073] There are several methods available and well known to those
skilled in the art to obtain full-length cDNAs, or extend short
cDNAs, for example those based on the method of Rapid Amplification
of cDNA ends (RACE) (see, Frohman et al., 1988). Recent
modifications of the technique, exemplified by the Marathon.TM.
technology (Clontech Laboratories Inc.) for example, have
significantly simplified the search for longer cDNAs. In the
Marathon.TM. technology, cDNAs have been prepared from mRNA
extracted from a chosen tissue and an "adaptor" sequence ligated
onto each end. Nucleic acid amplification (PCR) is then carried out
to amplify the "missing" 5' end of the cDNA using a combination of
gene specific and adaptor specific oligonucleotide primers. The PCR
reaction is then repeated using "nested" primers, that is, primers
designed to anneal within the amplified product (typically an
adaptor specific primer that anneals further 3' in the adaptor
sequence and a gene specific primer that anneals further 5' in the
known gene sequence). The products of this reaction can then be
analyzed by DNA sequencing and a full-length cDNA constructed
either by joining the product directly to the existing cDNA to give
a complete sequence, or carrying out a separate full-length PCR
using the new sequence information for the design of the 5'
primer.
[0074] To provide certain advantages in accordance with the present
invention, a preferred nucleic acid sequence employed for
hybridization studies or assays includes probe molecules that are
complementary to at least a 10 to 70 or so long nucleotide stretch
of a polynucleotide that encodes a P2T receptor polypeptide, such
as that shown in SEQ ID NO:2. A size of at least 10 nucleotides in
length helps to ensure that the fragment will be of sufficient
length to form a duplex molecule that is both stable and selective.
Molecules having complementary sequences over stretches greater
than 10 bases in length are generally preferred, though, in order
to increase stability and selectivity of the hybrid, and thereby
improve the quality and degree of specific hybrid molecules
obtained. One will generally prefer to design nucleic acid
molecules having gene-complementary stretches of 25 to 40
nucleotides, 55 to 70 nucleotides, or even longer where desired.
Such fragments can be readily prepared by, for example, directly
synthesizing the fragment by chemical means, by application of
nucleic acid reproduction technology, such as the PCR technology of
U.S. Pat. No. 4,683,202 (incorporated by reference herein in its
entirety) or by excising selected DNA fragments from recombinant
plasmids containing appropriate inserts and suitable restriction
enzyme sites.
[0075] In another aspect, the present invention contemplates an
isolated and purified polynucleotide comprising a base sequence
that is identical or complementary to a segment of at least 10
contiguous bases of SEQ ID NO:1, wherein the polynucleotide
hybridizes to a polynucleotide that encodes a P2T receptor
polypeptide. Preferably, the isolated and purified polynucleotide
comprises a base sequence that is identical or complementary to a
segment of at least 25 to 70 contiguous bases of SEQ ID NO:1. For
example, the polynucleotide of the invention can comprise a segment
of bases identical or complementary to 40 or 55 contiguous bases of
the disclosed nucleotide sequences.
[0076] Accordingly, a polynucleotide probe molecule of the
invention can be used for its ability to selectively form duplex
molecules with complementary stretches of the gene. Depending on
the application envisioned, one will desire to employ varying
conditions of hybridization to achieve varying degree of
selectivity of the probe toward the target sequence. For
applications requiring a high degree of selectivity, one will
typically desire to employ relatively stringent conditions to form
the hybrids (see Table 1 below).
[0077] Of course, for some applications, for example, where one
desires to prepare mutants employing a mutant primer strand
hybridized to an underlying template or where one seeks to isolate
a P2T receptor polypeptide coding sequence from other cells,
functional equivalents, or the like, less stringent hybridization
conditions are typically needed to allow formation of the
heteroduplex. Cross-hybridizing species can thereby be readily
identified as positively hybridizing signals with respect to
control hybridizations. In any case, it is generally appreciated
that conditions can be rendered more stringent by the addition of
increasing amounts of formamide, which serves to destabilize the
hybrid duplex in the same manner as increased temperature. Thus,
hybridization conditions can be readily manipulated, and thus will
generally be a method of choice depending on the desired
results.
[0078] The present invention also includes polynucleotides capable
of hybridizing under reduced stringency conditions, more preferably
stringent conditions, and most preferably highly stringent
conditions, to polynucleotides described herein. Examples of
stringency conditions are shown in the table below: highly
stringent conditions are those that are at least as stringent as,
for example, conditions A-F; stringent conditions are at least as
stringent as, for example, conditions G-L; and reduced stringency
conditions are at least as stringent as, for example, conditions
M-R. TABLE-US-00001 TABLE 1 Stringency Conditions Poly- Hybrid
Hybridization Wash Stringency nucleotide Length Temperature
Temperature Condition Hybrid (bp).sup.I and Buffer.sup.H and
BufferH A DNA:DNA >50 65.degree. C.; 1xSSC -or- 65.degree. C.;
42.degree. C.; 1xSSC, 50% 0.3xSSC formamide B DNA:DNA <50
T.sub.B; 1xSSC T.sub.B; 1xSSC C DNA:RNA >50 67.degree. C.; 1xSSC
-or- 67.degree. C.; 45.degree. C.; 1xSSC, 50% 0.3xSSC formamide D
DNA:RNA <50 T.sub.D; 1xSSC T.sub.D; 1xSSC E RNA:RNA >50
70.degree. C.; 1xSSC -or- 70.degree. C.; 50.degree. C.; 1xSSC, 50%
0.3xSSC formamide F RNA:RNA <50 T.sub.F; 1xSSC T.sub.f; 1xSSC G
DNA:DNA >50 65.degree. C.; 4xSSC -or- 65.degree. C.; 1xSSC
42.degree. C.; 4xSSC, 50% formamide H DNA:DNA <50 T.sub.H; 4xSSC
T.sub.H; 4xSSC I DNA:RNA >50 67.degree. C.; 4xSSC -or-
67.degree. C.; 1xSSC 45.degree. C.; 4xSSC, 50% formamide J DNA:RNA
<50 T.sub.J; 4xSSC T.sub.J; 4xSSC K RNA:RNA >50 70.degree.
C.; 4xSSC -or- 67.degree. C.; 1xSSC 50.degree. C.; 4xSSC, 50%
formamide L RNA:RNA <50 T.sub.L; 2xSSC T.sub.L; 2xSSC M DNA:DNA
>50 50.degree. C.; 4xSSC -or- 50.degree. C.; 2xSSC 40.degree.
C.; 6xSSC, 50% formamide N DNA:DNA <50 T.sub.N; 6xSSC T.sub.N;
6xSSC O DNA:RNA >50 55.degree. C.; 4xSSC -or- 55.degree. C.;
2xSSC 42.degree. C.; 6xSSC, 50% formamide P DNA:RNA <50 T.sub.P;
6xSSC T.sub.P; 6xSSC Q RNA:RNA >50 60.degree. C.; 4xSSC -or-
60.degree. C.; 2xSSC 45.degree. C.; 6xSSC, 50% formamide R RNA:RNA
<50 T.sub.R; 4xSSC T.sub.R; 4xSSC (bp).sup.I: The hybrid length
is that anticipated for the hybridized region(s) of the hybridizing
polynucleotides. When hybridizing a polynucleotide to a target
polynucleotide of unknown sequence, the hybrid length is assumed to
be that of the hybridizing polynucleotide. When polynucleotides of
known sequence are hybridized, the hybrid length can be determined
by aligning the sequences of the polynucleotides and identifying
the region or regions of optimal sequence complementarity.
Buffer.sup.H: SSPE (1xSSPE is 0.15M NaCl, 10 mM NaH.sub.2PO.sub.4,
and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1xSSC is
0.15M NaCl and 15 mM sodium citrate) in the hybridization and wash
buffers; washes are performed for 15 minutes after hybridization is
complete. T.sub.B through T.sub.R: The hybridization temperature
for hybrids anticipated to be less than 50 base pairs in length
should be 5-10.degree. C. less than the melting temperature
(T.sub.m) of the hybrid, where T.sub.m is determined according to
the following equations. For hybrids less than 18 base pairs in
length, T.sub.m(.degree. C.) = 2 (# of A + T bases) + 4(# of G + C
bases). For hybrids between 18 and 49 base pairs in length, #
T.sub.m(.degree. C.) '2 81.5 = 16.6 (log.sub.10[Na.sup.+]) + 0.41
(% G + C) - (600/N), where N is the number of bases in the hybrid,
and [Na.sup.+] is the concentration of sodium ions in the
hybridization buffer ([Na.sup.+] for 1xSSC = 0.165 M).
[0079] Additional examples of stringency conditions for
polynucleotide hybridization are provided in Sambrook et al., 1989,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and
Ausubel et al., 1995, Current Protocols in Molecular Biology, eds.,
John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4,
incorporated herein by reference.
[0080] In addition to the nucleic acid molecules encoding P2T
receptor polypeptides described above, another aspect of the
invention pertains to isolated nucleic acid molecules which are
antisense thereto. An "antisense" nucleic acid comprises a
nucleotide sequence which is complementary to a "sense" nucleic
acid encoding a protein, e.g., complementary to the coding strand
of a double-stranded cDNA molecule or complementary to an mRNA
sequence. Accordingly, an antisense nucleic acid can hydrogen bond
to a sense nucleic acid. The antisense nucleic acid can be
complementary to an entire P2T coding strand (e.g., SEQ ID NO:1),
or to only a fragment thereof. In one embodiment, an antisense
nucleic acid molecule is antisense to a "coding region" of the
coding strand of a nucleotide sequence encoding a P2T receptor
polypeptide.
[0081] The term "coding region" refers to the region of the
nucleotide sequence comprising codons which are translated into
amino acid residues, e.g., the entire coding region of SEQ ID NO:1,
comprises about nucleotides 45,167 to about 46,358. In another
embodiment, the antisense nucleic acid molecule is antisense to a
"noncoding region" of the coding strand of a nucleotide sequence
encoding a P2T receptor polypeptide. The term "noncoding region"
refers to 5' and 3' sequences which flank the coding region that
are not translated into amino acids (i.e., also referred to as 5'
and 3' untranslated regions).
[0082] Given the coding strand sequence encoding the P2T
polypeptide disclosed herein (e.g., SEQ ID NO:1), antisense nucleic
acids of the invention can be designed according to the rules of
Watson and Crick base pairing. The antisense nucleic acid molecule
can be complementary to the entire coding region of P2T mRNA, but
more preferably is an oligonucleotide which is antisense to only a
fragment of the coding or noncoding region of P2T mRNA. For
example, the antisense oligonucleotide can be complementary to the
region surrounding the translation start site of P2T mRNA.
[0083] An antisense oligonucleotide can be, for example, about 5,
10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An
antisense nucleic acid of the invention can be constructed using
chemical synthesis and enzymatic ligation reactions using
procedures known in the art. For example, an antisense nucleic acid
(e.g., an antisense oligonucleotide) can be chemically synthesized
using naturally occurring nucleotides or variously modified
nucleotides designed to increase the biological stability of the
molecules or to increase the physical stability of the duplex
formed between the antisense and sense nucleic acids, e.g.,
phosphorothioate derivatives and acridine substituted nucleotides
can be used. Examples of modified nucleotides which can be used to
generate the antisense nucleic acid include 5-fluorouracil,
5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
I-methylguanine, I-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[0084] Alternatively, the antisense nucleic acid can be produced
biologically using an expression vector into which a nucleic acid
has been subcloned in an antisense orientation (i.e., RNA
transcribed from the inserted nucleic acid will be of an antisense
orientation to a target nucleic acid of interest, described further
in the following subsection).
[0085] The antisense nucleic acid molecules of the invention are
typically administered to a subject or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding a P2T receptor polypeptide to thereby inhibit expression
of the polypeptide, e.g., by inhibiting transcription and/or
translation. The hybridization can be by conventional nucleotide
complementarity to form a stable duplex, or, for example, in the
case of an antisense nucleic acid molecule which binds to DNA
duplexes, through specific interactions in the major groove of the
double helix. An example of a route of administration of an
antisense nucleic acid molecule of the invention includes direct
injection at a tissue site. Alternatively, an antisense nucleic
acid molecule can be modified to target selected cells and then
administered systemically. For example, for systemic
administration, an antisense molecule can be modified such that it
specifically binds to a receptor or an antigen expressed on a
selected cell surface, e.g., by linking the antisense nucleic acid
molecule to a peptide or an antibody which binds to a cell surface
receptor or antigen. The antisense nucleic acid molecule can also
be delivered to cells using the vectors described herein.
[0086] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule. An .alpha.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .gamma.-units, the strands run parallel to each other
(Gaultier et al., 1987). The antisense nucleic acid molecule can
also comprise a 2'-o-methylribonucleotide (Inoue et al., 1987) or a
chimeric RNA-DNA analogue (Inoue et al., 1987).
[0087] In still another embodiment, an antisense nucleic acid of
the invention is a ribozyme. Ribozymes are catalytic RNA molecules
with ribonuclease activity which are capable of cleaving a
single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. Thus, ribozymes (e.g., hammerhead ribozymes
(described in Haselhoff and Gerlach, 1988)) can be used to
catalytically cleave P2T mRNA transcripts to thereby inhibit
translation of P2T mRNA. A ribozyme having specificity for a
P2T-encoding nucleic acid can be designed based upon the nucleotide
sequence of the P2T genomic DNA disclosed herein (i.e., SEQ ID
NO:1). For example, a derivative of a Tetrahymena L-19 IVS RNA can
be constructed in which the nucleotide sequence of the active site
is complementary to the nucleotide sequence to be cleaved in a
P2T-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071
and Cech et al. U.S. Pat. No. 5,116,742, both of which are
incorporated by reference herein in their entirety. Alternatively,
P2T mRNA can be used to select a catalytic RNA having a specific
ribonuclease activity from a pool of RNA molecules. See, e.g.,
Bartel and Szostak, 1993).
[0088] Alternatively P2T gene expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory
region of the P2T gene (e.g., the P2T gene promoter and/or
enhancers) to form triple helical structures that prevent
transcription of the P2T gene in target cells. See generally,
Helene, 1991; Helene et al., 1992; and Maher, 1992).
[0089] P2T gene expression can also be inhibited using RNA
interference (RNAi). This is a technique for post-transcriptional
gene silencing (PTGS), in which target gene activity is
specifically abolished with cognate double-stranded RNA (dsRNA).
RNAi resembles in many aspects PTGS in plants and has been detected
in many invertebrates including trypanosome, hydra, planaria,
nematode and fruit fly (Drosophila melangnoster). It may be
involved in the modulation of transposable element mobilization and
antiviral state formation. RNAi in mammalian systems is disclosed
in International Application No. WO 00/63364 which is incorporated
by reference herein in its entirety. Basically, dsRNA of at least
about 600 nucleotides, homologous to the target (P2T) is introduced
into the cell and a sequence specific reduction in gene activity is
observed.
B. P2T Receptor Polypeptides
[0090] In particular embodiments, the present invention provides
isolated and purified P2T polypeptides. Preferably, a P2T receptor
polypeptide of the invention is a recombinant polypeptide.
Typically, a P2T receptor is produced by recombinant expression in
a non-human cell. In certain embodiments, a P2T receptor
polypeptide of the present invention comprises an amino acid
sequence that has at least 95% identity to the amino acid sequence
of SEQ ID NO:2, a variant thereof or a fragment thereof.
[0091] A P2T receptor polypeptide according to the present
invention encompasses a polypeptide that comprises: 1) the amino
acid sequence shown in SEQ ID NO:2; 2) functional and
non-functional naturally occurring allelic variants of murine P2T
receptor polypeptides; 3) recombinantly produced variants of murine
P2T receptor polypeptides; and 4) P2T receptor polypeptides
isolated from organisms other than mice (orthologues of murine P2T
receptor polypeptide.)
[0092] An allelic variant of murine P2T receptor polypeptides
according to the present invention encompasses 1) a polypeptide
isolated from murine cells or tissues; 2) a polypeptide encoded by
the same genetic locus as that encoding the murine P2T receptor
polypeptide; and 3) a polypeptide that contains substantially
homology to a murine P2T receptor.
[0093] Allelic variants of murine P2T include both functional and
non-functional P2T receptor polypeptides. Functional allelic
variants are naturally occurring amino acid sequence variants of
the murine P2T receptor polypeptide that maintain the ability to
bind a P2T receptor ligand and transduce a signal within a cell.
Functional allelic variants will typically contain only
conservative substitution of one or more amino acids of SEQ ID
NO:2, or substitution, deletion or insertion of non-critical
residues in non-critical regions of the polypeptide.
[0094] Non-functional allelic variants are naturally occurring
amino acid sequence variants of murine P2T receptor polypeptide
that do not have the ability to either bind ligand and/or transduce
a signal within a cell. Non-functional allelic variants will
typically contain a non-conservative substitution, a deletion, or
insertion or premature truncation of the amino acid sequence of SEQ
ID NO:2, or a substitution, insertion or deletion in critical
residues or critical regions.
[0095] The present invention further provides non-murine
orthologues of the murine P2T receptor polypeptide. Orthologues of
murine P2T receptor polypeptide are polypeptides that are isolated
from non-murine P2T organisms and possess the same ligand binding
and signaling capabilities of the murine P2T polypeptide.
Orthologues of the murine P2T receptor polypeptide can readily be
identified as comprising an amino acid sequence that is
substantially homologous to SEQ ID NO:2.
[0096] Modifications and changes can be made in the structure of a
polypeptide of the present invention and still obtain a molecule
having P2T like receptor characteristics. For example, certain
amino acids can be substituted for other amino acids in a sequence
without appreciable loss of receptor activity. Because it is the
interactive capacity and nature of a polypeptide that defines that
polypeptide's biological functional activity, certain amino acid
sequence substitutions can be made in a polypeptide sequence (or,
of course, its underlying DNA coding sequence) and nevertheless
obtain a polypeptide with like properties.
[0097] In making such changes, the hydropathic index of amino acids
can be considered. The importance of the hydropathic amino acid
index in conferring interactive biologic function on a polypeptide
is generally understood in the art (Kyte & Doolittle, 1982). It
is known that certain amino acids can be substituted for other
amino acids having a similar hydropathic index or score and still
result in a polypeptide with similar biological activity. Each
amino acid has been assigned a hydropathic index on the basis of
its hydrophobicity and charge characteristics. Those indices are:
isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine
(+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8);
glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9);
tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate
(-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5);
lysine (-3.9); and arginine (-4.5).
[0098] It is believed that the relative hydropathic character of
the amino acid residue determines the secondary and tertiary
structure of the resultant polypeptide, which in turn defines the
interaction of the polypeptide with other molecules, such as
enzymes, substrates, receptors, antibodies, antigens, and the like.
It is known in the art that an amino acid can be substituted by
another amino acid having a similar hydropathic index and still
obtain a functionally equivalent polypeptide. In such changes, the
substitution of amino acids whose hydropathic indices are within
+/-2 is preferred, those which are within +/-1 are particularly
preferred, and those within +/-0.5 are even more particularly
preferred.
[0099] Substitution of like amino acids can also be made on the
basis of hydrophilicity, particularly where the biological
functional equivalent polypeptide or peptide thereby created is
intended for use in immunological embodiments. U.S. Pat. No.
4,554,101, incorporated by reference herein in its entirety, states
that the greatest local average hydrophilicity of a polypeptide, as
governed by the hydrophilicity of its adjacent amino acids,
correlates with its immunogenicity and antigenicity, i.e. with a
biological property of the polypeptide.
[0100] As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); proline (-0.5.+-.1); threonine (-0.4); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4). It is understood that an
amino acid can be substituted for another having a similar
hydrophilicity value and still obtain a biologically equivalent,
and in particular, an immunologically equivalent polypeptide. In
such changes, the substitution of amino acids whose hydrophilicity
values are within .+-.2 is preferred, those which are within .+-.1
are particularly preferred, and those within .+-.0.5 are even more
particularly preferred.
[0101] As outlined above, amino acid substitutions are generally
therefore based on the relative similarity of the amino acid
side-chain substituents, for example, their hydrophobicity,
hydrophilicity, charge, size, and the like. Exemplary substitutions
which take various of the foregoing characteristics into
consideration are well known to those of skill in the art and
include: arginine and lysine; glutamate and aspartate; serine and
threonine; glutamine and asparagine; and valine, leucine and
isoleucine (see Table 2, below). The present invention thus
contemplates functional or biological equivalents of a P2T receptor
polypeptide as set forth above. TABLE-US-00002 TABLE 2 Original
Exemplary Residue Residue Substitution Ala Gly; Ser Arg Lys Asn
Gln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Ala His Asn; Gln Ile
Leu; Val Leu Ile; Val Lys Arg Met Leu; Tyr Ser Thr Thr Ser Trp Tyr
Tyr Trp; Phe Val Ile; Leu
[0102] Biological or functional equivalents of a polypeptide can
also be prepared using site-specific mutagenesis. Site-specific
mutagenesis is a technique useful in the preparation of second
generation polypeptides, or biologically functional equivalent
polypeptides or peptides, derived from the sequences thereof,
through specific mutagenesis of the underlying DNA. As noted above,
such changes can be desirable where amino acid substitutions are
desirable. The technique further provides a ready ability to
prepare and test sequence variants, for example, incorporating one
or more of the foregoing considerations, by introducing one or more
nucleotide sequence changes into the DNA. Site-specific mutagenesis
allows the production of mutants through the use of specific
oligonucleotide sequences which encode the DNA sequence of the
desired mutation, as well as a sufficient number of adjacent
nucleotides, to provide a primer sequence of sufficient size and
sequence complexity to form a stable duplex on both sides of the
deletion junction being traversed. Typically, a primer of about 17
to 25 nucleotides in length is preferred, with about 5 to 10
residues on both sides of the junction of the sequence being
altered.
[0103] In general, the technique of site-specific mutagenesis is
well known in the art. As will be appreciated, the technique
typically employs a phage vector which can exist in both a single
stranded and double stranded form. Typically, site-directed
mutagenesis in accordance herewith is performed by first obtaining
a single-stranded vector which includes within its sequence a DNA
sequence which encodes all or a portion of the P2T polypeptide
sequence selected. An oligonucleotide primer bearing the desired
mutated sequence is prepared (e.g., synthetically). This primer is
then annealed to the single-stranded vector, and extended by the
use of enzymes such as E. coli polymerase I Klenow fragment, in
order to complete the synthesis of the mutation-bearing strand.
Thus, a heteroduplex is formed wherein one strand encodes the
original non-mutated sequence and the second strand bears the
desired mutation. This heteroduplex vector is then used to
transform appropriate cells such as E. coli cells and clones are
selected which include recombinant vectors bearing the mutation.
Commercially available kits come with all the reagents necessary,
except the oligonucleotide primers.
[0104] The P2T receptor polypeptide is a P2T receptor that
participates in signaling pathways within cells. As used herein, a
signaling pathway refers to the modulation (e.g., stimulated or
inhibited) of a cellular function/activity upon the binding of a
ligand to the P2T receptor. Examples of such functions include
mobilization of intracellular molecules that participate in a
signal transduction pathway, e.g., phosphatidylinositol
4,5-bisphosphate (PIP.sub.2), inositol 1,4,5-triphosphate,
adenylate cyclase, Ca.sup.2+, ADP and ATP; inhibition or activation
of adenylyl cyclase; neurotransmitters; polarization of the plasma
membrane; production or secretion of molecules; alteration in the
structure of a cellular component; cell proliferation, e.g.,
synthesis of DNA; cell migration; cell differentiation; and cell
survival.
[0105] Depending on the type of cell, the response mediated by the
P2T receptor polypeptide/ligand binding may be different. For
example, in some cells, binding of a ligand to a P2T receptor
polypeptide may stimulate an activity such as adhesion, migration,
differentiation, etc. through phosphatidylinositol or cyclic AMP
metabolism and turnover while in other cells, the binding of the
ligand to the P2T receptor polypeptide will produce a different
result. Regardless of the cellular activity modulated by P2T
receptor, it is universal that the P2T receptor polypeptide is a
P2T and interacts with a "G-protein" to produce one or more
secondary signals in a variety of intracellular signal transduction
pathways, e.g., through phosphatidylinositol or cyclic AMP
metabolism and turnover, in a cell. G-proteins represent a family
of heterotrimeric polypeptides composed of .alpha., .beta. and
.gamma. subunits, which bind guanine nucleotides. These
polypeptides are usually linked to cell surface receptors, e.g.,
receptors containing seven transmembrane domains, such as the
ligand receptors. Following ligand binding to the receptor, a
conformational change is transmitted to the G-protein, which causes
the .alpha.-subunit to exchange a bound GDP molecule for a GTP
molecule and to dissociate from the .beta. and .gamma.-subunits.
The GTP-bound form of the .alpha.-subunit typically functions as an
effector-modulating moiety, leading to the production of second
messengers, such as cyclic AMP (e.g., by activation of adenylate
cyclase), diacylglycerol or inositol phosphates. As well, the free
.beta..gamma.-subunit complex may function as an
effector-modulating moiety, leading to the production of second
messengers, such as cyclic AMP (e.g., by activation of adenylate
cyclase), diacylglycerol or inositol phosphates. Greater than 20
different types of .alpha.-subunits are known in man, which
associate with a smaller pool of .beta. and .gamma. subunits.
[0106] As used herein, "phosphatidylinositol turnover and
metabolism" refers to the molecules involved in the turnover and
metabolism of phosphatidylinositol 4,5-bisphosphate (PIP.sub.2) as
well as to the activities of these molecules. PIP.sub.2 is a
phospholipid found in the cytosolic leaflet of the plasma membrane.
Binding of a ligand to the GPCR, (e.g., P2T) activates, in some
cells, the plasma-membrane enzyme phospholipase C that in turn can
hydrolyze PIP.sub.2 to produce 1,2-diacylglycerol (DAG) and
inositol 1,4,5-triphosphate (IP.sub.3). Once formed IP.sub.3 can
diffuse to the endoplasmic reticulum surface where it can bind an
IP.sub.3 receptor, e.g., a calcium channel polypeptide containing
an IP.sub.3 binding site. IP.sub.3 binding can induce opening of
the channel, allowing calcium ions to be released into the
cytoplasm. IP3 can also be phosphorylated by a specific kinase to
form, a molecule which can cause calcium entry into the cytoplasm
from the extracellular medium. IP.sub.3 and inositol
1,3,4,5-tetraphosphate can subsequently be hydrolyzed very rapidly
to the inactive products inositol 1,4-biphosphate and inositol
1,3,4- triphosphate, respectively. These inactive products can be
recycled by the cell to synthesize PIP.sub.2. The other second
messenger produced by the hydrolysis of PIP.sub.2, namely
1,2-diacylglycerol (DAG), remains in the cell membrane where it can
serve to activate the enzyme polypeptide kinase C. Polypeptide
kinase C is usually found soluble in the cytoplasm of the cell, but
upon an increase in the intracellular calcium concentration, this
enzyme can move to the plasma membrane where it can be activated by
DAG. The activation of polypeptide kinase C in different cells
results in various cellular responses such as the phosphorylation
of glycogen synthase, or the phosphorylation of various
transcription factors, e.g., NF-kB. The language
"phosphatidylinositol activity," as used herein, refers to an
activity of PIP.sub.2 or one of its metabolites.
[0107] Another signaling pathway in which the P2T receptor
polypeptide may participate is the cAMP turnover pathway. As used
herein, "cyclic AMP turnover and metabolism" refers to the
molecules involved in the turnover and metabolism of cyclic AMP
(cAMP) as well as to the activities of these molecules. Cyclic AMP
is a second messenger produced in response to ligand induced
stimulation of certain G-protein-coupled receptors. In the ligand
signaling pathway, binding of ligand to a ligand receptor can lead
to the activation of the enzyme adenylate cyclase, which catalyzes
the synthesis of cAMP. The newly synthesized cAMP can in turn
activate a cAMP-dependent protein kinase. This activated kinase
can, for example, phosphorylate a voltage-gated potassium channel
polypeptide, or an associated polypeptide, and lead to the
inability of the potassium channel to open during an action
potential. The inability of the potassium channel to open results
in a decrease in the outward flow of potassium, which normally
repolarizes the membrane of a neuron, leading to prolonged membrane
depolarization. Of course, the activated cAMP-dependent protein
kinase can affect other molecules as well, such as enzymes (e.g.,
metabolic enzymes), transcription factors, adenylyl cyclase and the
like.
[0108] A P2T receptor polypeptide of the present invention is
understood to be any P2T polypeptide comprising substantial
sequence similarity, structural similarity and/or functional
similarity to a P2T polypeptide comprising the amino acid sequence
of SEQ ID NO:2. In addition, a P2T polypeptide of the invention is
not limited to a particular source. Thus, the invention provides
for the general detection and isolation of the genus of P2T
receptor polypeptides from a variety of sources.
[0109] It is contemplated in the present invention, that a P2T
polypeptide may advantageously be cleaved into fragments for use in
further structural or functional analysis, or in the generation of
reagents such as P2T-related polypeptides and P2T-specific
antibodies. This can be accomplished by treating purified or
unpurified P2T with a peptidase such as endopolypeptidease glu-C
(Boehringer, Indianapolis, Ind.). Treatment with CNBr is another
method by which P2T fragments may be produced from natural P2T.
Recombinant techniques also can be used to produce specific
fragments of P2T.
[0110] In addition, the invention also contemplates that compounds
sterically similar to a P2T may be formulated to mimic the key
portions of the peptide structure, called peptidomimetics. Mimetics
are peptide-containing molecules which mimic elements of
polypeptide secondary structure. See, for example, Johnson et al.
(1993). The underlying rationale behind the use of peptide mimetics
is that the peptide backbone of polypeptides exists chiefly to
orient amino acid side chains in such a way as to facilitate
molecular interactions, such as those of receptor and ligand.
[0111] Successful applications of the peptide mimetic concept have
thus far focused on mimetics of .beta.-turns within polypeptides.
Likely .beta.-turn structures within a P2T polypeptide can be
predicted by computer-based algorithms as discussed above. Once the
component amino acids of the turn are determined, mimetics can be
constructed to achieve a similar spatial orientation of the
essential elements of the amino acid side chains, as discussed in
Johnson et al. (1993).
[0112] "Fusion polypeptide" refers to a polypeptide encoded by two,
often unrelated, fused genes or fragments thereof. For example,
fusion polypeptides comprising various portions of constant region
of immunoglobulin molecules together with another human polypeptide
or part thereof have been described. In many cases, employing an
immunoglobulin Fc region as a part of a fusion polypeptide is
advantageous for use in therapy and diagnosis resulting in, for
example, improved pharmacokinetic properties (see, e.g., U.S. Pat.
No. 5,696,237). On the other hand, for some uses it would be
desirable to be able to delete the Fc part after the fusion
polypeptide has been expressed, detected and purified.
C. P2T Polynucleotide and Polypeptide Variants
[0113] "Variant" as the term is used herein, is a polynucleotide or
polypeptide that differs from a reference polynucleotide or
polypeptide respectively, but retains essential properties. A
typical variant of a polynucleotide differs in nucleotide sequence
from another, reference polynucleotide. Changes in the nucleotide
sequence of the variant may or may not alter the amino acid
sequence of a polypeptide encoded by the reference polynucleotide.
Nucleotide changes may result in amino acid substitutions,
additions, deletions, fusions and truncations in the polypeptide
encoded by the reference sequence, as discussed below. A typical
variant of a polypeptide differs in amino acid sequence from
another, reference polypeptide. Generally, differences are limited
so that the sequences of the reference polypeptide and the variant
are closely similar overall and, in many regions, identical. A
variant and reference polypeptide may differ in amino acid sequence
by one or more substitutions, additions, deletions in any
combination. A substituted or inserted amino acid residue may or
may not be one encoded by the genetic code. A variant of a
polynucleotide or polypeptide may be naturally occurring such as an
allelic variant, or it may be a variant that is not known to occur
naturally. Non-naturally occurring variants of polynucleotides and
polypeptides may be made by mutagenesis techniques or by direct
synthesis.
[0114] "Identity," as known in the art, is a relationship between
two or more polypeptide sequences or two or more polynucleotide
sequences, as determined by comparing the sequences. In the art,
"identity" also means the degree of sequence relatedness between
polypeptide or polynucleotide sequences, as the case may be, as
determined by the match between strings of such sequences.
"Identity" and "similarity" can be readily calculated by known
methods, including but not limited to those described in
(Computational Molecular Biology, Lesk, A. M., ed., Oxford
University Press, New York, 1988; Biocomputing: Informatics and
Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
Computer Analysis of Sequence Data, Part I, 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; and Carillo, H., and
Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Preferred
methods to determine identity are designed to give the largest
match between the sequences tested. Methods to determine identity
and similarity are codified in publicly available computer
programs. Preferred computer program methods to determine identity
and similarity between two sequences include, but are not limited
to, the GCG program package (Devereux, J., et al 1984), BLASTP,
BLASTN, and FASTA (Altschul, S. F., et al., 1990. The BLASTX
program is publicly available from NCBI and other sources (BLAST
Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894;
Altschul, S., et al., 1990). The well known Smith Waterman
algorithm may also be used to determine identity.
[0115] By way of example, a genomic polynucleotide sequence of the
present invention may be identical to the reference sequence of SEQ
ID NO:1, that is be 100% identical, or it may include up to a
certain integer number of nucleotide alterations as compared to the
reference sequence. Such alterations are selected from the group
consisting of at least one nucleotide deletion, substitution,
including transition and transversion, or insertion, and wherein
said alterations may occur at the 5' or 3' terminal positions of
the reference nucleotide sequence or anywhere between those
terminal positions, interspersed either individually among the
nucleotides in the reference sequence or in one or more contiguous
groups within the reference sequence. The number of nucleotide
alterations is determined by multiplying the total number of
nucleotides in SEQ ID NO:1, by the numerical percent of the
respective percent identity (divided by 100) and subtracting that
product from said total number of nucleotides in SEQ ID NO:1.
[0116] For example, an isolated murine P2T polynucleotide
comprising a polynucleotide sequence that has at least 95% identity
to the nucleic acid sequence of SEQ ID NO:1; a degenerate variant
thereof or a fragment thereof, wherein the polynucleotide sequence
may include up to n.sub.n nucleic acid alterations over the entire
polynucleotide region of the nucleic acid sequence of SEQ ID NO:1,
wherein n.sub.n is the maximum number of alterations and is
calculated by the formula: n.sub.n.ltoreq.x.sub.n-(x.sub.ny),
[0117] in which xn is the total number of nucleic acids of SEQ ID
NO:1 and y has a value of 0.95, wherein any non-integer product of
x.sub.n and y is rounded down to the nearest integer prior to
subtracting such product from x.sub.n. Of course, y may also have a
value of 0.80 for 80%, 0.85 for 85%, 0.90 for 90% 0.95 for 95%,
etc. Alterations of a polynucleotide sequence encoding a P2T
polypeptide of SEQ ID NO:2 may result in a functional P2T receptor
polypeptide or a non-functional P2T receptor polypeptide. A
functional P2T receptor polypeptide maintains the ability to bind a
P2T receptor ligand and transduce a signal within a cell. A
non-functional P2T receptor polypeptide lacks the ability to either
bind ligand and/or transduce a signal within a cell.
D. Vectors, Host Cells and Recombinant P2T Polypeptides
[0118] In an alternate embodiment, the present invention provides
expression vectors comprising polynucleotides that encode P2T
polypeptides. Preferably, the expression vectors of the present
invention comprise polynucleotides that encode polypeptides
comprising the amino acid residue sequence of SEQ ID NO:2. More
preferably, the expression vectors of the present invention
comprise polynucleotides comprising the nucleotide base sequence of
SEQ ID NO:1. Even more preferably, the expression vectors of the
invention comprise polynucleotides operatively linked to an
enhancer-promoter. In certain embodiments, the expression vectors
of the invention comprise polynucleotides operatively linked to a
prokaryotic promoter. Alternatively, the expression vectors of the
present invention comprise polynucleotides operatively linked to an
enhancer-promoter that is a eukaryotic promoter, and the expression
vectors further comprise a polyadenylation signal that is
positioned 3' of the carboxy-terminal amino acid and within a
transcriptional unit of the encoded polypeptide.
[0119] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, to the amino or carboxy terminus of the
recombinant protein. Such fusion vectors typically serve three
purposes: 1) to increase expression of recombinant protein; 2) to
increase the solubility of the recombinant protein; and 3) to aid
in the purification of the recombinant protein by acting as a
ligand in affinity purification. Often, in fusion expression
vectors, a proteolytic cleavage site is introduced at the junction
of the fusion moiety and the recombinant protein to enable
separation of the recombinant protein from the fusion moiety
subsequent to purification of the fusion protein. Such enzymes, and
their cognate recognition sequences, include Factor Xa, thrombin
and enterokinase.
[0120] Typical fusion expression vectors include pGEX (Pharmacia
Biotech Inc; Smith and Johnson,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.
[0121] In one embodiment, the coding sequence of the P2T gene is
cloned into a pGEX expression vector to create a vector encoding a
fusion protein comprising, from the N-terminus to the C-terminus,
GST-thrombin cleavage site-P2T polypeptide. The fusion protein can
be purified by affinity chromatography using glutathione-agarose
resin. Recombinant P2T polypeptide unfused to GST can be recovered
by cleavage of the fusion protein with thrombin.
[0122] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., 1988) and pET I I d (Studier et
al., 1990). Target gene expression from the pTrc vector relies on
host RNA polymerase transcription from a hybrid trp-lac fusion
promoter. Target gene expression from the pET I I d vector relies
on transcription from a T7 gn1 .beta.-lac fusion promoter mediated
by a coexpressed viral RNA polymerase J7 gnl. This viral polymerase
is supplied by host strains BL21 (DE3) or HMS I 74(DE3) from a
resident prophage harboring a T7 gnl gene under the transcriptional
control of the lacUV 5 promoter.
[0123] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacteria with an
impaired capacity to proteolytically cleave the recombinant
protein. Another strategy is to alter the nucleic acid sequence of
the nucleic acid to be inserted into an expression vector so that
the individual codons for each amino acid are those preferentially
utilized in E. coli. Such alteration of nucleic acid sequences of
the invention can be carried out by standard DNA mutagenesis or
synthesis techniques.
[0124] In another embodiment, the P2T polynucleotide expression
vector is a yeast expression vector. Examples of vectors for
expression in yeast S. cerivisae include pYepSec I (Baldari, et
al., 1987), pMFa (Kurjan and Herskowitz, 1982), pJRY88 (Schultz et
al., 1987), and pYES2 (Invitrogen Corporation, San Diego, Calif.),
p416GPD and p426GPD (Mumberg et al., 1995).
[0125] Alternatively, a P2T polynucleotide can 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., 1983) and the pVL series (Lucklow and Summers,
1989).
[0126] In yet another embodiment, a polynucleotide of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pCDM8
(Seed, 1987) and pMT2PC (Kaufman et al., 1987). When used in
mammalian cells, the expression vector's control functions are
often provided by viral regulatory elements.
[0127] For example, commonly used promoters are derived from
polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For
other suitable expression systems for both prokaryotic and
eukaryotic cells see chapters 16 and 17 of 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, incorporated herein by reference.
[0128] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al., 1987),
lymphoid-specific promoters (Calame and Eaton, 1988), in particular
promoters of T cell receptors (Winoto and Baltimore, 1989) and
immunoglobulins (Banerji et al., 1983), Queen and Baltimore (1983),
neuron-specific promoters (e.g., the neurofilament promoter; Byrne
and Ruddle, 1989), pancreas-specific promoters (Edlund et al.,
1985), and mammary gland-specific promoters (e.g., milk whey
promoter; U.S. Pat. No. 4,873,316 and International Application No.
EP 264,166). Developmentally-regulated promoters are also
encompassed, for example the murine hox promoters (Kessel and
Gruss, 1990) and the .alpha.-fetoprotein promoter (Campes and
Tilghman, 1989).
[0129] The present invention also relates to improved methods for
both the in vitro production of P2T polypeptides and for the
production and delivery of P2T polypeptides by gene therapy. The
present invention includes approaches which activate expression of
endogenous cellular genes, and further allows amplification of the
activated endogenous cellular genes, which does not require in
vitro manipulation and transfection of exogenous DNA encoding P2T
polypeptides. These methods are described in PCT International
Application WO 94/12650, U.S. Pat. No. 5,968,502, and Harrington et
al., 2001, all of which are incorporated in their entirety by
reference. These, and variations of them which one skilled in the
art will recognize as equivalent, may collectively be referred to
as "gene activation".
[0130] Thus, in certain embodiments, the invention relates to
transfected cells, both transfected primary or secondary cells
(i.e., non-immortalized cells) and transfected immortalized cells,
useful for producing proteins, methods of making such cells,
methods of using the cells for in vitro protein production and
methods of gene therapy. Cells of the present invention are of
vertebrate origin, particularly of mammalian origin and even more
particularly of human origin. Cells produced by the method of the
present invention contain exogenous DNA which encodes a therapeutic
product, exogenous DNA which is itself a therapeutic product and/or
exogenous DNA which causes the transfected cells to express a gene
at a higher level or with a pattern of regulation or induction that
is different than occurs in the corresponding nontransfected
cell.
[0131] The present invention also relates to methods by which
primary, secondary, and immortalized cells are transfected to
include exogenous genetic material, methods of producing clonal
cell strains or heterogeneous cell strains, and methods of
immunizing animals, or producing antibodies in immunized animals,
using the transfected primary, secondary, or immortalized
cells.
[0132] The present invention relates particularly to a method of
gene targeting or homologous recombination in cells of vertebrate,
particularly mammalian, origin. That is, it relates to a method of
introducing DNA into primary, secondary, or immortalized cells of
vertebrate origin through homologous recombination, such that the
DNA is introduced into genomic DNA of the primary, secondary, or
immortalized cells at a pre-selected site. The targeting sequences
used are determined by (selected with reference to) the site into
which the exogenous DNA is to be inserted. The genomic P2T
sequences provided by the present invention (i.e., SEQ ID NO:1) are
useful in these methods. The present invention further relates to
homologously recombinant primary, secondary, or immortalized cells,
referred to as homologously recombinant (HR) primary, secondary or
immortalized cells, produced by the present method and to uses of
the HR primary, secondary, or immortalized cells.
[0133] The present invention also relates to a method of activating
(i.e., turning on) a P2T gene present in primary, secondary, or
immortalized cells of vertebrate origin, which is normally not
expressed in the cells or is not expressed at physiologically
significant levels in the cells as obtained. According to the
present method, homologous recombination is used to replace or
disable the regulatory region normally associated with the gene in
cells as obtained with a regulatory sequence which causes the gene
to be expressed at levels higher than evident in the corresponding
nontransfected cell, or to display a pattern of regulation or
induction that is different than evident in the corresponding
nontransfected cell. The present invention, therefore, relates to a
method of making proteins by turning on or activating an endogenous
gene which encodes the desired product in transfected primary,
secondary, or immortalized cells.
[0134] In one embodiment, the activated gene can be further
amplified by the inclusion of a selectable marker gene which has
the property that cells containing amplified copies of the
selectable marker gene can be selected for by culturing the cells
in the presence of the appropriate selectable agent. The activated
endogenous gene which is near or linked to the amplified selectable
marker gene will also be amplified in cells containing the
amplified selectable marker gene. Cells containing many copies of
the activated endogenous gene are useful for in vitro protein
production and gene therapy.
[0135] In certain embodiments, the present invention relates also
to methods for activating the expression of an endogenous gene in a
cell or over-expressing an endogenous gene in a cell by
non-homologous or random activation of gene expression (RAGE). The
method comprises introducing a vector into the cell, allowing the
vector to integrate into the genome of the cell by non-homologous
recombination and allowing activation or over-expression of the
endogenous gene in the cell. The use of non-homologous or
"non-targeted" recombination does not require previous knowledge of
the endogenous gene sequence. The methods for expression of
endogenous genes via non-homologous recombination and preparing
vector constructs for non-homologous recombination are described in
International Patent Applications WO 99/15650 and WO 00/49162, both
of which are incorporated in their entirety by reference.
[0136] Vector constructs useful in non-homologous recombination
events should contain at least a transcriptional regulatory
sequence operably linked to an unpaired splice donor sequence and
one or more amplifiable markers. The transcriptional regulatory
sequence is typically, but not limited to, a promoter sequence. The
transcriptional regulatory sequence may further comprise an
enhancer sequence, in addition to the promoter sequence. The
transcriptional regulatory sequence is operatively linked to a
translational start codon, a signal secretion sequence and an
unpaired splice donor site. The transcriptional regulatory sequence
may additionally be operatively linked to a translational start
codon, an epitope tag and an unpaired splice donor site; or
operatively linked to a translational start codon, a signal
secretion sequence, an epitope tag and an unpaired splice donor
site; or operatively linked to a translational start codon, a
signal secretion sequence, an epitope tag, a sequence specific
protease site and an unpaired splice donor site.
[0137] Examples of amplifiable markers that may be used in the
above described vectors include, but are not limited to,
dihydrofolate reductase (DHFR), neomycin resistance (neo),
hypoxanthine phosphoribosyl transferase (HPRT), puromycin (pac),
adenosine deaminase (ada), aspartate transcarbamylase (ATC),
dihydro-orotase, histidine D (his D), multidrug resistance 1 (mdr
1), xanthine-guanine phosphoribosyl transferase (gpt), glutamine
synthetase (GS) and carbamyl phosphate synthase (CAD). The vector
could additionally comprise a screenable marker, such as a gene
encoding a cell surface protein, a fluorescent protein and/or an
enzyme. A signal secretion sequence may be included on the
"activation" vector construct, such that the activated gene
expression product is secreted.
[0138] The regulatory sequence of the vector construct can be a
constitutive promoter, an inducible promoter or a tissue specific
promoter or an enhancer. The use of an inducible promoter will
permit low basal levels of activated protein to be produced by the
cell during routine culturing and expansion. Subsequently, the
cells may then be induced to express large amounts of the desired
protein during production or screening. The regulatory sequence may
be isolated from cellular or viral genomes. Examples of cellular
regulatory sequences include, but are not limited to, the actin
gene, metallothionein I gene, collagen gene, serum albumin gene and
immunoglobulin genes. Examples of viral regulatory sequences
include, but are not limited to, regulatory elements from
Cytomegalovirus (CMV) immediate early gene, adenovirus late genes,
SV40 genes, retroviral LTRs and Herpesvirus genes (see Tables 3 and
4 for additional tissue specific and inducible regulatory
sequences, respectively).
[0139] Splicing of primary transcripts, the process by which
introns are removed, is directed by a splice donor site and a
splice acceptor site, located at the 5' and 3' ends introns,
respectively. The consensus sequence for splice donor sites is
(A/C)AGGURAGU (where R represents a purine nucleotide), with
nucleotides (A/C)AG in positions 1-3 located in the exon and
nucleotides GURAGU located in the intron.
[0140] An unpaired splice donor site is defined herein as a splice
donor site present on the vector construct without a downstream
splice acceptor site. When the vector is integrated by
non-homologous recombination into the genome of a host cell, the
unpaired splice donor site becomes paired with a splice acceptor
site from an endogenous gene. The splice donor site from the vector
construct, in conjunction with the splice acceptor site from the
endogenous gene, will then direct the excision of all of the
sequences between the vector splice donor site and the endogenous
splice acceptor site. Excision of these intervening sequences
removes sequences that interfere with translation of the endogenous
protein.
[0141] A promoter is a region of a DNA molecule typically within
about 100 nucleotide pairs in front of (upstream of) the point at
which transcription begins (i.e., a transcription start site). That
region typically contains several types of DNA sequence elements
that are located in similar relative positions in different genes.
As used herein, the term "promoter" includes what is referred to in
the art as an upstream promoter region, a promoter region or a
promoter of a generalized eukaryotic RNA Polymerase II
transcription unit.
[0142] Another type of discrete transcription regulatory sequence
element is an enhancer. An enhancer provides specificity of time,
location and expression level for a particular encoding region
(e.g., gene). A major function of an enhancer is to increase the
level of transcription of a coding sequence in a cell that contains
one or more transcription factors that bind to that enhancer.
Unlike a promoter, an enhancer can function when located at
variable distances from transcription start sites so long as a
promoter is present.
[0143] As used herein, the phrase "enhancer-promoter" means a
composite unit that contains both enhancer and promoter elements.
An enhancer-promoter is operatively linked to a coding sequence
that encodes at least one gene product. As used herein, the phrase
"operatively linked" means that an enhancer-promoter is connected
to a coding sequence in such a way that the transcription of that
coding sequence is controlled and regulated by that
enhancer-promoter. Means for operatively linking an
enhancer-promoter to a coding sequence are well known in the art.
As is also well known in the art, the precise orientation and
location relative to a coding sequence whose transcription is
controlled, is dependent inter alia upon the specific nature of the
enhancer-promoter. Thus, a TATA box minimal promoter is typically
located from about 25 to about 30 base pairs upstream of a
transcription initiation site and an upstream promoter element is
typically located from about 100 to about 200 base pairs upstream
of a transcription initiation site. In contrast, an enhancer can be
located downstream from the initiation site and can be at a
considerable distance from that site.
[0144] A coding sequence of an expression vector is operatively
linked to a transcription terminating region. RNA polymerase
transcribes an encoding DNA sequence through a site where
polyadenylation occurs. Typically, DNA sequences located a few
hundred base pairs downstream of the polyadenylation site serve to
terminate transcription. Those DNA sequences are referred to herein
as transcription-termination regions. Those regions are required
for efficient polyadenylation of transcribed messenger RNA (mRNA).
Transcription-terminating regions are well known in the art. A
preferred transcription-terminating region used in an adenovirus
vector construct of the present invention comprises a
polyadenylation signal of SV40 or the protamine gene.
TABLE-US-00003 TABLE 3 Tissue Specific Promoters PROMOTER Target
Tyrosinase Melanocytes Tyrosinase Related Protein, Melanocytes
TRP-1 Prostate Specific Antigen, Prostate Cancer PSA Albumin Liver
Apolipoprotein Liver Plasminogen Activator Liver Inhibitor Type-1,
PAI-1 Fatty Acid Binding Colon Epithelial Cells Insulin Pancreatic
Cells Muscle Creatine Kinase, Muscle Cell MCK Myelin Basic Protein,
MBP Oligodendrocytes and Glial Cells Glial Fibrillary Acidic Glial
Cells Protein, GFAP Neural Specific Enolase Nerve Cells
Immunoglobulin Heavy B-cells Chain Immunoglobulin Light Chain
B-cells, Activated T-cells T-Cell Receptor Lymphocytes HLA
DQ.alpha. and DQ.beta. Lymphocytes .beta.-Interferon Leukocytes;
Lymphocytes Fibroblasts Interlukin-2 Activated T-cells Platelet
Derived Growth Erythrocytes Factor E2F-1 Proliferating Cells Cyclin
A Proliferating Cells .alpha.-, .beta.-Actin Muscle Cells
Haemoglobin Erythroid Cells Elastase I Pancreatic Cells Neural Cell
Adhesion Neural Cells Molecule, NCAM
[0145] TABLE-US-00004 TABLE 4 Inducible Promoters Promoter Element
Inducer Early Growth Response-1 Radiation Gene, egr-1 Tissue
Plasmingen Radiation Activator, t-PA fos and jun Radiation Multiple
Drug Resistance Chemotherapy Gene 1, mdr-1 Heat Shock Proteins;
Heat hsp16, hs60, hps68, hsp70, human Plasminogen Tumor Necrosis
Factor, Activator Inhibitor type-1, TNF hPAI-1 Cytochrome P-450
Toxins CYP1A1 Metal-Responsive Heavy Metals Element, MRE Mouse
Mammary Tumor Glucocorticoids Virus Collagenase Phorbol Ester
Stromolysin Phorbol Ester SV40 Phorbol Ester Proliferin Phorbol
Ester .alpha.-2-Macroglobulin IL-6 Murine MX Gene Interferon,
Newcastle Disease Virus Vimectin Serum Thyroid Stimulating Thyroid
Hormone Hormone .alpha. Gene HSP70 Ela, SV40 Large T Antigen Tumor
Necrosis Factor FMA Interferon Viral Infection, dsRNA Somatostatin
Cylic AMP Fibronectin Cyclic AMP
[0146] The cell expressing or over-expressing the gene of interest
can be cultured in vitro under conditions favoring the production
of the desired amounts of the expression product of the endogenous
gene that has been activated or whose expression has been
increased. A cell containing a vector construct which has been
integrated into its genome may also be introduced into a eukaryote
(e.g., a vertebrate, preferably a mammal, more preferably a human)
under conditions favoring the activation or over-expression of the
gene by the cell in vivo in the eukaryote. In particular
embodiments, a genome-wide transcription library and protein
expression library are generated (Harrington et al., 2001).
Libraries are generated by random activation of gene expression
(RAGE) using the above described vector constructs for
non-homologous recombination.
[0147] Host cells can be derived from any eukaryotic species and
can be primary, secondary, or immortalized. Furthermore, the cells
can be derived from any tissue in the organism. Examples of useful
tissues which cells can be isolated and activated include, but are
not limited to, liver, spleen, kidney, bone marrow, thymus, heart,
muscle, lung, brain, testes, ovary, islet, intestinal, skin, gall
bladder, prostate, bladder and the immune hemapoietic systems.
[0148] The vector construct can be integrated into primary,
secondary, or immortalized cells. Primary cells are cells that have
been isolated from a vertebrate and have not been passaged.
Secondary cells are primary cells that have been passaged, but are
not immortalized. Immortalized cells are cell lines that can be
passaged, apparently indefinitely. Examples of immortalized cell
lines include, but are not limited to, HT1080, HeLa, Jurkat, 293
cells, KB carcinoma, T84 colonic epithelial cell line, Raji, Hep G2
or Hep 3B, hepatoma cell lines, A2058 melanoma, U937 lymphoma and
WI38 fibroblast cell line, somatic cell hybrids and hybridomas.
[0149] Thus, to activate an endogenous gene of the present by
non-homologous recombination, one would generate an "activation"
vector construct comprising a regulatory sequence, one or more
amplifiable markers, an epitope tag or a secretion signal sequence
and an unpaired splice donor sequence. The activation construct is
then introduced into a preferred eukaryotic host cell by any
transfection method known in the art. Following introduction of the
vector into the cell, the DNA is allowed to integrate into the host
cell genome via non-homologous recombination. Integration can occur
at spontaneous chromosome breaks or at artificially induced
chromosomal beaks (e.g., .gamma. irradiation, restriction enzymes).
Following integration of the vector into the genome of the host
cell, the genetic locus may be amplified in copy number by
simultaneous or sequential selection for the one or more
amplifiable markers located on the integrated vector construct.
This approach facilitates the isolation of clones of cells that
have amplified the locus containing the integrated vector. The
cells containing the activated genes are isolated, sorted and the
activated endogenous genes are isolated by PCR-based cloning (for a
detailed experimental protocol, see International Application WO
99/15650, which is incorporated in its entirety by reference). One
of ordinary skill in the art will appreciate, however, that any
art-known method of cloning genes may be equivalently used to
isolate activated genes from the sorted cells.
[0150] The invention further provides a recombinant expression
vector comprising a DNA molecule encoding a P2T polypeptide cloned
into the expression vector in an antisense orientation. That is,
the DNA molecule is operatively linked to a regulatory sequence in
a manner which allows for expression (by transcription of the DNA
molecule) of an RNA molecule which is antisense to P2T mRNA.
Regulatory sequences operatively linked to a nucleic acid cloned in
the antisense orientation can be chosen which direct the continuous
expression of the antisense RNA molecule in a variety of cell
types, for instance viral promoters and/or enhancers, or regulatory
sequences can be chosen which direct constitutive, tissue specific
or cell type specific expression of antisense RNA. The antisense
expression vector can be in the form of a recombinant plasmid,
phagemid or attenuated virus in which antisense nucleic acids are
produced under the control of a high efficiency regulatory region,
the activity of which can be determined by the cell type into which
the vector is introduced.
[0151] Another aspect of the invention pertains to host cells into
which a recombinant expression vector of the invention has been
introduced. The terms "host cell" and "recombinant host cell" are
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein. A host cell can be any
prokaryotic or eukaryotic cell. For example, P2T polypeptide can be
expressed in bacterial cells such as E. Coli insect cells, yeast or
mammalian cells (such as Chinese hamster ovary cells (CHO) or COS
cells). Other suitable host cells are known to those skilled in the
art.
[0152] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation, infection or transfection
techniques. As used herein, the terms "transformation" and
"transfection" are intended to refer to a variety of art-recognized
techniques for introducing foreign nucleic acid (e.g., DNA) into a
host cell, including calcium phosphate or calcium chloride
co-precipitation, DEAE-dextran-mediated transfection, lipofection,
or electroporation. Suitable methods for transforming or
transfecting host cells can be 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), and other laboratory manuals.
[0153] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest.
[0154] Preferred selectable markers include those which confer
resistance to drugs, such as G418, hygromycin and methotrexate. A
nucleic acid encoding a selectable marker can be introduced into a
host cell on the same vector as that encoding the P2T polypeptide
or can be introduced on a separate vector. Cells stably transfected
with the introduced nucleic acid can be identified by drug
selection (e.g., cells that have incorporated the selectable marker
gene will survive, while the other cells die).
[0155] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) P2T polypeptides. Accordingly, the invention further
provides methods for producing P2T polypeptides using the host
cells of the invention. In one embodiment, the method comprises
culturing the host cell of invention (into which a recombinant
expression vector encoding a P2T polypeptide has been introduced)
in a suitable medium until the P2T polypeptide is produced. In
another embodiment, the method further comprises isolating the P2T
polypeptide from the medium or the host cell.
[0156] A promoter is a region of a DNA molecule typically within
about 100 nucleotide pairs in front of (upstream of) the point at
which transcription begins (i.e., a transcription start site). That
region typically contains several types of DNA sequence elements
that are located in similar relative positions in different genes.
As used herein, the term "promoter" includes what is referred to in
the art as an upstream promoter region, a promoter region or a
promoter of a generalized eukaryotic RNA Polymerase II
transcription unit.
[0157] Another type of discrete transcription regulatory sequence
element is an enhancer. An enhancer provides specificity of time,
location and expression level for a particular encoding region
(e.g., gene). A major function of an enhancer is to increase the
level of transcription of a coding sequence in a cell that contains
one or more transcription factors that bind to that enhancer.
Unlike a promoter, an enhancer can function when located at
variable distances from transcription start sites so long as a
promoter is present.
[0158] As used herein, the phrase "enhancer-promoter" means a
composite unit that contains both enhancer and promoter elements.
An enhancer-promoter is operatively linked to a coding sequence
that encodes at least one gene product. As used herein, the phrase
"operatively linked" means that an enhancer-promoter is connected
to a coding sequence in such a way that the transcription of that
coding sequence is controlled and regulated by that
enhancer-promoter. Means for operatively linking an
enhancer-promoter to a coding sequence are well known in the art.
As is also well known in the art, the precise orientation and
location relative to a coding sequence whose transcription is
controlled, is dependent inter alia upon the specific nature of the
enhancer-promoter. Thus, a TATA box minimal promoter is typically
located from about 25 to about 30 base pairs upstream of a
transcription initiation site and an upstream promoter element is
typically located from about 100 to about 200 base pairs upstream
of a transcription initiation site. In contrast, an enhancer can be
located downstream from the initiation site and can be at a
considerable distance from that site.
[0159] An enhancer-promoter used in a vector construct of the
present invention can be any enhancer-promoter that drives
expression in a cell to be transfected. By employing an
enhancer-promoter with well-known properties, the level and pattern
of gene product expression can be optimized.
[0160] A coding sequence of an expression vector is operatively
linked to a transcription terminating region. RNA polymerase
transcribes an encoding DNA sequence through a site where
polyadenylation occurs. Typically, DNA sequences located a few
hundred base pairs downstream of the polyadenylation site serve to
terminate transcription. Those DNA sequences are referred to herein
as transcription-termination regions. Those regions are required
for efficient polyadenylation of transcribed messenger RNA (mRNA).
Transcription-terminating regions are well known in the art. A
preferred transcription-terminating region used in an adenovirus
vector construct of the present invention comprises a
polyadenylation signal of SV40 or the protamine gene.
[0161] An expression vector comprises a polynucleotide that encodes
a P2T polypeptide. Such a polypeptide is meant to include a
sequence of nucleotide bases encoding a P2T polypeptide sufficient
in length to distinguish said segment from a polynucleotide segment
encoding a non-P2T polypeptide. A polypeptide of the invention can
also encode biologically functional polypeptides or peptides which
have variant amino acid sequences, such as with changes selected
based on considerations such as the relative hydropathic score of
the amino acids being exchanged. These variant sequences are those
isolated from natural sources or induced in the sequences disclosed
herein using a mutagenic procedure such as site-directed
mutagenesis.
[0162] Preferably, the expression vectors of the present invention
comprise polynucleotides that encode polypeptides comprising the
amino acid residue sequence of SEQ ID NO:2. An expression vector
can include a P2T polypeptide coding region itself of any of the
P2T polypeptides noted above or it can contain coding regions
bearing selected alterations or modifications in the basic coding
region of such a P2T polypeptide. Alternatively, such vectors or
fragments can code larger polypeptides or polypeptides which
nevertheless include the basic coding region. In any event, it
should be appreciated that due to codon redundancy as well as
biological functional equivalence, this aspect of the invention is
not limited to the particular DNA molecules corresponding to the
polypeptide sequences noted above.
[0163] Exemplary vectors include the mammalian expression vectors
of the pCMV family including pCMV6b and pCMV6c (Chiron Corp.,
Emeryville Calif.). In certain cases, and specifically in the case
of these individual mammalian expression vectors, the resulting
constructs can require co-transfection with a vector containing a
selectable marker such as pSV2neo. Via co-transfection into a
dihydrofolate reductase-deficient Chinese hamster ovary cell line,
such as DG44, clones expressing P2T polypeptides by virtue of DNA
incorporated into such expression vectors can be detected.
[0164] A DNA molecule, gene or polynucleotide of the present
invention can be incorporated into a vector by a number of
techniques which are well known in the art. For instance, the
vector pUC18 has been demonstrated to be of particular value
Likewise, the related vectors M13mp18 and M13mp19 can be used in
certain embodiments of the invention, in particular, in performing
dideoxy sequencing.
[0165] An expression vector of the present invention is useful both
as a means for preparing quantities of the P2T polypeptide-encoding
DNA itself, and as a means for preparing the encoded polypeptide
and peptides. It is contemplated that where P2T polypeptides of the
invention are made by recombinant means, one can employ either
prokaryotic or eukaryotic expression vectors as shuttle systems.
However, in that prokaryotic systems are usually incapable of
correctly processing precursor polypeptides and, in particular,
such systems are incapable of correctly processing membrane
associated eukaryotic polypeptides, and since eukaryotic P2T
polypeptides are anticipated using the teaching of the disclosed
invention, one likely expresses such sequences in eukaryotic hosts.
However, even where the DNA segment encodes a eukaryotic P2T
polypeptide, it is contemplated that prokaryotic expression can
have some additional applicability. Therefore, the invention can be
used in combination with vectors which can shuttle between the
eukaryotic and prokaryotic cells. Such a system is described herein
which allows the use of bacterial host cells as well as eukaryotic
host cells.
[0166] Where expression of recombinant P2T polypeptides is desired
and a eukaryotic host is contemplated, it is most desirable to
employ a vector such as a plasmid, that incorporates a eukaryotic
origin of replication. Additionally, for the purposes of expression
in eukaryotic systems, one desires to position the P2T encoding
sequence adjacent to and under the control of an effective
eukaryotic promoter such as promoters used in combination with
Chinese hamster ovary cells. To bring a coding sequence under
control of a promoter, whether it is eukaryotic or prokaryotic,
what is generally needed is to position the 5' end of the
translation initiation side of the proper translational reading
frame of the polypeptide between about 1 and about 50 nucleotides
3' of or downstream with respect to the promoter chosen.
Furthermore, where eukaryotic expression is anticipated, one would
typically desire to incorporate into the transcriptional unit which
includes the P2T polypeptide, an appropriate polyadenylation
site.
[0167] The pCMV plasmids are a series of mammalian expression
vectors of particular utility in the present invention. The vectors
are designed for use in essentially all cultured cells and work
extremely well in SV40-transformed simian COS cell lines. The
pCMV1, 2, 3, and 5 vectors differ from each other in certain unique
restriction sites in the polylinker region of each plasmid. The
pCMV4 vector differs from these four plasmids in containing a
translation enhancer in the sequence prior to the polylinker. While
they are not directly derived from the pCMV1-5 series of vectors,
the functionally similar pCMV6b and c vectors are available from
the Chiron Corp. (Emeryville, Calif.) and are identical except for
the orientation of the polylinker region which is reversed in one
relative to the other.
[0168] The universal components of the pCMV plasmids are as
follows. The vector backbone is pTZ18R (Pharmacia), and contains a
bacteriophage f1 origin of replication for production of single
stranded DNA and an ampicillin-resistance gene. The CMV region
consists of nucleotides -760 to +3 of the powerful
promoter-regulatory region of the human cytomegalovirus (Towne
stain) major immediate early gene (Thomsen et al., 1984; Boshart et
al., 1985). The human growth hormone fragment (hGH) contains
transcription termination and poly-adenylation signals representing
sequences 1533 to 2157 of this gene (Seeburg, 1982). There is an
Alu middle repetitive DNA sequence in this fragment. Finally, the
SV40 origin of replication and early region promoter-enhancer
derived from the pcD-X plasmid (HindII to PstI fragment) described
in (Okayama et al., 1983). The promoter in this fragment is
oriented such that transcription proceeds away from the CMV/hGH
expression cassette.
[0169] The pCMV plasmids are distinguishable from each other by
differences in the polylinker region and by the presence or absence
of the translation enhancer. The starting pCMV1 plasmid has been
progressively modified to render an increasing number of unique
restriction sites in the polylinker region. To create pCMV2, one of
two EcoRI sites in pCMV1 were destroyed. To create pCMV3, pCMV1 was
modified by deleting a short segment from the SV40 region (StuI to
EcoRI), and in so doing made unique the PstI, SaII, and BamHI sites
in the polylinker. To create pCMV4, a synthetic fragment of DNA
corresponding to the 5'-untranslated region of a mRNA transcribed
from the CMV promoter was added C. The sequence acts as a
translational enhancer by decreasing the requirements for
initiation factors in polypeptide synthesis (Jobling et al., 1987;
Browning et al., 1988). To create pCMV5, a segment of DNA (Hpal to
EcoRI) was deleted from the SV40 origin region of pCMV1 to render
unique all sites in the starting polylinker.
[0170] The pCMV vectors have been successfully expressed in simian
COS cells, mouse L cells, CHO cells, and HeLa cells. In several
side by side comparisons they have yielded 5- to 10-fold higher
expression levels in COS cells than SV40-based vectors. The pCMV
vectors have been used to express the LDL receptor, nuclear factor
1, GS alpha polypeptide, polypeptide phosphatase, synaptophysin,
synapsin, insulin receptor, influenza hemagglutinin, androgen
receptor, sterol 26-hydroxylase, steroid 17- and 21-hydroxylase,
cytochrome P-450 oxidoreductase, beta-adrenergic receptor, folate
receptor, cholesterol side chain cleavage enzyme, and a host of
other cDNAs. It should be noted that the SV40 promoter in these
plasmids can be used to express other genes such as dominant
selectable markers. Finally, there is an ATG sequence in the
polylinker between the HindIII and PstI sites in pCMU that can
cause spurious translation initiation. This codon should be avoided
if possible in expression plasmids. A paper describing the
construction and use of the parenteral pCMV1 and pCMV4 vectors has
been published (Anderson et al., 1989b).
[0171] In yet another embodiment, the present invention provides
recombinant host cells transformed, infected or transfected with
polynucleotides that encode P2T polypeptides, as well as transgenic
cells derived from those transformed or transfected cells.
Preferably, the recombinant host cells of the present invention are
transfected with a polynucleotide of SEQ ID NO:1. Means of
transforming or transfecting cells with exogenous polynucleotide
such as DNA molecules are well known in the art and include
techniques such as calcium-phosphate- or DEAE-dextran-mediated
transfection, protoplast fusion, electroporation, liposome mediated
transfection, direct microinjection and adenovirus infection
(Sambrook, Fritsch and Maniatis, 1989).
[0172] The most widely used method is transfection mediated by
either calcium phosphate or DEAE-dextran. Although the mechanism
remains obscure, it is believed that the transfected DNA enters the
cytoplasm of the cell by endocytosis and is transported to the
nucleus. Depending on the cell type, up to 90% of a population of
cultured cells can be transfected at any one time. Because of its
high efficiency, transfection mediated by calcium phosphate or
DEAE-dextran is the method of choice for experiments that require
transient expression of the foreign DNA in large numbers of cells.
Calcium phosphate-mediated transfection is also used to establish
cell lines that integrate copies of the foreign DNA, which are
usually arranged in head-to-tail tandem arrays into the host cell
genome.
[0173] In the protoplast fusion method, protoplasts derived from
bacteria carrying high numbers of copies of a plasmid of interest
are mixed directly with cultured mammalian cells. After fusion of
the cell membranes (usually with polyethylene glycol), the contents
of the bacteria are delivered into the cytoplasm of the mammalian
cells and the plasmid DNA is transported to the nucleus. Protoplast
fusion is not as efficient as transfection for many of the cell
lines that are commonly used for transient expression assays, but
it is useful for cell lines in which endocytosis of DNA occurs
inefficiently. Protoplast fusion frequently yields multiple copies
of the plasmid DNA tandemly integrated into the host
chromosome.
[0174] The application of brief, high-voltage electric pulses to a
variety of mammalian and plant cells leads to the formation of
nanometer-sized pores in the plasma membrane. DNA is taken directly
into the cell cytoplasm either through these pores or as a
consequence of the redistribution of membrane components that
accompanies closure of the pores. Electroporation can be extremely
efficient and can be used both for transient expression of cloned
genes and for establishment of cell lines that carry integrated
copies of the gene of interest. Electroporation, in contrast to
calcium phosphate-mediated transfection and protoplast fusion,
frequently gives rise to cell lines that carry one, or at most a
few, integrated copies of the foreign DNA.
[0175] Liposome transfection involves encapsulation of DNA and RNA
within liposomes, followed by fusion of the liposomes with the cell
membrane. The mechanism of how DNA is delivered into the cell is
unclear but transfection efficiencies can be as high as 90%.
[0176] Direct microinjection of a DNA molecule into nuclei has the
advantage of not exposing DNA to cellular compartments such as
low-pH endosomes. Microinjection is therefore used primarily as a
method to establish lines of cells that carry integrated copies of
the DNA of interest.
[0177] The use of adenovirus as a vector for cell transfection is
well known in the art. Adenovirus vector-mediated cell transfection
has been reported for various cells (Stratford-Perricaudet, et al.
1992).
[0178] A transfected cell can be prokaryotic or eukaryotic.
Preferably, the host cells of the invention are eukaryotic host
cells. The recombinant host cells of the invention may be COS-1
cells. Where it is of interest to produce a human P2T polypeptide,
cultured mammalian or human cells are of particular interest.
[0179] In another aspect, the recombinant host cells of the present
invention are prokaryotic host cells. Preferably, the recombinant
host cells of the invention are bacterial cells of the DH5 a strain
of Escherichia coli. In general, prokaryotes are preferred for the
initial cloning of DNA sequences and constructing the vectors
useful in the invention. For example, E. coli K12 strains can be
particularly useful. Other microbial strains which can be used
include E. coli B, and E. coli.sub.x1976 (ATCC No. 31537). These
examples are, of course, intended to be illustrative rather than
limiting.
[0180] Prokaryotes can also be used for expression. The
aforementioned strains, as well as E. coli W3110 (ATCC No. 273325),
bacilli such as Bacillus subtilis, or other enterobacteriaceae such
as Salmonella typhimurium or Serratia marcesans, and various
Pseudomonas species can be used.
[0181] In general, plasmid vectors containing replicon and control
sequences which are derived from species compatible with the host
cell are used in connection with these hosts. The vector ordinarily
carries a replication site, as well as marking sequences which are
capable of providing phenotypic selection in transformed cells. For
example, E. coli can be transformed using pBR322, a plasmid derived
from an E. coli species (Bolivar, et al. 1977). pBR322 contains
genes for ampicillin and tetracycline resistance and thus provides
easy means for identifying transformed cells. The pBR plasmid, or
other microbial plasmid or phage must also contain, or be modified
to contain, promoters which can be used by the microbial organism
for expression of its own polypeptides.
[0182] Those promoters most commonly used in recombinant DNA
construction include the .beta.-lactamase (penicillinase) and
lactose promoter systems (Chang, et al. 1978; Itakura., et al.
1977, Goeddel, et al. 1979; Goeddel, et al. 1980) and a tryptophan
(TRP) promoter system (International Application No. EP 0036776;
Siebwenlist et al. 1980). While these are the most commonly used,
other microbial promoters have been discovered and utilized, and
details concerning their nucleotide sequences have been published,
enabling a skilled worker to introduce functional promoters into
plasmid vectors (Siebwenlist, et al. 1980).
[0183] In addition to prokaryotes, eukaryotic microbes such as
yeast can also be used. Saccharomyces cerevisiase or common baker's
yeast is the most commonly used among eukaryotic microorganisms,
although a number of other strains are commonly available. For
expression in Saccharomyces, the plasmid YRp7, for example, is
commonly used (Stinchcomb, et al. 1979; Kingsman, et al. 1979;
Tschemper, et al. 1980). This plasmid already contains the trpI
gene which provides a selection marker for a mutant strain of yeast
lacking the ability to grow in tryptophan, for example ATCC No.
44076 or PEP4-1 (Jones, 1977). The presence of the trpI lesion as a
characteristic of the yeast host cell genome then provides an
effective environment for detecting transformation by growth in the
absence of tryptophan.
[0184] Suitable promoter sequences in yeast vectors include the
promoters for 3-phosphoglycerate kinase (Hitzeman., et al. 1980) or
other glycolytic enzymes (Hess, et al. 1968; Holland, et al. 1978)
such as enolase, glyceraldehyde-3-phosphate dehydrogenase,
hexokinase, pyruvate decarboxylase, phosphofructokinase,
glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate
kinase, triosephosphate isomerase, phosphoglucose isomerase, and
glucokinase. In constructing suitable expression plasmids, the
termination sequences associated with these genes are also
introduced into the expression vector downstream from the sequences
to be expressed to provide polyadenylation of the mRNA and
termination. Other promoters, which have the additional advantage
of transcription controlled by growth conditions are the promoter
region for alcohol dehydrogenase 2, isocytochrome C, acid
phosphatase, degradative enzymes associated with nitrogen
metabolism, and the aforementioned glyceraldehyde-3-phosphate
dehydrogenase, and enzymes responsible for maltose and galactose
utilization. Any plasmid vector containing a yeast-compatible
promoter, origin or replication and termination sequences is
suitable.
[0185] In addition to microorganisms, cultures of cells derived
from multicellular organisms can also be used as hosts. In
principle, any such cell culture is workable, whether from
vertebrate or invertebrate culture. However, interest has been
greatest in vertebrate cells, and propagation of vertebrate cells
in culture (tissue culture) has become a routine procedure in
recent years. Examples of such useful host cell lines are AtT-20,
VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and
W138, BHK, COSM6, COS-7, 293 and MDCK cell lines. Expression
vectors for such cells ordinarily include (if necessary) an origin
of replication, a promoter located upstream of the gene to be
expressed, along with any necessary ribosome binding sites, RNA
splice sites, polyadenylation site, and transcriptional terminator
sequences.
[0186] For use in mammalian cells, the control functions on the
expression vectors are often derived from viral material. For
example, commonly used promoters are derived from polyoma,
Adenovirus 2, Cytomegalovirus and most frequently Simian Virus 40
(SV40). The early and late promoters of SV40 virus are particularly
useful because both are obtained easily from the virus as a
fragment which also contains the SV40 viral origin of replication
(Fiers, et al. 1978). Smaller or larger SV40 fragments can also be
used, provided there is included the approximately 250 bp sequence
extending from the HindIII site toward the BgII site located in the
viral origin of replication. Further, it is also possible, and
often desirable, to utilize promoter or control sequences normally
associated with the desired gene sequence, provided such control
sequences are compatible with the host cell systems.
[0187] An origin of replication can be provided with by
construction of the vector to include an exogenous origin, such as
can be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV,
BPV, CMV) source, or can be provided by the host cell chromosomal
replication mechanism. If the vector is integrated into the host
cell chromosome, the latter is often sufficient.
[0188] In yet another embodiment, the present invention
contemplates a process or method of preparing P2T polypeptides
comprising transfecting cells with a polynucleotide that encodes
P2T polypeptides to produce transformed host cells, and maintaining
the transformed host cells under biological conditions sufficient
for expression of the polypeptide. Preferably, the transformed host
cells are eukaryotic cells. Alternatively, the host cells are
prokaryotic cells. More preferably, the prokaryotic cells are
bacterial cells of the DH5-.alpha. strain of Escherichia coli. Even
more preferably, the polynucleotide transfected into the
transformed cells comprise the nucleic acid sequence of SEQ ID
NO:1. Additionally, transfection is accomplished using an
expression vector disclosed above.
[0189] A host cell used in the process is capable of expressing a
functional, recombinant P2T polypeptide. A preferred host cell is a
Chinese hamster ovary cell. However, a variety of cells are
amenable to a process of the invention, for instance, yeast cells,
human cell lines, and other eukaryotic cell lines known well to
those of skill in the art.
[0190] Following transfection, the cell is maintained under culture
conditions for a period of time sufficient for expression of a P2T
receptor polypeptide. Culture conditions are well known in the art
and include ionic composition and concentration, temperature, pH
and the like. Typically, transfected cells are maintained under
culture conditions in a culture medium. Suitable medium for various
cell types are well known in the art. In a preferred embodiment,
temperature is from about 20.degree. C. to about 50.degree. C.,
more preferably from about 30.degree. C. to about 40.degree. C.
and, even more preferably about 37.degree. C.
[0191] pH is preferably from about a value of 6.0 to a value of
about 8.0, more preferably from about a value of about 6.8 to a
value of about 7.8 and, most preferably about 7.4. Osmolality is
preferably from about 200 milliosmols per liter (mosm/L) to about
400 mosm/l and, more preferably from about 290 mosm/L to about 310
mosm/L. Other biological conditions needed for transfection and
expression of an encoded polypeptide are well known in the art.
[0192] Transfected cells are maintained for a period of time
sufficient for expression of a P2T polypeptide. A suitable time
depends inter alia upon the cell type used and is readily
determinable by a skilled artisan. Typically, maintenance time is
from about 2 to about 14 days.
[0193] Recombinant P2T polypeptide is recovered or collected either
from the transfected cells or the medium in which those cells are
cultured. Recovery comprises isolating and purifying the P2T
polypeptide. Isolation and purification techniques for polypeptides
are well known in the art and include such procedures as
precipitation, filtration, chromatography, electrophoresis and the
like.
E. P2T Antibodies
[0194] In another embodiment, the present invention provides
antibodies immunoreactive with P2T polypeptides. Preferably, the
antibodies of the invention are monoclonal antibodies.
Additionally, the P2T polypeptides comprise the amino acid residue
sequence of SEQ ID NO:2. Means for preparing and characterizing
antibodies are well known in the art (see, e.g., Antibodies "A
Laboratory Manual, E. Howell and D. Lane, Cold Spring Harbor
Laboratory, 1988). In yet other embodiments, the present invention
provides antibodies immunoreactive with P2T polynucleotides.
[0195] Briefly, a polyclonal antibody is prepared by immunizing an
animal with an immunogen comprising a polypeptide or polynucleotide
of the present invention, and collecting antisera from that
immunized animal. A wide range of animal species can be used for
the production of antisera. Typically an animal used for production
of anti-antisera is a rabbit, a mouse, a rat, a hamster or a guinea
pig. Because of the relatively large blood volume of rabbits, a
rabbit is a preferred choice for production of polyclonal
antibodies.
[0196] As is well known in the art, a given polypeptide or
polynucleotide may vary in its immunogenicity. It is often
necessary therefore to couple the immunogen (e.g., a polypeptide or
polynucleotide) of the present invention with a carrier. Exemplary
and preferred carriers are keyhole limpet hemocyanin (KLH) and
bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse
serum albumin or rabbit serum albumin can also be used as
carriers.
[0197] Means for conjugating a polypeptide or a polynucleotide to a
carrier polypeptide are well known in the art and include
glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester,
carbodiimide and bis-biazotized benzidine.
[0198] As is also well known in the art, immunogencity to a
particular immunogen can be enhanced by the use of non-specific
stimulators of the immune response known as adjuvants. Exemplary
and preferred adjuvants include complete Freund's adjuvant,
incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
[0199] The amount of immunogen used for the production of
polyclonal antibodies varies inter alia, upon the nature of the
immunogen as well as the animal used for immunization. A variety of
routes can be used to administer the immunogen (subcutaneous,
intramuscular, intradermal, intravenous and intraperitoneal). The
production of polyclonal antibodies is monitored by sampling blood
of the immunized animal at various points following immunization.
When a desired level of immunogenicity is obtained, the immunized
animal can be bled and the serum isolated and stored.
[0200] In another aspect, the present invention contemplates a
process of producing an antibody immunoreactive with a P2T
polypeptide comprising the steps of (a) transfecting recombinant
host cells with a polynucleotide that encodes a P2T polypeptide;
(b) culturing the host cells under conditions sufficient for
expression of the polypeptide; (c) recovering the polypeptides; and
(d) preparing the antibodies to the polypeptides. Preferably, the
host cell is transfected with the polynucleotide of SEQ ID NO:1.
Even more preferably, the present invention provides antibodies
prepared according to the process described above.
[0201] A monoclonal antibody of the present invention can be
readily prepared through use of well-known techniques such as those
exemplified in U.S. Pat. No. 4,196,265, herein incorporated by
reference. Typically, a technique involves first immunizing a
suitable animal with a selected antigen (e.g., a polypeptide or
polynucleotide of the present invention) in a manner sufficient to
provide an immune response. Rodents such as mice and rats are
preferred animals. Spleen cells from the immunized animal are then
fused with cells of an immortal myeloma cell. Where the immunized
animal is a mouse, a preferred myeloma cell is a murine NS-1
myeloma cell.
[0202] The fused spleen/myeloma cells are cultured in a selective
medium to select fused spleen/myeloma cells from the parental
cells. Fused cells are separated from the mixture of non-fused
parental cells, e.g., by the addition of agents that block the de
novo synthesis of nucleotides in the tissue culture media.
Exemplary and preferred agents are aminopterin, methotrexate, and
azaserine. Aminopterin and methotrexate block de novo synthesis of
both purines and pyrimidines, whereas azaserine blocks only purine
synthesis. Where aminopterin or methotrexate is used, the media is
supplemented with hypoxanthine and thymidine as a source of
nucleotides. Where azaserine is used, the media is supplemented
with hypoxanthine.
[0203] This culturing provides a population of hybridomas from
which specific hybridomas are selected. Typically, selection of
hybridomas is performed by culturing the cells by single-clone
dilution in microtiter plates, followed by testing the individual
clonal supernatants for reactivity with an antigen-polypeptide. The
selected clones can then be propagated indefinitely to provide the
monoclonal antibody.
[0204] By way of specific example, to produce an antibody of the
present invention, mice are injected intraperitoneally with between
about 1-200 .mu.g of an antigen comprising a polypeptide of the
present invention. B lymphocyte cells are stimulated to grow by
injecting the antigen in association with an adjuvant such as
complete Freund's adjuvant (a non-specific stimulator of the immune
response containing killed Mycobacterium tuberculosis). At some
time (e.g., at least two weeks) after the first injection, mice are
boosted by injection with a second dose of the antigen mixed with
incomplete Freund's adjuvant.
[0205] A few weeks after the second injection, mice are tail bled
and the sera titered by immunoprecipitation against radiolabeled
antigen. Preferably, the process of boosting and titering is
repeated until a suitable titer is achieved. The spleen of the
mouse with the highest titer is removed and the spleen lymphocytes
are obtained by homogenizing the spleen with a syringe. Typically,
a spleen from an immunized mouse contains approximately
5.times.10.sup.7 to 2.times.10.sup.8 lymphocytes.
[0206] Mutant lymphocyte cells known as myeloma cells are obtained
from laboratory animals in which such cells have been induced to
grow by a variety of well-known methods. Myeloma cells lack the
salvage pathway of nucleotide biosynthesis. Because myeloma cells
are tumor cells, they can be propagated indefinitely in tissue
culture, and are thus denominated immortal. Numerous cultured cell
lines of myeloma cells from mice and rats, such as murine NS-1
myeloma cells, have been established.
[0207] Myeloma cells are combined under conditions appropriate to
foster fusion with the normal antibody-producing cells from the
spleen of the mouse or rat injected with the antigen/polypeptide of
the present invention. Fusion conditions include, for example, the
presence of polyethylene glycol. The resulting fused cells are
hybridoma cells. Like myeloma cells, hybridoma cells grow
indefinitely in culture.
[0208] Hybridoma cells are separated from unfused myeloma cells by
culturing in a selection medium such as HAT media (hypoxanthine,
aminopterin, thymidine). Unfused myeloma cells lack the enzymes
necessary to synthesize nucleotides from the salvage pathway
because they are killed in the presence of aminopterin,
methotrexate, or azaserine. Unfused lymphocytes also do not
continue to grow in tissue culture. Thus, only cells that have
successfully fused (hybridoma cells) can grow in the selection
media.
[0209] Each of the surviving hybridoma cells produces a single
antibody. These cells are then screened for the production of the
specific antibody immunoreactive with an antigen/polypeptide of the
present invention. Single cell hybridomas are isolated by limiting
dilutions of the hybridomas. The hybridomas are serially diluted
many times and, after the dilutions are allowed to grow, the
supernatant is tested for the presence of the monoclonal antibody.
The clones producing that antibody are then cultured in large
amounts to produce an antibody of the present invention in
convenient quantity.
[0210] By use of a monoclonal antibody of the present invention,
specific polypeptides and polynucleotide of the invention can be
recognized as antigens, and thus identified. Once identified, those
polypeptides and polynucleotide can be isolated and purified by
techniques such as antibody-affinity chromatography. In
antibody-affinity chromatography, a monoclonal antibody is bound to
a solid substrate and exposed to a solution containing the desired
antigen. The antigen is removed from the solution through an
immunospecific reaction with the bound antibody. The polypeptide or
polynucleotide is then easily removed from the substrate and
purified.
[0211] Additionally, examples of methods and reagents particularly
amenable for use in generating and screening an antibody display
library can be found in, for example, U.S. Pat. No. 5,223,409;
International Application No. WO 92/18619; International
Application No. WO 91/17271; International Application No. WO
92/20791; International Application No. WO 92/15679; International
Application No. WO 93/01288; International Application No. WO
92/01047; International Application No. WO 92/09690; International
Application No. WO 90/02809.
[0212] Additionally, recombinant anti-P2T antibodies, such as
chimeric and humanized monoclonal antibodies, comprising both human
and non-human fragments, which can be made using standard
recombinant DNA techniques, are within the scope of the invention.
Such chimeric and humanized monoclonal antibodies can be produced
by recombinant DNA techniques known in the art, for example using
methods described in U.S. Pat. No. 6,054,297; European Application
Nos. EP 184,187; EP 171,496; EP 173,494; International Application
No. WO 86/01533; U.S. Pat. No. 4,816,567; and European Application
No. EP 125,023.
[0213] An anti-P2T antibody (e.g., monoclonal antibody) can be used
to isolate P2T polypeptides by standard techniques, such as
affinity chromatography or immunoprecipitation. An anti-P2T
antibody can facilitate the purification of a natural P2T
polypeptides from cells and recombinantly produced P2T polypeptide
expressed in host cells. Moreover, an anti-P2T antibody can be used
to detect P2T polypeptide (e.g., in a cellular lysate or cell
supernatant) in order to evaluate the abundance and pattern of
expression of the P2T polypeptide. The detection of circulating
fragments of a P2T polypeptide can be used to identify P2T
polypeptide turnover in a subject. Anti-P2T antibodies can be used
diagnostically to monitor protein levels in tissue as part of a
clinical testing procedure, e.g., to, for example, determine the
efficacy of a given treatment regimen. Detection 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, P-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, dichlorotriazinylarnine
fluorescein, dansyl chloride or phycoerythrin; an example of a
luminescent material includes luminol; examples of bioluminescent
materials include luciferase, luciferin, and acquorin, and examples
of suitable radioactive material include .sup.125I, .sup.131I,
.sup.15S or .sup.3H.
F. Transgenic Animals
[0214] In certain preferred embodiments, the invention pertains to
nonhuman animals with somatic and germ cells having a functional
disruption of at least one, and more preferably both, alleles of an
endogenous P2T gene of the present invention. Accordingly, the
invention provides viable animals having a mutated P2T gene, and
thus lacking P2T activity. These animals will produce substantially
reduced amounts of a P2T in response to stimuli that produce normal
amounts of a P2T in wild type control animals. The animals of the
invention are useful, for example, as standard controls by which to
evaluate P2T inhibitors, as recipients of a normal human P2T gene
to thereby create a model system for screening human P2T inhibitors
in vivo, and to identify disease states for treatment with P2T
inhibitors. The animals are also useful as controls for studying
the effect of ligands on P2T receptors.
[0215] In the transgenic nonhuman animal of the invention, the P2T
gene preferably is disrupted by homologous recombination between
the endogenous allele and a mutant P2T polynucleotide, or portion
thereof, that has been introduced into an embryonic stem cell
precursor of the animal. The embryonic stem cell precursor is then
allowed to develop, resulting in an animal having a functionally
disrupted P2T gene. As used herein, a "transgenic animal" is a
non-human animal, preferably a mammal, more preferably a rodent
such as a rat or mouse, in which one or more of the cells of the
animal include a transgene. Other examples of transgenic animals
include non-human primates, sheep, dogs, cows, goats, chickens,
amphibians, and the like. The animal may have one P2T gene allele
functionally disrupted (i.e., the animal may be heterozygous for
the mutation), or more preferably, the animal has both P2T gene
alleles functionally disrupted (i.e., the animal can be homozygous
for the mutation).
[0216] In one embodiment of the invention, functional disruption of
both P2T gene alleles produces animals in which expression of the
P2T gene product in cells of the animal is substantially absent
relative to non-mutant animals. In another embodiment, the P2T gene
alleles can be disrupted such that an altered (i.e., mutant) P2T
gene product is produced in cells of the animal. A preferred
nonhuman animal of the invention having a functionally disrupted
P2T gene is a mouse. Given the essentially complete inactivation of
P2T function in the homozygous animals of the invention and the
about 50% inhibition of P2T function in the heterozygous animals of
the invention, these animals are useful as positive controls
against which to evaluate the effectiveness of P2T inhibitors. For
example, a stimulus that normally induces production or activity of
P2T can be administered to a wild type animal (i.e., an animal
having a non-mutant P2T gene) in the presence of a P2T inhibitor to
be tested and production or activity of P2T by the animal can be
measured. The P2T response in the wild type animal can then be
compared to the P2T response in the heterozygous and homozygous
animals of the invention, similarly administered the P2T stimulus,
to determine the percent of maximal P2T inhibition of the test
inhibitor.
[0217] Additionally, the animals of the invention are useful for
determining whether a particular disease condition involves the
action of P2T and thus can be treated by a P2T inhibitor. For
example, an attempt can be made to induce a disease condition in an
animal of the invention having a functionally disrupted P2T gene.
Subsequently, the susceptibility or resistance of the animal to the
disease condition can be determined. A disease condition that is
treatable with a P2T inhibitor can be identified based upon
resistance of an animal of the invention to the disease condition.
Another aspect of the invention pertains to a transgenic nonhuman
animal having a functionally disrupted endogenous P2T gene but
which also carries in its genome, and expresses, a transgene
encoding a heterologous P2T (i.e., a P2T from another species).
Preferably, the animal is a mouse and the heterologous P2T is a
human P2T. An animal of the invention which has been reconstituted
with human P2T can be used to identify agents that inhibit human
P2T in vivo. For example, a stimulus that induces production and/or
activity of P2T can be administered to the animal in the presence
and absence of an agent to be tested and the P2T response in the
animal can be measured. An agent that inhibits human P2T in vivo
can be identified based upon a decreased P2T response in the
presence of the agent compared to the P2T response in the absence
of the agent. As used herein, a "transgene" is exogenous DNA which
is integrated into the genome of a cell from which a transgenic
animal develops and which remains in the genome of the mature
animal, thereby directing the expression of an encoded gene product
in one or more cell types or tissues of the transgenic animal.
[0218] Yet another aspect of the invention pertains to a
polynucleotide construct for functionally disrupting a P2T gene in
a host cell. The nucleic acid construct comprises: a) a
nonhomologous replacement portion; b) a first homology region
located upstream of the nonhomologous replacement portion, the
first homology region having a nucleotide sequence with substantial
identity to a first P2T gene sequence; and c) a second homology
region located downstream of the nonhomologous replacement portion,
the second homology region having a nucleotide sequence with
substantial identity to a second P2T gene sequence, the second P2T
gene sequence having a location downstream of the first P2T gene
sequence in a naturally occurring endogenous P2T gene.
Additionally, the first and second homology regions are of
sufficient length for homologous recombination between the nucleic
acid construct and an endogenous P2T gene in a host cell when the
nucleic acid molecule is introduced into the host cell. As used
herein, a "homologous recombinant animal" is a non-human animal,
preferably a mammal, more preferably a mouse, in which an
endogenous P2T gene has been altered by homologous recombination
between the endogenous gene and an exogenous DNA molecule
introduced into a cell of the animal, e.g., an embryonic cell of
the animal, prior to development of the animal.
[0219] In a preferred embodiment, the nonhomologous replacement
portion comprises a positive selection expression cassette,
preferably including a neomycin phosphotransferase gene operatively
linked to a regulatory element(s). In another preferred embodiment,
the nucleic acid construct also includes a negative selection
expression cassette distal to either the upstream or downstream
homology regions. A preferred negative selection cassette includes
a herpes simplex virus thymidine kinase gene operatively linked to
a regulatory element(s). Another aspect of the invention pertains
to recombinant vectors into which the nucleic acid construct of the
invention has been incorporated.
[0220] Yet another aspect of the invention pertains to host cells
into which the nucleic acid construct of the invention has been
introduced to thereby allow homologous recombination between the
nucleic acid construct and an endogenous P2T gene of the host cell,
resulting in functional disruption of the endogenous P2T gene. The
host cell can be a mammalian cell that normally expresses P2T, such
as a human neuron, or a pluripotent cell, such as a mouse embryonic
stem cell. Further development of an embryonic stem cell into which
the nucleic acid construct has been introduced and homologously
recombined with the endogenous P2T gene produces a transgenic
nonhuman animal having cells that are descendant from the embryonic
stem cell and thus carry the P2T gene disruption in their genome.
Animals that carry the P2T gene disruption in their germline can
then be selected and bred to produce animals having the P2T gene
disruption in all somatic and germ cells. Such mice can then be
bred to homozygosity for the P2T gene disruption.
[0221] It is contemplated that in some instances the genome of a
transgenic animal of the present invention will have been altered
through the stable introduction of one or more of the P2T
polynucleotide compositions described herein, either native,
synthetically modified or mutated. As described herein, a
"transgenic animal" refers to any animal, preferably a non-human
mammal (e.g. mouse, rat, rabbit, squirrel, hamster, rabbits, guinea
pigs, pigs, micro-pigs, prairie dogs, baboons, squirrel monkeys and
chimpanzees, etc.), a bird or an amphibian, in which one or more
cells contain heterologous nucleic acid introduced by way of human
intervention, such as by transgenic techniques well known in the
art. The nucleic acid is introduced into the cell, directly or
indirectly, by introduction into a precursor of the cell, by way of
deliberate genetic manipulation, such as by microinjection or by
infection with a recombinant virus. The term genetic manipulation
does not include classical cross-breeding, or in vitro
fertilization, but rather is directed to the introduction of a
recombinant DNA molecule. This molecule may be integrated within a
chromosome, or it may be extrachromosomally replicating DNA.
[0222] The host cells of the invention can also be used to produce
non-human transgenic animals. The non-human transgenic animals can
be used in screening assays designed to identify agents or
compounds, e.g., drugs, pharmaceuticals, etc., which are capable of
ameliorating detrimental symptoms of selected disorders such as
nervous system disorders, e.g., psychiatric disorders or disorders
affecting circadian rhythms and the sleep-wake cycle. For example,
in one embodiment, a host cell of the invention is a fertilized
oocyte or an embryonic stem cell into which P2T polypeptide-coding
sequences have been introduced. Such host cells can then be used to
create non-human transgenic animals in which exogenous P2T gene
sequences have been introduced into their genome or homologous
recombinant animals in which endogenous P2T gene sequences have
been altered. Such animals are useful for studying the function
and/or activity of a P2T polypeptide and for identifying and/or
evaluating modulators of P2T polypeptide activity.
[0223] A transgenic animal of the invention can be created by
introducing P2T polypeptide encoding nucleic acid into the male
pronuclei of a fertilized oocyte, e.g., by microinjection,
retroviral infection, and allowing the oocyte to develop in a
pseudopregnant female foster animal. The murine P2T cDNA sequence
of SEQ ID NO:1 can be introduced as a transgene into the genome of
a non-human animal.
[0224] Moreover, a non-human homologue of the murine P2T gene, such
as a rabbit P2T gene, can be isolated based on hybridization to the
murine P2T cDNA (described above) and used as a transgene. Intronic
sequences and polyadenylation signals can also be included in the
transgene to increase the efficiency of expression of the
transgene. A tissue-specific regulatory sequence(s) can be operably
linked to the P2T transgene to direct expression of a P2T
polypeptide to particular cells. 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, 4,870,009, and
U.S. Pat. No. 4,873,191, and in Hogan, 1986. Similar methods are
used for production of other transgenic animals. A transgenic
founder animal can be identified based upon the presence of the P2T
transgene in its genome and/or expression of P2T mRNA in tissues or
cells of the animals. A transgenic founder animal can then be used
to breed additional animals carrying the transgene. Moreover,
transgenic animals carrying a transgene encoding a P2T polypeptide
can further be bred to other transgenic animals carrying other
transgenes.
[0225] To create a homologous recombinant animal, a vector is
prepared which contains at least a fragment of a P2T gene into
which a deletion, addition or substitution has been introduced to
thereby alter, e.g., functionally disrupt, the P2T gene. The P2T
gene is preferably a mouse gene (e.g., SEQ ID NO:1). The mouse P2T
gene then can be used to construct a homologous recombination
vector suitable for altering an endogenous P2T gene in the mouse
genome. In a preferred embodiment, the vector is designed such
that, upon homologous recombination, the endogenous P2T gene is
functionally disrupted (i.e., no longer encodes a functional
protein; also referred to as a "knock out" vector.
[0226] Alternatively, the vector can be designed such that, upon
homologous recombination, the endogenous P2T gene is mutated or
otherwise altered but still encodes functional protein (e.g., the
upstream regulatory region can be altered to thereby alter the
expression of the endogenous P2T polypeptide). In the homologous
recombination vector, the altered fragment of the P2T gene is
flanked at its 5' and 3' ends by additional nucleic acid of the P2T
gene to allow for homologous recombination to occur between the
exogenous P2T gene carried by the vector and an endogenous P2T gene
in an embryonic stem cell. The additional flanking P2T nucleic acid
is of sufficient length for successful homologous recombination
with the endogenous gene.
[0227] Typically, several kilobases of flanking DNA (both at the 5'
and 3' ends) are included in the vector (see e.g., Thomas and
Capecchi, 1987, for a description of homologous recombination
vectors). The vector is introduced into an embryonic stem cell line
(e.g., by electroporation) and cells in which the introduced P2T
gene has homologously recombined with the endogenous P2T gene are
selected (see e.g., Li et al., 1992). The selected cells are then
injected into a blastocyst of an animal (e.g., a mouse) to form
aggregation chimeras (see e.g., Bradley, 1987, pp. 113-152). A
chimeric embryo can then be implanted into a suitable
pseudopregnant female foster animal and the embryo brought to term.
Progeny harboring the homologously recombined DNA in their germ
cells can be used to breed animals in which all cells of the animal
contain the homologously recombined DNA by germline transmission of
the transgene. Methods for constructing homologous recombination
vectors and homologous recombinant animals are described further in
Bradley, 1991; and in PCT International Publication Nos. WO
90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.
[0228] In another embodiment, transgenic non-human animals can be
produced which contain selected systems which allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage PL. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al., 1992.
Another example of a recombinase system is the FLP recombinase
system of Saccharomyces cerevisiae (O'Gonnan et al., 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 are required. Such animals can
be provided through the construction of "double" transgenic
animals, e.g., by mating two transgenic animals, one containing a
transgene encoding a selected protein and the other containing a
transgene encoding a recombinase.
[0229] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut
et al., 1997, and PCT International Application 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 borne of this
female foster animal will be a clone of the animal from which the
cell, e.g., the somatic cell, is isolated.
G. Uses and Methods of the Invention
[0230] The nucleic acid molecules, polypeptides, polypeptide
homologues, modulators, and antibodies described herein can be used
in one or more of the following methods: a) drug screening assays;
b) diagnostic assays particularly in disease identification,
allelic screening and pharmocogenetic testing; c) methods of
treatment; d) pharmacogenomics; and e) monitoring of effects during
clinical trials. A P2T polypeptide of the invention can be used as
a drug target for developing agents to modulate the activity of the
P2T polypeptide. The isolated nucleic acid molecules of the
invention can be used to express P2T polypeptide (e.g., via a
recombinant expression vector in a host cell or in gene therapy
applications), to detect P2T mRNA (e.g., in a biological sample) or
a naturally occurring or recombinantly generated genetic mutation
in a P2T gene, and to modulate P2T polypeptide activity, as
described further below. In addition, the P2T polypeptides can be
used to screen drugs or compounds which modulate P2T polypeptide
activity. Moreover, the anti-P2T antibodies of the invention can be
used to detect and isolate a P2T polypeptide, particularly
fragments of a P2T polypeptides present in a biological sample, and
to modulate P2T polypeptide activity.
[0231] Drug Screening Assays
[0232] The invention provides methods for identifying compounds or
agents that can be used to treat disorders characterized by (or
associated with) aberrant or abnormal P2T nucleic acid expression
and/or P2T polypeptide activity. These methods are also referred to
herein as drug screening assays and typically include the step of
screening a candidate/test compound or agent to identify compounds
that are an agonist or antagonist of a P2T polypeptide, and
specifically for the ability to interact with (e.g., bind to) a P2T
polypeptide, to modulate the interaction of a P2T polypeptide and a
target molecule, and/or to modulate P2T nucleic acid expression
and/or P2T polypeptide activity. Candidate/test compounds or agents
which have one or more of these abilities can be used as drugs to
treat disorders characterized by aberrant or abnormal P2T nucleic
acid expression and/or P2T polypeptide activity. Candidate/test
compounds include, for example, 1) peptides such as soluble
peptides, including Ig-tailed fusion peptides and members of random
peptide libraries 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.,
1993; 3) antibodies (e.g., polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric, and single chain antibodies as well as
Fab, F(ab')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). In one embodiment, the invention provides
assays for screening candidate/test compounds which interact with
(e.g., bind to) a P2T polypeptide. Typically, the assays are
recombinant cell based or cell-free assays which include the steps
of combining a cell expressing a P2T polypeptide or a bioactive
fragment thereof, or an isolated P2T polypeptide, and a
candidate/test compound, e.g., under conditions which allow for
interaction of (e.g., binding of) the candidate/test compound to
the P2T polypeptide or fragment thereof to form a complex, and
detecting the formation of a complex, in which the ability of the
candidate compound to interact with (e.g., bind to) the P2T
polypeptide or fragment thereof is indicated by the presence of the
candidate compound in the complex. Formation of complexes between
the P2T polypeptide and the candidate compound can be detected
using competition binding assays, and can be quantitated, for
example, using standard immunoassays.
[0233] In another embodiment, the invention provides screening
assays to identify candidate/test compounds which modulate (e.g.,
stimulate or inhibit) the interaction (and most likely P2T
polypeptide activity as well) between a P2T polypeptide and a
molecule (target molecule) with which the P2T polypeptide normally
interacts. Examples of such target molecules include proteins in
the same signaling path as the P2T polypeptide, e.g., proteins
which may function upstream (including both stimulators and
inhibitors of activity) or downstream of the P2T polypeptide in,
for example, a cognitive function signaling pathway or in a pathway
involving P2T polypeptide activity, e.g., a G protein or other
interactor involved in cAMP or phosphatidylinositol turnover,
and/or adenylate cyclase or phospholipase C activation. Typically,
the assays are recombinant cell based assays which include the
steps of combining a cell expressing a P2T polypeptide, or a
bioactive fragment thereof, a P2T polypeptide target molecule
(e.g., a P2T ligand) and a candidate/test compound, e.g., under
conditions wherein but for the presence of the candidate compound,
the P2T polypeptide or biologically active fragment thereof
interacts with (e.g., binds to) the target molecule, and detecting
the formation of a complex which includes the P2T polypeptide and
the target molecule or detecting the interaction/reaction of the
P2T polypeptide and the target molecule.
[0234] Detection of complex formation can include direct
quantitation of the complex by, for example, measuring inductive
effects of the P2T polypeptide. A statistically significant change,
such as a decrease, in the interaction of the P2T polypeptide and
target molecule (e.g., in the formation of a complex between the
P2T polypeptide and the target molecule) in the presence of a
candidate compound (relative to what is detected in the absence of
the candidate compound) is indicative of a modulation (e.g.,
stimulation or inhibition) of the interaction between the P2T
polypeptide and the target molecule. Modulation of the formation of
complexes between the P2T polypeptide and the target molecule can
be quantitated using, for example, an immunoassay.
[0235] To perform cell free drug screening assays, it is desirable
to immobilize either the P2T polypeptide 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. Interaction (e.g., binding of) of the P2T polypeptide to a
target molecule, in the presence and absence of a candidate
compound, can be accomplished in any vessel suitable for containing
the reactants. Examples of such vessels include microtitre plates,
test tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided which adds a domain that allows the protein
to be bound to a matrix. For example, glutathione-S-transferase/
P2T 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 P2T-binding protein found in the bead
fraction quantitated from the gel using standard electrophoretic
techniques.
[0236] Other techniques for immobilizing proteins on matrices can
also be used in the drug screening assays of the invention. For
example, either the P2T polypeptide or its target molecule can be
immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated P2T polypeptide molecules can be prepared from
biotin-NHS (N-hydroxy-succinimide) using techniques well known in
the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,
Ill.), and immobilized in the wells of streptavidin-coated 96 well
plates (Pierce Chemical). Alternatively, antibodies reactive with a
P2T polypeptide but which do not interfere with binding of the
protein to its target molecule can be derivatized to the wells of
the plate, and P2T polypeptide trapped in the wells by antibody
conjugation. As described above, preparations of a P2T-binding
protein and a candidate compound are incubated in the P2T
polypeptide-presenting wells of the plate, and the amount of
complex trapped in the well can be quantitated. Methods for
detecting such complexes, in addition to those described above for
the GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with the P2T polypeptide target molecule,
or which are reactive with P2T polypeptide and compete with the
target molecule; as well as enzyme-linked assays which rely on
detecting an enzymatic activity associated with the target
molecule.
[0237] In yet another embodiment, the invention provides a method
for identifying a compound (e.g., a screening assay) capable of use
in the treatment of a disorder characterized by (or associated
with) aberrant or abnormal P2T nucleic acid expression or P2T
polypeptide activity. This method typically includes the step of
assaying the ability of the compound or agent to modulate the
expression of the P2T nucleic acid or the activity of the P2T
polypeptide thereby identifying a compound for treating a disorder
characterized by aberrant or abnormal P2T nucleic acid expression
or P2T polypeptide activity. Methods for assaying the ability of
the compound or agent to modulate the expression of the P2T nucleic
acid or activity of the P2T polypeptide are typically cell-based
assays. For example, cells which are sensitive to ligands which
transduce signals via a pathway involving a P2T polypeptide can be
induced to overexpress a P2T polypeptide in the presence and
absence of a candidate compound.
[0238] Candidate compounds which produce a statistically
significant change in P2T polypeptide-dependent responses (either
stimulation or inhibition) can be identified. In one embodiment,
expression of the P2T nucleic acid or activity of a P2T polypeptide
is modulated in cells and the effects of candidate compounds on the
readout of interest (such as cAMP or phosphatidylinositol turnover)
are measured. For example, the expression of genes which are up- or
down-regulated in response to a P2T polypeptide- dependent signal
cascade can be assayed. In preferred embodiments, the regulatory
regions of such genes, e.g., the 5' flanking promoter and enhancer
regions, are operably linked to a detectable marker (such as
luciferase) which encodes a gene product that can be readily
detected. Phosphorylation of a P2T polypeptide or P2T polypeptide
target molecules can also be measured, for example, by
immunoblotting.
[0239] Alternatively, modulators of P2T gene expression (e.g.,
compounds which can be used to treat a disorder characterized by
aberrant or abnormal P2T nucleic acid expression or P2T polypeptide
activity) can be identified in a method wherein a cell is contacted
with a candidate compound and the expression of P2T mRNA or protein
in the cell is determined. The level of expression of P2T mRNA or
protein in the presence of the candidate compound is compared to
the level of expression of P2T mRNA or protein in the absence of
the candidate compound. The candidate compound can then be
identified as a modulator of P2T nucleic acid expression based on
this comparison and be used to treat a disorder characterized by
aberrant P2T nucleic acid expression. For example, when expression
of P2T mRNA or protein is greater (statistically significantly
greater) in the presence of the candidate compound than in its
absence, the candidate compound is identified as a stimulator of
P2T nucleic acid expression. Alternatively, when P2T nucleic acid
expression is less (statistically significantly less) in the
presence of the candidate compound than in its absence, the
candidate compound is identified as an inhibitor of P2T nucleic
acid expression. The level of P2T nucleic acid expression in the
cells can be determined by methods described herein for detecting
P2T mRNA or protein.
[0240] In certain aspects of the invention, P2T polypeptides or
portions thereof 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;
U.S. Statutory Invention Registration No. H1,892; Zervos et al.,
1993; Madura et al., 1993; Bartel et al., 1993(a); Iwabuchi et al.,
1993; International Application No. WO94/10300), to identify other
proteins, which bind to or interact with P2T ("P2T-binding
proteins" or "P2T-bp") and are involved in P2T activity. Such
P2T-binding proteins are also likely to be involved in the
propagation of signals by the P2T polypeptides or P2T targets as,
for example, downstream elements of a P2T-mediated signaling
pathway. Alternatively, such P2T-binding proteins may be P2T
inhibitors.
[0241] Thus, in certain embodiments, the invention contemplates
determining protein:protein interactions, e.g., P2T and a P2T
binding protein. The yeast two-hybrid system is extremely useful
for studying protein:protein interactions. Variations of the system
are available for screening yeast phagemid (Harper et al., 1993;
Elledge et al., 1991) or plasmid (Bartel et al., 1993(a),(b);
Finley and Brent, 1994) cDNA libraries to clone interacting
proteins, as well as for studying known protein pairs. Recently, a
two-hybrid method for high volume screening for specific inhibitors
of protein:protein interactions and a two-hybrid screen that
identifies many different interactions between protein pairs at
once have been described (see, U.S. Statutory Invention
Registration No. H1,892).
[0242] The success of the two-hybrid system relies upon the fact
that the DNA binding and polymerase activation domains of many
transcription factors, such as GAL4, can be separated and then
rejoined to restore functionality (Morin et al., 1993). Briefly,
the assay utilizes two different DNA constructs. In one construct,
the gene that codes for a P2T polypeptide 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
P2T-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 P2T
polypeptide.
[0243] Modulators of P2T polypeptide activity and/or P2T nucleic
acid expression identified according to these drug screening assays
can be used to treat, for example, nervous system disorders. These
methods of treatment include the steps of administering the
modulators of P2T polypeptide activity and/or nucleic acid
expression, e.g., in a pharmaceutical composition as described
herein, to a subject in need of such treatment, e.g., a subject
with a disorder described herein.
[0244] Diagnostic Assays
[0245] The invention further provides a method for detecting the
presence of a P2T polypeptide or P2T nucleic acid molecule, or
fragment thereof, in a biological sample. The method involves
contacting the biological sample with a compound or an agent
capable of detecting P2T polypeptide or mRNA such that the presence
of P2T polypeptide/encoding nucleic acid molecule is detected in
the biological sample. A preferred agent for detecting P2T mRNA is
a labeled or labelable nucleic acid probe capable of hybridizing to
P2T mRNA. The nucleic acid probe can be, for example, the
full-length P2T cDNA of SEQ ID NO: 1, or a fragment thereof, such
as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500
nucleotides in length and sufficient to specifically hybridize
under stringent conditions to P2T mRNA. A preferred agent for
detecting P2T polypeptide is a labeled or labelable antibody
capable of binding to P2T polypeptide. Antibodies can be
polyclonal, or more preferably, monoclonal. An intact antibody, or
a fragment thereof (e.g., Fab or F(ab')2) can be used. The term
"labeled or labelable," with regard to the probe or antibody, is
intended to encompass direct labeling of the probe or antibody by
coupling (i.e., physically linking) a detectable substance to the
probe or antibody, as well as indirect labeling of the probe or
antibody by reactivity with another reagent that is directly
labeled. Examples of indirect labeling include detection of a
primary antibody using a fluorescently labeled secondary antibody
and end-labeling of a DNA probe with biotin such that it can be
detected with fluorescently labeled streptavidin. The term
"biological sample" is intended to include tissues, cells and
biological fluids isolated from a subject, as well as tissues,
cells and fluids present within a subject. That is, the detection
method of the invention can be used to detect P2T mRNA or protein
in a biological sample in vitro as well as in vivo. For example, in
vitro techniques for detection of P2T mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detection of P2T polypeptide include enzyme linked immunosorbent
assays (ELISAs), Western blots, immunoprecipitations and
immunofluorescence. Alternatively, P2T polypeptide can be detected
in vivo in a subject by introducing into the subject a labeled
anti-P2T antibody. 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 which detect the allelic variant of a P2T polypeptide
expressed in a subject and methods which detect fragments of a P2T
polypeptide in a sample.
[0246] The invention also encompasses kits for detecting the
presence of a P2T polypeptide in a biological sample. For example,
the kit can comprise reagents such as a labeled or labelable
compound or agent capable of detecting P2T polypeptide or mRNA in a
biological sample; means for determining the amount of P2T
polypeptide in the sample; and means for comparing the amount of
P2T polypeptide 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 P2T mRNA or
protein.
[0247] The methods of the invention can also be used to detect
naturally occurring genetic mutations in a P2T gene, thereby
determining if a subject with the mutated gene is at risk for a
disorder characterized by aberrant or abnormal P2T nucleic acid
expression or P2T polypeptide activity as described herein. In
preferred embodiments, the methods include detecting, in a sample
of cells from the subject, the presence or absence of a genetic
mutation characterized by at least one of an alteration affecting
the integrity of a gene encoding a P2T polypeptide, or the
misexpression of the P2T gene. For example, such genetic mutations
can be detected by ascertaining the existence of at least one of 1)
a deletion of one or more nucleotides from a P2T gene; 2) an
addition of one or more nucleotides to a P2T gene; 3) a
substitution of one or more nucleotides of a P2T gene, 4) a
chromosomal rearrangement of a P2T gene; 5) an alteration in the
level of a messenger RNA transcript of a P2T gene, 6) aberrant
modification of a P2T gene, such as of the methylation pattern of
the genomic DNA, 7) the presence of a non-wild type splicing
pattern of a messenger RNA transcript of a P2T gene, 8) a non-wild
type level of a P2T-protein, 9) allelic loss of a P2T gene, and 10)
inappropriate post-translational modification of a P2T-protein. As
described herein, there are a large number of assay techniques
known in the art that can be used for detecting mutations in a P2T
gene.
[0248] In certain embodiments, detection of the mutation involves
the use of a probe/primer in a polymerase chain reaction (PCR)
(see, e.g. U.S. Pat. No. 4,683,195 and U.S. Pat. No. 4,683,202),
such as anchor PCR or RACE PCR, or, alternatively, in a ligation
chain reaction (LCR), the latter of which can be particularly
useful for detecting point mutations in the P2T-gene (see Abravaya
et al., 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 P2T gene under conditions such that hybridization
and amplification of the P2T-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.
[0249] In an alternative embodiment, mutations in a P2T gene from a
sample cell can be identified by alterations in restriction enzyme
cleavage patterns. For example, sample and control DNA is isolated,
amplified (optionally), digested with one or more restriction
endonucleases, and fragment length sizes are determined by gel
electrophoresis and compared. Differences in fragment length sizes
between sample and control DNA indicates mutations in the sample
DNA. Moreover, the use of sequence specific ribozymes (see U.S.
Pat. No. 5,498,531 hereby incorporated by reference in its
entirety) can be used to score for the presence of specific
mutations by development or loss of a ribozyme cleavage site.
[0250] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the P2T
gene and detect mutations by comparing the sequence of the sample
P2T gene with the corresponding wild-type (control) sequence.
Examples of sequencing reactions include those based on techniques
developed by Maxim and Gilbert (1977) or Sanger (1977). A variety
of automated sequencing procedures can be utilized when performing
the diagnostic assays, including sequencing by mass spectrometry
(see, e.g., International Application No. WO 94/16101; Cohen et
al., 1996; and Griffin et al. 1993).
[0251] Other methods for detecting mutations in the P2T 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., 1985; Cotton et al., 1988; Saleeba et al., 1992),
electrophoretic mobility of mutant and wild type nucleic acid is
compared (Orita et al., 1989; Cotton, 1993; and Hayashi, 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., 1985). Examples of
other techniques for detecting point mutations include selective
oligonucleotide hybridization, selective amplification, and
selective primer extension.
[0252] Methods of Treatment
[0253] Another aspect of the invention pertains to methods for
treating a subject, e.g., a human, having a disease or disorder
characterized by (or associated with) aberrant or abnormal P2T
nucleic acid expression and/or P2T polypeptide activity. These
methods include the step of administering a P2T polypeptide/gene
modulator (agonist or antagonist) to the subject such that
treatment occurs. The language "aberrant or abnormal P2T
polypeptide expression" refers to expression of a non-wild-type P2T
polypeptide or a non-wild-type level of expression of a P2T
polypeptide. Aberrant or abnormal P2T polypeptide activity refers
to a non-wild-type P2T polypeptide activity or a non-wild-type
level of P2T polypeptide activity. As the P2T polypeptide is
involved in a pathway involving signaling within cells, aberrant or
abnormal P2T polypeptide activity or expression interferes with the
normal regulation of functions mediated by P2T polypeptide
signaling. The terms "treating" or "treatment," as used herein,
refer to reduction or alleviation of at least one adverse effect or
symptom of a disorder or disease, e.g., a disorder or disease
characterized by or associated with abnormal or aberrant P2T
polypeptide activity or P2T nucleic acid expression.
[0254] As used herein, a P2T polypeptide/gene modulator is a
molecule which can modulate P2T nucleic acid expression and/or P2T
polypeptide activity. For example, a P2T gene or protein modulator
can modulate, e.g., upregulate (activate/agonize) or downregulate
(suppress/antagonize), P2T nucleic acid expression. In another
example, a P2T polypeptide/gene modulator can modulate (e.g.,
stimulate/agonize or inhibit/antagonize) P2T polypeptide activity.
If it is desirable to treat a disorder or disease characterized by
(or associated with) aberrant or abnormal (non-wild-type) P2T
nucleic acid expression and/or P2T polypeptide activity by
inhibiting P2T nucleic acid expression, a P2T modulator can be an
antisense molecule, e.g., a ribozyme, as described herein. Examples
of antisense molecules which can be used to inhibit P2T nucleic
acid expression include antisense molecules which are complementary
to a fragment of the 5' untranslated region of SEQ ID NO: 1, which
also includes the start codon and antisense molecules which are
complementary to a fragment of a 3' untranslated region of SEQ ID
NO: 1.
[0255] A P2T modulator that inhibits P2T nucleic acid expression
can also be a small molecule or other drug, e.g., a small molecule
or drug identified using the screening assays described herein,
which inhibits P2T nucleic acid expression. If it is desirable to
treat a disease or disorder characterized by (or associated with)
aberrant or abnormal (non-wild-type) P2T nucleic acid expression
and/or P2T polypeptide activity by stimulating P2T nucleic acid
expression, a P2T modulator can be, for example, a nucleic acid
molecule encoding a P2T polypeptide (e.g., a nucleic acid molecule
comprising a nucleotide sequence homologous to the nucleotide
sequence of SEQ ID NO: 1 or a small molecule or other drug, e.g., a
small molecule (peptide) or drug identified using the screening
assays described herein, which stimulates P2T nucleic acid
expression.
[0256] Alternatively, if it is desirable to treat a disease or
disorder characterized by (or associated with) aberrant or abnormal
(non-wild-type) P2T nucleic acid expression and/or P2T polypeptide
activity by inhibiting P2T polypeptide activity, a P2T modulator
can be an anti-P2T antibody or a small molecule or other drug,
e.g., a small molecule or drug identified using the screening
assays described herein, which inhibits P2T polypeptide activity.
If it is desirable to treat a disease or disorder characterized by
(or associated with) aberrant or abnormal (non-wild- type) P2T
nucleic acid expression and/or P2T polypeptide activity by
stimulating P2T polypeptide activity, a P2T modulator can be an
active P2T polypeptide or fragment thereof (e.g., a P2T polypeptide
or fragment thereof having an amino acid sequence which is
homologous to the amino acid sequence of SEQ ID NO:2 or a fragment
thereof) or a small molecule or other drug, e.g., a small molecule
or drug identified using the screening assays described herein,
which stimulates P2T polypeptide activity.
[0257] Other aspects of the invention pertain to methods for
modulating a P2T polypeptide mediated cell activity. These methods
include contacting the cell with an agent (or a composition which
includes an effective amount of an agent) which modulates P2T
polypeptide activity or P2T nucleic acid expression such that a P2T
polypeptide mediated cell activity is altered relative to normal
levels (for example, cAMP or phosphatidylinositol metabolism). As
used herein, "a P2T polypeptide mediated cell activity" refers to a
normal or abnormal activity or function of a cell. Examples of P2T
polypeptide mediated cell activities include phosphatidylinositol
turnover, production or secretion of molecules, such as proteins,
contraction, proliferation, migration, differentiation, and cell
survival. The term "altered" as used herein refers to a change,
e.g., an increase or decrease, of a cell associated activity
particularly cAMP or phosphatidylinositol turnover, and adenylate
cyclase or phospholipase C activation.
[0258] In one embodiment, the agent stimulates P2T polypeptide
activity or P2T nucleic acid expression. In another embodiment, the
agent inhibits P2T polypeptide activity or P2T nucleic acid
expression. These modulatory methods can be performed in vitro
(e.g., by culturing the cell with the agent) or, alternatively, in
vivo (e.g., by administering the agent to a subject). In a
preferred embodiment, the modulatory methods are performed in vivo,
i.e., the cell is present within a subject, e.g., a mammal, e.g., a
human, and the subject has a disorder or disease characterized by
or associated with abnormal or aberrant P2T polypeptide activity or
P2T nucleic acid expression.
[0259] A nucleic acid molecule, a protein, a P2T modulator, a
compound etc. used in the methods of treatment can be incorporated
into an appropriate pharmaceutical composition described below and
administered to the subject through a route which allows the
molecule, protein, modulator, or compound etc. to perform its
intended function.
[0260] A modulator of P2T polynucleotide expression and/or P2T
polypeptide activity may be used in the treatment of various
diseases or disorders including, but not limited to, the
cardiopulmonary system such as acute heart failure, hypotension,
hypertension, angina pectoris, myocardial infarction and the like;
the gastrointestinal system; the central nervous system; kidney
diseases; liver diseases; hyperproliferative diseases, such as
cancers and psoriasis; apoptotic diseases; pain; endometriosis;
anorexia; bulimia; asthma; osteoporosis; neuropsychiatric disorders
such as schizophrenia, delirium, bipolar, depression, anxiety,
panic disorders; urinary retention; ulcers; allergies; benign
prostatic hypertrophy; and dyskinesias, such as Huntington's
disease or Gilles dela Tourett's syndrome.
[0261] Disorders involving the brain include, but are not limited
to, disorders involving neurons, and disorders involving glia, such
as astrocytes, oligodendrocytes, ependymal cells, and microglia;
cerebral edema, raised intracranial pressure and herniation, and
hydrocephalus; malformations and developmental diseases, such as
neural tube defects, forebrain anomalies, posterior fossa
anomalies, and syringomyelia and hydromyelia; perinatal brain
injury; cerebrovascular diseases, such as those related to hypoxia,
ischemia, and infarction, including hypotension, hypoperfusion, and
low-flow states--global cerebral ischemia and focal cerebral
ischemia--infarction from obstruction of local blood supply,
intracranial hemorrhage, including intracerebral (intraparenchymal)
hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms,
and vascular malformations, hypertensive cerebrovascular disease,
including lacunar infarcts, slit hemorrhages, and hypertensive
encephalopathy; infections, such as acute meningitis, including
acute pyogenic (bacterial) meningitis and acute aseptic (viral)
meningitis, acute focal suppurative infections, including brain
abscess, subdural empyema, and extradural abscess, chronic
bacterial meningoencephalitis, including tuberculosis and
mycobacterioses, neurosyphilis, and neuroborreliosis (Lyme
disease), viral meningoencephalitis, including arthropod-borne
(Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes
simplex virus Type 2, Varicella-zoster virus (Herpes zoster),
cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency
virus 1, including FHV-I meningoencephalitis (subacute
encephalitis), vacuolar myelopathy, AIDS-associated myopathy,
peripheral neuropathy, and AIDS in children, progressive multifocal
leukoencephalopathy, subacute sclerosing panencephalitis, fungal
meningoencephalitis, other infectious diseases of the nervous
system; transmissible spongiform encephalopathies (prion diseases);
demyelinating diseases, including multiple sclerosis, multiple
sclerosis variants, acute disseminated encephalomyelitis and acute
necrotizing hemorrhagic encephalomyelitis, and other diseases with
demyelination; degenerative diseases, such as degenerative diseases
affecting the cerebral cortex, including Alzheimer disease and Pick
disease, degenerative diseases of basal ganglia and brain stem,
including Parkinsonism, idiopathic Parkinson disease (paralysis
agitans), progressive supranuclear palsy, corticobasal
degeneration, multiple system atrophy, including striatonigral
degenration, Shy-Drager syndrome, and olivopontocerebellar atrophy,
and Huntington disease; spinocerebellar degenerations, including
spinocerebellar ataxias, including Friedreich ataxia, and
ataxia-telanglectasia, degenerative diseases affecting motor
neurons, including amyotrophic lateral sclerosis (motor neuron
disease), bulbospinal atrophy (Kennedy syndrome), and spinal
muscular atrophy; inborn errors of metabolism, such as
leukodystrophies, including Krabbe disease, metachromatic
leukodystrophy, adrenoleukodystrophy, Elizaeus-Merzbacher disease,
and Canavan disease, mitochondrial encephalomyopathies, including
Leigh disease and other mitochondrial encephalomyopathies; toxic
and acquired metabolic diseases, including vitamin deficiencies
such as thiamine (vitamin BI) deficiency and vitamin B12
deficiency, neurologic sequelae of metabolic disturbances,
including hypoglycernia, hyperglycemia, and hepatic encephatopathy,
toxic disorders, including carbon monoxide, methanol, ethanol, and
radiation, including combined methotrexate and radiation-induced
injury; tumors, such as gliomas, including astrocytoma, including
fibrillary (diffuse) astrocytoma and glioblastorna multiforme,
pilocytic astrocytoma, pleomorphic xanthoastrocytorna, and brain
stem glioma, oligodendrogliorna, and ependymoma and related
paraventricular mass lesions, neuronal tumors, poorly
differentiated neoplasms, including medulloblastoma, other
parenchymal tumors, including primary brain lymphoma, germ cell
tumors, and pineal parenchymal tumors, meningiomas, metastatic
tumors, paraneoplastic syndromes, peripheral nerve sheath tumors,
including schwannoma, neurofibroma, and malignant peripheral nerve
sheath tumor (malignant schwannoma), neurocutaneous syndromes
(phakomatoses), including neurofibromotosis, including Type I
neurofibromatosis (NFI) and TYPE 2 neurofibromatosis (NF2),
tuberous sclerosis, and Von Hippel-Lindau disease, and
neuropsychiatric disorders, such as schizophrenia, bipolar,
depression, anxiety and panic disorders.
[0262] Pharmacogenomics
[0263] Test/candidate compounds, or modulators which have a
stimulatory or inhibitory effect on P2T polypeptide activity (e.g.,
P2T gene expression) as identified by a screening assay described
herein can be administered to individuals to treat
(prophylactically or therapeutically) disorders (e.g., neurological
disorders) associated with aberrant P2T polypeptide activity. In
conjunction with such treatment, the pharmacogenomics (i.e., the
study of the relationship between an individual's genotype and that
individual's response to a foreign compound or drug) of the
individual may be considered. Differences in metabolism of
therapeutics can lead to severe toxicity or therapeutic failure by
altering the relation between dose and blood concentration of the
pharmacologically active drug. Thus, the pharmacogenomics of the
individual permit the selection of effective compounds (e.g.,
drugs) for prophylactic or therapeutic treatments based on a
consideration of the individual's genotype. Such pharmacogenomics
can further be used to determine appropriate dosages and
therapeutic regimens. Accordingly, the activity of P2T polypeptide,
expression of P2T nucleic acid, or mutation content of P2T genes in
an individual can be determined to thereby select appropriate
compound(s) for therapeutic or prophylactic treatment of the
individual.
[0264] 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,
1996 and Linder, 1997. In general, two types of pharmacogenetic
conditions can be differentiated. Genetic conditions transmitted as
a single factor altering the way drugs act on the body (altered
drug action) or genetic conditions transmitted as single factors
altering the way the body acts on drugs (altered drug metabolism).
These pharmacogenetic conditions can occur either as rare defects
or as polymorphisms. For example, glucose-6-phosphate dehydrogenase
deficiency (GOD) is a common inherited enzymopathy in which the
main clinical complication is haemolysis after ingestion of oxidant
drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and
consumption of fava beans.
[0265] As an illustrative embodiment, the activity of drug
metabolizing enzymes is a major determinant of both the intensity
and duration of drug action. The discovery of genetic polymorphisms
of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2)
and cytochrome P450 enzymes CYP2136 and CYP2C 19) has provided an
explanation as to why some patients do not obtain the expected drug
effects or show exaggerated drug response and serious toxicity
after taking the standard and safe dose of a drug.
[0266] These polymorphisms are expressed in two phenotypes in the
population, the extensive metabolizer (EM) and poor metabolizer
(PM). The prevalence of PM is different among different
populations. For example, the gene coding for CYP2136 is highly
polymorphic and several mutations have been identified in PM, which
all lead to the absence of functional CYP2D6. Poor metabolizers of
CYP2136 and CYP2C 19 quite frequently experience exaggerated drug
response and side effects when they receive standard doses.
[0267] If a metabolite is the active therapeutic moiety, PM shows
no therapeutic response, as demonstrated for the analgesic effect
of codeine mediated by its CYP2136-formed metabolite morphine. The
other extreme is the so called ultra-rapid metabolizers who do not
respond to standard doses. Recently, the molecular basis of
ultra-rapid metabolism has been identified to be due to CYP2D6 gene
amplification.
[0268] Thus, the activity of P2T polypeptide, expression of P2T
nucleic acid, or mutation content of P2T genes in an individual can
be determined to thereby select appropriate agent(s) for
therapeutic or prophylactic treatment of a subject. In addition,
pharmacogenetic studies can be used to apply genotyping of
polymorphic alleles encoding drug-metabolizing enzymes to the
identification of a subject's drug responsiveness phenotype. This
knowledge, when applied to dosing or drug selection, can avoid
adverse reactions or therapeutic failure and thus enhance
therapeutic or prophylactic efficiency when treating a subject with
a P2T modulator, such as a modulator identified by one of the
exemplary screening assays described herein.
[0269] Monitoring of Effects During Clinical Trials
[0270] Monitoring the influence of compounds (e.g., drugs) on the
expression or activity of P2T polypeptide/gene can be applied not
only in basic drug screening, but also in clinical trials. For
example, the effectiveness of an agent determined by a screening
assay, as described herein, to increase P2T gene expression,
protein levels, or up-regulate P2T activity, can be monitored in
clinical trials of subjects exhibiting decreased P2T gene
expression, protein levels, or down-regulated P2T polypeptide
activity. Alternatively, the effectiveness of an agent, determined
by a screening assay, to decrease P2T gene expression, protein
levels, or down-regulate P2T polypeptide activity, can be monitored
in clinical trials of subjects exhibiting increased P2T gene
expression, protein levels, or up-regulated P2T polypeptide
activity. In such clinical trials, the expression or activity of a
P2T polypeptide and, preferably, other genes which have been
implicated in, for example, a nervous system related disorder can
be used as a "read out" or markers of the ligand responsiveness of
a particular cell.
[0271] For example, and not by way of limitation, genes, including
a P2T gene, which are modulated in cells by treatment with a
compound (e.g., drug or small molecule) which modulates P2T
polypeptide/gene activity (e.g., identified in a screening assay as
described herein) can be identified. Thus, to study the effect of
compounds on CNS disorders, for example, in a clinical trial, cells
can be isolated and RNA prepared and analyzed for the levels of
expression of a P2T gene and other genes implicated in the
disorder. The levels of gene expression (i.e., a gene expression
pattern) can be quantified by Northern blot analysis or RT-PCR, as
described herein, or alternatively by measuring the amount of
protein produced, by one of the methods described herein, or by
measuring the levels of activity of a P2T polypeptide or other
genes. In this way, the gene expression pattern can serve as an
marker, indicative of the physiological response of the cells to
the compound. Accordingly, this response state may be determined
before, and at various points during, treatment of the individual
with the compound.
[0272] In a preferred embodiment, the present invention provides a
method for monitoring the effectiveness of treatment of a subject
with a compound (e.g., an agonist, antagonist, peptidomimetic,
protein, peptide, nucleic acid, small molecule, or other drug
candidate identified by the screening assays described herein)
comprising the steps of (i) obtaining a pre-administration sample
from a subject prior to administration of the compound; (ii)
detecting the level of expression of a P2T polypeptide, mRNA, or
genomic DNA in the preadministration sample; (iii) obtaining one or
more post- administration samples from the subject; (iv) detecting
the level of expression or activity of the P2T polypeptide, mRNA,
or genomic DNA in the post-administration samples; (v) comparing
the level of expression or activity of the P2T polypeptide, mRNA,
or genomic DNA in the pre-administration sample with the P2T
polypeptide, mRNA, or genomic DNA in the post administration sample
or samples; and (vi) altering the administration of the compound to
the subject accordingly. For example, increased administration of
the compound may be desirable to increase the expression or
activity of a P2T polypeptide/gene to higher levels than detected,
i.e., to increase the effectiveness of the agent.
[0273] Alternatively, decreased administration of the agent may be
desirable to decrease expression or activity of P2T to lower levels
than detected, i.e. to decrease the effectiveness of the
compound.
[0274] Pharmaceutical Compositions
[0275] The P2T nucleic acid molecules, P2T polypeptides
(particularly fragments of P2T), modulators of a P2T polypeptide,
and anti-P2T antibodies (also referred to herein as "active
compounds") of the invention can be incorporated into
pharmaceutical compositions suitable for administration to a
subject, e.g., a human. Such compositions typically comprise the
nucleic acid molecule, protein, modulator, or antibody and a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. The use of
such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, such media can be
used in the compositions of the invention. Supplementary active
compounds can also be incorporated into the compositions.
[0276] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), transmucosal, and rectal administration.
Solutions or suspensions used for parenteral, intradermal, or
subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
[0277] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor ELTM(BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0278] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a P2T polypeptide or
anti-P2T antibody) in the required amount in an appropriate solvent
with one or a combination of ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle which contains a basic dispersion medium and the
required other ingredients from those enumerated above. In the case
of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying
and freeze-drying which yields a powder of the active ingredient
plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0279] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0280] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from a pressured
container or dispenser which contains a suitable propellant, e.g.,
a gas such as carbon dioxide, or a nebulizer. Systemic
administration can also be by transmucosal or transdermal means.
For transmucosal or transdermal administration, penetrants
appropriate to the barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art, and
include, for example, for transmucosal administration, detergents,
bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0281] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery. In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems.
[0282] Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes targeted to infected cells with
monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art, for
example, as described in U.S. Pat. No. 4,522,811 which is
incorporated by reference herein in its entirety.
[0283] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0284] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (see U.S. Pat. No. 5,328,470) or by
stereotactic injection (see e.g., Chen et al., 1994). The
pharmaceutical preparation of the gene therapy vector can include
the gene therapy vector in an acceptable diluent, or can comprise a
slow release matrix in which the gene delivery vehicle is imbedded.
Alternatively, where the complete gene delivery vector can be
produced intact from recombinant cells, e.g. retroviral vectors,
the pharmaceutical preparation can include one or more cells which
produce the gene delivery system. The pharmaceutical compositions
can be included in a container, pack, or dispenser together with
instructions for administration.
H. Uses of Partial P2T Sequences
[0285] Fragments or fragments of the cDNA sequences identified
herein (and the corresponding complete gene sequences) can be used
in numerous ways as polynucleotide reagents. For example, these
sequences can be used to: (a) map their respective genes on a
chromosome; and, thus, locate gene regions associated with genetic
disease; (b) identify an individual from a minute biological sample
(tissue typing); and (c) aid in forensic identification of a
biological sample. These applications are described in the
subsections below.
[0286] Chromosome Mapping
[0287] Once the sequence (or a fragment of the sequence) of a gene
has been isolated, this sequence can be used to map the location of
the gene on a chromosome. This process is called chromosome
mapping. Accordingly, fragments of a P2T nucleic acid sequences can
be used to map the location of the P2T gene, respectively, on a
chromosome. The mapping of the P2T sequence to chromosomes is an
important first step in correlating these sequence with genes
associated with disease.
[0288] Briefly, the P2T gene can be mapped to a chromosome by
preparing PCR primers (preferably 15-25 bp in length) from the P2T
gene sequence. Computer analysis of the P2T gene sequence can be
used to rapidly select primers that do not span more than one exon
in the genomic DNA, thus complicating the amplification process.
These primers can then be used for PCR screening of somatic cell
hybrids containing individual human chromosomes. Only those hybrids
containing the human gene corresponding to the P2T gene sequence
will yield an amplified fragment.
[0289] Somatic cell hybrids are prepared by fusing somatic cells
from different mammals (e.g., human and mouse cells). As hybrids of
human and mouse cells grow and divide, they gradually lose human
chromosomes in random order, but retain the mouse chromosomes. By
using media in which mouse cells cannot grow, because they lack a
particular enzyme, but human cells can, the one human chromosome
that contains the gene encoding the needed enzyme, will be
retained. By using various media, panels of hybrid cell lines can
be established. Each cell line in a panel contains either a single
human chromosome or a small number of human chromosomes, and a full
set of mouse chromosomes, allowing easy mapping of individual genes
to specific human chromosomes. (D'Eustachio et al., 1983). Somatic
cell hybrids containing only fragments of human chromosomes can
also be produced by using human chromosomes with translocations and
deletions.
[0290] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular sequence to a particular chromosome. Three
or more sequences can be assigned per day using a single thermal
cycler. Using the P2T gene sequence to design oligonucleotide
primers, sublocalization can be achieved with panels of fragments
from specific chromosomes. Other mapping strategies which can
similarly be used to map a P2T gene sequence to its chromosome
include in situ hybridization (described in Fan et al., 1990),
pre-screening with labeled flow-sorted chromosomes, and
pre-selection by hybridization to chromosome specific cDNA
libraries.
[0291] Fluorescence in situ hybridization (FISH) of a DNA sequence
to a metaphase chromosomal spread can further be used to provide a
precise chromosomal location in one step. Chromosome spreads can be
made using cells whose division has been blocked in metaphase by a
chemical like colcemid that disrupts the mitotic spindle. The
chromosomes can be treated briefly with trypsin, and then stained
with Giemsa. A pattern of light and dark bands develops on each
chromosome, so that the chromosomes can be identified individually.
The FISH technique can be used with a DNA sequence as short as 500
or 600 bases. However, clones larger than 1,000 bases have a higher
likelihood of binding to a unique chromosomal location with
sufficient signal intensity for simple detection. Preferably 1,000
bases, and more preferably 2,000 bases will suffice to get good
results at a reasonable amount of time.
[0292] Reagents for chromosome mapping can be used individually to
mark a single chromosome or a single site on that chromosome, or
panels of reagents can be used for marking multiple sites and/or
multiple chromosomes. Reagents corresponding to noncoding regions
of the genes actually are preferred for mapping purposes. Coding
sequences are more likely to be conserved within gene families,
thus increasing the chance of cross hybridizations during
chromosomal mapping.
[0293] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data (such data are found, for
example, above). McKusick, Mendelian Inheritance in Man, available
on-line through Johns Hopkins University Welch Medical Library. The
relationship between genes and disease, mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes).
[0294] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
the P2T gene, can be determined. If a mutation is observed in some
or all of the affected individuals but not in any unaffected
individuals, then the mutation is likely to be the causative agent
of the particular disease. Comparison of affected and unaffected
individuals generally involves first looking for structural
alterations in the chromosomes, such as deletions or translocations
that are visible from chromosome spreads or detectable using PCR
based on that DNA sequence. Ultimately, complete sequencing of
genes from several individuals can be performed to confirm the
presence of a mutation and to distinguish mutations from
polymorphisms.
[0295] Tissue Typing
[0296] The P2T gene sequences of the present invention can also be
used to identify individuals from minute biological samples. The
United States military, for example, is considering the use of
restriction fragment length polymorphism (RFLP) for identification
of its personnel. In this technique, an individual's genomic DNA is
digested with one or more restriction enzymes, and probed on a
Southern blot to yield unique bands for identification. This method
does not suffer from the current limitations of "Dog Tags" which
can be lost, switched, or stolen, making positive identification
difficult. The sequences of the present invention are useful as
additional DNA markers for RFLP (described in U.S. Pat. No.
5,272,057).
[0297] Furthermore, the sequences of the present invention can be
used to provide an alternative technique which determines the
actual base-by-base DNA sequence of selected fragments of an
individual's genome. Thus, the P2T sequences described herein can
be used to prepare two PCR primers from the 5' and 3' ends of the
sequences. These primers can then be used to amplify an
individual's DNA and subsequently sequence it.
[0298] Panels of corresponding DNA sequences from individuals
prepared in this manner can provide unique individual
identifications, as each individual will have a unique set of such
DNA sequences due to allelic differences. The sequences of the
present invention can be used to obtain such identification
sequences from individuals and from tissue. The P2T gene sequences
of the invention uniquely represent fragments of the human genome.
Allelic variation occurs to some degree in the coding regions of
these sequences, and to a greater degree in the noncoding regions.
It is estimated that allelic variation between individual humans
occurs with a frequency of about once per each 500 bases. Each of
the sequences described herein can, to some degree, be used as a
standard against which DNA from an individual can be compared for
identification purposes. Because greater numbers of polymorphisms
occur in the noncoding regions, fewer sequences are necessary to
differentiate individuals. The noncoding sequence of SEQ ID NO:1
can comfortably provide positive individual identification with a
panel of perhaps 10 to 1,000 primers which each yield a noncoding
amplified sequence of 100 bases. If a predicted coding sequence,
such as that in SEQ ID NO:1 is used, a more appropriate number of
primers for positive individual identification would be
500-2,000.
[0299] If a panel of reagents from the P2T gene sequences described
herein is used to generate a unique identification database for an
individual, those same reagents can later be used to identify
tissue from that individual. Using the unique identification
database, positive identification of the individual, living or
dead, can be made from extremely small tissue samples.
EXAMPLES
[0300] The following examples are carried out using standard
techniques, which are well known and routine to those of skill in
the art, except where otherwise described in detail. The following
examples are presented for illustrative purpose, and should not be
construed in any way limiting the scope of this invention.
Example 1
Materials and Methods
[0301] Materials
[0302] Purinergic ligands and 3-aminotriazole were purchased from
Sigma RBI. CPRG was purchased from Boehringer Mannheim. Mouse
genomic DNA and multiple tissue northern blots were purchased from
Clontech. The chemiluminescent .E-backward.-galactosidase assay kit
GAL-SCREEN.TM. was purchased from TROPIX.
[0303] Identification of Genomic Sequences Encoding the Mouse P2T
Receptor
[0304] A BLAST search (Altschul et al., 1990) of the Genbank cDNA
sequence database with the human EBI-2 receptor sequence (U.S. Pat.
No. 6,060,272) identified a full length cDNA sequence encoding the
mouse P2T receptor (Accession No. AK013804) (Adachi et al., 2001).
A similar search of the Celera mouse genome sequence database with
the mouse cDNA sequence identified a 46,358 base pair segment
encoding the mouse P2T promoter and transcript. The mouse P2T mRNA
is encoded in four exons (SEQ ID NO:1), three of which comprise the
5' untranslated region. The mouse P2T open reading frame is encoded
in a single uninterrupted exon from nucleotide 45,167 to nucleotide
46,358 of SEQ ID NO:1.
[0305] Cloning of the Mouse P2T Receptor
[0306] The mouse P2T receptor protein coding sequences were
amplified from mouse genomic DNA using oligos MPO720 (5' AA GGATCC
AAA ATG GAT GTG CCT GGT GTC; SEQ ID NO:7) and MPO721 (5' AA CTCGAG
CTA CAT TGG GGT CTC TTC GC; SEQ ID NO:8) that add BamHI to the 5'
end and a XhoI site to the 3' end. The fragment was cloned into
corresponding sites in the multicopy yeast expression vector,
p426GPD (Mumberg et al., 1995) producing pMP344.
[0307] Yeast Expression
[0308] The EC323 yeast expression plasmids were introduced into
variants of MPY578 cells (MATa ura3 his3 trp1 leu2 lys2 ade2
far1::LYS2 fus1::FUS1-HIS3 sst2::SST2-G418.sup.R ste2::LEU2)
(Pausch et al., 1998) using LiOAc and selected for ura prototrophy.
In order to facilitate coupling of the P2T receptor to G protein,
MPY578 variant cells express chimeric G alpha proteins coupled to
the mating signal transduction pathway. The chimeric constructs are
expressed from the GPA1 locus and are composed of Gpa1 sequences in
which the 5 C-terminal amino acids have been replaced with those of
all the mammalian G.alpha. proteins. A multicopy FUS1-LacZ reporter
gene plasmid, pMP283, was introduced into P2T receptor-containing
cells and selected on media lacking trp and ura. The resulting
yeast strains were used for further analysis.
[0309] Yeast Cell-Based Assay of Mouse P2T Receptor Agonist
Activation
[0310] MPY578 cells that express Gpa1-Gat chimeric G alpha protein
and containing pMP344 and pMP283 were diluted in assay medium
(SCD-ura-trp, pH 6.8, 25 mM PIPES, 1 mM 3-aminotriazole) dispensed
(5.times.10.sup.6/ml, 200 .mu.l/well) to the wells of 96-well
microtiter dishes containing purinergic ligands. The plates were
incubated with shaking (600 rpm) at 30.degree. C. for 3 hours.
Samples (25 .mu.l) were transferred to Wallac B & W isoplates
for .beta.-galactosidase assay. An equal volume of GAL-SCREEN lysis
and chemiluminescent .beta.-galactosidase assay mixture was added.
The plates were incubated for 30 min at 30.degree. C. and light
emission measured using a Wallac Victor II. Assays were conducted
in quadruplicate and results were plotted using GraphPad Prizm.
[0311] Northern Blotting Analysis of mRNA Expression
[0312] A 400 bp fragment corresponding to position +70 to +470 with
respect to ATG was PCR amplified from the murine P2T receptor clone
using oligos F2 (CAG AGA CTA CAA GAT CAC CCA GGT; SEQ ID NO:9) and
R3 (GM GGC CCA GAT GAC MC AGA AAG A; SEQ ID NO:10). The DNA
fragment was labeled with .sup.32P-dCTP by random priming (Life
Technologies, Rockville, Md.) and used as probe to screen a mouse
multiple tissue northern blot (Clontech, Palo Alto, Calif.). The
blot was washed at final stringency of 0.5.times.SSC, 0.5% SDS, at
60.degree. C. and exposed to film. The resulting image was captured
electronically for documentation (Scion Corp. Bethesda, Md.).
[0313] In Situ Hybridization
[0314] Distribution of murine P2T receptor mRNA within the mouse
brain was assessed in situ as described previously (Kwak et al.,
1993). Briefly, frozen mouse brains from C57/BL6 strain were
sectioned on a cryostat (Bright-hacker, Fairfield, N.J.). Coronal
as well as para-sagittal 15 .mu.m sections were thaw-mounted on
polylysine-coated slides and stored at -70.degree. C. On the day of
experimentation, the slides were posffixed in 4% paraformaldehyde
for 1 hour. Riboprobe complementary to region +70 to +470 bp of
murine P2T was sythesized by an in vitro transcription reaction
which incorporated [.sup.33p] UTP. Approximately 2 million cpm were
applied per slide for hybridization. The sections were incubated at
55.degree. C. overnight, treated with RNase A and then washed in
0.5.times.SSC at 65.degree. C. for 1 hour. The sections were
initially exposed on film and subsequently dipped in emulsion for
further analysis.
Example 2
Clining of the Murine P2T Receptor
[0315] Queries of the Celera mouse genome DNA sequence database
using the DNA sequence of the human orphan P2T, EBI-2, revealed
fragments that once assembled into a single contiguous sequence,
appeared to encode a presumptive murine ortholog. Comparison of the
genomic sequence with a recently reported cDNA sequence identified
in a search of the Genbank indicates that the murine genomic
sequence encodes the apparent protein coding sequence in a single
uninterrupted exon (data not shown). The genomic sequence appears
to encode an intron in the untranslated region of the mRNA
immediately upstream of the initiator codon. A complete description
of the mouse EBI-2 locus awaits complete assembly of the mouse
genome. The predicted protein was 347 amino acids in length and was
86% identical to the human version and 95% identical to the rat
(FIG. 1). The similarity between the three proteins is even greater
in the transmembrane domains were only four conservative
substitutions were found. The greatest divergences were found in
the amino terminal extracellular domain and carboxy terminal
intracellular domains. The P2T receptors exhibit the greatest
degree of similarity to a subset of the P2Y receptors including the
UDP-glucose receptor (Chambers et al., 2000) and H963 (Jacobs et
al., 1997). Oligonucleotides corresponding to the 5' and 3' ends of
the predicted protein coding region were used to amplify a fragment
from mouse genomic DNA.
Example 3
Pharmacological Analysis
[0316] The murine P2T was expressed in yeast cells modified to
permit agonist-induced expression of a .beta.-galactosidase
reporter gene (Pausch et al, 1998; Pausch, 1997). Agonist
activation of the P2T with various purinergic ligands induces a
dose-dependent activation of the murine receptor that responds with
pharmacological properties comparable to the cloned human P2T
receptor (Zhang et al., 2001; Hollopeter et al., 2001) (FIG. 2).
The EC50 for ADP stimulation of the mouse P2T assayed in yeast was
256 nM. In Xenopus oocytes injected with P2T receptor cRNA and
coupled to stimulation of coinjected Kir3.1/Kir3.2 inwardly
rectifying K.sup.+ currents, the EC50 was 300 nM (Hollopeter et
al., 2001).
[0317] In NIH3T3 cells, the P2T receptor transiently transfected
along with a chimeric Gq5 construct produced and EC50 of 74 nM
measured by FLIPR (Boyer et al., 1993). Assays of ADP dependent
inhibition of cAMP accumulation gave an EC50 of 61 nM (Zhang et
al., 2001). The rank order of potency of agonist stimulation of the
mouse P2T receptor
(2MeSADP>2MeSATP>ADP=ADP.beta.S>ATP.gamma.S>2CIATP)
agrees well with reported values for the human P2T receptor
measured in FLIPR
(2MeSADP=2MeSATP>ADP=ADP.beta.S>2CIATP>ATP.gamma.S) and in
adenylyl cyclase inhibition assays
(2MeSATP>2MeSADP>ADP>ATP.gamma.S>ADP.beta.S>2CIATP)(Zhang
et al, 2001).
[0318] Mammalian cell expression and measurement of intracellular
calcium. NIH3T3 cells were stably transfected with
pCDNA-G.alpha.q/i3 by electroporation and cultured in the presence
of 800 .mu.g/ml neomycin. The NIH3T3 G.alpha.q/i3 line was
subsequently stably transfected with pCDNA3.1-mP2Y12 by
electroporation and cultured in the presence of 800 .mu.g/ml
neomycin and 400 .mu.g/ml hygromycin. Cells were plated without
selection and grown overnight at 37.degree. C. The cells were
washed with Hank's balanced salt solution without phenol red plus
20 mM HEPES, 2.5 mM probenecid, pH 7.4 and loaded with wash
solution plus 5 .mu.M Fluo-4, 0.04% pluronic acid and 1% fetal
bovine serum for 1 hour at 37.degree. C. After removing the dye
solution and washed with wash buffer, agonists were added and
analyzed using the FLIPR (PE Biosystems) to measure accumulation of
intracellular calcium in response to compound treatments.
[0319] Agonist activation of the P2Y12 with various purinergic
ligands induces a dose-dependent activation of the murine receptor
that responds with pharmacological properties comparable to the
cloned human P2Y12 receptor. The EC50 for ADP stimulation of the
mouse P2Y12 dependent release of intracellular calcium is detected
in FLIPR with an EC50 of 27 nM (FIG. 4). In published reports,
Xenopus oocytes injected with the human P2Y12 receptor cRNA and
coupled to stimulation of coinjected Kir3.1/Kir3.2 inwardly
rectifying K+ currents, the EC50 was 300 nM (Hollopeter, 2001). In
NIH3T3 cells, the human P2Y12 receptor expressed along with a
chimeric Gq/i3 construct produced an EC50 of 74 nM measured by
FLIPR (Zhang, 2001). Assays of ADP dependent inhibition of cAMP
accumulation gave an EC50 of 61 nM (Zhang, 2001). The rank orders
of potency of agonist stimulation of the mouse P2Y12 receptor in
FLIPR (2MeSADP>ADP=AD.beta.S>ATP.gamma.S) agree well with
reported values for the human P2Y12 receptor measured in FLIPR
(2MeSADP=2MeSATP>ADP=AD.beta.S>2CIATP>ATP.gamma.S) and in
adenylyl cyclase inhibition assays
(2MeSATP>2MeSADP>ADP>ATP.gamma.S>ADP.beta.S>2CIATP)(Zhang,
2001).
Example 4
Expression Analysis of the P2T Receptor
[0320] Northern blotting analysis was performed on mRNA extracted
from various murine tissues (FIG. 3). A single prominent 2.4 kb
band was detected in brain. Lesser amounts were present in liver,
spleen and testis. A 2.6 kb band was detected as well in heart,
lung and kidney. Notably, spleen RNA contain both the 2.4 kb and
2.6 kb species.
[0321] In situ hybridization using a murine P2T probe (data not
shown) reveals broadly diffuse and uniform signal throughout the
brain. Unlike the neuron-specific Wave-1 mRNA, which is expressed
in discrete layers of pyramidal and granule neurons within the
hippocampus, the P2T receptor signal is in all major fiber tracts
including the corpus callosum and brain stem pyramidal tracts
suggesting that the mRNA is in glia. Examination at high
magnification of emulsion-dipped slides counter stained with cresyl
violet confirms that murine P2T receptor mRNAs are expressed
exclusively in astroglial or microglial cells of the brain and not
in neurons.
Example 5
Identification of Ligards for Mouse P2T in Mammalian Cells
[0322] Cell Line Generation
[0323] The open reading frame of mouse P2T is ligated into the
mammalian expression vector pCDNA3.1+ zeo (Invitrogen, 1600 Faraday
Avenue, Carlsbad, Calif. 92008). HEK293 or NIH3T3 cells are
transfected with the plasmid and selected with 500 .mu.g/ml zeocin.
Zeocin resistant clones are tested for expression of mouse P2T by
RT-PCR and then tested for their ability to stimulate or inhibit
cAMP production.
[0324] Adenylylcyclase Assay
[0325] 4.times.10.sup.5 cells are plated into 96 well Biocoat cell
culture plates (Becton Dickinson, 1 Becton Drive, Franklin Lakes,
N.J. 07417-1886) 24 hours prior to assay. The cells are then
incubated in Krebs-bicarbonate buffer at 37.degree. C. for 15
minutes. A 5 minute pretreatment with 500 .mu.M isobutylmethyl
xanthine (IBMX) precedes addition of test compounds. To test for
stimulation of adenylylcyclase, the test chemical compounds (10
.mu.M) are added and incubated with the cells for 12 minutes at
37.degree. C. and cAMP levels are determined using the SPA assay
(Amersham Pharmacia Biotech, 800 Centennial Avenue, Pistcataway,
N.J. 08855). An increase in cAMP reflects stimulation of
adenylylcyclase. To test for inhibition, adenylylcyclase is
stimulated with 1 .mu.M forskolin for 12 minutes at 37.degree. C.
prior to addition of the test chemical compounds. Test compounds
(10 .mu.M) are added and incubated with the cells for 12 minutes at
37.degree. C. and cAMP levels are determined using the SPA assay
(Amersham Pharmacia Biotech, 800 Centennial Avenue, Pistcataway,
N.J. 08855). A decrease in forskolin stimulated cAMP levels
reflects inhibition of adenylylcyclase.
Example 6
Inhibition of P2T Production
[0326] Desiqn of RNA Molecules as Compositions of the Invention
[0327] All RNA molecules in this experiment are approximately 600
nucleotides in length, and all RNA molecules are designed to be
incapable of producing functional P2T protein. The molecules have
no cap and no poly-A sequence; the native initiation codon is not
present, and the RNA does not encode the full-length product. The
following RNA molecules are designed: [0328] (1) a single-stranded
(ss) sense RNA polynucleotide sequence homologous to a portion of
P2T murine messenger RNA (mRNA); [0329] (2) a ss anti-sense RNA
polynucleotide sequence complementary to a portion of P2T murine
mRNA, [0330] (3) a double-stranded (ds) RNA molecule comprised of
both sense and anti-sense a portion of P2T murine mRNA
polynucleotide sequences, [0331] (4) a ss sense RNA polynucleotide
sequence homologous to a portion of P2T murine heterogeneous RNA
(hnRNA), [0332] (5) a ss anti-sense RNA polynucleotide sequence
complementary to a portion of P2T murine hnRNA, [0333] (6) a ds RNA
molecule comprised of the sense and anti-sense P2T murine hnRNA
polynucleotide sequences, [0334] (7) a ss murine RNA polynucleotide
sequence homologous to the top strand of the a portion of P2T
promoter, [0335] (8) a ss murine RNA polynucleotide sequence
homologous to the bottom strand of the a portion of P2T promoter,
and [0336] (9) a ds RNA molecule comprised of murine RNA
polyriucleotide sequences homologous to the top and bottom strands
of the P2T promoter.
[0337] The various RNA molecules of (1)-(9) above may be generated
through T7 RNA polymerase transcription of PCR products bearing a
T7 promoter at one end. In the instance where a sense RNA is
desired, a T7 promoter is located at the 5' end of the forward PCR
primer. In the instance where an antisense RNA is desired, the T7
promoter is located at the 5' end of the reverse PCR primer. When
dsRNA is desired both types of PCR products may be included in the
T7 transcription reaction. Alternatively, sense and anti-sense RNA
may be mixed together after transcription, under annealing
conditions, to form ds RNA.
[0338] Construction of Expression Plasmid Encoding a Fold-Back Type
of RNA
[0339] An expression plasmid encoding an inverted repeat of a
portion of the P2T gene may be constructed using the information
disclosed in this application. A DNA fragment encoding a P2T
foldback transcript may be prepared by PCR amplification and
introduced into suitable restriction sites of a vector which
includes the elements required for transcription of the P2T
foldback transcript. The DNA fragment would encode a transcript
that contains a fragment of the P2T gene of approximately at least
600 nucleotides in length, followed by spacer sequence of at least
10 bp but not more than 200 bp, followed by the reverse complement
of the P2T sequence chosen. CHO cells transfected with the
construct will produce only fold-back RNA in which complementary
target gene sequences form a double helix.
[0340] Assay
[0341] Balb/c mice (5 mice/group) may be injected intercranially
with the murine P2T chain specific RNAs described above or with
controls at doses ranging between 10 .mu.g and 500 .mu.g. Brains
are harvested from a sample of the mice every four days for a
period of three weeks and assayed for P2T levels using the
antibodies as disclosed herein or by northern blot analysis for
reduced RNA levels.
[0342] According to the present invention, mice receiving ds RNA
molecules derived from both the P2T mRNA, P2T hnRNA and ds RNA
derived from the P2T promoter demonstrate a reduction or inhibition
in P2T production. A modest, if any, inhibitory effect is observed
in sera of mice receiving the single stranded P2T derived RNA
molecules, unless the RNA molecules have the capability of forming
some level of double-strandedness.
Example 7
Production of Ttransferic Cell Strains by Gene Targeting
[0343] Gene targeting occurs when transfecting DNA either
integrates into or partially replaces chromosomal DNA sequences
through a homologous recombinant event. While such events can occur
in the course of any given transfection experiment, they are
usually masked by a vast excess of events in which plasmid DNA
integrates by nonhomologous, or illegitimate, recombination.
[0344] Generation of a Construct Useful for Selection of Gene
Targeting Events in Human Cells
[0345] One approach to selecting the targeted events is by genetic
selection for the loss of a gene function due to the integration of
transfecting DNA. The human HPRT locus encodes the enzyme
hypoxanthine-phosphoribosyl transferase. HPRT-cells can be selected
for by growth in medium containing the nucleoside analog
6-thioguanine (6-TG): cells with the wild-type (HPRT+) allele are
killed by 6-TG, while cells with mutant (hprt-) alleles can
survive. Cells harboring targeted events which disrupt HPRT gene
function are therefore selectable in 6-TG medium.
[0346] To construct a plasmid for targeting to the HPRT locus, the
6.9 kb HindIII fragment extending from positions 11,960-18,869 in
the HPRT sequence (Genebank name HUMHPRTB; Edwards, A. et al.,
Genomics 6:593-608 (1990)) and including exons 2 and 3 of the HPRT
gene, may be subdcloned into the HindIII site of pUC12. The
resulting clone is cleaved at the unique XhoI site in exon 3 of the
HPRT gene fragment and the 1.1 kb SaII-XhoI fragment containing the
neo gene from pMC1Neo (Stratagene) is inserted, disrupting the
coding sequence of exon 3. One orientation, with the direction of
neo transcription opposite that of HPRT transcription was chosen
and designated pE3Neo. The replacement of the normal HPRT exon 3
with the neo-disrupted version will result in an HPRT-, 6-TG
resistant phenotype. Such cells will also be G418 resistant.
[0347] Generation of a Construct for Targeted Insertion of a Gene
of Therapeutic Interest into the Human Genome and its use in Gene
Targeting
[0348] A variant of pE3Neo, in which a P2T gene is inserted within
the HPRT coding region, adjacent to or near the neo gene, can be
used to target the P2T gene to a specific position in a recipient
primary or secondary cell genome. Such a variant of pE3Neo can be
constructed for targeting the P2T gene to the HPRT locus.
[0349] A DNA fragment containing the P2T gene and linked mouse
metallothionein (mMT) promoter is constructed. Separately, pE3Neo
is digested with an enzyme which cuts at the junction of the neo
fragment and HPRT exon 3 (the 3' junction of the insertion into
exon 3). Linearized pE3Neo fragment may be ligated to the P2T-mMT
fragment.
[0350] Bacterial colonies derived by transfection with the ligation
mixture are screened by restriction enzyme analysis for a single
copy insertion of the P2T-mMT fragment. An insertional mutant in
which the P2T DNA is transcribed in the same direction as the neo
gene is chosen and designated pE3Neo/P2T. pE3Neo/P2T is digested to
release a fragment containing HPRT, neo and mMT-P2T sequences.
Digested DNA is treated and transfected into primary or secondary
human fibroblasts. G418.sup.r TG.sup.r colonies are selected and
analyzed for targeted insertion of the mP2T and neo sequences into
the HPRT gene. Individual colonies may be assayed for P2T
expression using antibodies as described elsewhere herein.
[0351] Secondary human fibroblasts may be transfected with
pE3Neo/P2T and thioguanine-resistant colonies analyzed for stable
P2T expression and by restriction enzyme and Southern hybridization
analysis.
[0352] The use of homologous recombination to target a P2T gene to
a specific position in a cell's genomic DNA can be expanded upon
and made more useful for producing products for therapeutic
purposes (e.g., pharmaceuticals, gene therapy) by the insertion of
a gene through which cells containing amplified copies of the gene
can be selected for by exposure of the cells to an appropriate drug
selection regimen. For example, pE3neo/P2T can be modified by
inserting the dhfr, ada, or CAD gene at a position immediately
adjacent to the P2T or neo genes in pE3neo/P2T. Primary, secondary,
or immortalized cells are transfected with such a plasmid and
correctly targeted events are identified. These cells are further
treated with increasing concentrations of drugs appropriate for the
selection of cells containing amplified genes (for dhfr, the
selective agent is methotrexate, for CAD the selective agent is
N-(phosphonacetyl)-L-aspartate (PALA), and for ada the selective
agent is an adenine nucleoside (e.g., alanosine). In this manner
the integration of the gene of therapeutic interest will be
coamplified along with the gene for which amplified copies are
selected. Thus, the genetic engineering of cells to produce genes
for therapeutic uses can be readily controlled by preselecting the
site at which the targeting construct integrates and at which the
amplified copies reside in the amplified cells.
[0353] Construction of Targetinq Plasmids for Placing the P2T Gene
Under the Control of the Mouse Metallothionein Promoter in Primary,
Secondary and Immortalized Human Fibroblasts
[0354] The following serves to illustrate one embodiment of the
present invention, in which the normal positive and negative
regulatory sequences upstream of the P2T gene are altered to allow
expression of P2T in primary, secondary or immortalized human
fibroblasts or other cells which do not express P2T in significant
quantities.
[0355] Unique sequences of SEQ ID NO:1 are selected which are
located upstream from the P2T coding region and ligated to the
mouse metallothionein promoter as targeting sequences. Typically,
the 1.8 kb EcoRI-BgIII from the mMT-I gene (containing no mMT
coding sequences; Hamer, D. H. and Walling M., J. Mol. Appl. Gen.
1:273 288 (1982); this fragment can also be isolated by known
methods from mouse genomic DNA using PCR primers designed from
analysis of mXT sequences available from Genbank; i.e., MUSMTI,
MUSMTIP, MUSMTIPRM) is made blunt-ended by known methods and
ligated with the 5' P2T sequences. The orientations of resulting
clones are analyzed and suitable DNAs are used for targeting
primary and secondary human fibroblasts or other cells which do not
express P2T in significant quantities.
[0356] Additional upstream sequences are useful in cases where it
is desirable to modify, delete and/or replace negative regulatory
elements or enhancers that lie upstream of the initial target
sequence.
[0357] The cloning strategies described above allow sequences
upstream of P2T to be modified in vitro for subsequent targeted
transfection of primary, secondary or immortalized human
fibroblasts or other cells which do not express P2T in significant
quantities. The strategies describe simple insertions of the mMT
promoter, and allow for deletion of the negative regulatory region,
and deletion of the negative regulatory region and replacement with
an enhancer with broad host-cell activity.
[0358] Targeting to Sequences Flanking the P2T Gene and Isolation
of Targeted Primary, Secondary and Immortalized Human Fibroblasts
by Screening
[0359] Targeting fragment containing the mMT promoter and P2T
upstream sequences may be purified by phenol extraction and ethanol
precipitation and transfected into primary or secondary human
fibroblasts. Transfected cells are plated onto 150 mm dishes in
human fibroblast nutrient medium. 48 hours later the cells are
plated into 24 well dishes at a density of 10,000 cells/cm.sup.2
(approximately 20,000 cells per well) so that, if targeting occurs
at a rate of 1 event per 10.sup.6 clonable cells then about 50
wells would need to be assayed to isolate a single expressing
colony. Cells in which the transfecting DNA has targeted to the
homologous region upstream of P2T will express P2T under the
control of the mMT promoter. After 10 days, whole well supernatants
are assayed for P2T expression. Clones from wells displaying P2T
synthesis are isolated using known methods, typically by assaying
fractions of the heterogenous populations of cells separated into
individual wells or plates, assaying fractions of these positive
wells, and repeating as needed, ultimately isolating the targeted
colony by screening 96-well microtiter plates seeded at one cell
per well. DNA from entire plate lysates can also be analyzed by PCR
for amplification of a fragment using primers specific for the
targeting sequences. Positive plates are trypsinized and replated
at successively lower dilutions, and the DNA preparation and PCR
steps repeated as needed to isolate targeted cells.
[0360] Targeting to Sequences Flanking the Human P2T Gene and
Isolation of Targeted Primary, Secondary and Immortalized Human
Fibroblasts by a Positive or a Combined Positive/Negative Selection
System
[0361] Construction of 5' P2T-mMT targeting sequences and
derivatives of such with additional upstream sequences can include
the additional step of inserting the neo gene adjacent to the mMT
promoter. In addition, a negative selection marker, for example,
gpt (from PMSG (Pharmacia) or another suitable source), can be
inserted. In the former case, G418.sup.r colonies are isolated and
screened by PCR amplification or restriction enzyme and Southern
hybridization analysis of DNA prepared from pools of colonies to
identify targeted colonies. In the latter case, G418.sup.r colonies
are placed in medium containing 6-thioxanthine to select against
the integration of the gpt gene (Besnard, C. et al., Mol. Cell.
Biol. 7:4139-4141 (1987)). In addition, the HSV-TK gene can be
placed on the opposite side of the insert to gpt, allowing
selection for neo and against both gpt and TK by growing cells in
human fibroblast nutrient medium containing 400 .mu.g/ml G418, 100
.mu.M 6-thioxanthine, and 25 .mu.g/ml gancyclovir. The double
negative selection should provide a nearly absolute selection for
true targeted events and Southern blot analysis provides an
ultimate confirmation.
[0362] The targeting schemes herein described can also be used to
activate P2T expression in immortalized human cells (for example,
HT1080 fibroblasts, HeLa cells, MCF-7 breast cancer cells, K-562
leukemia cells, KB carcinoma cells or 2780AD ovarian carcinoma
cells) for the purposes of producing P2T for conventional
pharmaceutical delivery.
[0363] The targeting constructs described and used in this example
can be modified to include an amplifiable selectable marker (e.g.,
ada, dhfr, or CAD) which is useful for selecting cells in which the
activated endogenous gene, and the amplifiable selectable marker,
are amplified. Such cells, expressing or capable of expressing the
endogenous gene encoding a P2T product can be used to produce
proteins for conventional pharmaceutical delivery or for gene
therapy.
[0364] Equivalents: Those skilled in the art will recognize, or be
able to ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
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Sequence CWU 1
1
10 1 46358 DNA Mus musculus misc_feature (7632)..(7651) unsure
misc_feature (11838)..(11885) unsure 1 aaaagtgcat ctacttcccc
ctaagggcag ggattccctc caccatctcc attgctgcag 60 tgccaatggc
tactcaggct ggcctttctg ttattttaaa gaaccttcca aagcgttgta 120
tagaaagcaa atgcccatct aatactgctt cttagagact aggtagtgtt gtaacacaag
180 ttttactaaa tactgcttac tcagcaaatg aaaattctta tcagcacaca
ttttcttata 240 taaacatagc ttgtttccta tcctgatgta aaataataat
tttattttac ttttatttct 300 tatgctaagc tgttacttaa gtttttggta
gattctagaa atgccctgaa gtccagataa 360 gcattttaat tgggagatct
ttgaagataa tggctttttt ctttttcttt ttgcaatgcc 420 aatagctatt
gtgctttgat gacaaaaatt ataccactga aaattaagaa aagtcacttg 480
gcttctataa ctagatatat ttatatatgt gggaaaaaac atttactctt gcctgttttt
540 tatcaataga gtcaaatcca gcaattttgt ttccctttgt atcgatcaaa
gaacccatgg 600 gttcacacgt gatgacatcg caggtctgct ttggcaaagg
cagcaactgt cgaactttgt 660 caatgctttc tgacctcttg tctcctagtt
ctttctagaa gaaagagggg ggaaaaatgt 720 aaaagcaaga gagaagtgag
gcaaaaattc tgactttaca atgaagcaaa agctaacaag 780 aaggaagttc
cccagtgaca catttccttt tttttagggc tagtatgtct ttagtcctta 840
gcgcagtcta tggaacagct gcaaattgga ctggtgaaat gtttcatttc ttctattcaa
900 gtagaaaatc agggtgcaca caaaggcagt cttgttctcc atgacaaatc
agagtgctga 960 aatgacaaaa cgccagtgtc atttgctgtc acacagcagg
agctgccgca cggacacttt 1020 cccgtatcca gggtcacagt gcaaggtaac
catcagcttt tttactgtaa cgtcttcaga 1080 ttatgtattt gcaatgtcta
tttgccgttc atgtccagga ctgttagtgg atttacaaat 1140 tcaatgcaga
gctcattttt ttttttccca cttgcatgtg taaatccaag catgggttca 1200
aaggaaatct gacactggag ttttggttag atatctgata atgcattttt aaaatcctta
1260 aaaattaaaa cacattttat ttgagcacac cttttcagag aaaaaaatcg
ttcaaatatc 1320 atgcttgtaa ttttgaagga ctgacagagt aggtaagata
acaaataacc acaatctgct 1380 gaatcagcta cacatgtaga ttaatgagga
ggaaattaag acaataatcc ataaacaaca 1440 tgatttcaga gtagaggctg
atcacaagct tactttcagt ttctttacta agttcatgta 1500 tgcacgtttg
ttctcttctg atcccccggg ggaagcactt gataggtcag aagccagggt 1560
tccattgatt aaaacaccca gcatgtcaag aaccgttgtg aataattcac tgagaagaaa
1620 gaaaacccca ttttattctc acaaacaaaa cccggacaca cagaattgcc
taagcatttc 1680 tactgaaagt gctaagtttt taatgacagc taaatcagta
agtggcagaa tttcctgtca 1740 gtagacaaaa gcttgtacca tgcatgcatg
caggggaaaa aaaaaggtat aaaatgataa 1800 aaaacaaaat aaaacaaaac
aaaaacccaa catacttgtt agtgtgcatg tcaacagttc 1860 ccgaagtaat
tatctggagg agtaggaggg cccagtcggt ggtcccctgc gtgctccgct 1920
gtaccgtgtc aaacattcct cctacctagc atacaaaata tgataaagat tttgtattgc
1980 tacccctatt accatatgtg cccaaatgtt gatttgttaa accagaagtg
atgttaagaa 2040 ctggttcttg ctagccacac tatggattat cctacaactg
tctcttctag ctatgtggtt 2100 atcactgttt atggttcttt ccacagaggc
taggcattca aaacttaaca gaaagtgtaa 2160 cacttgaatg tgggcagtca
caaaattacc tttctcttct catcagtctc tcatggtact 2220 tagttttaga
cataactgca gttaaacctg aatagcatca aaactctgtc tcatcacctg 2280
caattgtgtg atattcgatt attattgcat ctgagcctgc cactataata tccctggaga
2340 tgccctttaa cgtggaagga atgaggagga gaaggtattg ccacaggtct
gccagctcct 2400 gggaggaggt cagagtgctg aaggtgaggt gaacaactgc
ccagggaggt cagattgcca 2460 aaagccagaa aagcaattgc ccagggcacg
ttcaggattt gtcaagagga aatttaaggg 2520 aatacaaaac atgctgaaac
tttcagattt ctgtaaaata ttcttgtgag atttgtcttt 2580 tttttttctg
atcaatttaa cagaatgtct ctgagaaaaa ttgagagttg acagatttct 2640
gtggttgtaa aatagaacac agtgaaagcc aacactccta ttaaaggttt gtcggacctc
2700 cccttagcac tgcacaggag gaggaggaga gatctgccag acggagcctc
tactccctgg 2760 tggaaaacac aaataggtgc ttacaactct actgtctaga
attcaaccca gacaggaagg 2820 acggcagcta aggaaacaga aggtttaaga
gaaaaggaag taggtataga gtgagaaaga 2880 ggaaggcttc cctctgtcat
gcagattctg tccacagatg ttgaaagctg gtattacttt 2940 cacacagaga
gaggtgtgac agaaatgggt ctgccacatc cttaccagat tcaggcggag 3000
ctgtagggcc tcatgcatca tctggcgggc tttcgtatca tcttggtatc tttcttccct
3060 ccagttactt aaaatctaag attgggaaca aacaaagaat aacaacagaa
atccctttgt 3120 tctttgctat gacgactttt acaggtttaa aatgaagctc
tacagtaaca gctaacactt 3180 cttagcttaa acattaacaa agttattttt
tttttttact cttcctcttc ttccagacag 3240 acaaaatccc agttatttta
taatttcctg ggtagctggg agaagaaagt cttacacaca 3300 cacacacaca
cacacacaca cacacacaca cggaaaaagg ccacacagaa atgcctatgg 3360
caaggacaaa gggaggcaag gtgaactgag actcgcagaa aggcagagca ggtctgctta
3420 acatctgaca tggatccttc caggcaacaa aagaaggaag tgctcaggca
cctaaaccag 3480 gacactggcc cagcccagag cggcttgaag gaaaggataa
acccactgga agagacagca 3540 aatgcccaat caggagtggc tctatcagtt
cagccctggc atttgtagtc taggtatgaa 3600 gagaaccctg agtgctttgt
atttaaatgg ctccctctgg aagctagaca gagaaaagac 3660 ctgtccaaag
tgtgggtgtg gtgacaaagg aaaccgagta agcctggtgc cattgtgcat 3720
gttatcagct ctcatttaat gccaccgggc agggttcacc tggctttgtg tcaggataat
3780 ccaactcaca aaaccaatct gacatctttc cttgattcct tgcttctcgg
taggcgcaac 3840 agagatcggt aagctagctg ctaagcagtg tgcaacaact
tacttttata gtctttacat 3900 atctctcaat gcataattag catcagcttt
gctggccttc tggtgaccat gatgctaggc 3960 agaatgggac atggactgtg
tttcttagca tctcagcgac agaacattaa tctaatgtca 4020 cattaggaca
ctatcatgaa atcacaaaag ggaaataatg aataccatca gaaagatact 4080
ggccgagttt agtactccac gctttacctg attgacttga ttctgcagag atgttagaag
4140 gccttcccgt tgctcatctt gccccttaag gcaggtaagg accaatgaga
ggaaaggctg 4200 ctggctcaaa agagacatac tgcaaaggaa gtattaacag
agctagtcag aaaaaatggc 4260 ttgcccctgc tgcaacagct gctctttcta
gtagactcac tcactgctct tgtttattta 4320 cgactacatg aaggacaggg
tgggaaacca ccagagaaca tttgggaagg tgacaccttc 4380 ctgtactggc
aagattaaaa accactggta aagcttttaa atgacaattc tctattgctc 4440
tggaaggaaa tcccaaagca acagaaacgt gcagggtatg tgatgagctt gctttctgtt
4500 actaggggga agtttttaaa tatacaggac aaagaatata aatgtatgca
gagaaaaaca 4560 aaacaaagaa caaacaaaaa ataccggaga gattgctcag
agaaaactct tccttaggaa 4620 gcggctctct gtggaccttg actatttccg
aggcctgtgt ttcttggttg tatatttctc 4680 aaaggccaac agcagagtgt
tggtaaagtt aggggggagc acaacaaggt ctgaggcctc 4740 caaaaagcaa
gatacttgct aggaacaccg tgtgccaggc cccgacactg cccatggcct 4800
ccgctagaaa gggggcaaaa ttctatttac aatgttcacg ttaacaagat atctcccatt
4860 agagaacatt ggtcaaactg attgatatag tgaaaagaaa attttcctaa
agaacaggtc 4920 tatgccagaa tgtctgaatc aggagccctg taggtagcca
ataaagaaca ctcagttgtg 4980 gtagtattat tagctctatc tttaagttta
tagaatgaag aaattatgac ctttctcttt 5040 aaaacttctt gggtttcaaa
tgtatagtat gaatattcat cctcaatacg agatgatctg 5100 tttttcaaaa
acaatgatgt gaaaccattt cttgggcatg aaataatgaa atagttttgg 5160
cagttgttat ggctagtttt aatcaaaaat ctacaacaag gttcctaaat taagaaaatg
5220 aaaatcagta attatttcaa ggtctagcag gtcagaatta acaacaagcc
tgtgtacgtc 5280 aatagctttg cagtgatttg gacacacagg cagggatggg
acctggtgcc ctatacagat 5340 ccttgctaaa cagagagttg gccgttctac
aagccccacc ccgtggagct ggctgcgatg 5400 tgactttgat ggaggcttct
aggcttcctc cgggtccttc ggatgccagc cttgacgcga 5460 ggcacctgcc
agcgtacctt ttctgcttct gcctatctct ttccttcttt gaagaggaac 5520
ccaggtgctg ccccttctcc agctcttccc cagcggcttt caacactctt ccctgcacag
5580 atgttggcag cctggcgatg aggggggcca ccagccacac acccctgcgt
tcagaggaac 5640 taaaatcaga aacaggcgtt acacccgaga acgcagctgc
ctctgctctc acagacgatg 5700 cagatgccac agtcaaacac cggtgcacaa
ggcacagact agagttctca tttcatgtct 5760 gcactttctc cttttgctaa
agaccaggca tttgcttacg ctttataccc acatctgctg 5820 aggagtaaaa
ggtgctcttc tgggaaggca ttttttgcat tgtaaactgt tttcagtgac 5880
tcatggatcc tgcctttctg accatgaaac accatatgtt ctctctatga acactatacg
5940 tttcagtcat ggtgatgata tgcaatggga ttgtgttcaa gctaaggaat
aagcgatctt 6000 taaattctaa aaccccctat ttacctttta ccaaaatgat
gacatgaaaa ccaatctgtc 6060 tttcatgatg aaatacaaat tactgtattt
gtacaaaact atcattaaag acacatagat 6120 agtatgttat gtttagtgaa
tgacccaaag acagagatac cacacatcaa acttaagtcc 6180 atctgtgtca
ctgaggtacc tggacctaca acaaaaaatg taacgtgcca agacatcact 6240
caccccactt ttccctccta cctggctgag taacagaaac tgtcacttgt aagttattat
6300 ttcccttaca tttcctgaag cttaataaaa aatgaattat gctaatcaag
gctattgtga 6360 catatgaaat atcaacggta gcccaacaaa actataaata
ccttaagaat gtctttattc 6420 cattctgtct tgtgctactt ggatcaacac
ttccaatagt gtttgggttg aagaggctca 6480 tgccagaatt ggaggcatta
ttatttaggt ctgcagattg ctggaacact tctatcgttg 6540 cctttgcgat
attgtccagc aaattattca tttcggccac agaaccagag ctctagtatc 6600
aggaaaaaga aagctaactt gtctacacac ttcttgattt tcaagcacca atgacattct
6660 gcttttctat tcagttcaca cgtgtgctgt caatatatac tcacagggtc
cttcaagcac 6720 tgtttgatca tgagctggag ctccagccag gactgtctca
acgtccactg ctcaagattc 6780 tggagaaaat tcgaagaatt gtcacatttt
gaatactgaa tgtatgctaa cacagcctta 6840 cagtaattaa tactcatacc
cagcaaggaa actcacaagc cttaaagact tccaatccca 6900 tgcctgataa
aaatgaacat gcacaacaaa gtagattcaa gcatcaaaat acgttcacca 6960
acaagccagg aagtagaaat cagacaagta tggcttctct cagaatccat ttaccaatta
7020 gccaacctca ctccaaaatg atgaatctct taaataacac atttaaaatt
gagctatcat 7080 aaatactatg tatcaagtaa atataccact gacttaaaat
acaatatatg ccctttgcta 7140 ttgactgtat ctactgagta gccagcagtg
gcaatgtcac tgtaaagaaa attacagaat 7200 aaaacaaaag tcagaattaa
aatcaaggtc aggagaaagt gggcagcctt gtctagtccc 7260 tgattttagt
gggattgctt ccagcttctc tccatttact ttgatgttgg ctactggttt 7320
gctgtaaatt gcttttatca tgtttaggta tgggccttga attcctgatc tttccaacaa
7380 tggatggatc acagggctcc caatggagga gctagagaaa gtagccaagg
agctaaaggg 7440 atctgcaacc ctataggcgg aacaacatta tgaactaacc
agtaccccgg agctcttgac 7500 tctagctgca tatgtatcaa aagatggcct
agtcggccat cactggaaag agaggcccat 7560 tggacatgca aactttatat
gccccagtac aggggaacac cagggccaaa aagggggagt 7620 gggtgggtag
gnnnnnnnnn nnnnnnnnnn nacggggtcc agtatcagtt ctttgtctgt 7680
acacagtcgt tctggttctt ttaagcagtg ttctcccacc cattcctaaa aacaagccgg
7740 cagttagact ggcacacaat gattctacct ggctgttgat tttattctca
tttctcaggg 7800 taaaatgata aaaggctagt aaagacaatc taatgaaatg
aaattaatga aaaaaatggg 7860 tggagaaaag ctggcatgta gatatatgct
ggttcttttg ttattgttct atttttcttt 7920 gagacaaggt ctcaggtatt
gcaggacggc ctctaagtca ctatgtggtc aagaataacc 7980 ttgaactact
gatccttctg ctctgtccag tcctgggata acaggcctaa atttaatgcc 8040
atgtcttcct gcctaccaga gctccctgca tgttcagcaa gcactcttta tcaaataagc
8100 cacattgcca gctaatggaa aaatttcaaa acaaaattat aacacctgac
atctttaaaa 8160 aaaaatccaa ggaggggagg gcttgagtta aaccagaaac
cacttaaatc tgtgggacag 8220 agttaaaatt tgggattttt aaaaaataag
gacttactct aaggtactat tttgtaacca 8280 ggtttttgtt tatttgtttg
ctttgactta atatttaggg acaagaattt catgctttat 8340 ataactttca
taattcttat gtgatatatt ctgctaaaca ggtaaagtgt ataaatactt 8400
agaaggttac ttcttttgac actaacatct ttctttagca ttttttcttt tattaagtgg
8460 cattaagaaa ctatcagtat cagagctcaa tctgaaccca aatctgtttc
agatgaagta 8520 agtctccctt tacactgtgt cagctgctca gctccgggta
tttactgaag ttacataatc 8580 ccatgttcta tattagctgc attaaatcaa
cctcagacca aggcacccag atgcagtgtg 8640 ttgcttttct aggtatcaca
ttattattta ctggcaaact gattaatgtt ttttgttttt 8700 gttttgtttg
gggttttttt ttgttttttt ggttttgttt ttgtttttgt ttattggata 8760
ctggcactgt agggcaggcc agaggcttcc catagggctt gcagcagagc ctcttgggac
8820 actaaacctg gccagctcca tctggttcca ggcatctaga gaaccataca
cagtccagtg 8880 gcaccactgt gtgctgacag ttggggactg atccacaatg
gccatcaata gctgtaatgt 8940 cagttaaaca ccacagtgaa aacaaatgta
aggaaccgtg cacttaatgc acgctacaaa 9000 aatacgtaat atacagcatt
tttaaaataa aagatttctg aaagtatgga aagataatca 9060 gaagatactg
aagctagtct tcggagcaga gccagatagg gagaaggcaa caggtgagga 9120
caggttattg tgtgcacttt tatgagagct tttgaaggta tgctatgata caaacattag
9180 atgagaccca aaacagacaa aacaaacaaa caaaataaaa caaagtaaat
aaaagtgatt 9240 ctaattttaa agaaagaata tgttcaccat aagtgagaca
actgtaaaaa acatttttct 9300 ttatttttcc cttcctgttt ctccaaataa
tgtttgaatg cattatcgta aacccttcag 9360 ataaatgaac cctcagtgct
ctcagagccc cttttcctaa tggtgcatgt ataacaactc 9420 tgaaaccatg
ctagctgctg tatgggggac aaggcaagaa tgtaattact aatatgaagc 9480
gagagtatgt aaaaatacag ctttaaaata ttagattaga actagaagta tcaatatgaa
9540 tttataatca cacacatgca cgcatatgta cacatatgct cacacacgca
catgtatgca 9600 aaggctcaca catgtgcacg cacagctctg tctgctggga
gagttcaaaa acagtgatac 9660 tcgatagcag tggggattta taacccagac
cttgctttcc tcttttagtg tgatgcggcc 9720 tgtcatgtta cacattgcat
gcgtgcctgt atgcatgtgt gcatacatac tgaacatgat 9780 ggataatgcc
catgtgtgaa tgtgtgtaga ggacagacat tgatgtcagg tatcttctta 9840
gatccttgtc caccttatac accaagaaag catctcttag atgaactcag agcttaccta
9900 tttagtcatt ctctctagcc tgcttaccct aggatcctgt atctccattt
ctgagttctg 9960 ggattacagg tgggctgcca cagcctccca gtgcatacat
gaatataggg atctcgattc 10020 taggctttat gctagcacag caagcatgtc
accattgatc tgtccctcag ccctcagact 10080 ctgctttctg agtaccagtc
tcttcacagg cagaaatggc actcctcaga atggctgatt 10140 ataactatga
ggcaaggaat atggggggac gggtaaagta aggcaatagt tccctaaagg 10200
atagaaatat tccaattaca acaaaaggta ggatgacaga ttgctaatat gtcagcttga
10260 acgagctgga cattaaagaa gctataggca atgtgttata tatccacata
gataactaaa 10320 taaataaata caaatgaaca acaacataag aagagtattt
gtagagcata aactgatcat 10380 gtggaggaac aagaggaact cagaagaaaa
acacaacatt ttgcagccgt catagtaaag 10440 actgagttag acaagaatca
caagggattc ttatgaggaa tggggtgtgt aacaaggatc 10500 agaatatttg
catcatctca gagcatcttt ctgaaggtta ctagttaact gagagcaaag 10560
catactaaca ctctagtgga tcctgaatct ttagatgcct tcatcaaagc taacattacc
10620 tactggtgaa gagggcaaat gtgtgcaatc agtagtggtc acctaaataa
tatagtttca 10680 gctctaaact ctccccgact ccaatgacct gaagttaatc
tctagaccct tatgcaactc 10740 taacaacact agaataaatc agaaaatccc
ctagagatgg ggaaaagatt attgcaccaa 10800 agtttagatt atgattttta
ttaactaaac tttggttgta acaagaaatg agaatgtaag 10860 tattcagaag
taaagcagag aaccaaaaag agatgggaca gatactacca tctacttgtc 10920
aactgttgag aaaaatacaa aatgtccaga attagtgaat ctgtataaag gatacactaa
10980 aagtctccat tatcacagct ctttaacaaa tttgaaatta ttggggaaaa
aaggggggga 11040 atacttaaat acaataataa aaaaaatagt ggatagttgc
ctccccaaaa tcaggtacat 11100 attcaaagta gaagccattt gaaattggat
aagtacctgc tggcagatag tcctcagtac 11160 gtatctagca tattctctga
gattggcagt ttctatggaa atacttttcc catacgattt 11220 tgagttctgc
gaggtggccc aggcatcttc agcattccca tcacgtcgta aacccctcat 11280
gccgaagtct tcattcttca tcgtgttgac attgttactg ccaattttgg catcgcctaa
11340 atagcagaat cacaaaatca aagtcacaga cattcaaaat attgtcccaa
atatgtaata 11400 agcaaattag gctaggtttc agggactcga gaggtgtgaa
aattgtcact tctacatagc 11460 cagttgtcca atgcgtagat caagaaatac
ataaaaggaa acaaaaccat tggatatgga 11520 gaaaagtcaa agtcaaggtt
cactccttct tttggtttac atgatttaat gctcttttgc 11580 tagctttgaa
agagccatta cggatgacac ttaataaccc aaacacattg cgttcaccaa 11640
aacttcaggc atgcgtgtac tcaagtttat ctgagttata gacaagaaaa tctacagcat
11700 ccagcgtaga cctgacccga ccaaactcta atacactgag taagatgaag
ctaagggagg 11760 tatttgtctc cccaaaacac tatgcaaagc aggagcatac
ttggatgaaa taaaaatctc 11820 aaaaaaaaaa aaaaagtnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11880 nnnnnacaag caaacaacac
ccaaccaaaa caaacaaaca aacaaacaaa caaacacaac 11940 ttaaaactca
aggccaatcc ccaaaaggtt ttgactctgt tgaaaaaaaa aaaacaaaaa 12000
acaaaaaaca aaaccccaaa aactcttttc ttgggaggaa gagtaatcaa aatgtcctaa
12060 taaatgtcta ctctcaaaaa tcttagaagc agaggggcta aggctgtcac
tcagtggcag 12120 agtacttgtc taccatgcct gagaccttag gttctctcct
cagcaccatc agtaccacca 12180 cacattaact aagcagaaaa accatagggg
taaaaataag caagacatct caacttacca 12240 atgtgttttc atacactcat
catgtacctt aaaatataaa acctagtagc tgtacccatg 12300 agagtttatt
tatttcaaca gcacttttca aaattagcag acacgtgtat tctctacact 12360
tagatgatgt aaatatctat ctctatttca agttaaaata ctatgcaaat taggagcaaa
12420 gttcacgata atagtcctat tttcctgtct taatatatat ttggtagcta
agagttggga 12480 aaaaattggt taaaagcact agagtggata aggcaagagc
aaaaattagc tcatttctta 12540 gaccccttgt agacctccat ttactttgga
tatggggtac tgaagtctgt gtaactatgt 12600 tagaacctcc gcaattgtgc
agaaaaaaaa ccagaatatt ctatgtgaat ctatgtgaat 12660 ctgtctaagg
cttcactggc atataactgt gctgattata atttttcaga ccaagaacct 12720
cccacattat ttgaatatct gaaaagtctg gcaactttac ttgggattag tccagttctg
12780 caaacaaagg gctcactgtc tagatgagac tcatcttgtg ggtgccactg
aaacatgtgg 12840 tgggcattca tacttccaac ataagtttat tgaaaatata
tttcttactt tctttgatga 12900 gacttaaaaa tatctaaaga taaatctaat
tcctgtcttc atgaccccaa tgattactaa 12960 tcagtaaagt ggaccagctc
tccttgccct ggctgaaatc agcagtgagt attggctgta 13020 taatatttat
ggccctaagc ccattcttcc cccttcctag atctctctcc tattttccta 13080
accagccatt tccatgatcc ttctcaaaaa aaaaaaaaaa aatccaaaac caaggaaaga
13140 aaatctaaac aacataatca ataaaataac acaaacaaac aaacaaaagc
agaatacact 13200 ttgtgttggc taaccattct ggggcatggg gcttgctctg
gagtgtggct gatataaata 13260 acattgtatt ggagaaaact gaaaagattt
gtatattcat tttcatgtat attcttttac 13320 atttattttt aaaaggctgc
tttataaaca ctgctattat tctttgccat gcacaaatac 13380 catttgtcaa
gcaaggcaag gctatatttt tatctgtgtt atctaaggct cacctaaatg 13440
gaaacctgtc tatcctgtaa ttctgtttaa atcatgaact actttataac atgatgtggc
13500 aactattgtt tggttatggc caaaatgagg tacccaaagt taaaagtagg
caatattgga 13560 gttctcctaa ttcaaatgct ttatggttta tataaaaata
tcctgaggtg tcaccctgcc 13620 gatgaacaga aggcagcact attactcttg
ttaaatgtgt accagtaaga atgtctaggg 13680 aatcttgggc actgtgaatt
agaggaaaaa cctgtgcccc tcctgctgaa catcagcaat 13740 acaagcacca
ttgcagctcc atttgcttaa taaaaacaat atggcagaga atacaaactt 13800
gttcagagtc acagcagatg gagaaacacg aaattaaaat ccagcttttc tttgtttttg
13860 cacctggaat ataatctggc tgaaacataa agcttttatt aaagatatga
agtacaattt 13920 ccgtcagtaa ataaaggcaa acgatcctgt gggaatgagg
acctgattta catcacaatg 13980 aagaagcaca caattataac caacacgcag
tacttctatt acctttctcc cagggtcacc 14040 gtagttaaga gacagaatcc
ttgaagtgcc cattacttgg gcttagggaa agccaaccaa 14100 tttttttccc
ccaggacaaa caagcaaaca aaagatatgt ttccattaac attgcttaga 14160
atttacagtg caattgcata tgccagcaag attttgctga agggaccctg tgatagctgt
14220 ctcctgtgag gctatgccag ttcctggcaa atacagaagt ggatgctcac
agtcatctat 14280 aggatggaac acagggcccc caatggagaa gctagagaaa
gcacccaagg agcttaggga 14340 tctgcaaccc tataggtgga acaacaatat
gaactaacca gtacccccag agctcgtatc 14400 tctagctgca tatgtagcag
aagatggcct agtcagccat cactgggaag agaggcccct 14460 gggtcttgca
aacgttatat gccccagtac aggggaaagc cagggccaag aagcaggagt 14520
aggtgggtag ggaagcagga caggggagct tttaggatag catttgaaat gtatataatg
14580 aaaacatcta aaaaaaaatc tcatagctta aaaaaattac agtgcatata
aattattttt 14640 gcctccactt ccggtgatta gccaagaagc taaaggccag
gatagtctta ccaagcatca 14700 taattgcctt taagacagca aaaactgctc
ccacttctat gctgttgtga gctgcagcta 14760 agaggtgtct atcacaagat
gatcttattc cggggaaagg tttgcctata aaaacaacaa 14820 caaaccttca
gtggcctctt cattgccatt aaccaccttc tgcaatcatt cacagtcagt 14880
aagacttttc attggttgtt tgtgggcact taattgacat gtttgcatta cctgatgccc
14940 cacagaaaca ctgccagtat tcttacttaa
cctttcctct ttaattatta ttaaaacaac 15000 gttcctattc ctcaaagaga
tgtggtccaa agattagcag gattcccaca cagatgcatc 15060 catctctata
tggaaatgat ttagcaccag acttcactaa taccctccat tgcctcagga 15120
aagataaata aatgagttgt catccgctta agctgagatg gcatggctca gaatcaacac
15180 agggtataca agagactgag acttcccacc agcacactat ttcctaacag
cccaggcatt 15240 tttattgttt attaattcct catttttatt tcccaatata
tccgcatatt acatcatgga 15300 aggtgccgac agcccttcaa tggcatgatc
tcactcctcc gtcatgcgca gctcaagccc 15360 atgaaaatca gggaggaaat
gacaaactgg acagctcact tggtcacagt agaaccaaaa 15420 gtgcccaata
aaccccaaag cagagctttc tcttgaggcc agctcacctg ttccctgagg 15480
gaaaaagcag gcctgggggg ctctgaagag gtgaagcagg aggcggcacg tcatccttgc
15540 cccgggctct gcatccgcgt ctccacaagc tggaaagagg aggaggcttt
aaaacaagct 15600 tcctcacaaa ctccacaaca agcttttcgg gtttgctact
actaacgttt ctgtaataca 15660 ttaaataacc cttctgttta gtaaggccat
tagttatatc gttctgaaga accttcatgc 15720 tgccttacac cccgaataaa
gcaatggcag tttcctgtcc cagagttcag tgtcactctg 15780 cataaaacag
ctactacagc tctaccatct cttgcaggta caatgccatc ccacaatgca 15840
cacatcctta aaaatggccc tttccctgtt cttaagaggt aacaggtgac taccaaccag
15900 gaaacaatag cagacgcagc tcaggaccag cttacctgct gctaggagag
agggaagtgc 15960 gacgtgttgc acgacatctt ccagggagaa acactgtcgt
gctatcagga tagcaatgaa 16020 agtagctaat gaatcatgga atgaaaggtc
actcaccttt aagaaaaaca ttgacaggtt 16080 ttaatacccg gcatccttta
gtgcaatatc aaagacctaa gagaatacca gcgataccac 16140 taagatctca
aacctccata tgaaccatca gagggaaaag acttgcgatt atcttcttag 16200
gcctattgct actatgataa gacaaagcac gtttactcat attctagtgt tctaggtaga
16260 tgttggcctt taacctgtca gttgcatgtg catgcaactg agagaagaca
acacaagagc 16320 aaaattgaga tgctaataaa aggtgtatca taactgatgc
ttgccaatgg aatacatacg 16380 ccatttatgg gaaaggcagg aaggagaagg
tgaagcctct agcaggaaaa caggacccac 16440 taacacctga accttctttc
ctgtcacaac taatgcattt tttttttaat gagagaggat 16500 tcatttaaaa
gaactctgaa agttttcaat tggctgctat atttaaaaat gaaaaatgag 16560
agaaaggaag gggagaaaaa ttagttcagc tacagggaaa tctctacatt ccagatctca
16620 caaaatatta gaagaaaata ctaagagaac acaattacca caatagcaat
cttcctagtg 16680 actggtacta ggtgcagcaa gtcacaaata tgtatttcag
aaaaaaaata acttaggtct 16740 ttgttggtgt ggaaaagcag cctattatta
atgtattttg cacaaataat atcttaatcc 16800 acggaatgtg gataaggagg
caggcctgtc cattcagcac tgaagcagca gaaagaggaa 16860 ataagctttc
agcattacaa ttgtcaagtt tctttaataa catatagagc tctgacaaag 16920
atcaccccaa gcaatgataa aggcataaat ggatagagaa tctacagaaa tgtttaagga
16980 acgaatatat atctctatct ctatctctat ctctatctct atctctatct
ctatctctat 17040 ctctatctct atctctatct ctatctctat ctctatctct
atcatgggtg caacatggaa 17100 gacgaggaag tcaaggaaca tgatttgggc
aattattcca tcactctaca ggcatagcct 17160 gagaatactg aatatcctga
ggctgcacgt ggattttctt atgcaaaaca acaaaatctg 17220 tacacttaga
cttaagtgta tggtggttct cctcatgcat gaaacgtttt atgtactgtg 17280
gtgagggatc cacgctgcaa aaaaacgaag cagctgagat agtttaaaac cttagcttct
17340 agaaatcagg caacagggaa cacactggaa ttccttctgt tacgtctatt
ttgttttgtt 17400 ttaatttatt gaatttgtgg tattgaacat cacttttgga
aatagagaaa tatgttgctg 17460 tgttagagtt tagttaatta agcagtaaaa
gatgggtaat agtaaattag taattgataa 17520 gttaagaaaa cttacatcca
ccgtgcagag gacatcatta aatccccaca catgatttga 17580 agaacaacaa
agagccttca gaacccccag ccactccgaa ctcagaactg tacagcaggc 17640
agttagctca gaggagaaat tggctatgtc atttatccta ggggggaaaa aaaaagaaga
17700 tagcagaaag tgaactggaa caaataagaa ctgattacac ctaaccccca
agaaactgga 17760 ggccccaggg agtttagagg tctattggga tggaggtgtg
gggtgtggag gaggtatagg 17820 gtgtgaaaca gtcggagggt ggaccaggag
gggaagaaaa tctgaagttt ttttttaaaa 17880 aaagatttaa taaaaagaag
gaatatatga aaaaaacaaa atgattacac atggtttgtt 17940 cactgaatac
atacttcatt atcattaagc atcacttaga tttcaggata taataccctc 18000
ttgtatttat acaaggtata attatacaca gcaacatata attataagaa atgaattagc
18060 atatttttga ttctctgaat aaaccttgtc tacaatttta cattttatag
ggatcaaaag 18120 ttaaggacac aagcttgggg atattctatg agaaagatcc
tggaatcagt cggtaataac 18180 accaggataa atagttaaag acattatgtg
atgtaactag gcccaagttt atatcaaaag 18240 atagtgctgt ctttaaaatt
agatatgctt ttcaaaatgg accaccaata aaaaatgtct 18300 acagccaaac
caagaacccc aaagccatga tttctcaggc taagtgttta gttaggagag 18360
agtggggtag aatcagaaat tactaaagaa actccaaagc agctcgccct tgatagctgc
18420 tgcactcagg gaaaagcagc tcaccggccg gcatcctggt gtcccatgca
cacattcatg 18480 agggtgttgc acacaaagct gtaccggttg gcagcattgt
cattgaggat cttgcccagc 18540 attgaataat tgatgcttct tgccgagggg
ttctcaataa aatccatcat aaagtctgga 18600 tcccagcgta agttggaatt
tgcaggcatc acattattgt atatggtctg ctttactttt 18660 gaacaggcac
tactaaggtt agaaaacgac agttaaaaaa ggaaaaaaaa aaaaaaagaa 18720
caaagcaacc aagaaaatca cttcacttac agacatgcta agtggcaact actctaagac
18780 tttcctagac catctatatc ctactattac taatagcaaa atctttgtaa
accagcattg 18840 gtacaatatc tgagatagag taattgctta atgaagagat
ttttaattta tagatggaaa 18900 caaaaatcaa tgtatttact gagccttcta
acaacttttt ctttcttaag agtagttgta 18960 aaatgttata acctaaaaat
aaaaatgggt ccaattacac acaaggaaaa gttcagtgtt 19020 ttaactccga
ctgtaagccc taaaccctat ttctttgggt gtagtatgcc cagtaatagc 19080
cactgtgcca aagctactgt gagctctaag ctacagctgg gacaaatgga ggtaaaactc
19140 cggcatctgt gtagctcatg ctctcctagg ggaatacaga tgatacattc
tatagtgtga 19200 agaaacttac aacatgccaa atggagaggg ctaagttccc
acctagctgg gcatgtgccc 19260 tgaacaagga agcaaacatg ttttaggaaa
tgagtgtttt cagtgggcaa catctgaaca 19320 gatacggaat actggaaata
agagaaatta gaatgtattc tccaatgttt ctctgcagac 19380 aagacgagca
atggtgtgat cccaagcttt tgattccata tgagctccgt tctttgtgtg 19440
ctctagctgt gtacagtaaa tccacaagca gaaatccatt tcctgctcaa gagctttact
19500 atgtagtaca attttttgga ccaaaatcct ggcaggtgaa ggttctgtca
ttggggtgac 19560 acataaatct cctctattaa actgcagaag cgtttcttct
tttccttaag ttttgagatc 19620 tactccactc tgctgtcaaa ttcagacttc
tctggagaac ctctctggcg ggcatccgag 19680 cttctctaac ctctgagtct
ctgcaccttt cctttctaga ctctttctct tctttaccct 19740 acttcccaag
aggagcctct ctgttttgat aagatgctgg ctgccactga gaatgtgttc 19800
tcattcttcc cactctctgc tgcccacgcg tggggaattg agccaaaaat tcaggcatgc
19860 tccttctgta actgtgtgtc tcaggaaaac ctgatagagt aatgaaagca
acgcatgcct 19920 tgaggtcaga aaaacttgtt ttaaatctga gcttcatcag
tatgtcaaaa ataatcagga 19980 acaatcattt gtagctaaag aaactcactc
ttcttcccag gataacgaca attcaaaact 20040 ttaatggaaa aactatcaca
gttttgtggc acgcagaagc tcaagcagca ggagatgtga 20100 ttagtgtgac
ctgtccctca aatttcctcc ctacacaaag ttacagggtt ttagttaaaa 20160
ctgttacaat gtttaattgc tattgtacta accaaaacct cactgaacat gtagcttctc
20220 tgtctatgtt tttcctaacc tccaaactgg cctctgactg tacactatgc
ccagagagct 20280 gcactccttt tccgtttcct gttgaggact gtgcattcat
tatctgtgat gcctctgctc 20340 ctggtgttag gagcacctta gtccactggt
aatcacttaa aaataaatcc tgtgctctta 20400 atgaactcac ttagaccacc
tggtacttac aattgtaaga ataatagggg ctgcccacca 20460 ctaaaaccag
ggctgttgca aaaaagccta tctttgagaa gcgtaaactg aaaactcaaa 20520
atgaagcaat agaaggaaat gaagtatttt tattgttact tcattcaaat attttacaaa
20580 gggctatttt aaatcaaatg gtaacatttt gtcagtttta tttattaatg
ccactcatat 20640 tttaccaaca gcagtgcaac agtgttaaca gtagtgtaga
taaattgctt ttgtgtaaaa 20700 tacaatatta ttgggataaa agagaaaaat
ataagctctt aggagatttg catcccatag 20760 aggagccaaa ctaatatata
tatgtgtgtg tatgtatgta tatatatata tatatattat 20820 ggagagagag
agagagagag agagagagag agagagagag agagagagag agagagagaa 20880
ggctagccaa tactgaggtt tgcagaatga ctgactgaga atgggttgtc actgaacaca
20940 aggctgccag caagcaaggc cacagacact gtgtaacaag gcataaggac
tacacaaagg 21000 aagagagcca gagcagcagc aagtagtcag tgctccgggt
gtgaggacca gaagtgggtt 21060 tctggagaca gaagagaata cactaaagga
aaggaacaca ggagagatgg aagccaacac 21120 atctcaatgt ccatcagagc
tgtgagtttg atgctgccta gatgacaacc atgggtgcct 21180 cacaatcatc
cctggttgaa agagtcacct cagcagatgt gctgagaata tgctatagtg 21240
agtctgagta aaggcaatga agccaatgag aaggactctg caagttggtc caagcaagag
21300 atgcctatga tggccgtgac tggggtattg aagagaggtc aaatgtgatc
tatacatgga 21360 agtcagagct catggtattt ctaagcaaac tgaacacaga
gcaaggagga gcaaagaaaa 21420 ccattactga catatcttgt ggatttcaat
accctatgcc acattatttc ctcctgtaac 21480 ttcccaacag gcacttaata
tctggcacac acaatgctaa gcaacagatg aaaggaactg 21540 gagactctac
aagactttac agacaaaaaa gtagagacac aaatgaaaca agcttgcaaa 21600
aatctctgaa agaatattca aagtgaggtg aaagcaaagg caaaatacag aatgtttgta
21660 ctcaagaaac tgtaaccata gttacacaaa gagcgatgtg cactaacacg
aggctctaac 21720 ttctgcaaaa attaatttgg tataagctct gagtaagagt
tctgagaaga taaaagtaca 21780 aagcttctaa cactaaaaac gtttggactt
gttttaaaaa tgttccccaa gtaacacaat 21840 taatgtacag gagtaaaatt
cttggcagga gaatggctgt catctaaagg cttactaaca 21900 agccggtcag
ttgtccataa tcaacacacg tactcttgca tcatgccgca aacataggga 21960
tttatcacag tgtaactcag ttgagtaaaa acaggccatg caaataactg cttttaattc
22020 gtgttggtta aacggtattt agagaagtgc tagccttcca tttaaagctc
attcatgcat 22080 gggcaaatga tttgctaaag actaaactga ttttaaaaag
agaagaaata aattttaaaa 22140 taccaggttg ggaggtggta taaaattacg
gatatagagc cttaaagaat cactaagaag 22200 ggggaagatt tgcaactttg
tgacccagaa agagtgtgga agccaatgta gcaggaaaag 22260 cctgctaaaa
aggcaagatg ggaacaaaga ggacatgaca caaaagcatt atgagacttt 22320
caaattttaa attatatttt aaatcaaaat ataagcatat gctagatttt tatcatgact
22380 tcaatatggc tcatctagaa cttctaaaag aatgtgaaat cttaaataag
agaccctttc 22440 acaggtgttg tacagcttgc cacctaaaat cactgttatg
tacatacgaa aaaaaaatct 22500 tgtactatta tcattaagta ttttagtatc
tgtctaaatg agtctcgggt agatcaaaca 22560 cttcagattg gcttacatca
gtacatagaa tataaaccac aatcagacca ttaacaaacg 22620 gaagtgtgat
catgatggaa gtataatcat gtctgtaggt ttttttgttt tttcgttttt 22680
tgttttttta ctttttcctg ctcacctaaa aaggtctcca aatttacttc tgaggtggct
22740 gcaggatacg tagaggtcat agaggtaggc caagatacat ctctctgggg
aagaacattc 22800 tgaggggttg acaacgtgct tcaccacacc acacaacctg
aggacagaga cacaggaata 22860 aaaaagtcgt gtcttgatag tatgtgaata
gtgagtaatg aattctttaa tgtctaccca 22920 gcacttagaa gtatgtatca
aacataaaaa gaaactcgat tgctcataaa actatgaagt 22980 tattaaatat
caatttttgt tacactgaat actagaaaag ctttaaaaaa ccctcagaaa 23040
catcacttaa aagaaacata tgtaatgaaa tgaattctcc acactgcaat taacactcaa
23100 aactccatca cttaggctga tttgactaga aaaaaaaaag aaaaggctag
gcagagaagt 23160 ttaagtcaac acaaagtttc acaacttcca ttagttgaca
tggactgcca agttcactag 23220 tttggaaaga agtaactctt cagtgcagga
aatctgctga gtttgatgaa gccgtggttt 23280 ggaatcccat tcctcttctt
tgtactacag acaaatttat cttggcttct tatgtacgaa 23340 gatgaaaatc
cattccctgt agaggcatcc tttataataa tgccagcctt tcaagagtac 23400
caacaccaca gccactttct tcccttccaa accatggctc ctctgtccat acaagatggc
23460 catcagtatg gcatagaaga caacacctgt ggcccatcca tacatgtgtc
tgactccacc 23520 accaccctgt atccgacaca ggactccaca taacatgtca
cccatagtgt caccaatggc 23580 tgagactctg aaaagagttt aataatattc
ttccatctta ccaccatcac tgcacaaaga 23640 aggctattag gtgacctgaa
cagagaaagg aaaggggagg ctgggtgcat agtggaccac 23700 tctccattat
tcagaagata aggaagatca agaggtggta gtctgcaatg ttcaggccca 23760
ctagggacag gcattctcca gcatcagaca tggatgccag gaaaataagg aaccgcagtg
23820 caatatccca ggtagtctga aggatgtcac ctgatgggga acttggtaat
tataaaggag 23880 gacaacatgc tgtttgtctg ggttacctca ctcgggatga
tttgttgttg ttattattcc 23940 atccatttgc ctgcaaattt tatgatgtca
tttttttttc taacagcgaa ctaatacttt 24000 attgtataaa tgtaccatat
tttctttatc tagtttttgg ttaagggaca tctaggttat 24060 ttccagtttc
tggctattat gaagaaagcc acaataaaca tagttgagca agtgtccatg 24120
tggtaggagg cagcatcttt tgagcatatg tccagaagtg gttcttgagg tagattctga
24180 gaaacaacca cagtgacttc aatagtggct gtacaagttg gtgctcccac
cagcaatgga 24240 ggagtgctcc ccttgctcca catcctcacc agcatgagct
gtcacttgtg gggtttttgt 24300 ttgtttgttt gtttgtttgt ttgttaccat
agtgattctg acagtataag atagaatctc 24360 tagatcattt tgatttgtat
ttccctgatg actaaggatg ttgaactttt tttttctcat 24420 ttgatatttt
tttttcattt gatattcctc tgttgaaagt tctgtttaga actgtacccc 24480
atttttaaat tggataattt ggtttgttga tgtttagttt cttgagttct tttatatatt
24540 ttagatgtaa gctctcggtt agatagagtt ggtggatatc ttttcctatt
ctgtagactg 24600 ctgttttgtc ctattgaggt gtgtactcac tcataagtgg
atattagctg taaagtaaaa 24660 gacaatcacg ctacaatcca cagacccaga
gaggctgagt aacaaggaaa gacacgtgga 24720 cctccctgcg cagaggaaac
agaagatttt gcaagggact gaggggagat gggcatggga 24780 acaggaggga
tcaggtgtgg gatggagtac taggagagag tactgggaaa gataactatg 24840
gggggggcat ttgaggggca aggtggaaat atagtgcaat gaaaactccc tggaatctac
24900 aagggagacc ctagcgaaga ctcctagtaa taggagattt ggagcctgaa
ctggctatct 24960 tccgcaacca ggcaaggcgt ccagaggcag gactgggaca
ccgatccagc cacaaatcgt 25020 ccacctacaa tttgtcttgc ttacaagatg
tcctggggta aaggtgacac agaacttttg 25080 ggagtggcca accagtgact
ggtccagctt aagagccact ctatgagaag cccacacctg 25140 aactgcctgg
gtggccaaga accagaggct agatagccca gagacctagg acagaaccaa 25200
acacaactgg aaaaaagtag ggaatgaaat gattcctaag gataattctg ctattgatcg
25260 gagcctagca taactgtcat cagagaggat tcacccggca actaatgggg
ccagatacag 25320 agacccatag ccaaagatta ggaagaacac ggggaatcac
gcagaagaag aggagggaga 25380 aaggacagga gaagatagag gggtcaaacc
attgcataca ctagcaagat tttattgaaa 25440 ggacccagat gtagctgtct
cttgtgagac tatgccgggg cctagaaaac acagaagtgg 25500 atgatcacag
tcagctaatg gatggatcat agggctccca atggaggagc tagagaaagt 25560
agccaaggag ctaaagggat ctgcaaccct ataggtggat caacattatg aactaaccag
25620 taccccggag ctcttgactc tagctgcata tatatcaaaa gatggcctag
tcggccatca 25680 ctggaaagag aggcccattg gacttgcaaa ctttatatgc
cccagtacag gggaacacca 25740 gggccaaaaa gggggagtgg gtgggcaggg
gagtgggggt gggtggatat gggggacttt 25800 tggtatagca ttggaaatgt
aaatgagcta aatacctaat aaaaaatgga aaaaaaaaaa 25860 gatagaggga
tcaagcacac cgcaagaaca tggcctacat aatcaactaa gcagtgctca 25920
tgggggctga cagagactga accagcaatt aagcaacctg gatgagtcgg atctaggtcc
25980 cctgcatata tgttacggtg tgtaactagg tgttctggta ggattcctta
tagcaggagt 26040 gcatgctatc tctgactctt ttgcctgctt ttgggaccca
ttttctccca ctgagttaac 26100 cttgtccgct ttgatataag ggatgtgcct
agttgtgttg taacttgatc atttgatatc 26160 cctgggaggc ctgctctttt
ctgaaggaaa atggaaaagg agtggatttg gagggaaggg 26220 cctgataaag
gggataactg ggaagagttg agggagggaa aaaaaataaa aagatttttt 26280
aaatggaatg ttttttaaaa aggggggggg gcaacaggta cagacaatgg aaccatcgtt
26340 taagagggaa gcaacacctg aatctgctgg gtatgcagaa gcaaacaaat
cccaacagag 26400 catggaaagt aaagacttat cctgcgacta aagtaaacaa
atgcaaatgg gtaggtgcgt 26460 ccatcttaca gtagttttca ctttctaaga
ctaaagcctt ggactgtggt ctgatactct 26520 cagtcacatt aaggggggga
ttgtgatttt gaaaaaaaaa aagtcatacg gtgtgtcaat 26580 agttaagaca
gctatctatg tgacacaacc tgagtcactt gggaatgacg ggggggaggg 26640
ggggggcgga cagacgtgct ttatttctgc ttcacaaggg gagggtccag tttcctgtag
26700 cctgtgccat ctctaggcag gcagacctgg gttgtatagg atagctagct
gaccaaggca 26760 aagggagcat gccagtaagt tcctgcatcc agagtttttc
ctcgagctct ggtcctggtg 26820 tgcttcaatg atacaactac aggctataaa
ctgaaaaaca aaacaaaaca aaaaaacaaa 26880 aaacaaaaaa acaacctttg
ataaagttga atttggtcac agcgtttacc gtagcaacaa 26940 cgagcaaact
agggcattat aaataaggta aaaatataac aaagttaagc caaatataat 27000
tttatgagta tttaagttaa tattttaaat gttaaaatat taaaacaatt tcaaactaac
27060 aatcaaagaa caaccaaaca aaaacacttt ttccaactaa gtatgtatga
gttcatgatc 27120 agtgaatgtg ttgttaaatg aattattatt ttaaaattga
atgattttaa aagaatagtg 27180 ctgaaaaaac agtaaggagt caacttcaat
ttgcaacata tactagctgt gtaatgaata 27240 tggggcacat cacttccaaa
ctcttcacct gtcgaacgat ataaaagagc gaggcctcac 27300 ataaatacac
accttctctc actgtaaagc ttttgaggct tgtaatattc tacttcttaa 27360
gttagatgta catgtaaatc aaagaaaagc ctctgcaaag tagatgccac tagagaaata
27420 ctgtttataa attaaatgca tgtgtaaggt tacagagggg atgggtgaaa
accaagaaga 27480 caaatgagga gacagaggaa taaagaatag gttgtctttt
ttttttttta ataaaaggcc 27540 ctagaaatgc aggtgaggag caggcaacac
gatgtttgca cagagctccc tgcatttccc 27600 tggcctttta agcacagcct
cttgcagaca gttccttagc ccgactttat gagcgacagc 27660 agaagctttt
gtccgttaga cactcagacg aacaactttc atgagtcaga aagaagatgg 27720
ccaagtttct acagtgcaaa gaatccttgc agtgttgcaa ggactatgta gtgtctgatt
27780 gttttttcaa aagtacactg aaaatagatg accagatccc agtattcact
gctcgcagta 27840 agttgttggg cggcataaat gagaagagag ctgtgcatga
ccaggaggtg aagaggaagc 27900 cagtaagtaa gtagaaaaaa aaaaaaaatc
aaacacactt cctccattta tatctctttc 27960 tctacaaaat attttggtaa
gaccattgtt gggatgtttt cattgaaact ttaataagac 28020 atctaaaact
tcaaaatgag tggtatcaaa acttcagacc aacagtctaa acaaaaatac 28080
ttttttagaa ctcagtataa tttttctccc aaatgcactg tatttcacta tgaattctac
28140 cttttaggat aagaaaaaca aacaaacaaa caaacaaaca aacaaacaaa
aaacaaaaac 28200 attgctactt atatacccac ccttcaaaca cctgggctgt
ctgatcagga tttaggatca 28260 ggcaactatg gtaccgcctg agcacggcca
caatgcagac acatagtcct gtagtgtaac 28320 ttcctgccag gctagaagac
ttcaagagga gctctgcttc caccacactc agttcattca 28380 gcagctgcag
cagaagtggg gttggggggg gggtgaaggg agggttggag gggagggaag 28440
aagacaaaca gggaaaccgt tcagtgaacc cctagtgcac atttcctata cgctacggaa
28500 tcagaaagca cacaagcttt tgctggcatt tacttttaag cctttactaa
tttaaacgaa 28560 tgtttttctg tctagacacc agcatgcacc caaccaggat
gtagtcagtg ctgaaacagt 28620 caccaaaact ttctgaaagc ccagagcctg
gaactaagcg tttaataaac aaccccattc 28680 tttatttaat tttttttcct
ttttttcttt ttttcttttt cattttggtt gttgtgcttc 28740 tttttttttt
tttcctttca atgcaacaca actgataact ataataacta cattcttatt 28800
tataaagctt ggagtaactg tacaatagaa aaaaatcaaa cctaatgtct atttattaac
28860 acaaggaata aatgatctaa aatgtgtgta actacagtgg gacaaatgaa
attataaaga 28920 gtattttaac aatcatttgt gcttttcact gcttaactct
actagagaga atatatcatt 28980 tccttattcc caacgcaaaa taataaggta
acaacaaccg acaccaaaac aaaaacatct 29040 ttcacgtata catagtttta
aataagttgt gtattactaa caccaactat agcttaacag 29100 taaaactatt
aactcaaaat gtctcaaaca ctttaaaatg tactaggtgc ttaaaatagt 29160
tttgttttat ttgttttgtt ttagggattt tgttttgttt tgttttttgg ttttgttttt
29220 ttcgagacag ggtttctctg tgtagccctg gctgtcctgg aactcactct
gtagaccagg 29280 ctggcctcaa actcagaaat ctgcctgcct cggcctccca
agtgctggga ttaaaggcac 29340 gtgccaccac tgcccagctt aaatagtttt
aatgtaacag aactggcaca tgttcaaagg 29400 catgaagttc caagaggcca
aagagagttg ggacagagaa gtgattaggg aaagttaatc 29460 agtggggact
aagttcttac aggagcaaga tatcttcacg tgtaatatgt atggcagggc 29520
ggctataggt aacaggatgc actgtaccta gcaaacgtag ggaaaaggac tcttgacagt
29580 ttctaccatc aagaaatgat gtttgaagaa taggtatgtt taatttgcta
taagaatata 29640 cataacatat acatgtttta tggcatcata tggtatttca
taaaactaca tttttgcata 29700 tttatgcaag tttaaaaaat aagaggcttg
agagatgact cagtagttaa gtaccactag 29760 ctgctctttc agaggaccca
tgttcagtac ccaacacccc caagatgtct cataagtatc 29820 cgtaacgccc
ttccaaggga gccaatgccc tcttttgaac tctgcaggca ccaggcacac 29880
acatagtgca catctgtagg caagacaccc atgcacataa aataaatcta aaaatatttc
29940 atataaatgt aagccaggtc acaagcttgt agccttaggt acttgggggc
tgagtagggt 30000 ggtcacttca gatggaaagt tcagacaagc
tcaacagtaa gggatagcct ggtttttaaa 30060 tcaacaaata aagagtatga
agaagcaaga taaactctag tgtctggttg gtttgctttc 30120 ctaaatgtga
gaaaggcaca gtatagtctc tgtggcacct gtatggcaaa gtcaatcagc 30180
ccattgatgt tcagcgctgg ctccatgaga tcaaagatga gctgaatatg gtgagccaac
30240 gggaggtgat aggatgttcc agacgcaaag cttgtgattt gttccagcac
attgttagag 30300 atctgctcag acaaaagcaa ggaaaatcac acaaccaaca
gtctgggtga ggcttcactt 30360 cgatatatca cttcgattgt tttcgtttta
ggtttttttc cagcaacaca tatgaattca 30420 tgtgattcta cttagaaatt
agtatttccg tggaagtcaa aacatataca aatcagcatt 30480 cttacaaaca
tcatttgaaa atttcaatat aatctagata ctggtgaccc caaagggaaa 30540
tcactcaaaa gtgtgagtca tcagaaactg acatgttaac tgaataacaa tggatttctt
30600 ctaaatttcc cagtatttta attaaatcat caaaaagttt ctccttcacc
aatagtgatg 30660 ctgggtcagc atgttatata ataagtatat taggaattac
ctttatgaat taataattac 30720 tataaagcaa aaatacttac ataataaaaa
caggtgttga aggaaacacc taatactcac 30780 aaatgaagct tgttctgctt
ccaagtctat aagggtcatt tgtaaaagtt aaggctggaa 30840 gtacactgct
tgagctttta agggaggaat attcaaagca tgtgacaaac agccctgaat 30900
ggatacctga gatgttactt gatgctgatc gaagtatgag aggagttgga gtttcgtgaa
30960 cacattttcc ggggttggaa aaacttcctg cttggtcttc ctggctttct
gtccttcgtc 31020 cccaactaaa gaaacatcag atgtattact ttccatttaa
attaatctct tcatatttct 31080 aaagattttt ttgatgtgtg tgtgtgtgtg
tgtgtgtgtg tatgtgtgtg catgttcact 31140 tgtttgtaca cgtgtgtgga
ggccagaggg aaatacctgg tgtcatcctt caggcaatca 31200 caccattgga
gtttaccctc tctgatggta ctctacaaca gagtctctta ctggccaaaa 31260
ctcaccaagt gagcttgcct ggctgtttaa ggaacacaag gggtctgcct atatccatac
31320 ctccacagca atgggattac aaccctgctc catcatgccc agctattttt
atgtgggact 31380 tggaacgtta ctgagttacc tccccatccc atctttatat
gggtattttt tactgaagta 31440 tggcacaata atagatgaaa gcaatatata
ctctatgcat tttgacaaac agaatgagat 31500 ccaaatcgtg aaatagtacc
tcaaaagtcc acctacctga ctctaatctc ctctccctcc 31560 ccaaggggcc
aaaggtttgg acagcttcca tcgtgggtgg gtggaaactt tcactcaaca 31620
ctatgtttta agatagcctc attatttagg taattttaat gtgtactaat tgttagtaat
31680 tttcacatac tttattccaa tgcataaaaa tatgccacta cttattcttg
caaataatga 31740 cttgattctg gctattataa agtgtctttg caagcattca
tcattaagta actttaaaga 31800 tatagtaatg aatcaccacg gttgggtggg
gaacaccaag gcccactgtg actaaacagg 31860 atacattaca ggctttttaa
aaaaatatgg attatttatt tattttgtat tcatatgcac 31920 tcatcaggga
acatttcggt aaaccaagaa aaatttaagg gtgttctctc ctccagacat 31980
caaattcaga ggatgaggct tggtagcaag atcctttgcc ttctgagcca tttcactgac
32040 ttttagactc acctctgtac ttttataaat cagtaaatat atgtatattt
ttgcttattt 32100 gaggaaaact ggttttctta acaactaaga gcaaaattaa
agtctctgga aacaggattt 32160 catctatgta tatacaaaga caaattttta
aatgaaagta atggttgggt tacagtactt 32220 ctacagcact gtgcgccatc
tggttagagg gagcactcca actcagtttc atgtgggaat 32280 tgttagctca
gaaaacaaaa tgtttcttat tgctctagaa gttggcaaga ctccaagctc 32340
ctccacatca aaagtggacc tccctttgaa cagtaagtga gatacaaaat ccaagtgagt
32400 tgtcacgttt ctgctttgta caatggatgg tgataccaga atgaacagag
tagtaaaaag 32460 agctggtact gggcagggga gatagcacgg ttagcaaatg
gcttgccctg caagcatgag 32520 gctctgagtg cggaatccag aacccatgtt
ttaaaaaaaa aaaaaaaaaa aaaaaagcta 32580 ggcgtagtga caccggcctg
cgatcacagc ccctgggagg cagagatggg acaagtcatg 32640 cttcgctgag
ttacaagcca ataaaagacc ctgtttcaaa gtacaaacaa cacagtggag 32700
gacactagaa gaatgacatt tgagtttgtt ttctgaccct ccatgctcac atacattaag
32760 gaagtgctgg taaaacacat gacaaaaact tccttctgtc attagacctc
cttctcttcc 32820 agcatctatt ctcataagca caagttaaac gtgggatgag
gccttctctc tcactgctct 32880 tatatgtatc ataagtatag ttgagatacc
caagttttga cttagttttc aattccctag 32940 aatcattgct ccaaaactga
aagggtccaa cctactactg tcttgtgttc atattaatca 33000 tttagtctcc
cttggatcga aataaacaca gaagacttgg ggaagtaggc ctagctcaga 33060
actcccaagt gcattttcct ttgctcattt gtataaacag gttccattac tgaacatgta
33120 aggtcataaa agctcaactt gatataactg ggtcttgtaa cctagaaagg
aaaccactga 33180 taactaatga tgctttgaaa tgaaagattt cagtgtcgtt
tgcataagta acaatacact 33240 ggggatataa agaggttttg ttttgtattg
ttttattctt ttgcccgttt ccttgttttc 33300 ctggcatgca ccactgctct
ttcaatactt aatttttaca taaaaaaatt aagcctttaa 33360 attctgaaag
tattggcctt tttagacaat gttatagtga agttgacatg aaatttggct 33420
gtttaactgg aaatagtgtt taagctttaa gttaggggag cagaggttgc tcaggcagca
33480 gactgtctgc ttagcatcac acaggcctga ggtcaaccct tagcagcaca
taaacaggca 33540 tggtggtggg agcccataat cctagcacat gggagacaga
agcaggggga agagactttc 33600 gggttcatcc ctcaaattcc agcctgcctg
gaaaacagaa gactccatct caaaaagaga 33660 caaaacataa caacagactt
ccagttacaa atcccctagg ggaagactgg ctggttctgt 33720 aagtgtccta
aagggttctt caaaagtcta atttttgttt tctcaacatt aaagcacatg 33780
ctagtccaag ggatagtttc cttttttcct ttccaatctt cacattgcta agtaaagtag
33840 ttgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt atacaagttt
cctgtagcct 33900 caaatacagt tggctaactt cttgtataca aagtcttaaa
taataactta aaatgaagac 33960 tgtttaccaa gtggctgtgc actgtggctg
agattaaatc gaggaagatg tcagtttgaa 34020 gtggtgtttg tgtgactaat
actttgaaag cccgcacagt gaatatctct tttgtagtcc 34080 atacggaaga
atatctactt aaggggttgg cacaaagaca aaacaagact agtttggcat 34140
taaagcaaaa ctgtgaagga aggaaggaag gaaggaagct tgacagcaag actgcttctt
34200 tccccttccc acttcctttg gtctttctcc accataaact tcagaaatct
gcaaaggccc 34260 tctacactaa tttagatgaa ttaagcgtga ctgtgcctct
tatagacatc agcacctccc 34320 accgcattcc aactcccccg aaagctactt
caaatgccct tggctaggga cttgtgagcc 34380 ttccatattc aggcttctat
cgcaacttat ttcctgtttt ctctgaggga gaaaaaaaat 34440 agcacaagga
gagttttctt agacagagca gtttcttttt caagctcaat gagaagaagc 34500
caacagaaga cagtttcatt tccctttctc ggctcttggt tgcttcttgt ggcgctggca
34560 tattgaaggt cctaaacagg caagggaagg gagccagggg tggagacaga
tcacatgagg 34620 gcaacttccc cacagtgttc tccaaagccc atccagcaga
cagcctgcgc tcaggtaggc 34680 ttacccatgc tgatggcaga caaaagttac
aattgtttcc acctgtaggg aaaaaatcta 34740 gcatacacaa acaagcccta
ataatgcacc ccattaacgt cataaaaatg gaaaggttcc 34800 taggggtttc
tactcaagta ggaaaaccag ctgtgaacat agggcaaacg caaccctgtc 34860
ctccttcata ggaatgacca tgctagagag ggagcgagca gtggcagggc taaggtagaa
34920 aatgatgcca tgttttaaac catgattatg aaatgacgta aaccttaaag
agagtaacta 34980 ggcaaatatg ataatagata atagataatt ataggacttc
aattatgcaa cacaatgacg 35040 cactatgagg gccaagctct gaaagaaaca
acaacaacaa caacaaaaac atgtgagaag 35100 aacagggaaa gaaaattcca
agtcaaaaag atcaaattgc ctctttcact tttctgtggt 35160 gacagactta
gccatggcta ctatggcaat taggttttgg ctaatgtgga atcagcatct 35220
atatgatgac ctgccactca tgtgaaacac agagcaaaaa caaatggtgc aaaggaaagg
35280 tgaactggct gtttgcaggc agagtatttc tcctcctcgt cactcagaac
atcacccaca 35340 gcaatgacca tagaaagctt ggcacccaga tgacaaagta
caaatctcac ctgtatctcc 35400 ctacaaaagg cggtcaatgc gaatgctact
aataatgaac gcaaggcctt agagtgtaaa 35460 tgtgcctatt catatttcag
tgtgacagtc attcatgaga ggaaacactg gggacagtga 35520 catggagcgc
tctgataagg cctgtgggca gagggagcac aaacgcaatg ggagccaggg 35580
cactgaggcg ccttaccccc tgattctgtg gtgcccttct tgttcaggat tttgaggata
35640 tctttggtaa tcttcttcag ctgatgcctg gcttcatcac gctcttttcc
gactccatag 35700 agaaggattg tgcgctggtt acattcatga cttgaagatt
catcctgaaa aacagcaaca 35760 gcccctgaaa ccagtttgca gagctggggg
tacattttct tctcctgcac cacactaatc 35820 acatcctttc catgcctgct
tgcaggggtg gcatctacct gcttggcaac tcaccttcac 35880 cggcaccacc
cctgtttttc cagtttggaa catgctaaag taaaataata acgtgctacc 35940
cgacctgaag cacttgatgt gggcgtaccc tacagagtaa gttctggctg ttcaaagact
36000 ttttgttgtc gctgtcgtct aaatttgacc atggcataag atgatgttac
tatgtcctta 36060 aggaaaatgc catttcctta gtgagcttgg tcttagatat
atggcattag catgtgtttt 36120 ataattagga aaaaaaatat cttgctagga
ccttcaagca tcttcagaaa aaaaatgtac 36180 gacacttacg tcaacatgac
cgtataaggt atattagtat ttatttccag atgacaaaaa 36240 gcaaagtgtt
gggatcacta ggcgtgagct aagtggctag gaattcagaa tcggagatga 36300
tcgagtgagg ttttagacct cggtctcctc taatgatcac tgcaaatgca agcactcctt
36360 tttttccttg tgtgaaaata aatgacctga gtaatgggca ggaatccaac
acagaactcc 36420 atttaccttt agccaaatgt aagtatccaa ggcagagcct
cactcttatg actgccttaa 36480 atctctcagt gctaatctct ccctgggatc
ttttgttttc cattttctga gcaaagttat 36540 ttacttttct gttggacctt
tcaatatacc tccccctttg tcattctttg tataaagccc 36600 tctaaaaggc
atcacctctt atcctaaaga actcctccca gggaaaatgg attgcatatg 36660
ttcaccatta ctgatggctg caactataga acgaaccctt cagaagtggg ggagccacag
36720 tgacactcac tggtagaact ctgtgtgtcc tgtgaacaca gttatcagta
ccatccttct 36780 gcagctgagg aagcaccatg tccactctcc aattctgtca
ggttttatat ttccttgaag 36840 tacacacttg ccttcaggcc ccaactcagt
taccctagtt ttattttttt atatatatac 36900 agtgctagaa ataatacaca
tatttgcata tcctgttgac ttatctttct ctacccctct 36960 gctaattctt
catgttccaa gtatttacag aggcctacgg agatcctaac agggatggtg 37020
agagataaaa gatccatgta ccaataacaa ctcaagacag ccccatccca atagtacacc
37080 atacttaggg aagaaggagc tttgtaattt atcctagtca tgacattcaa
caaccccata 37140 ctcctgagta ataataagcc tagtaccttg cctcagatcc
ctctcctgaa aacagtgtga 37200 tgccaccttc catttaagga tctgtgagaa
taaacaagac ataacaatac acgcaatgcc 37260 caagagtact gtgtacatac
agggtgctca ctcttcacat tatagactcc cattcttcca 37320 catgcatcca
gagtccacac tgttttgcat tagccaatac ctcccataac ccactatgcc 37380
tttcctgcca tctaagatgc atgatttcaa tggctgacat ctgtctacag gtgcagtgtt
37440 atggccccat caatgtccac agcacatggg gattaggaaa ggaaagaaga
gtttgcagca 37500 ttcatcagac acaattcagt gattttctag gttattggag
aaggggtggc aggcatcaat 37560 ggctatcttc cctgagtcct ctatttattc
tcttttgtaa gcacaaagtt ggcaagcaca 37620 gcaagcacaa aggaagccag
tagactaagg aacaaactca tggacagaag acagagggga 37680 agaaaagaag
acaaaaatca ttgttttata ggaaacaatt aggttgctaa agtgctgaga 37740
agactgaaga agtgagagac aggtagacag ctcatgtctg tgtagtgcat taagtcttag
37800 ttgtgaggat gaggaactga aagcagaatt tcagaggcac ttggggaaga
gaagagtcag 37860 acattagggt ccaatgagtg aacatgcatc tctctgaatt
tcactgatcc actttgaaaa 37920 agttgttgga gaaaaaccat gaaaatgttt
agtttttttt ttaaaaggaa aatgtatagt 37980 taatatgtat ttagtgtgat
gtagaaccaa tgcaggtcag aagtgaggca gtaagggaac 38040 actgagttct
gagggaagtg caaaccatcc acatcctaca agaacagtag gtaggtagct 38100
aaggtagtgg ggaatacaag caagccatta aatgatgctt atgcgtgctt cttcatttac
38160 taggatgctc taaccataca aagcctagaa attaggcagg gagacacagt
aataggaacc 38220 atcaaccaat gatccaatag gtaatagagg taaggagtca
gtttagatgg gatgtctttg 38280 ggttttgtag tttgtggttt ttcttttgat
ttgtttgttt tcataaaaac acctgaatgt 38340 ctcaaaagag gagtttctcc
agactagttg tgaagatagc acaacaggaa gtgatggggt 38400 gaagtaacgg
ctactatgac aggagtaagg aacagggggt ggcagagagt ttactagaag 38460
ctgggcatga agtacaaatg aagggaaaag agaaccttga cttgggtaat aagcaggaaa
38520 tccagtttca aagatatgac acagataaag aagtctccat agaggacatc
acgtttgcag 38580 agcagattac tggtaagagc tgggacacaa tggagtattg
tttcgattaa caggagagac 38640 aaggacactg ctttacccaa ccacagggaa
tgggggagaa caaactaagg aaaatagaag 38700 tatggagatg aagaagtgag
aaacttcaaa ttctaattag cctactgatg tccttgggca 38760 actgagccag
gaagcacagc cctgatggag agagatgaaa ttgagccact ccagatcagc 38820
atgcaatgaa aagggaggga tttgccaggg agaagaccat actgacttaa tatcaggaaa
38880 atagtagtaa cgattactgg ctttccacca gcaagggaga gacattttga
agttcccagc 38940 aggctaaatg attcatttat gtctattttt ttgtacaaat
agaaaagtgt acaaaacctc 39000 aaactgaatt gatcagataa gcagctcaac
ttacaattat atattaaata cttgcaaatg 39060 tggcagtgga tttctactat
ttcatcttag catccccata ctgcattctt tgagtaacat 39120 taatgatctg
agaccagaac caaaaattat cctagtatct gtttcctgaa gagtatattc 39180
actgggcata aaaggaccaa ccgaaccttc tacagaagtc caagtcttta tttttaaggt
39240 aaatgctgag agtttaccat gtctctgtta actgagcttt gataagaaca
tcctccagat 39300 caatgacttc attctctagg cattacatca tgctcacagg
gacctatctg gctatgaagc 39360 tagaaggctg aaccttgaga atcacagcac
tctcacccac catagaaagt ctgacagagt 39420 cttctcgggt agacatcctt
aggaggaaat aatagagctg gccaaattaa ggtcttctct 39480 aagtcacctt
gatttcttta aaataaatgt cgataataac tgccaaaata tacatatttg 39540
tggtgtgtgt gtgtgtgtgt gtgtgtgtct cacattgtgt gataataggg ttgagaccaa
39600 cagaaagatg tgttctgctt cccctggtcc tactttctat atatattttt
atttataaag 39660 agattctatg agcaaagaat attagaatta gtggttatgg
ataagtcttt gaaaaatatt 39720 ctgagtctgt tcctttctct gggacactta
ggacagatga gttattgcaa ttatcctcat 39780 aacacactac gtagaagtcg
catcttaaga ttgtatttct gaatattcaa gtgggagccc 39840 accatgaacc
acagtaaata cgaacaaaga attagggaca gagagtaaag cacagcatta 39900
gagcatctgt caagcatctg agaagatctg ggttcaattc cctgtactac atgagtaatg
39960 tatacattat attcatgaat gaacaaaaaa gcaaacatct acatcttcaa
atataaatta 40020 ttaaaacaaa ttattaaaaa cttgccattg aacatgacct
ctttcggctg aatctcacta 40080 taaccacact aatttgttca agaaataact
caaatgtttt attcgtagtt agccttgtgc 40140 tggaagagtg aatgagtaca
cagaaggata atggctgtac cataggacaa tgtaactgtc 40200 agcttctttt
tattggtaac tgttcctaaa tggtagtgcc ctttctcata aatcaagaaa 40260
gcagataaca tatttttgaa ggaaatacct gttctccagt agcagagtct gtgatacact
40320 atttctattc tttccagcaa agacaatgtt ggctctttct aaatattccc
aatgcccaag 40380 atgacttatt tttgaaagct attgtttata taacctaaat
tactatgatc aataaagcta 40440 agcaacacta atatatgtcc tcaataaaaa
aaatcattga tataaaaata aaagtcatga 40500 caattgcaag atcccaactg
ataatcaaat gtccaattag ctcttcattc tccctctgaa 40560 acctagattt
tcaaggacca catgcacttc aaggcatggc ctctttggct tagagtaagt 40620
aagctatgag ttatgtatga gctgtaagtt gtaagcataa gtaagtcata tgccatgtat
40680 gagttgaata gatttcagat gaacaggaag tattgcttat cttcaaggga
tttgtcaaat 40740 aattaaccat ttaaaataaa aattctcctc aattttccaa
attatcttgg ttagaaatga 40800 tcaaattaaa tttcatttcc ctataataca
gttataaacc gtatgtgatg atgctgttta 40860 ctgatggggg aagatggtgt
gtccgactaa ttatgctata tgataatgga gttttaggat 40920 gagtcacttt
ctatctaaag ctttcagttg ctttcatggt ggtcaaaggg gaaattggca 40980
attgtctaca aggagaaaat gtgataagtt cagaaagagg tgtttgtggt tgtcacttca
41040 attctcagga aagatgaagc tagcttaggt cctaagtttg ttttattact
tcaaatgcct 41100 gctgagaatg ttttagggat ttgaaatata tataaatata
catatacata cacacacaca 41160 cacacacaca cacacacaca cacacacaca
catattctca gtaaagaaag acttagaaat 41220 cttgaattga ttttctttta
atgtgggaga ctaattcact gaagaaacct gtaattcccc 41280 ttcagaagac
tggtttcaac cgagatctca gaaatactat aaatatccca gagtttagaa 41340
tttgttataa aggcagttat agtggttatt tgatcaaaac agagtgcagc cacttcacga
41400 gccattcatc cctcagtcct gggccctgaa ggggaaagag actacagaga
tggcatgagg 41460 atggactcag agacaagggt ggctccagag cagactttgt
aggtttgagt agggcctcca 41520 attaacctgg ctattctact aagttccctt
cattctgatg ggaaaaactg aagataaaat 41580 gagtactagc taatatgata
aatgtgggtt atttcatggc agcataagaa atgaaatgtt 41640 gccaattatc
gttttgaagc ataaagctcc tttaaaaaat atttcagggg ctggagagat 41700
ggctcagcgg tcaggaaaat tggctgctct tccagaggac ctgggttcat tccccagtac
41760 ccacatggca actcacaaat aactataact ccagttgcag agtatctgac
accttcacac 41820 aaatatccat gcaggaaaaa caccaatgca catgaaataa
aagtaaatta ttttttaaaa 41880 agtatctcaa atatccaaac caacaaataa
gacaagttta attgactgtt tatgtacaaa 41940 ggtaaagcag cccatataaa
ctttctgaaa agtttaaaat caaaccaata cttctttcta 42000 ggtaaaaaat
attgcaccta ctatgtattt gaatgttgct aaagtgaatg agatcaatag 42060
cttgccctca aattttagtg acaataaaag tccaatgtcc atttaatcaa gtggtcagca
42120 ctctttactg tgcctaggca tcacatagaa gggaggttaa acacaaatgg
gggtttctgg 42180 ttcacagaaa ctaggcagtg gcccctaagc atgtttttaa
aaaaatgttc aagtgatgct 42240 gagcttccat gatacatcat taaattgatt
taataggtat aaacacactc aaaattcaga 42300 atatatggaa tatatgttcc
aagtagcagc acctcctcat gtggttttat agctaacggc 42360 catttcagtt
ggaaatactg tgcttgtgcc ttttctccat cgcgggagcc agaaatagtt 42420
acgcaaatat acagcagtaa caaacttaac ctacaatttc actccaagtg tatcttgcct
42480 ttttcgttta aaagttaaaa actgcatgta atagttacca agctgattaa
ttcaatatta 42540 tctttaacag taaactcaat tcataaagaa acactgtctg
ttacctaagt tctgttcacg 42600 gaaaccatga aggccgcttc acacaatcag
cctggcactg tatacaactg ttttttccct 42660 aatgtaattt ggggaactaa
taatttataa tgggatgatg gtgttgtttt acccacaaag 42720 acaataatca
aacccagatt gcaaataatg aaattatgaa ctaggataaa acgagaagca 42780
gaaggcttac cctcccccca tgggagcaca tattttgaga tgatacaatt acttgtcaga
42840 cagtgacaca tcgtccactt tgagcatagt atgctttcag gtgaacagtc
atctgcgaaa 42900 gcgagcctgc agcattctcc aggaacacat ttagaagaca
tcctctcaac ttggatttat 42960 catttatgga aaatagccaa accaaccaac
caaacaaaca aacaaacaaa caaaacccct 43020 ctactttaat gatactattt
caagttttag aaaaaaaatt tataaaaaca aaaacccaca 43080 atatcttaaa
ttgcttttta aaagacaact tctcagccac cttcgcctca atgttttcta 43140
cagaataaaa ggagaagtcc taaaaacaga cagactggag tctgcagagc tctgaatatt
43200 cacagaaggt aaacaatctt ctactggggg ctgttttcct cccctctctg
cagacagaca 43260 cggagtctgc actgctctga gcctgcatgg aaggtgtgct
ttcttccctt cttcctttct 43320 ctttctctag aacactcaag gctgccttgc
tgaagtctct gcagagtctc tatcacagag 43380 ggctttggga acttatgcaa
gtcactgaga agaaagcaac agatgccagt ctgcaagttc 43440 cactaactag
tattcccgga gacactcata tccttcagat tcagcagaac caggtaatgt 43500
tgttatatta gcatttaaca gaaatctact tatattctga aaaaatatgt tatatacata
43560 tatatattat atacactcaa gtttgttctt gaaattgaaa gccttatata
gcaattaata 43620 aaaccagaag catatgtttg gaatggcatt aagttaatta
aaccaagata taagtctcag 43680 tggtgtggga gacgaaattc ttcaatgaaa
atatgctaag catggagaaa tattgatgct 43740 ttgaccctaa aatgagaatt
aaaaatagat gaaaggagtg gaaactccca aagtcaagaa 43800 cattgttaaa
tgatttgcct actctacaca aggagcattc ttaaggtaga gtggtcagaa 43860
ctatccagga aatgagtaat taaaaggaac taacaattgc aaagaggttt gaactcagtg
43920 gtgaactcag tcactctgag gtgttggatg aggggctgag gactgagctc
atacttctgg 43980 tattattctt aactgtatcg ttttcagtaa ttaaaggatc
ccattgacat tagagggaag 44040 tatgtgctat cttcagttat cacgttaaat
ctcccatgag agttttgtgg ggttttcaaa 44100 ctgtatattt gaatatgagg
aatcccattt tatgaaaaat cattttattt atttgatgga 44160 aatttgaaaa
gttttaaatt tcatcttaaa tttcaagtta gatacaagac agccaagtca 44220
actgctccta ttccttcccc cgtctgttgt gtgataatct aggatatcag ataaggatcc
44280 gattctgcta ttttatttta gctaccatta aatatgccag gaatgatgct
ctggctgcct 44340 tgagaaatat caagtaatga tcacagatag agaatctatt
gcctgcatca tcattattac 44400 cattttagga ttcactctga atgatattca
aaaacatata caaacttcaa atataatgaa 44460 tgagctgaaa atgagaaatg
catgggctaa aagcactcta aatcatgagt ctctgtgagg 44520 ctaatatttt
attgcatgac tgaacacagg caaagatatt tctcaatgca aaggaaaatt 44580
ttttggatta ttattttcta ctatttacta tttaatatat gaattcattt cccagaagtg
44640 attttatacc aatctctttg aaattctcca tttatgattc caactcataa
ttaatcactc 44700 ctctattgaa ttcatcaatg atactaaaga gtgaaaccag
gaatatgggc agagaaggaa 44760 aggcagctgt aaacagacac aaaaggctac
tcggccatgt aaaagaagat gcaggggaaa 44820 taggcaaagc atcactagag
tgcctgtcat taggcttttc gaaattaaca caggggtgct 44880 gacaccaaga
ccacagtgtt gtaaaataca gccttcaacc aaaacaagca caaagctctc 44940
atgacatctc acaacacacc acctcgtttt aagttgctag agatggaatt cttgtgtaga
45000 tgagatattc agggattcgg ttgtctattc acccctacta actgccacag
taaagataaa 45060 aaaaactcaa gttttaagca acgctgtaat
tctaaaatgt atgtatgaag ataacaacca 45120 aaccaatgac ctgtgatcac
cctccctctc tctctttctt tctactacag gaccatggat 45180 gtgcctggtg
tcaacaccac ctcagccaat accaccttct cccctgggac cagcaccctg 45240
tgcgtcagag actacaagat cacccaggtt ctcttcccat tgctgtacac cgtcctgttc
45300 tttgctgggc tcatcacgaa cagcttggca atgaggattt tctttcagat
ccgcagtaaa 45360 tccaacttca tcatttttct taagaacacg gtcatctctg
atctactaat gattctaact 45420 tttccattta aaattcttag tgatgctaaa
ctgggagccg ggcctctgag aaccttggtg 45480 tgccaagtta cttcagtcac
attttatttt acaatgtata tcagtatatc gttcctgggg 45540 ttgataacca
ttgaccgcta cctgaagacc accaggccat ttaaaacgtc cagccccagc 45600
aatctcttgg gtgcaaagat tctttctgtt gtcatctggg ccttcatgtt cttaatttca
45660 ctgcctaaca tgattctcac caacaggagg ccaaaagata aggacgtaac
aaaatgttct 45720 ttcttaaagt cagagtttgg tctagtttgg cacgaaatag
tcaattacat ctgccaagtc 45780 attttctgga ttaatttttt aattgtcatc
gtttgttata gcctcattac caaagaactc 45840 tatcggtctt atgtcagaac
aaggggttca gccaaagttc ccaagaaaaa ggtaaacgtc 45900 aaggttttca
tcatcattgc tgtattcttt atttgctttg ttcccttcca ctttgcacgg 45960
attccctaca ccctgagcca aactcgggcc gtctttgact gcagtgctga gaacaccctg
46020 ttctacgtga aggagagcac cctatggctg acgtcactga acgcctgcct
tgatccattc 46080 atctactttt ttctttgcaa gtctttcaga aattccttga
caagcatgct gaggtgctca 46140 aactctacat caacatctgg gacaaacaag
aagaaaggac aagaaggtgg cgaaccaagc 46200 gaagagaccc caatgtagaa
cattacccaa ggggctgctt cagtctttaa tatccagact 46260 gctccaagga
aatcaccata caaatatatt aacattcact aaaaagaagt tgagttaatg 46320
attctttaaa taatcaataa agtaagaaaa taattttt 46358 2 347 PRT Mus
musculus 2 Met Asp Val Pro Gly Val Asn Thr Thr Ser Ala Asn Thr Thr
Phe Ser 1 5 10 15 Pro Gly Thr Ser Thr Leu Cys Val Arg Asp Tyr Lys
Ile Thr Gln Val 20 25 30 Leu Phe Pro Leu Leu Tyr Thr Val Leu Phe
Phe Ala Gly Leu Ile Thr 35 40 45 Asn Ser Leu Ala Met Arg Ile Phe
Phe Gln Ile Arg Ser Lys Ser Asn 50 55 60 Phe Ile Ile Phe Leu Lys
Asn Thr Val Ile Ser Asp Leu Leu Met Ile 65 70 75 80 Leu Thr Phe Pro
Phe Lys Ile Leu Ser Asp Ala Lys Leu Gly Ala Gly 85 90 95 Pro Leu
Arg Thr Leu Val Cys Gln Val Thr Ser Val Thr Phe Tyr Phe 100 105 110
Thr Met Tyr Ile Ser Ile Ser Phe Leu Gly Leu Ile Thr Ile Asp Arg 115
120 125 Tyr Leu Lys Thr Thr Arg Pro Phe Lys Thr Ser Ser Pro Ser Asn
Leu 130 135 140 Leu Gly Ala Lys Ile Leu Ser Val Val Ile Trp Ala Phe
Met Phe Leu 145 150 155 160 Ile Ser Leu Pro Asn Met Ile Leu Thr Asn
Arg Arg Pro Lys Asp Lys 165 170 175 Asp Val Thr Lys Cys Ser Phe Leu
Lys Ser Glu Phe Gly Leu Val Trp 180 185 190 His Glu Ile Val Asn Tyr
Ile Cys Gln Val Ile Phe Trp Ile Asn Phe 195 200 205 Leu Ile Val Ile
Val Cys Tyr Ser Leu Ile Thr Lys Glu Leu Tyr Arg 210 215 220 Ser Tyr
Val Arg Thr Arg Gly Ser Ala Lys Val Pro Lys Lys Lys Val 225 230 235
240 Asn Val Lys Val Phe Ile Ile Ile Ala Val Phe Phe Ile Cys Phe Val
245 250 255 Pro Phe His Phe Ala Arg Ile Pro Tyr Thr Leu Ser Gln Thr
Arg Ala 260 265 270 Val Phe Asp Cys Ser Ala Glu Asn Thr Leu Phe Tyr
Val Lys Glu Ser 275 280 285 Thr Leu Trp Leu Thr Ser Leu Asn Ala Cys
Leu Asp Pro Phe Ile Tyr 290 295 300 Phe Phe Leu Cys Lys Ser Phe Arg
Asn Ser Leu Thr Ser Met Leu Arg 305 310 315 320 Cys Ser Asn Ser Thr
Ser Thr Ser Gly Thr Asn Lys Lys Lys Gly Gln 325 330 335 Glu Gly Gly
Glu Pro Ser Glu Glu Thr Pro Met 340 345 3 59 DNA Mus musculus 3
gtcacacagc aggagctgcc gcacggacac tttcccgtat ccagggtcac agtgcaagg 59
4 133 DNA Mus musculus 4 aggggtggca tctacctgct tggcaactca
ccttcaccgg caccacccct gtttttccag 60 tttggaacat gctaaagtaa
aataataacg tgctacccga cctgaagcac ttgatgtggg 120 cgtaccctac aga 133
5 164 DNA Mus musculus 5 aacactcaag gctgccttgc tgaagtctct
gcagagtctc tatcacagag ggctttggga 60 acttatgcaa gtcactgaga
agaaagcaac agatgccagt ctgcaagttc cactaactag 120 tattcccgga
gacactcata tccttcagat tcagcagaac cagg 164 6 1190 DNA Mus musculus 6
caggaccatg gatgtgcctg gtgtcaacac cacctcagcc aataccacct tctcccctgg
60 gaccagcacc ctgtgcgtca gagactacaa gatcacccag gttctcttcc
cattgctgta 120 caccgtcctg ttctttgctg ggctcatcac gaacagcttg
gcaatgagga ttttctttca 180 gatccgcagt aaatccaact tcatcatttt
tcttaagaac acggtcatct ctgatctact 240 aatgattcta acttttccat
ttaaaattct tagtgatgct aaactgggag ccgggcctct 300 gagaaccttg
gtgtgccaag ttacttcagt cacattttat tttacaatgt atatcagtat 360
atcgttcctg gggttgataa ccattgaccg ctacctgaag accaccaggc catttaaaac
420 gtccagcccc agcaatctct tgggtgcaaa gattctttct gttgtcatct
gggccttcat 480 gttcttaatt tcactgccta acatgattct caccaacagg
aggccaaaag ataaggacgt 540 aacaaaatgt tctttcttaa agtcagagtt
tggtctagtt tggcacgaaa tagtcaatta 600 catctgccaa gtcattttct
ggattaattt tttaattgtc atcgtttgtt atagcctcat 660 taccaaagaa
ctctatcggt cttatgtcag aacaaggggt tcagccaaag ttcccaagaa 720
aaaggtaaac gtcaaggttt tcatcatcat tgctgtattc tttatttgct ttgttccctt
780 ccactttgca cggattccct acaccctgag ccaaactcgg gccgtctttg
actgcagtgc 840 tgagaacacc ctgttctacg tgaaggagag caccctatgg
ctgacgtcac tgaacgcctg 900 ccttgatcca ttcatctact ttttctttgc
aagtctttca gaaattcctt gacaagcatg 960 ctgaggtgct caaactctac
atcaacatct gggacaaaca agaagaaagg acaagaaggt 1020 ggcgaaccaa
gcgaagagac cccaatgtag aacattaccc aaggggctgc ttcagtcttt 1080
aatatccaga ctgctccaag gaaatcacca tacaaatata ttaacattca ctaaaaagaa
1140 gttgagttaa tgattcttta aataatcaat aaagtaagaa aataattttt 1190 7
29 DNA Mus musculus 7 aaggatccaa aatggatgtg cctggtgtc 29 8 28 DNA
Mus musculus 8 aactcgagct acattggggt ctcttcgc 28 9 24 DNA Mus
musculus 9 cagagactac aagatcaccc aggt 24 10 25 PRT Mus musculus 10
Gly Ala Ala Gly Gly Cys Cys Cys Ala Gly Ala Thr Gly Ala Cys Ala 1 5
10 15 Ala Cys Ala Gly Ala Ala Ala Gly Ala 20 25
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