U.S. patent application number 12/334289 was filed with the patent office on 2009-08-27 for anktm1, a cold-activated trp-like channel expressed in nociceptive neurons.
This patent application is currently assigned to THE SCRIPPS RESEARCH INSTITUTE. Invention is credited to Stuart Bevan, Ardem Patapoutian, Gina M. Story.
Application Number | 20090214532 12/334289 |
Document ID | / |
Family ID | 32595284 |
Filed Date | 2009-08-27 |
United States Patent
Application |
20090214532 |
Kind Code |
A1 |
Bevan; Stuart ; et
al. |
August 27, 2009 |
ANKTM1, A Cold-Activated TRP-Like Channel Expressed in Nociceptive
Neurons
Abstract
The methods and compositions of the invention are based on a
method for measuring nociceptive responses in vertebrates,
including humans and other mammals utilizing a newly discovered
thermoreceptor belonging to the Transient Receptor Potential (TRP)
family of non-selective cation channels that participates in
thermosensation and pain. This receptor, designated ANKTM1, is
associated with nociceptive pain, such as hyperalgesia.
Accordingly, the invention provides isolated polypeptides and
polynucleotides associated with nociception as well as methods for
identifying or screening agents that modulate nociception.
Inventors: |
Bevan; Stuart; (London,
GB) ; Patapoutian; Ardem; (San Diego, CA) ;
Story; Gina M.; (San Marcos, CA) |
Correspondence
Address: |
DLA PIPER LLP (US)
4365 EXECUTIVE DRIVE, SUITE 1100
SAN DIEGO
CA
92121-2133
US
|
Assignee: |
THE SCRIPPS RESEARCH
INSTITUTE
NOVARTIS AG
|
Family ID: |
32595284 |
Appl. No.: |
12/334289 |
Filed: |
December 12, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10539377 |
Jan 18, 2006 |
7465581 |
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PCT/EP2003/014403 |
Dec 17, 2003 |
|
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12334289 |
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60434540 |
Dec 18, 2002 |
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Current U.S.
Class: |
514/1.1 ;
424/9.2; 435/7.21; 514/44R; 536/23.5 |
Current CPC
Class: |
A01K 2217/075 20130101;
A61P 25/00 20180101; G01N 33/5088 20130101; A01K 2217/05 20130101;
A01K 2227/10 20130101; C07K 14/705 20130101 |
Class at
Publication: |
424/133.1 ;
536/23.5; 424/9.2; 435/7.21; 514/2; 514/44.R |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07H 21/00 20060101 C07H021/00; A61K 49/00 20060101
A61K049/00; G01N 33/567 20060101 G01N033/567; A61K 38/02 20060101
A61K038/02; A61K 31/7088 20060101 A61K031/7088; A61P 25/00 20060101
A61P025/00 |
Goverment Interests
GRANT INFORMATION
[0002] This invention was made with government support under NINDS
Grant No. R01NS42822 awarded by the National Institutes of Health.
The federal government has certain rights in this invention.
Claims
1. An isolated nucleic acid sequence encoding an ANKTM1-related
polypeptide, characterized as encoding a temperature/pain sensitive
non-selective cation channel protein that is activated by
temperature below 20.degree. C.; being expressed in Calcitonin
gene-related peptide- and substance P-positive neurons; and
comprising more than five ankyrin domains and a six transmembrane
domain.
2. (canceled)
3. The isolated nucleic acid sequence of claim 1, wherein the
ANKTM1-related polypeptide comprises the amino acid sequence of SEQ
ID NO:1, or a conservation variation thereof.
4. (canceled)
5. A method for identifying an agent that modulates nociceptive
response, the method comprising: (a) contacting an organism
containing an ANKTM1-related polypeptide encoded by the sequence
set forth in claim 1 with an agent suspected of having nociceptive
pain modulating activity under conditions that allow the agent and
the polypeptide to interact; (b) measuring a nociceptive response
to administration of a nociceptive stimulus to the organism; (c)
and comparing the nociceptive response to a nociceptive response to
the stimulus in the organism when not administered the agent,
wherein a change in the nociceptive response indicates an agent
that modulates the nociceptive response.
6. The method for claim 5, wherein the agent is selected from the
group consisting of a peptide, a peptidomimetic, a chemical, and a
nucleic acid sequence.
7. The method for claim 5, wherein modulation of the nociceptive
response is a decrease in pain and the change in the nociceptive
response indicates that the agent decreases nociceptive pain.
8. The method for claim 5, wherein the organism is selected from a
non-human organism, a vertebrate, especially a murine species, and
a mammal, especially a human.
9. The method for claim 8, wherein the non-human organism is a
mammalian cell.
10. The method for claim 9, wherein stimulus is noxious cold and
the response comprises a rise in [Ca.sup.2+].sub.i or
[Mg.sup.2+].sub.i in the cell.
11. A method for modulating nociceptive pain in an organism, the
method comprising contacting a sentient organism containing a
polypeptide sequence comprising an amino acid sequence selected
from SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:4, and conservative
variations thereof, with an effective amount of an agent that
modulates operation of the polypeptide under conditions that allow
the agent and the polypeptide to interact, thereby modulating
nociceptive pain in the organism.
12. The method for claim 11, wherein the polypeptide contains an
ion channel and the agent modifies operation of the ion
channel.
13. The method for claim 11, wherein the modulating is a decrease
in the nociceptive pain.
14. The method for claim 11, wherein the organism is a human.
15. A method for identifying an agent that modulates nociceptive
pain, the method comprising: (a) contacting an organism containing
the polynucleotide sequence of claim 1 with a candidate agent under
conditions that allow the agent and the polynucleotide to interact;
(b) measuring a nociceptive response to administration of a
nociceptive stimulus to the organism; and (c) comparing the
nociceptive response to a nociceptive response in the organism when
not administered the nociceptive stimulus, wherein a change in the
nociceptive response indicates the agent modulates nociceptive
pain.
16. (canceled)
17. A method for reducing nociceptive pain in an organism, the
method comprising contacting an organism containing the
polynucleotide sequence of claim 1 with an effective amount of an
agent that blocks function of the polynucleotide sequence under
conditions that allow the agent and the polynucleotide to interact,
thereby reducing nociceptive pain in the organism.
18. The method of claim 11 or 17, wherein the agent is selected
from the group consisting of a peptide, a peptidomimetic, a
chemical, and a nucleic acid sequence.
19. The method of claim 11, wherein the organism is a transgenic
organism.
20. The method for claim 17, wherein the organism is a mammal.
21. An isolated polypeptide comprising an amino acid sequence
selected from SEQ ID NO:1, SEQ ID NO:2 and conservative variations
thereof.
22. A binding molecule which is capable of binding to the
polypeptide according to claim 21 with a dissociation constant
<1000 nM.
23. The binding molecule according to claim 22, which is a chimeric
or humanized monoclonal antibody.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 10/539,377 filed Jan. 18, 2006, now issued as
U.S. Pat. No. 7,465,581; which is a 35 USC .sctn. 371 National
Stage application of International Application No.
PCT/EP2003/014403 filed Dec. 17, 2003; which claims the benefit
under 35 USC .sctn. 119(e) to U.S. Application Ser. No. 60/434,540
filed Dec. 18, 2002, now abandoned. The disclosure of each of the
prior applications is considered part of and is incorporated by
reference in the disclosure of this application.
BACKGROUND OF THE INVENTION
[0003] 1. Field of Invention
[0004] The invention relates generally to nociceptive pain and
disorders associated with pain and more specifically to
polynucleotides encoding polypeptides that affect nociceptive pain
and methods for use therefor.
[0005] 2. Background Information
[0006] Pain has been defined in a variety of ways. For example,
pain can be defined as the perception by a subject of noxious
stimuli that produces a withdrawal reaction by the subject. The
most commonly experienced form of pain may be defined as the effect
of a stimulus on nerve endings, which results in the transmission
of impulses to the cerebrum. This somatic sensation and normal
function of pain, referred to as nociception or nociceptive pain,
informs the organism of impending tissue damage. Somatic and
visceral tree nerve endings, termed nociceptors, initially process
such pain signals.
[0007] Pain is a subjective experience related to perception of
inputs to the central nervous system by a specific class of sensory
receptors known as nociceptors. Nociceptors fire in response to
noxious thermal, mechanical and chemical stimuli. Coding of a
stimulus as painful occurs at several levels in the nervous system.
The first is at the level of transduction of the noxious stimulus
in the peripheral nerve terminals of the nociceptors. During the
transduction step, the noxious stimulus is converted to an
electrical stimulus in the form of an action potential. In mammals
the vanilloid receptors (VR-1 and VRL-1) are proposed to function
during transduction of a noxious heat stimulus. Candidate molecules
for transducing noxious mechanical stimuli have yet to be
identified.
[0008] The second level of coding occurs in the dorsal horn of the
spinal cord. The cell bodies of nociceptive neurons are found in
the dorsal root ganglia and send projections both to the periphery
and to the dorsal horn. Upon stimulation nociceptors release the
excitatory neurotransmitter glutamate, among others, which produces
action potential sin post-synaptic cells of the dorsal horn, which
project to the brain where pain is perceived. The higher level
processing involved in pain perception is poorly understood. High
intensity pain is signaled through increased release of substance P
by the afferent nociceptive terminals in the dorsal horn. This
peptide function through the G-protein couples substance P
receptor, NK-1.
[0009] In general, while brain pathways governing the perception of
pain are still incompletely understood, sensory afferent synaptic
connections to the spinal cord, termed "nociceptive pathways" have
been studied. The nociceptive pathway, which exists for protection
of the organism (such as the pain experienced in response to a burn
or noxious cold), is inactive. Activity is initiated by the
application of a high intensity, potentially damaging stimulus.
This stimulus serves to depolarize certain classes of afferent
(sensory) axons of the small unmyelinated category, designed C
fibers.
[0010] The signal carried by the C fibers travels up the peripheral
nerve and into the spinal cord where synapses are made on second
order and higher order neurons, which then transmit the pain signal
up the spinal cord in the spinothalamic tract ending in the
thalamus. Polysynaptic junctions in the dorsal horn of the spinal
cord are involved in the relay and modulation of sensations of pain
to various regions of the brain, including the periaqueductal grey
region. The ventrolateral and ventromedial thalamic nuclei project
to the cortex where the pain is then processed with regard to
localization and other integrative characteristics.
[0011] Analgesia, or the reduction of pain perception, can be
affected directly by decreasing transmission along such nociceptive
pathways. Analgesic opiates are thought to act by mimicking the
effects of endorphin or enkephalin peptide-containing neurons,
which synapse presynaptically at the C-fiber terminal and which,
when they fire, inhibit release of substance P from the C-fiber.
Descending pathways from the brain are also inhibitory to C-fiber
firing. Thus, CNS-mediated analgesia leads to an overall inhibition
of the pain transmission.
[0012] While neuropathic pain is known to have a number of
underlying etiologies, it is characterized by a distinct set of
symptoms. As described in greater detail below, these can include
enhanced sensitivity to innocuous thermal-mechanical stimuli,
abnormal sensitivity to noxious stimuli, tenderness, and
spontaneous burning pain. Neuropathic pain is also progressive in
nature, in that it generally worsens over time. Known treatment
methods treat the symptoms without necessarily lessening the
underlying pathology.
[0013] Typically, chronic nociceptive pain results from changes in
the peripheral sensory terminal secondary to local tissue damage.
Mild damage, such as abrasions or burns, and inflammation in the
cutaneous receptive fields or joints will produce significant
increases in the excitability of polymodal nociceptors (C fibers)
and high threshold mechanoreceptors. This increased excitability
leads to increased spontaneous activity and an exaggerated response
to otherwise minimal stimuli.
[0014] These events have several consequences. First, the magnitude
of the pain state in humans and animals is proportional to the
discharge rate in such sensory afferent. The facilitated response
secondary to the local peripheral injury may lead to an exaggerated
pain state simply because of the increased afferent activity.
Secondly, spontaneous activity in small sensory afferent causes
central neurons in the spinal cord to develop an exaggerated
response to subsequent input. Both of these events, secondary to
the increased spontaneous activity and reactivity in small sensory
afferent generated by the peripheral injury leads to a behavioral
state referred to as hyperalgesia. Thus, where the pain response is
the result of an exaggerated response to a given stimulus, the
organism is hyperalgesic. The importance of me hyperalgesia state
in the post injury pain state has been repeatedly demonstrated and
this facilitated processing appears to account for a major
proportion of the post-injury/inflammatory pain state.
[0015] Despite numerous definitions, the brain pathways, mechanisms
and intermediates governing the perception of pain are not
completely understood. A number of analgesics and opiates are
currently on the market to address the discomforts associated with
pain. However, many of these agents are addictive or have side
effects mat often provide additional discomforts to a subject when
taken over a long period of time. For example, side effects
associated with a number of opiates include sedation, depression of
respiration, constipation, nausea and emesis, abuse liability and
the development of addiction. These effects serve to limit the
utility of opiates for controlling post injury pain. Addiction
liability can occur secondary to medical uses of the drug where the
central effects lead to an addicted and dependent state.
[0016] The ability to sense cold as a distinct presence resides in
specialized neurons within the peripheral nervous system that
detect varied environmental temperature. The cell bodies of these
sensory neurons reside in the vertebral column and their
projections extend for long distances to peripheral tissues such as
the skin. It is hypothesized that channels present at the end of
these projections are activated by physical stimuli such as
temperature and pressure (Hensel, Monogr Physiol Soc 38:1-321,
1981).
[0017] The cloning of TRPV1 (VR1: a capsaicin- and heat-activated
channel) has proven this hypothesis and ignited research into
thermosensation at the molecular level (Caterina et al., Nature
389:816-824, 1997). TRPV1 is activated near 43.degree. C., a
temperature most mammals find noxious. Three other TRPV channels
with greater than 40% amino-acid level identity to TRPV1 have since
been cloned and characterized as thermosensors. These channels are
activated at various heat thresholds, ranging from 33.degree. C.
(warm) for TRPV3 to 55.degree. C. (high-threshold noxious heat) for
TRPV2 (Caterina et al., Nature 398, 436-441, 1999; Peier et al.,
Science 296:2046-2049, 2002b; Smith et al., Nature 418:186-190,
2002; Xu et al., Nature 418:181-186, 2002). TRPV4, originally
described as an osmo-sensor, has also been shown to be activated by
warm temperatures. Much less is known about channels that sense
cold. Recently, the cloning of a menthol- and cold-activated
channel, TRPM8 (CMR1) was described (McKemy et al., Nature
416:52-58, 2002; Peier et al., Cell 108:705-715, 2002a). The
threshold of TRPM8 activation is reported to be either 28.degree.
C. or 23.degree. C., consistent with the pleasant/cool feeling that
menthol products convey. Since TRPM8 is mostly activated at cool
rather than cold temperatures, it has been postulated that other
cold-activated channels exist.
[0018] The known thermoreceptors all belong to the Transient
Receptor Potential (TRP) family of non-selective cation channels.
TRP channels are divided into three subclasses designated TRPC,
TRPV, and TRPM (Montell et al., Mol Cell 9:229-231, 2002). All have
six putative transmembrane domains with a proposed pore region
between transmembrane domains five and six. TRP channels are
thought to have cytoplasmic N- and C-termini. The three classes of
TRP channels are distinguished according to overall homology as
well as a few unique characteristics. TRPV and TRPC members contain
two to four N-terminal ankyrin domains thought to be involved in
linking transmembrane proteins to the cytoskeleton, TRPC and TRPM
members have a TRP box (with unknown function) following the sixth
transmembrane domain. The involvement of TRP channels in sensory
function has been evident from the beginning. It is now recognized
that several other classes of ion channels are homologous to the
classical TRP channels and a new nomenclature system has been
proposed to reflect this relationship (Montell, 2001, supra). The
new subtypes include TRPP for PKD2-like channels (PKD2 is mutated
in polycystic kidney disease), TRPML for Mucolipidin-like channels
(Mucolipidin mutations are responsible for some lysosomal storage
disorders), and TRPN for NOMPC-like channels, which are
distinguished by a large number of N-terminal ankyrin repeats
(NOMPC is required for mechanosensory function in flies).
[0019] Pain is a major problem for the individual sufferer and for
society because of the high costs involved in managing pain. Pain
is often a part of numerous disorders or diseases including, for
example, cancer pathology. Terminally ill subjects often suffer
immensely because our ability to effectively manage pain is
inadequate. Therefore, strategies to identify molecules that
function in pain sensation are needed.
SUMMARY OF THE INVENTION
[0020] In one embodiment, the invention provides isolated nucleic
acid sequences encoding an ANKTM1-related polypeptide, which are
characterized as encoding a temperature/pain sensitive
non-selective cation channel protein that is activated by
temperature below 20.degree. C.; being expressed in Calcitonin
gene-related peptide- and substance P-positive neurons. The
invention nucleic acid sequences comprise more than five ankyrin
domains and a six transmembrane domain.
[0021] In another embodiment, the invention provides methods for
identifying an agent that modulates nociceptive response by
contacting an organism containing an ANKTM1-related polypeptide
encoded by an invention nucleic acid sequence with an agent
suspected of having nociceptive pain modulating activity under
conditions that allow the agent and the polypeptide to interact. A
nociceptive stimulus is then administered to the organism and any
nociceptive response is measured and compared with the nociceptive
response of the organism to the stimulus when not administered the
agent. A change in the nociceptive response indicates the agent
modulates the nociceptive response to the stimulus.
[0022] In yet another embodiment, the invention provides methods
for modulating nociceptive pain in a sentient organism that
contains a polypeptide sequence comprising an amino acid sequence
selected from SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:4, and
conservative variations thereof by contacting the organism with an
effective amount of an agent that modulates operation of the
polypeptide under conditions that allow the agent and the
polypeptide to interact, thereby modulating nociceptive pain in the
organism.
[0023] In still another embodiment, the invention provides methods
for reducing nociceptive pain in an organism by contacting an
organism containing an invention isolated nucleic acid sequence
with an effective amount of an agent that blocks function of the
polynucleotide sequence under conditions that allow the agent and
the polynucleotide to interact, thereby reducing nociceptive pain
in the organism.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1A is a representation of a phylogenetic tree of TRP
channels showing ANKTM1 grouped together with an emerging group of
TRP-like channels that have multiple ankyrin domains (more than
eight) and a six unit transmembrane (TM) domain. To date, ANKTM1 is
the only vertebrate member of this family (mouse=mANKTM1 (SEQ ID
NO:1) and human=hANKTM1 (SEQ ID NO:2). A close homologue exists in
Drosophila that is designated dmANKTM1 (SEQ ID NO:3). Overall, four
drosophila (dm) (including NOMPC) members and two C. elegans (ce)
family members belong to this family (dmNOMPC (SEQ ID NO:4);
ceNOMPC (SEQ ID NO:5); ce(NP.sub.--502249) (SEQ ID NO:6);
dm(CG1-409) (SEQ ID NO:7); dm(CG17142) (SEQ ID NO:8). Similarities
are calculated in Blast2.
[0025] FIGS. 1B and 1C are an alignment comparison of amino acid
sequences from the putative transmembrane domains of proteins in
the phylogenetic tree shown in FIG. 1A. The alignment shows strong
similarity in areas such as transmembrane domain six (TM6) among
all members, and more specific pockets of similarity common to the
three ANKTM1 homologues peppered throughout the region. The
alignment is generated using MEGALIGN.TM. and BOXSHADE.TM.
alignment programs. Identical and conserved areas are shaded.
Transmembrane domains of mANKTM1 are marked as TM.
[0026] FIG. 2A is a Northern blot analysis of mouse tissue showing
ANKTM is not expressed in tissues tested.
[0027] FIG. 2B is a Northern blot analysis of rat tissues showing
expression of ANKTM1 in the dorsal root ganglia (DRG). Blots were
hybridized with a .sup.32P-labeled fragment of ANKTM1 cDNA (top
panel) and with control cDNAs (bottom panel).
[0028] FIGS. 3A through 3M show the results of in-situ
hybridization and immunostaining analyses, which demonstrate ANKTM1
expression in a subset of TRPV1-positive nociceptive DRG
neurons.
[0029] (A-C) ANKTM1 mRNA is not present in heavily myelinated
neurons marked by Neurofilament (NF 150) antibody. (D-F) ANKTM1 and
calcitonin-gene-related peptide (CGRP) are coexpressed (arrows
D-F). (G-L) Double-in situ hybridization shows ANKTM1 is present in
a sub-population of thermosensitive neurons expressing TRPV1
(arrows G-I), but not TRPM8. Size bar is 50 .mu.m. (M) A schematic
representation showing that ANKTM1 is expressed in a subset of
TRPV1-positive nociceptive DRG neurons and in a sub-population of
thermosensitive neurons expressing TRPV1, but not TRPM8.
[0030] FIG. 4A is a graph showing that ANKTM1 is a calcium
permeable cation channel that responds to a cold stimulus by
eliciting a rise in [Ca.sup.2+].sub.i in ANKTM1-expressing CHO
cells. A schematic representation of the stimulus temperature is
indicated below the graph. CHO=untransfected CHO cells;
RR=ruthenium red blocker. Values shown are average increase in
ratio 340/380.+-.SEM of 30-40 cells from representative
experiments.
[0031] FIG. 4B is a graph showing average fold increase in 340
nm/380 nm ratio of ANKTM1-, TPRM8-, and TRPV1-expressing CHO cells
in response to hot-(capsaicin) and cold-(menthol, icilin) inducing
compounds.
[0032] FIGS. 5A and 5B show that ANKTM1 is activated at colder
temperatures compared to TRPM8.
[0033] FIG. 5A shows representative images taken from calcium
imaging experiments comparing responses to gradual cooling of
Fura-2 loaded ANKTM1- and TRPM8-expressing CHO cells. Increases in
fluorescence correspond to increases in intracellular calcium
levels and are indicated by a change in color where
red>yellow>green>blue. Note that the majority of
ANKTM1-expressing cells (left) are not active until temperatures
fall below 20.degree. C., while many TRPM8 expressing cells (right)
are already strongly activated at 20.degree. C.
[0034] FIG. 5B is a graph showing responses of individual ANKTM1-
and TRPM8-expressing CHO cells to gradual cooling from 31.degree.
C. to 9.degree. C. Responses shown are from representative
cells.
[0035] FIGS. 6A through 6D are a series of graphs showing currents
evoked by decreasing temperatures in CHO cells expressing
ANKTM1.
[0036] FIG. 6A provides an example of an inward current evoked by
cooling of a cell voltage clamped at -60 mV.
[0037] FIG. 6B is a graph showing a comparison of temperature
current relationship for ANKTM1 and TRPM8. The threshold for
activation of ANKTM1 is lower than that of TRPM8.
[0038] FIG. 6C is a graph showing current-voltage relationship for
ANKTM1 (voltage ramp is from -100 mV to +80 mV with two second
ramps). The current is outwardly rectifying and there is an
increase in both inward and outward currents when the temperature
is lowered (30.degree. C.-18.degree. C.).
[0039] FIG. 6D is a graph showing current-voltage relationships for
ANKTM1 in various external solutions. The main charge ion is (1)
140 mM NaCl (2) 40 mM NaCl/100 mM choline (3) 1 mM CaCl (4) 30 mM
CaCl. E.sub.revs for these examples are, +4, -6, -2 and +11 mV,
respectively.
[0040] FIGS. 7A through 7D are a series of graphs showing that
ANKTM1 currents are desensitized to repeated cold stimuli.
[0041] FIGS. 7A and 7C show, respectively, inward currents recorded
in response to first and second cold steps from Xenopus oocytes
expressing ANKTM1.
[0042] FIGS. 7B and 7D show, respectively, inward currents recorded
in response to cold steps from Xenopus oocytes expressing TRPM8.
The oocytes were held at -70 mV during the recordings. ANKTM1
current responses were markedly reduced in the second cold
step.
[0043] FIGS. 8A and 8B show the nucleotide sequence (SEQ ID NO:13)
of the 3.378 kb mouse homologue (mANKTM) of human ANKTM
(hANKTM).
DETAILED DESCRIPTION OF THE INVENTION
[0044] In humans and other vertebrates, painful stimuli are sensed
by specialized neurons known as nociceptors, which fire in response
to noxious temperature and mechanical or chemical stimuli, all of
which have the potential to cause tissue damage. The signals are in
turn processed by the central nervous system and perceived as pain,
serving an indispensable protective role. Nociceptors are also
involved in pathological pain states caused by inflammation, nerve
damage, or cancer. An increased understanding of nociception
therefore is of wide interest, and model systems for molecular
genetic analysis are desirable.
[0045] The methods and compositions of the invention are based on a
method, described herein, to measure nociceptive responses in
vertebrates such as, for example, mammals including humans,
utilizing a new member of the TRPN family that participates in
thermosensation and pain. This receptor, designated ANKTM1, is
associated with nociceptive pain. Accordingly, the invention
provides isolated polypeptides and polynucleotides associated with
nociception as well as methods for identifying or screening agents
that modulate nociception.
[0046] As used herein, "hyperalgesia" or a "hyperalgesia state"
refers to a condition in which a warm-blooded animal is extremely
sensitive to mechanical, chemical or thermal stimulation that,
absent the condition, would be painless. Typical models for such a
hyperalgesic state include the inflamed rat paw compression model
and the compression of the inflamed knee joint.
[0047] Hyperalgesia is known to accompany certain physical injuries
to the body, for example the injury inevitably caused by surgery.
Hyperalgesia is also known to accompany certain inflammatory
conditions in man such as arthritic and rheumatic disease.
Hyperalgesia, thus refers to mild to moderate pain to severe pain
such as the pain associated with, but not limited to, inflammatory
conditions (e.g., such as rheumatoid arthritis and osteoarthritis),
postoperative pain, post-partum pain, the pain associated with
dental conditions (e.g., dental caries and gingivitis), the pain
associated with burns, including but not limited to sunburns,
abrasions, contusions and the like, the pain associated with sports
injuries and sprains, inflammatory skin conditions, including but
not limited to poison ivy, and allergic rashes and dermatitis, and
other such pain that increases sensitivity to mild stimuli, such as
cold.
[0048] To date, all five thermo-activated channels identified
belong to the TRP family, characterized by six transmembrane
domains (all) and N-terminal ankyrin domains (most) (Montell, 2001,
supra). In the invention, a combined bioinformatic and expression
analysis approach has been used to identify additional TRP channels
involved in sensory detection. A search in PFAM protein sequence
HMM data base for predicted cDNA sequences that contain both
ankyrin domains (PFAM00023) and six-transmembrane domains
(PFAM00520) led to ANKTM1. Human ANKTM1 is an uncharacterized
putative channel cloned from cultured fibroblasts. A previous study
had shown no significant expression of ANKTM1 in various tissues
examined, although EST sequences suggest that ANKTM1 is upregulated
in many human tumor cells (Jaquemar et al., J Biol Chem
274:7325-7333, 1999).
[0049] To characterize human ANKTM1, full-length mouse ANKTM1 was
amplified from mouse dorsal root ganglia (DRG) and trigeminal
ganglia that contain somatic sensory neurons using RT-PCR.
Theoretical translation of the mouse nucleotide sequence predicts a
protein of 1125 amino acid residues (SEQ ID NO:1), very similar to
human ANKTM1 (1119 amino acids) (SEQ ID NO:2) (FIG. 1A). Mouse
ANKTM1 has 14 predicted N-terminal ankyrin domains followed by a
six transmembrane (6.TM.) domain.
[0050] A phylogenetic tree analysis of all TRP-like channels
grouped ANKTM1 with Drosophila NOMPC, which has 29 N-terminal
ankyrin domains and is required for mechanosensation (Walker et
al., Science 287:2229-2234, 2000). Further searches for proteins
that contain both a 6.TM. domain and more than five ankyrin domains
(to exclude TRPV and TRPC members, which have two to four ankyrin
domains) demonstrated that ANKTM1 is the only mammalian member of
this group. Drosophila has four such proteins dmANKTM1 (SEQ ID
NO:3, dmNOMPC (SEQ ID NO:4), dmCG10409 (SEQ ID NO:7) and dmCG17142
(SEQ ID NO:8). In C. elegans two such proteins have been found,
ceNOMPC (SEQ ID NO:5) and ceNP-502249 (SEQ ID NO:6) (FIGS. 1A and
B). These putative channels form a branch of TRP proteins named
TRPN after the founding member, NOMPC. Some of the TRPN family
members do not have high similarity at the primary amino acid
level: mANKTM1 (SEQ ID NO:1) shares only 25% amino acid identity to
dmNOMPC (SEQ ID NO:4). By comparison, TRPV1 and TRPV3 are 43%
similar. The clustering of mANKTM1 and NOMPC in the phylogenetic
tree reflects their shared numerous N-terminal ankyrin domains.
Indeed, if 6TM domains alone are compared, ANKTM1 is more similar
to TRPV3 than to NOMPC. Therefore, the phylogenetic tree
organization should be carefully interpreted.
[0051] However, at the amino-acid level, the two cold-receptors
surprisingly have no significant amino acid sequence identity. This
finding is in contrast to the four heat-activated TRPV channels,
which have at least 40% amino acid sequence identity. The
transmembrane domain of ANKTM1 is more similar to TRPV members than
to any other mammalian proteins. This amino acid similarity is
likely significant at the structural level since ruthenium red, a
potent blocker of all TRPV channels, also blocks ANKTM1. The 16
N-terminal ankyrin domains show further correlation with TRPV
channels (two to four ankyrin domains). However, in phylogenetic
tree prediction programs, ANKTM1 is grouped with the emerging TRPN
family of NOMPC-like channels, probably due to the large number of
N-terminal ankyrin domains reported in this family. Within this
group of putative channels, the uncharacterized protein CG5751 is
shown to be the Drosophila orthologue of ANKTM1, since the two
proteins are more similar to each other than to any other
protein.
[0052] Most TRP channels are non-selective cation channels and
readily let calcium into cells when activated, permitting both
electrophysiological studies and intracellular calcium imaging.
Therefore, in the studies described herein a series of
intracellular calcium imaging and electrophysicological studies
were applied to ANKTM1. Fluorescence microscopy was used to monitor
increases in intracellular calcium concentration in response to
various sensory stimuli by loading ANKTM1-expressing CHO cells with
the calcium indicator fluorescent dye, Fura-2. Hypo-osmotic
solutions are known to elicit calcium influx in TRPV4-expressing
cells. However, when ANKTM1 was subjected to hypo-osmotic solution,
activation did not occur. Heat stimuli of up to 42.degree. C. and
52.degree. C. are strong activators of TRPV1 and TRPV2 respectively
(Caterina et al., 1999, supra; Caterina et al., 1997, supra).
However, ANKTM1-expressing CHO cells, when stimulated with heat in
this range, were not activated. Menthol or cool stimuli in the
range of 23-28.degree. C., the threshold of activation of TRPM8
(McKemy et al., 2002, supra; Peier et al., 2002b, supra), also
failed to activate ANKTM1.
[0053] However, it was discovered that lowering temperature to
10.degree. C. caused a large influx of calcium in ANKTM1-expressing
cells, but not in Tet-treated CHO cell controls (FIG. 4A). This
response to cold stimulus by ANKTM1 was abolished upon removal of
extracellular calcium, showing that ANKTM1 is acting as a calcium
permeable channel rather than releasing calcium from intracellular
stores (FIG. 4A). Similar responses to cold were obtained from CHO
cells transiently transfected with ANKTM1 and from Tet-inducible
human embryonic kidney (HEK 293) cells stably expressing
ANKTM1.).
[0054] Ruthenium red, a blocker of thermosensitive TRPV channels,
also blocked cold responses by ANKTM1. Pre-incubation of
ANKTM1-expressing cells in 5 .mu.M ruthenium red followed by
application of cold completely blocked responses, which were not
entirely recovered after a ten minute washout (FIG. 4). Similar
experiments performed with 1 .mu.m ruthenium red caused complete
block of the cold response in 90% of ANKTM1-containing cells from
multiple experiments.
[0055] Icilin, an ultra-cooling agent and potent activator of
TRPM8, also activated ANKTM1-expressing cells (FIG. 4B) (McKemy et
al, 2002, supra), but Icilin did not activate TRPV1-expressing
cells. In additional tests, capsaicin, a potent activator of TRPV1,
did not activate TRPM8- or ANKTM1-expressing cells (FIG. 4B).
Higher concentrations of Icilin were required for ANKTM1 activation
compared to TRPM8, and the response was relatively delayed in
ANKTM1 cells. There was a 15-60 second delay for ANKTM1 as compared
with a 15 seconds delay for TRPM8. Therefore, it is not clear if
Icilin directly binds to and activates ANKTM1.
[0056] These experiments show that both "TRPM8 and ANKTM1 respond
to cold. However, to confirm that threshold for activation of
ANKTM1 is lower than that of TRPM8, as suggested in the
above-described experimental results, further tests were conducted.
The activation temperature of TRPM8 is reported at approximately 23
CC (Peier et al., 2002a, supra). Therefore, to further compare the
activation temperatures of ANKTM1 and TRPM8, calcium imaging
experiments were conducted wherein incubation temperature of both
types of cells was elevated to 33.degree. C. for several minutes
before the bath temperature was lowered gradually to 10.degree. C.
The majority of TRPM8-expressing cells exhibited increased
intracellular calcium concentration when the temperature was
lowered to 20.degree. C. (FIG. 5A). However, the majority of
ANKTM1-expressing cells were not activated until the temperature
was cooled to 10.degree. C. (FIG. 5A). Quantitative analysis showed
that TRPM8-expressing cells exhibited an activation threshold
(defined as 20% increase from baseline fluorescence) at
temperatures ranging from 19-24.degree. C. with a mean activation
temperature of 22.5.+-.1.degree. C. (mean.+-.SD, n=60). By
comparison, ANKTM1-expressing cells exhibited a broader range of
activation temperatures (12.degree. C.-24.degree. C.) with an
average activation temperature of 17.5.+-.3.5.degree. C.
(mean.+-.SD, n=100). FIG. 5B illustrates traces of typical
responses of individual ANKTM1 and TRPM8-expressing cells as
temperature is gradually decreased.
[0057] ANKTM1- and TRPM8-expressing CHO cells were also assayed
electrophysiologically using the whole cell voltage clamp
technique. The cells were clamped at -60 mV and the temperature of
the perfused bath solution was decreased from 32.degree. C. to
10.degree. C. In ANKTM1-expressing cells small and slowly
developing inward currents were observed followed by rapid and
larger phase currents with an average peak amplitude of
0.55.+-.0.07 nA (n=35) (FIGS. 6A and 6B). The activation
temperature for the large evoked currents varied from cell to cell,
ranging from 8-28.degree. C., yielding results similar to those
obtained by calcium imaging (n=35). A comparison of representative
currents from ANKTM1- and TRPM8-expressing CHO cells shown in FIG.
6B demonstrates a lower threshold of activation for ANKTM1 than for
TRPM8.
[0058] Currents evoked by decreasing the temperature in
ANKTM1-expressing cells show outward rectification, but with
substantial current in the inward direction (FIGS. 6C and 6D). A
reversal potential of +7.7.+-.1.2 mV was observed in an external
solution containing 140 mM NaCl (n=6). Reducing the NaCl in the
external solution to 40 mM (by equimolar replacement with 100 mM
choline chloride) caused a negative shift in the reversal
potential. This result is consistent with ANKTM1 being a cation
channel. Differences in reversal potentials were used to determine
the ionic selectivity of ANKTM1. The shift in reversal potential of
-18 mV to -10.35.+-.1.6 mV (n=7) seen upon replacing 100 mM NaCl
with 100 mM choline Cl gives a relative permeability ratio of
P.sub.choline/P.sub.Na=0.28. The reversal potentials of the
cold-activated currents were similar in simplified external
solutions containing 100 mM Choline and 40 mM NaCl, KCl, or CsCl.
The measured reversal potentials yield relative permeability ratios
of P.sub.K/P.sub.Na=1-19 and P.sub.Cs/P.sub.Na=1.43 (NaCl,
E.sub.rev=-10.35.+-.1.6 mV, n=7; KCl, E.sub.rev=-7.65.+-.1.5 mV,
n=8; CsCl, E.sub.rev=-4.75.+-.0.75 mV, n=7). The relative
permeability of Ca.sup.2+ and Mg.sup.2+ were estimated from the
shift in reversal potentials when their concentrations were raised
from 1 mM to 30 mM in a 40 mM NaCl/100 mM choline Cl solution
containing the divalent cation under investigation. In these tests,
the reversal potential shifted from +0.54.+-.3.3 (1 mM CaCl.sub.2,
n=6) to +14.16.+-.3.9 mV (30 mM CaCl.sub.2, n=6) for Ca.sup.2+ and
from -14.36.+-.1.1 (1 mM MgCl.sub.2, n=6) to +7.01.+-.2.9 mV (30 mM
MgCl.sub.2, n=6) for Mg.sup.2+, corresponding to
P.sub.Ca/P.sub.Na=0.84 and P.sub.Mg/P.sub.Na=1.23 (FIG. 6D). These
results indicate that ANKTM1 is a non-selective cation permeable
channel, similar to many previously described TRP channels.
[0059] To investigate the properties of ANKTM1 in another
heterologous system, Xenopus oocytes were injected with ANKTM1
cRNA. Large currents were observed in response to cold
temperatures, similar to the activity of ANKTM1-expressing CHO
cells (FIGS. 7A and C). In both Xenopus and CHO systems, a strong
desensitization of ANKTM1 to cold stimuli was observed. Cold
activation of ANKTM1 showed a marked desensitization during a first
cold pulse, and desensitization to repeated cold pulses (FIGS. 7B
and D). On average, the second cold pulse resulted in a current
that was 26% of the first pulse in ANKTM1-injected oocytes (SD=6.5,
n=5), compared to 78% for TRPM8 (SD=5.9, n=4).
[0060] The above-described characterization studies show that
ANKTM1 belongs to the superfamily of TRP channels as does the
menthol- and cold-activated receptor, TRPM8, despite the lack of
amino acid sequence similarity between the two. Functional studies
of ANKTM1 agree with the phylogenetic characterization of this
channel as TRP-like. There is now strong evidence that a group of
TRP channels play a role in temperature detection in the mammalian
peripheral nervous system. ANKTM1 belongs to the third TRP
subfamily shown to play a role in temperature detection. Like other
thermosensitive TRPs, ANKTM1 is a non-selective cation channel.
However, ANKTM1 displays several unique characteristics compared to
previously characterized temperature-activated TRP channels. The
variability in activation threshold temperature of ANKTM1 from cell
to cell is broader when compared to other TRPs. Furthermore, the
current through ANKTM1 rapidly desensitizes to cold, a property not
seen to such an extent in other temperature-activated TRPs.
Finally, long-term overexpression of ANKTM1 is detrimental to
cells, making it necessary to for cell lines to conditionally
express ANKTM1. The mechanisms underlying the threshold
variability, strong desensitization, and cell toxicity are not yet
clear.
[0061] ANKTM1 is activated at lower temperatures than TRPM8,
starting at near 17.degree. C., which approximates the threshold of
noxious cold for humans (.about.15.degree. C.). A role for ANKTM1
in noxious cold detection is also suggested by its expression
pattern. Mouse ANKTM1 is specifically expressed in somatic sensory
neurons. Within this population, ANKTM1 is not expressed in neurons
that express TRPM8. Instead, the vast majority of ANKTM1-positive
cells also express TRPV1 and CGRP, markers for pain-sensing
neurons. Recent reports describe the presence of two separate
populations of cold-sensitive DRG neurons: one population that
expresses TRPM8 and is menthol-sensitive, and a distinct population
that is menthol-insensitive and is activated at even colder
temperatures. It is likely that ANKTM1 marks this second population
of cold sensitive neurons.
[0062] The major overlap of ANKTM1 and TRPV1 tissue expression
raises the question of how temperature/pain information is decoded
in the brain and spinal cord. Electrophysiological characterization
of DRG neurons has led to the labeled line hypothesis of sensory
perception (Scott, 1992). According to this hypothesis, the nervous
system interprets the environment by monitoring the electrical
activity of distinct groups of sensory neurons that are specialized
to detect a unique sensory modality. However, this hypothesis does
not incorporate the observation of single neurons known to transmit
more than one sensory modality, for example in this case cold and
pain. Indeed, there are so-called polymodal nociceptors that
respond to a variety of noxious stimuli (Julius and Basbaum, Nature
413:203-210, 2001). Here molecular evidence is shown that a
cold-activated channel (ANKTM1) is expressed in a subset of
heat-sensitive (TRPV1-expressing) neurons.
[0063] The identification of ANKTM1 as a channel activated by
noxious cold increases the range of temperatures "sensed" by TRP
channels, but does not explain how the quality of the sensation
changes from pleasantly cool to aching or burning, i.e., pain,
depending on whether ANKTM1, TRPM8, or both of these cold channels
are active.
[0064] The term "isolated" means altered "by the hand of man" from
its natural state; i.e., if it occurs in nature, it has been
changed or removed from its original environment, or both. For
example, a naturally occurring polynucleotide or a polypeptide
naturally present in a living animal in its natural state 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. As part of or following isolation,
a polynucleotide can be joined to other polynucleotides, such as
for example DNAs, for mutagenesis studies, to form fusion proteins,
and for propagation or expression of the polynucleotide in a host.
The isolated polynucleotides, alone or joined to other
polynucleotides, such as vectors, can be introduced into host
cells, in culture or in whole organisms. Such polynucleotides, when
introduced into host cells in culture or in whole organisms, still
would be isolated, as the term is used herein, because they would
not be in their naturally occurring form or environment. Similarly,
the polynucleotides and polypeptides may occur in a composition,
such as a media formulation (solutions for introduction of
polynucleotides or polypeptides, for example, into cells or
compositions or solutions for chemical or enzymatic reactions).
[0065] "Polynucleotide" or "nucleic acid sequence" refers to a
polymeric form of nucleotides. In some instances a polynucleotide
refers to a sequence that is not immediately contiguous with either
of the coding sequences with which it is immediately contiguous
(one on the 5' end and one on the 3' end) in the naturally
occurring genome of the organism from which it is derived. The term
therefore includes, for example, a recombinant DNA which is
incorporated into a vector; into an autonomously replicating
plasmid or virus; or into the genomic DNA of a prokaryote or
eukaryote, or which exists as a separate molecule (e.g., a cDNA)
independent of other sequences. The nucleotides of the invention
can be ribonucleotides, deoxyribonucleotides, or modified forms of
either nucleotide. In addition, the polynucleotide sequence
involved in producing a polypeptide chain can include regions
preceding and following the coding region (leader and trailer) as
well as intervening sequences (introns) between individual coding
segments (exons) depending upon the source of the polynucleotide
sequence.
[0066] The term "polynucleotide(s)" generally refers to any
polyribonucleotide or polydeoxyribonucleotide, which may be
unmodified RNA or DNA or modified RNA or DNA. Thus, for instance,
polynucleotides as used herein refers to, among others, single- and
double-stranded DNA, DNA that is a mixture of single- and
double-stranded regions, single- and double-stranded RNA, and RNA
that is mixture of single- and double-stranded regions, hybrid
molecules comprising DNA and RNA that may be single-stranded or,
more typically, double-stranded or a mixture of single- and
double-stranded regions.
[0067] In addition, polynucleotide as used herein refers to
triple-stranded regions comprising RNA or DNA or both RNA and DNA.
The strands in such regions may be from the same molecule or from
different molecules. The regions may include all of one or more of
the molecules, but more typically involve only a region of some of
the molecules. One of the molecules of a triple-helical region
often is an oligonucleotide.
[0068] In addition, the polynucleotides or nucleic acid sequences
may contain one or more modified bases. Thus, DNAs or RNAs with
backbones modified for stability or for other reasons are
"polynucleotides" as that term is intended herein. Moreover, DNAs
or RNAs comprising unusual bases, such as inosine, or modified
bases, such as tritylated bases, to name just two examples, are
polynucleotides as the term is used herein.
[0069] Nucleic acid sequences can be created which encode a fusion
protein and can be operatively linked to expression control
sequences. "Operatively linked" refers to a juxtaposition wherein
the components so described are in a relationship permitting them
to function in their intended manner. For example, a coding
sequence is "operably linked" to another coding sequence when RNA
polymerase will transcribe the two coding sequences into a single
mRNA, which is then translated into a single polypeptide having
amino acids derived from both coding sequences. The coding
sequences need not be contiguous to one another so long as the
expressed sequences ultimately process to produce the desired
protein. An expression control sequence operatively linked to a
coding sequence is ligated such that expression of the coding
sequence is achieved under conditions compatible with the
expression control sequences. As used herein, the term "expression
control sequences" refers to nucleic acid sequences that regulate
the expression of a nucleic acid sequence to which it is
operatively linked. Expression control sequences are operatively
linked to a nucleic acid sequence when the expression control
sequences control and regulate the transcription and, as
appropriate, translation of the nucleic acid sequence. Thus,
expression control sequences can include appropriate promoters,
enhancers, transcription terminators, a start codon (i.e., ATG) in
front of a protein-encoding gene, splicing signals for introns,
maintenance of the correct reading frame of that gene to permit
proper translation of the mRNA, and stop codons. The term "control
sequences" is intended to include, at a minimum, components whose
presence can influence expression, and can also include additional
components whose presence is advantageous, for example, leader
sequences and fusion partner sequences. Expression control
sequences can include a promoter.
[0070] By "promoter" is meant minimal sequence sufficient to direct
transcription. Also included in the invention are those promoter
elements which are sufficient to render promoter-dependent gene
expression controllable for cell-type specific, tissue-specific, or
inducible by external signals or agents; such elements may be
located in the 5' or 3' regions of the of the polynucleotide
sequence. Both constitutive and inducible promoters, are included
in the invention (see e.g., Bitter et al., Methods in Enzymology
153:516-544, 1987). For example, when cloning in bacterial systems,
inducible promoters such as pL of bacteriophage, plac, ptrp, ptac
(ptrp-lac hybrid promoter) and the like may be used. When cloning
in mammalian cell systems, promoters derived from the genome of
mammalian cells (e.g., metallothionein promoter) or from mammalian
viruses (e.g., the retrovirus long terminal repeat; the adenovirus
late promoter; the vaccinia virus 7.5K promoter) may be used.
Promoters produced by recombinant DNA or synthetic techniques may
also be used to provide for transcription of the nucleic acid
sequences of the invention.
[0071] A nucleic acid sequence of the invention including, for
example, a polynucleotide encoding a fusion protein, may be
inserted into a recombinant expression vector. A recombinant
expression vector generally refers to a plasmid, virus or other
vehicle known in the art that has been manipulated by insertion or
incorporation of a nucleic acid sequences. For example, a
recombinant expression vector of the invention includes a
polynucleotide sequence encoding an ANKTM1-related polypeptide
involved in nociception or a fragment thereof. The expression
vector typically contains an origin of replication, a promoter, as
well as specific genes that allow phenotypic selection of the
transformed cells. Vectors suitable for use in the invention
include, but are not limited to the T7-based expression vector for
expression in bacteria (Rosenberg, et al., Gene 56:125, 1987), the
pMSXND expression vector for expression in mammalian cells (Lee and
Nathans, J. Biol. Chem. 263:3521, 1988), baculovirus-derived
vectors for expression in insect cells, cauliflower mosaic virus,
CaMV; tobacco mosaic virus, TMV. The nucleic acid sequences of the
invention can also include a localization sequence to direct the
indicator to particular cellular sites by fusion to appropriate
organellar targeting signals or localized host proteins. For
example, a polynucleotide encoding a localization sequence, or
signal sequence, can be used as a repressor and thus can be ligated
or fused at the 5' terminus of a polynucleotide encoding a
polypeptide of the invention such that the localization or signal
peptide is located at the amino terminal end of a resulting
polynucleotide/polypeptide. The construction of expression vectors
and the expression of genes in transfected cells involves the use
of molecular cloning techniques also well known in the art. (See,
for example, Sambrook et al., Molecular Cloning--A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
1989, and Current Protocols in Molecular Biology, M. Ausubel et
al., eds., (Current Protocols, a joint venture between Greene
Publishing Associates, Inc. and John Wiley & Sons, Inc., most
recent Supplement)). These methods include in vitro recombinant DNA
techniques, synthetic techniques and in vivo recombination/genetic
recombination. (See also, Maniatis, et al., Molecular Cloning A
Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1989).
[0072] In yeast, a number of vectors containing constitutive or
inducible promoters may be used. For a review, see Current
Protocols in Molecular Biology, Vol. 2, Ed. Ausubel, et al., Greene
Publish. Assoc. & Wiley Interscience, Ch. 13, 1988; Grant, et
al., "Expression and Secretion Vectors for Yeast," in Methods in
Enzymology, Eds. Wu & Grossman, 1987, Acad. Press, N.Y., Vol.
153, pp. 516-544, 1987; Glover, DNA Cloning, Vol. H, IRL Press,
Wash., D.C., Ch. 3, 1986; and Bitter, "Heterologous Gene Expression
in Yeast," Methods in Enzymology, Eds. Berger & Kimmel, Acad.
Press, N.Y., Vol. 152, pp. 673-684, 1987; and The Molecular Biology
of the Yeast Saccharomyces, Eds. Strathern et al., Cold Spring
Harbor Press, Vols. I and II, 1982. A constitutive yeast promoter
such as ADH or LEU2 or an inducible promoter such as GAL may be
used ("Cloning in Yeast," Ch. 3, R. Rothstein In: DNA Cloning Vol.
11, A Practical Approach, Ed. DM Glover, IRL Press, Wash., D.C.,
1986). Alternatively, vectors may be used which promote integration
of foreign DNA sequences into the yeast chromosome.
[0073] An alternative expression system that could be used to
express an invention ANKTM1-related polypeptide is an insect
system. In one such system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
or mutated polynucleotide sequences. The virus grows in Spodoptera
frugiperda cells. The sequence encoding an invention protein may be
cloned into non-essential regions (for example, the polyhedrin
gene) of the virus and placed under control of an AcNPV promoter
(for example the polyhedrin promoter). Successful insertion of the
sequences coding for a protein of the invention will result in
inactivation of the polyhedrin gene and production of non-occluded
recombinant virus (i.e., virus lacking the proteinaceous coat coded
for by the polyhedrin gene). These recombinant viruses are then
used to infect S. frugiperda cells in which the inserted gene is
expressed, see Smith, et al., J. Viol. 46:584, 1983; Smith, U.S.
Pat. No. 4,215,051.
[0074] The vectors of the invention can be used to transform a host
cell. By transform or transformation is meant a permanent or
transient genetic change induced in a cell following incorporation
of new DNA (i.e., DNA exogenous to the cell). Where the cell is a
mammalian cell, a permanent genetic change is generally achieved by
introduction of the DNA into the genome of the cell.
[0075] A transformed cell or host cell generally refers to a cell
(e.g., prokaryotic or eukaryotic) into which (or into an ancestor
of which) has been introduced, by means of recombinant DNA
techniques, a DNA molecule encoding an invention ANKTM1-related
polypeptide or a fragment thereof.
[0076] Transformation of a host cell with recombinant DNA may be
carried out by conventional techniques as are well known to those
skilled in the art. Where the host is prokaryotic, such as E. coli,
competent cells which are capable of DNA uptake can be prepared
from cells harvested after exponential growth phase and
subsequently treated by the CaCl.sub.2 method by procedures well
known in the art. Alternatively, MgCl.sub.2 or RbCl can be used.
Transformation can also be performed after forming a protoplast of
the host cell or by electroporation.
[0077] When the host is a eukaryote, methods for transfection or
transformation with DNA include calcium phosphate co-precipitates,
conventional mechanical procedures such as microinjection,
electroporation, insertion of a plasmid encased in liposomes, or
virus vectors, as well as others known in the art, may be used.
Eukaryotic cells can also be cotransfected with DNA sequences
encoding an invention ANKTM1-related polypeptide and a second
foreign DNA molecule encoding a selectable marker, such as the
herpes simplex thymidine kinase gene. Another method is to use a
eukaryotic viral vector, such as simian virus 40 (SV40) or bovine
papilloma virus, to transiently infect or transform eukaryotic
cells and express the protein. (Eukaryotic Viral Vectors, Cold
Spring Harbor Laboratory, Gluzman ed., 1982). Typically, a
eukaryotic host will be utilized as the host cell. The eukaryotic
cell may be a yeast cell (e.g., Saccharomyces cerevisiae), an
insect cell (e.g., Drosophila sp.) or may be a mammalian cell,
including a human cell.
[0078] Eukaryotic systems, and mammalian expression systems, allow
for post-translational modifications of expressed mammalian
proteins to occur. Eukaryotic cells which possess the cellular
machinery for processing of the primary transcript, glycosylation,
phosphorylation, and, advantageously secretion of the gene product
should be used. Such host cell lines may include, but are not
limited to, CHO, VERO, BHK, HeLa, COS, MDCK, Jurkat, HEK-293, and
W138.
[0079] Mammalian cell systems that utilize recombinant viruses or
viral elements to direct expression may be engineered. For example,
when using adenovirus expression vectors, a polynucleotide encoding
an invention ANKTM1-related polypeptide may be ligated to an
adenovirus transcription/translation control complex, e.g., the
late promoter and tripartite leader sequence. This chimeric
sequence may then be inserted in the adenovirus genome by in vitro
or in vivo recombination. Insertion in a non-essential region of
the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing an
invention ANKTM1-related polypeptide or a fragment thereof in
infected hosts (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci.
USA, 81:3655-3659, 1984). Alternatively, the vaccinia virus 7.5K
promoter may be used. (e.g., see, Mackett, et al., Proc. Natl.
Acad. Sci. USA, 79:7415-7419, 1982; Mackett, et at, J. Virol.
49:857-864, 1984; Panicali, et al., Proc. Natl. Acad. Sci. USA
79:4927-4931, 1982). Of particular interest are vectors based on
bovine papilloma virus, which have the ability to replicate as
extrachromosomal elements (Sarver, et al., Mol. Cell. Biol. 1:486,
1981). Shortly after entry of this DNA into mouse cells, the
plasmid replicates to about 100 to 200 copies per cell.
Transcription of me inserted cDNA does not require integration of
the plasmid into the host's chromosome, thereby yielding a high
level of expression. These vectors can be used for stable
expression by including a selectable marker in the plasmid, such as
the neo gene. Alternatively, the retroviral genome can be modified
for use as a vector capable of introducing and directing the
expression of an invention nociception-related gene in host cells
(Cone & Mulligan, Proc. Natl. Acad. Sci. USA, 81:6349-6353,
1984). High-level expression may also be achieved using inducible
promoters, including, but not limited to, the metallothionein IIA
promoter and heat shock promoters.
[0080] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. Rather man using
expression vectors that contain viral origins of replication, host
cells can be transformed with the cDNA encoding an invention
ANKTM1-related polypeptide controlled by appropriate expression
control elements (e.g., promoter, enhancer, sequences,
transcription terminators, polyadenylation sites, etc.), and a
selectable marker. The selectable marker in the recombinant vector
confers resistance to the selection and allows cells to stably
integrate the plasmid into their chromosomes and grow to form foci,
which in turn can be cloned and expanded into cell lines. For
example, following the introduction of foreign DNA, engineered
cells may be allowed to grow for 1-2 days in an enriched media, and
then are switched to a selective media. A number of selection
systems may be used, including, but not limited to, the herpes
simplex virus thymidine kinase (Wigler, et al., Cell, 11:223,
1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska
& Szybalski, Proc. Natl. Acad. Sci. USA, 48:2026, 1962), and
adenine phosphoribosyltransferase (Lowy, et al., Cell, 22:817,
1980) genes can be employed in tk-, hgprt- or aprt- cells
respectively. Also, anti-metabolite resistance can be used as the
basis of selection for dhfr, which confers resistance to
methotrexate (Wigler, et al., Proc. Natl. Acad. Sci. USA, 77:3567,
1980; O'Hare, et al., Proc. Natl. Acad. Sci. USA, 8:1527, 1981);
gpt, which confers resistance to mycophenolic acid (Mulligan &
Berg, Proc. Natl. Acad. Sci. USA, 78:2072, 1981; neo, which confers
resistance to the aminoglycoside G-418 (Colberre-Garapin, et al.,
J. Mol. Biol. 150:1, 1981); and hygro, which confers resistance to
hygromycin (Santerre, et al., Gene 30:147, 1984) genes. Recently,
additional selectable genes have been described, namely trpB, which
allows cells to utilize indole in place of tryptophan; hisD, which
allows cells to utilize histinol in place of histidine (Hartman
& Mulligan, Proc. Natl. Acad. Sci. USA 85:8047, 1988); and ODC
(ornithine decarboxylase) which confers resistance to the ornithine
decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO
(McConlogue L., In: Current Communications in Molecular Biology,
Cold Spring Harbor Laboratory, ed., 1987).
[0081] The term "primer" as used herein refers to an
oligonucleotide, whether natural or synthetic, which is capable of
acting as a point of initiation of synthesis when placed under
conditions in which primer extension is initiated or possible.
Synthesis of a primer extension product that is complementary to a
nucleic acid strand is initiated in the presence of nucleoside
triphosphates and a polymerase in an appropriate buffer at a
suitable temperature. For instance, if a nucleic acid sequence is
inferred from a protein sequence, a primer generated to synthesize
nucleic acid sequence encoding the protein sequence is actually a
collection of primer oligonucleotides containing sequences
representing all possible codon variations based on the degeneracy
of the genetic code. One or more of the primers in this collection
will be homologous with the end of the target sequence. Likewise,
if a "conserved" region shows significant levels of polymorphism in
a population, mixtures of primers can be prepared that will amplify
adjacent sequences.
[0082] A polypeptide or protein refers to a polymer in which the
monomers are amino acid residues that are joined together through
amide bonds. When the amino acids are alpha-amino acids, either the
L-optical isomer or the D-optical isomer can be used, the L-isomers
being typical. An invention ANKTM1-related polypeptide is intended
to encompass any amino acid sequence and include modified sequences
such as glycoproteins, which provides a polypeptide having
nociception modulating activity. Accordingly, the polypeptides of
the invention are intended to cover naturally occurring proteins,
as well as those that are recombinantly or synthetically
synthesized. In addition, an invention ANKTM1-related polypeptide
can occur in at least two different conformations wherein both
conformations have the same or substantially the same amino acid
sequence but have different three-dimensional structures so long as
they have a biological activity related to nociception. Polypeptide
or protein fragments are also encompassed by the invention.
Fragments can have the same or substantially the same amino acid
sequence as the naturally occurring protein. A polypeptide or
peptide having substantially the same sequence means that an amino
acid sequence is largely, but not entirely, the same, but retains a
functional activity of the sequence to which it is related. In
general polypeptides of the invention include peptides, or
full-length protein, that contains substitutions, deletions, or
insertions into the protein backbone, that would still have an
approximately 70%-90% homology to the original protein over the
corresponding portion. A yet greater degree of departure from
homology is allowed if like-amino acids, i.e. conservative amino
acid substitutions, do not count as a change in the sequence
[0083] A polypeptide may be substantially related but for a
conservative variation, such polypeptides being encompassed by the
invention. A conservative variation denotes the replacement of an
amino acid residue by another, biologically similar residue.
Examples of conservative variations include the substitution of one
hydrophobic residue such as isoleucine, valine, leucine or
methionine for another, or the substitution of one polar residue
for another, such as the substitution of arginine for lysine,
glutamic for aspartic acids, or glutamine for asparagine, and the
like. Other illustrative examples of conservative substitutions
include the changes of: alanine to serine; arginine to lysine;
asparagine to glutamine or histidine; aspartate to glutamate;
cysteine to serine; glutamine to asparagine; glutamate to
aspartate; glycine to proline; histidine to asparagine or
glutamine; isoleucine to leucine or valine; leucine to valine or
isoleucine; lysine to arginine, glutamine, or glutamate; methionine
to leucine or isoleucine; phenylalanine to tyrosine, leucine or
methionine; serine to threonine; threonine to serine; tryptophan to
tyrosine; tyrosine to tryptophan or phenylalanine; valine to
isoleucine to leucine. The term "conservative variation" also
includes the use of a substituted amino acid in place of an
unsubstituted parent amino acid provided that antibodies raised to
the substituted polypeptide also immunoreact with the unsubstituted
polypeptide.
[0084] Modifications and substitutions are not limited to
replacement of amino acids. For a variety of purposes, such as
increased stability, solubility, or configuration concerns, one
skilled in the art will recognize the need to introduce, (by
deletion, replacement, or addition) other modifications. Examples
of such other modifications include incorporation of rare amino
acids, dextra-amino acids, glycosylation sites, cytosine for
specific disulfide bridge formation. The modified peptides can be
chemically synthesized, or the isolated gene can be site-directed
mutagenized, or a synthetic gene can be synthesized and expressed
in bacteria, yeast, baculovirus, tissue culture and so on.
[0085] In one embodiment, the invention provides' an isolated
polynucleotide sequence encoding an invention ANKTM1-related
polypeptide. An invention ANKTM1-related polypeptide can be
characterized by its ability to modulate nociceptive responses.
Polynucleotide sequences of the invention include DNA, cDNA and RNA
sequences that encode a nociceptive-related polypeptide of the
invention as well as complementary sequences thereof. It is
understood that all polynucleotides encoding all or a portion of an
invention ANKTM1-related polypeptide are also included herein, so
long as they encode a polypeptide with nociceptive activity (e.g.,
modulation of nociceptive responses). Such polynucleotides include
naturally occurring, synthetic, and intentionally manipulated
polynucleotides. For example, a nociceptive polynucleotide of the
invention may be subjected to site-directed mutagenesis. The
polynucleotides of the invention include sequences that are
degenerate as a result of the genetic code. There are 20 natural
amino acids, most of which are specified by more than one codon.
Therefore, all degenerate nucleotide sequences are included in the
invention so long as the amino acid sequence of an invention
ANKTM1-related polypeptide encoded by the nucleotide sequence is
functionally unchanged. In addition, polypeptide fragments of an
invention ANKTM1-related polypeptide, and their corresponding
polynucleotide sequences are encompassed by the current invention,
so long as the polypeptides retain some biological activity related
to nociception. A biological activity related to nociception
includes for example, antigenicity or the ability to modulate
nociceptive responses. Assays described in the examples below are
capable of identifying such fragments or modified polypeptides
having a biological activity related to nociception. For example, a
polypeptide that modulates a nociceptive response (e.g.,
intracellular calcium imaging tests) is encompassed by the
invention whether it is expressed in vivo by the organism or
administered to the organism.
[0086] The polynucleotides and polypeptides of this invention were
originally recovered from human and mouse tissue. Thus, the present
invention provides means for isolating the nucleic acid molecules
from other organisms, encoding the invention ANKTM1-related
polypeptides. For example, one may probe a gene library with a
natural or artificially designed probe using art recognized
procedures (see, for example: Current Protocols in Molecular
Biology, Ausubel F. M. et al., (Eds.) Green Publishing Company
Assoc, and John Wiley Interscience, New York, 1989, 1992). It is
appreciated by those skilled in the art that probes can be designed
based on the degeneracy of the genetic code to a sequences
corresponding to a polypeptide or polynucleotide of the
invention.
[0087] In addition, sequencing algorithms can be used to measure
homology or identity between known and unknown sequences. Such
methods and algorithms are useful in identifying corresponding
sequences present in other organisms. Homology or identity is often
measured using sequence analysis software (e.g., Sequence Analysis
Software Package of the Genetics Computer Group, University of
Wisconsin Biotechnology Center, 1710 University Avenue, Madison,
Wis. 53705). Such software matches similar sequences by assigning
degrees of homology to various deletions, substitutions and other
modifications. The terms "homology" and "identity" in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same when compared and aligned for maximum correspondence over
a comparison window or designated region as measured using any
number of sequence comparison algorithms or by manual alignment and
visual inspection.
[0088] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0089] Methods for alignment of sequence for comparison are well
known in the art Optimal alignment of sequences for comparison can
be conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method for Person & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection.
[0090] On example of a useful algorithm is BLAST and BLAST 2.0
algorithms, which are described in Altschul et al., Nuc. Acids Res.
25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410
(1990), respectively. Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information. The BLAST algorithm also performs a statistical
analysis of the similarity between two sequences (see, e.g., Karlin
& Altschul, Proc. Natl. Acad. Sci. USA 90:5873 (1993)). Other
algorithms for determining homology or identity are well known in
the art. Such alignment programs can also be used to screen genome
databases to identify polynucleotide sequences having substantially
identical sequences.
[0091] A "substantially pure polypeptide" is typically pure when it
is at least 60%, by weight, free from the proteins and naturally
occurring organic molecules with which it is naturally associated.
Preferably, the preparation is at least 75%, more preferably at
least 90%, and most preferably at least 99%, by weight, an
invention ANKTM1-related polypeptide. A substantially pure
ANKTM1-related polypeptide may be obtained, for example, by
extraction from a natural source (e.g., a mammalian cell); by
expression of a recombinant nucleic acid encoding an ANKTM1-related
polypeptide; or by chemically synthesizing the protein. Purity can
be measured by any appropriate method, e.g., by column
chromatography, polyacrylamide gel electrophoresis, or by HPLC
analysis.
[0092] In addition to polypeptides of the invention, specifically
disclosed herein is a DNA sequence for an invention ANKTM1-related
polypeptide. DNA sequences of the invention can be obtained by
several methods. For example, the DNA can be isolated using
hybridization or computer-based techniques that are well known in
the art. These include, but are not limited to: 1) hybridization of
genomic libraries with probes to detect homologous nucleotide
sequences; 2) antibody screening of expression libraries to detect
cloned DNA fragments with shared structural features; 3) polymerase
chain reaction (PCR) on genomic DNA using primers capable of
annealing to the DNA sequence of interest; and 4) computer searches
of sequence databases for similar sequences as described above.
[0093] The polynucleotide encoding an invention ANKTM1-related
polypeptide includes complementary polynucleotide sequences, as
well as splice variants thereof. When the sequence is RNA, the
deoxyribonucleotides A, G, C, and T are replaced by ribonucleotides
A, G, C, and U, respectively. Also included in the invention are
fragments (portions) of the above-described nucleic acid sequences
that are at least 15 bases in length, which is sufficient to permit
the fragment to selectively hybridize to DNA that encodes a
polypeptide sequence of the invention. "Selective hybridization" as
used herein refers to hybridization under moderately stringent or
highly stringent physiological conditions (See, for example, the
techniques described in Maniatis et al., 1989 Molecular Cloning A
Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.,
incorporated herein by reference), which distinguishes related from
unrelated nucleotide sequences.
[0094] In nucleic acid hybridization reactions, the conditions used
to achieve a particular level of stringency will vary, depending on
the nature of the nucleic acids being hybridized. For example, the
length, degree of complementarity, nucleotide sequence composition
(e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA)
of the hybridizing regions of the nucleic adds can be considered in
selecting hybridization conditions. An additional consideration is
whether one of the nucleic acids is immobilized, for example, on a
filter.
[0095] An example of progressively higher stringency conditions is
as follows: 2.times.SSC/0.1% SDS at about room temperature
(hybridization conditions); 0.2.times.SSC/0.1% SDS at about room
temperature (low stringency conditions); 0.2.times.SSC/0.1% SDS at
about 42.degree. C. (moderate stringency conditions); and
0.1.times.SSC at about 68.degree. C. (high stringency conditions).
Washing can be carried out using only one of these conditions,
e.g., high stringency conditions, or each of the conditions can be
used, e.g., for 10-15 minutes each, in the order listed above,
repeating any or all of the steps listed. However, as mentioned
above, optimal conditions will vary, depending on the particular
hybridization reaction involved, and can be determined
empirically.
[0096] Oligonucleotides encompassed by the present invention are
also useful as primers for nucleic acid amplification reactions. In
general, the primers used according to the method for the invention
embrace oligonucleotides of sufficient length and appropriate
sequence to provide specific initiation of polymerization of a
significant number of nucleic acid molecules containing the target
nucleic acid under the conditions of stringency for the reaction
utilizing the primers. In this manner, it is possible to
selectively amplify the specific target nucleic acid sequence
containing the nucleic acid of interest.
[0097] Amplified products may be detected by Southern blot
analysis, without using radioactive probes. In such a process, for
example, a small sample of DNA containing a very low level of a
nucleotide sequence is amplified and analyzed via a Southern
blotting technique known to those of skill in the art. The use of
non-radioactive probes or labels is facilitated by the high level
of the amplified signal.
[0098] Screening procedures that rely on nucleic acid hybridization
make it possible to isolate any gene sequence from any organism,
provided the appropriate probe is available. For example, it is
envisioned that such probes can be used to identify other homologs
of the ANKTM1-related polynucleotide family of factors in mammals
or, alternatively, in other organisms such as invertebrates. In
accomplishing this, alignment algorithms (as described above) can
be used to screen genome databases. Alternatively, oligonucleotide
probes, which correspond to a part of the sequence encoding the
protein in question, can be synthesized chemically. This requires
that short, oligopeptide stretches of amino acid sequence must be
known. The DNA sequence encoding the protein can be deduced from
the genetic code, however, the degeneracy of the code must be taken
into account. It is possible to perform a mixed addition reaction
when the sequence is degenerate. This includes a heterogeneous
mixture of denatured double-stranded DNA. For such screening,
hybridization is preferably performed on either single-stranded DNA
or denatured double-stranded DNA. Hybridization is particularly
useful in the detection of DNA clones derived from sources where an
extremely low amount of mRNA sequences relating to the polypeptide
of interest are present. In other words, by using stringent
hybridization conditions directed to avoid non-specific binding, it
is possible, for example, to allow the autoradiographic
visualization of a specific cDNA clone by the hybridization of the
target DNA to that single probe in the mixture which is its
complete complement (Wallace, et al., Nucl. Acid Res., 9:879,
1981).
[0099] When the entire sequence of amino acid residues of the
desired polypeptide is not known, the direct synthesis of DNA
sequences is not possible and the method for choice is use of cDNA
sequences. Among the standard procedures for isolating cDNA
sequences of interest is the formation of plasmid- or
phage-carrying cDNA libraries, which are derived from reverse
transcription of mRNA, which is abundant in donor cells that have a
high level of genetic expression. When used in combination with
polymerase chain reaction technology, even rare expression products
can be cloned.
[0100] DNA sequences encoding an invention ANKTM1-related
polypeptide can be expressed in vitro by DNA transfer into a
suitable host cell, as described above.
[0101] The invention ANKTM1-related polynucleotide sequences may be
inserted into a recombinant expression vector. The term
"Recombinant expression vector" refers to a plasmid, virus or other
vehicle known in the art that has been manipulated by insertion or
incorporation of the nociception-related genetic sequences. Such
expression vectors contain a promoter sequence that facilitates the
efficient transcription of the inserted genetic sequence of the
host. The expression vector typically contains an origin of
replication, a promoter, as well as specific genes that allow
phenotypic selection of the transformed cells. Vectors suitable for
use in the present invention include those described above.
[0102] Polynucleotide sequences encoding a nociception-related
polypeptide can be expressed in either prokaryotes or eukaryotes.
Hosts can include microbial, yeast, insect and mammalian organisms.
Such vectors are used to incorporate DNA sequences of the
invention.
[0103] Methods that are well known to those skilled in the art can
be used to construct expression vectors containing the
ANKTM1-related polypeptide coding sequence and appropriate
transcriptional/translational control signals. These methods
include in vitro recombinant DNA techniques, synthetic techniques,
and in vivo recombination/genetic techniques. (See, for example,
the techniques described in Maniatis et al., 1989, Molecular
Cloning A Laboratory Manual, Cold Spring Harbor Laboratory,
N.Y.).
[0104] The genetic construct can be designed to provide additional
benefits, such as, for example addition of C-terminal or N-terminal
amino acid residues that would facilitate purification by trapping
on columns or by use of antibodies. All those methodologies are
cumulative. For example, a synthetic gene can later be mutagenized.
The choice as to the method for producing a particular construct
can easily be made by one skilled in the art based on practical
considerations: size of the desired peptide, availability and cost
of starting materials, etc. All the technologies involved are well
established and well known in the art. See, for example, Ausubel et
al., Current Protocols in Molecular Biology, Volumes 1 and 2
(1987), with supplements, and Maniatis et al., Molecular Cloning, a
Laboratory Manual, Cold Spring Harbor Laboratory (1989). Yet other
technical references are known and easily accessible to one skilled
in the art.
[0105] In another embodiment, the invention provides antibodies
that bind to an invention ANKTM1-related polypeptide. Such
antibodies are useful for research and diagnostics in the study of
pain, nociceptive responses, central nervous system regulation and
modulation of pain, and nociceptive-associated pathologies in
general. For example, the invention allows for die diagnosis in a
subject of hyperalgesia associated with improper nociceptive pain
regulation. Preferably the subject is a human.
[0106] Such antibodies may be administered alone or contained in a
pharmaceutical composition comprising antibodies against an
invention ANKTM1-related polypeptide and other reagents effective
as modulators of nociceptive pain and associated pain disorders
both in vitro and in vivo.
[0107] The term "epitope", as used herein, refers to an antigenic
determinant on an antigen, such as an invention ANKTM1-related
polypeptide, to which the paratope of an antibody, such as an
antibody that binds to an invention ANKTM1-related polypeptide.
Antigenic determinants usually consist of chemically active surface
groupings of molecules, such as amino acids or sugar side chains,
and can have specific three-dimensional structural characteristics,
as well as specific charge characteristics.
[0108] Antibodies that bind to a polypeptide of the invention can
be prepared using an intact polypeptide or fragments containing
small peptides of interest as the immunizing antigen. The
polypeptide or a peptide used to immunize an animal can be derived
from translated cDNA or chemical synthesis, which can be conjugated
to a carrier protein, if desired. Such commonly used carriers,
which are chemically coupled to the peptide, include keyhole limpet
hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and
tetanus toxoid. The coupled peptide is then used to immunize the
animal (e.g., a mouse, a rat, or a rabbit).
[0109] If desired, polyclonal or monoclonal antibodies can be
further purified, for example, by binding to and elution from a
matrix to which the polypeptide or a peptide to which the
antibodies were raised is bound. Those of skill in the art will
know of various techniques common in the immunology arts for
purification and/or concentration of polyclonal antibodies, as well
as monoclonal antibodies (See for example, Coligan, et al., Unit 9,
Current Protocols in Immunology, Wiley Interscience, 1991,
incorporated by reference).
[0110] It is also possible to use the anti-idiotype technology to
produce monoclonal antibodies that mimic an epitope. For example,
an anti-idiotypic monoclonal antibody made to a first monoclonal
antibody will have a binding domain in the hypervariable region
that is the "image" of the epitope bound by the first monoclonal
antibody.
[0111] Antibodies of the invention include polyclonal antibodies,
monoclonal antibodies, and fragments of polyclonal and monoclonal
antibodies.
[0112] The preparation of polyclonal antibodies is well known to
those skilled in the art. See, for example, Green et al.,
Production of Polyclonal Antisera, in Immunochemical Protocols
(Manson, ed.), pages 1-5 (Humana Press 1992); Coligan et al.,
Production of Polyclonal Antisera in Rabbits, Rats, Mice and
Hamsters, in Current Protocols in Immunology, section 2.4.1 (1992),
which are hereby incorporated by reference.
[0113] The preparation of monoclonal antibodies likewise is
conventional. See, for example, Kohler & Milstein, Nature,
256:495 (1975); Coligan et al., sections 2.5.1-2.6.7; and Harlow et
al., Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor
Pub. 1988), which are hereby incorporated by reference. Briefly,
monoclonal antibodies can be obtained by injecting mice with a
composition comprising an antigen, verifying the presence of
antibody production by removing a serum sample, removing the spleen
to obtain B lymphocytes, fusing the B lymphocytes with myeloma
cells to produce hybridomas, cloning the hybridomas, selecting
positive clones that produce antibodies to the antigen, and
isolating the antibodies from the hybridoma cultures. Monoclonal
antibodies can be isolated and purified from hybridoma cultures by
a variety of well-established techniques. Such isolation techniques
include affinity chromatography with Protein-A Sepharose,
size-exclusion chromatography, and ion-exchange chromatography.
See, e.g., Coligan et al., sections 2.7.1-2.7.12 and sections
2.9.1-2.9.3; Barnes et al., Purification of Immunoglobulin G (IgG),
in Methods in Molecular Biology, Vol. 10, pages 79-104 (Humana
Press 1992). Methods for in vitro and in vivo multiplication of
monoclonal antibodies is well known to those skilled in the art
Multiplication in vitro may be carried out in suitable culture
media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium,
optionally replenished by a mammalian serum such as fetal calf
serum or trace elements and growth-sustaining supplements such as
normal mouse peritoneal exudate cells, spleen cells, bone marrow
macrophages. Production in vitro provides relatively pure antibody
preparations and allows scale-up to yield large amounts of the
desired antibodies. Large-scale hybridoma cultivation can be
carried out by homogenous suspension culture in an airlift reactor,
in a continuous stirrer reactor, or in immobilized or entrapped
cell culture. Multiplication in vivo may be carried out by
injecting cell clones into mammals histocompatible with the parent
cells, e.g., osyngeneic mice, to cause growth of antibody-producing
tumors. Optionally, the animals are primed with a hydrocarbon,
especially oils such as pristane tetramethylpentadecane prior to
injection. After one to three weeks, the desired monoclonal
antibody is recovered from the body fluid of the animal.
[0114] Therapeutic applications for antibodies disclosed herein are
also part of the present invention. For example, antibodies of the
present invention may also be derived from subhuman primate
antibody. General techniques for raising therapeutically useful
antibodies in baboons can be found, for example, in Goldenberg et
al., International Patent Publication WO 91/11465 (1991) and Losman
et al., Int. J. Cancer, 46:310 (1990), which are hereby
incorporated by reference.
[0115] Alternatively, an anti-ANKTM1-related polypeptide antibody
may be derived from a "humanized" monoclonal antibody. Humanized
monoclonal antibodies are produced by transferring mouse
complementarity determining regions from heavy and light variable
chains of the mouse immunoglobulin into a human variable domain,
and then substituting human residues in the framework regions of
the murine counterparts. The use of antibody components derived
from humanized monoclonal antibodies obviates potential problems
associated with the immunogenicity of murine constant regions.
General techniques for cloning murine immunoglobulin variable
domains are described, for example, by Orlandi et al., Proc. Nat'l
Acad. Sci. USA, 86:3833 (1989), which is hereby incorporated in its
entirety by reference. Techniques for producing humanized
monoclonal antibodies are described, for example, by Jones et al.,
Nature, 321:522 (1986); Riechmann et al., Nature, 332:323 (1988);
Verhoeyen et al., Science, 239:1534 (1988); Carter et al., Proc.
Nat'l Acad. Sci. USA, 89:4285 (1992); Sandhu, Crit. Rev. Biotech.,
12:437 (1992); and Singer et al., J. Immunol., 150:2844 (1993),
which are hereby incorporated by reference.
[0116] Antibodies of the invention also may be derived from human
antibody fragments isolated from a combinatorial immunoglobulin
library. See, for example, Barbas et al., Methods: A Companion to
Methods in Enzymology, Vol. 2, page 119 (1991); Winter et al., Ann.
Rev. Immunol. 12:433 (1994), which are hereby incorporated by
reference. Cloning and expression vectors that are useful for
producing a human immunoglobulin phage library can be obtained, for
example, from Stratagene Cloning Systems (La Jolla, Calif.).
[0117] In addition, antibodies of the present invention may be
derived from a human monoclonal antibody. Such antibodies are
obtained from transgenic mice that have been "engineered" to
produce specific human antibodies in response to antigenic
challenge. In this technique, elements of the human heavy and light
chain loci are introduced into strains of mice derived from
embryonic stem cell lines that contain targeted disruptions of the
endogenous heavy and light chain loci. The transgenic mice can
synthesize human antibodies specific for human antigens, and the
mice can be used to produce human antibody-secreting hybridomas.
Methods for obtaining human antibodies from transgenic mice are
described by Green et al., Nature Genet., 7:13 (1994); Lonberg et
al., Nature, 368:856 (1994); and Taylor et al., Int. Immunol.,
6:579 (1994), which are hereby incorporated by reference.
[0118] Antibody fragments of the invention can be prepared by
proteolytic hydrolysis of the antibody or by expression in E. coli
of DNA encoding the fragment. Antibody fragments can be obtained by
pepsin or papain digestion of whole antibodies by conventional
methods. For example, antibody fragments can be produced by
enzymatic cleavage of antibodies with pepsin to provide a 5S
fragment denoted F(ab').sub.2. This fragment can be further cleaved
using a thiol reducing agent, and optionally a blocking group for
the sulfhydryl groups resulting from cleavage of disulfide
linkages, to produce 3.5S Fab' monovalent fragments. Alternatively,
an enzymatic cleavage using pepsin produces two monovalent Fab'
fragments and an Fc fragment directly. These methods are described,
for example, by Goldenberg in U.S. Pat. No. 4,036,945 and No.
4,331,647, and references contained therein. These patents are
hereby incorporated in their entireties by reference. See also
Nisonhoff et al., Arch. Biochem. Biophys., 89:230 (1960); Porter,
Biochem. J., 73:119 (1959); Edelman et al., Methods in Enzymology,
Vol. 1, page 422 (Academic Press 1967); and Coligan et al., at
sections 2.8.1-2.8.10 and 2.10.1-2.10.4.
[0119] Other methods for cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical, or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody,
[0120] For example, Fv fragments comprise an association of V.sub.H
and V.sub.L chains. This association may be noncovalent, as
described in Inbar et al., Proc. Nat'l Acad. Sci. USA, 69:2659
(1972). Alternatively, the variable chains can be linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
glutaraldehyde. See, e.g., Sandhu, supra. Preferably, the Fv
fragments comprise V.sub.H and V.sub.L chains connected by a
peptide linker. These single-chain antigen binding proteins (sFv)
are prepared by constructing a structural gene comprising DNA
sequences encoding the V.sub.H and V.sub.L domains connected by an
oligonucleotide. The structural gene is inserted into an expression
vector, which is subsequently introduced into a host cell such as
E. coli. The recombinant host cells synthesize a single polypeptide
chain with a linker peptide bridging the two V domains. Methods for
producing sFvs are described, for example, by Whitlow et al.,
Methods: A Companion to Methods in Enzymology, Vol. 2, page 97
(1991); Bird et al., Science, 242:423 (1988); Ladner et al., U.S.
Pat. No. 4,946,778; Pack et al., Bio/Technology, 11:1271 (1993);
and Sandhu, supra.
[0121] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing
cells. See, for example, Larrick et al., Methods: A Companion to
Methods in Enzymology, Vol. 2, page 106 (1991).
[0122] In one embodiment, the invention provides a method for
modulating (e.g., inhibiting) nociceptive pain in a subject by
administering to a cell or subject an effective amount of a
composition which contains an invention ANKTM1-related polypeptide,
or biologically functional fragment thereof, or an agent (e.g., an
antibody, ribozyme, antisense molecule, or double-stranded
interfering RNA molecules) that interacts with or inhibits
expression of an invention ANKTM1-related polypeptide.
[0123] As used herein, an "effective amount" of a composition
containing ANKTM1-related polypeptide or an invention
ANKTM1-related polypeptide-modulating agent is defined as that
amount that is effective in modulating nociceptive pain or a
nociceptive response in a subject. For example, an
inhibitory-effective amount would be that amount of the composition
or agent sufficient to inhibit a nociceptive pain response. One
skilled in the art can easily identify agents that modulate as well
as the effective amount of an agent that modulates nociceptive
response by using, for example, the intracellular calcium imaging
tests described in the Examples herein and others as are known in
the art. Briefly, a determination can be made as to the
effectiveness or effective concentration or amount of an agent by
contacting a cell expressing an invention ANKTM1-related
polypeptide, such as mANKTM1 or hANKTM1 with the test agent or
concentration and then exposing the cell to a noxious agent (e.g.,
a temperature of about 10-15.degree. C.) and determining the cell's
response (e.g., using electrophysiological studies or intracellular
calcium imaging techniques as described herein) in the presence and
absence of the agent.
[0124] In another embodiment, the present invention provides a
method for modulating expression of an invention ANKTM1-related
polypeptide as well as methods for screening for agents that
modulate ANKTM1-related polypeptide gene expression. In this
embodiment, a cell or subject is contacted with an agent suspected
or known to have ANKTM1-related polypeptide expression modulating
activity. The change in ANKTM1-related polypeptide gene expression
is then measured as compared to a control or standard sample. The
control or standard sample can be the baseline expression of the
cell or subject prior to contact with the agent. An agent that
modulates ANKTM1-related polypeptide gene expression may be a
polynucleotide, for example, the polynucleotide may be an
antisense, a triplex agent, a ribozyme, or a double-stranded
interfering RNA. For example, an antisense molecule may be directed
to the structural gene region or to the promoter region of
ANKTM1-related polypeptide gene. The agent may be an agonist,
antagonist, peptide, peptidomimetic, antibody, or chemical.
[0125] Double-stranded interfering RNA molecules are especially
useful to inhibit expression of a target gene. For example,
double-stranded RNA molecules can be injected into a target cell or
organism to inhibit expression of a gene and the resultant gene
products activity. It has been found that such double-stranded RNA
molecules are more effective at inhibiting expression than either
RNA strand alone. (Fire et al., Nature, 1998,
19:391(6669):806-11).
[0126] When a disorder is associated with abnormal expression of an
invention ANKTM1-related polypeptide (e.g., overexpression, or
expression of a mutated form of the protein), a therapeutic
approach that directly interferes with the translation of an
invention ANKTM1-related polypeptide is possible. Alternatively,
similar methodology may be used to study gene activity. For
example, antisense nucleic acid, double-stranded interfering RNA or
ribozymes could be used to bind to the invention ANKTM1-related
polypeptide mRNA sequence or to cleave it. Antisense RNA or DNA
molecules bind specifically with a targeted gene's RNA message,
interrupting the expression of that gene's protein product. The
antisense binds to the messenger RNA forming a double stranded
molecule that cannot be translated by the cell. Antisense
oligonucleotides of about 15-25 nucleotides are preferred since
they are easily synthesized and have an inhibitory effect just like
antisense RNA molecules. In addition, chemically reactive groups,
such as iron-linked ethylenediaminetetraacetic acid (EDTA-Fe) can
be attached to an antisense oligonucleotide, causing cleavage of
the RNA at the site of hybridization. Antisense nucleic acids are
DNA or RNA molecules that are complementary to at least a portion
of a specific mRNA molecule (Weintraub, Scientific American,
262:40, 1990). In the cell, the antisense nucleic acids hybridize
to the corresponding mRNA, forming a double-stranded molecule. The
antisense nucleic adds interfere with the translation of the mRNA,
since the cell will not translate an mRNA that is double-stranded.
Antisense oligomers of about 15 nucleotides are preferred, since
they are easily synthesized and are less likely to cause problems
than larger molecules when introduced into the target
ANKTM1-related polypeptide-producing cell. The use of antisense
methods to inhibit the in vitro translation of genes is well known
in the art (Marcus-Sakura, Anal. Biochem., 172:289, 1988).
[0127] Use of an oligonucleotide to stall transcription is known as
the triplex strategy since the oligomer winds around double-helical
DNA, forming a three-strand helix. Therefore, these triplex
compounds can be designed to recognize a unique site on a chosen
gene (Maher, et al., Antisense Res. And Dev., 1:227, 1991; Helene,
Anticancer Drug Design, 6:569, 1991).
[0128] Ribozymes are RNA molecules possessing the ability to
specifically cleave other single-stranded RNA in a manner analogous
to DNA restriction endonucleases. Through the modification of
nucleotide sequences that encode these RNAs, it is possible to
engineer molecules that recognize specific nucleotide sequences in
an RNA molecule and cleave it (Cech, J. Amer. Med. Assn., 260:3030,
1988). A major advantage of this approach is that, because they are
sequence-specific, only mRNAs with particular sequences are
inactivated.
[0129] There are two basic types of ribozymes namely,
tetrahymena-type (Hasselhoff, Nature, 334:585, 1988) and
"hammerhead"-type. Tetrahymena-type ribozymes recognize sequences
that are four bases in length, while "hammerhead"-type ribozymes
recognize base sequences 11-18 bases in length. The longer the
recognition sequence, the greater the likelihood that the sequence
will occur exclusively in the target mRNA species. Consequently,
hammerhead-type ribozymes are preferable to tetrahymena-type
ribozymes for inactivating a specific mRNA species and 18-base
recognition sequences are preferable to shorter recognition
sequences.
[0130] These and other uses of antisense and ribozymes methods to
inhibit the in vivo translation of genes are known in the art
(e.g., De Mesmaeker, et al., Curr. Opin. Struct. Biol., 5:343,
1995; Gewirtz, A. M., et al., Proc. Natl. Acad. Sci. U.S.A.,
93:3161, 1996b; Stein, C. A., Chem. and Biol. 3:319, 1996).
[0131] Delivery of antisense, triplex agents, ribozymes,
competitive inhibitors, double-stranded interfering RNA and the
like can be achieved using a recombinant expression vector such as
a chimeric virus or a colloidal dispersion system or by injection.
Various viral vectors which can be utilized for gene therapy as
taught herein include adenovirus, herpes virus, vaccinia, or,
preferably, an RNA virus such as a retrovirus. Preferably, the
retroviral vector is a derivative of a murine or avian retrovirus.
Examples of retroviral vectors in which a single foreign gene can
be inserted include, but are not limited to: Moloney murine
leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),
murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A
number of additional retroviral vectors can incorporate multiple
genes. All of these vectors can transfer or incorporate a gene for
a selectable marker so that transduced cells can be identified and
generated. By inserting a polynucleotide sequence of interest into
the viral vector, along with another gene that encodes the ligand
for a receptor on a specific target cell, for example, the vector
is now target specific. Retroviral vectors can be made target
specific by inserting, for example, a polynucleotide encoding a
sugar, a glycolipid, or a protein. Preferred targeting is
accomplished by using an antibody to target the retroviral vector.
Those of skill in the art will know of, or can readily ascertain
without undue experimentation, specific polynucleotide sequences
which can be inserted into the retroviral genome to allow target
specific delivery of the retroviral vector containing the antisense
polynucleotide.
[0132] Another targeted delivery system for polynucleotides is a
colloidal dispersion system. Colloidal dispersion systems include
macromolecule complexes, nanocapsules, microspheres, beads, and
lipid-based systems including oil-in-water emulsions, micelles,
mixed micelles, and liposomes. The preferred colloidal system of
this invention is a liposome. Liposomes are artificial membrane
vesicles that are useful as delivery vehicles in vitro and in vivo.
It has been shown that large unilamellar vesicles (LUV), which
range in size from 0.2-4.0 urn can encapsulate a substantial
percentage of an aqueous buffer containing large macromolecules.
RNA, DNA and intact virions can be encapsulated within the aqueous
interior and be delivered to cells in a biologically active form
(Fraley, et al., Trends Biochem. Sci., 6:77, 1981). In addition to
mammalian cells, liposomes have been used for delivery of
polynucleotides in plant, yeast and bacterial cells. In order for a
liposome to be an efficient gene transfer vehicle, the following
characteristics should be present: (1) encapsulation of the genes
of interest at high efficiency while not compromising their
biological activity; (2) preferential and substantial binding to a
target cell in comparison to non-target cells; (3) delivery of the
aqueous contents of the vesicle to the target cell cytoplasm at
high efficiency; and (4) accurate and effective expression of
genetic information (Mannino, et al., Biotechniques, 6:682,
1988).
[0133] The composition of the liposome is usually a combination of
phospholipids, particularly high-phase-transition-temperature
phospholipids, usually in combination with steroids, especially
cholesterol. Other phospholipids or other lipids may also be used.
The physical characteristics of liposomes depend on pH, ionic
strength, and the presence of divalent cations.
[0134] Examples of lipids useful in liposome production include
phosphatidyl compounds, such as phosphatidylglycerol,
phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
sphingolipids, cerebrosides, and gangliosides. Particularly useful
are diacylphosphatidyl-glycerols, where the lipid moiety contains
from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and
is saturated. Illustrative phospholipids include egg
phosphatidylcholine, dipalmitoylphosphatidylcholine and
distearoylphosphatidylcholine.
[0135] The targeting of liposomes has been classified based on
anatomical and mechanistic factors. Anatomical classification is
based on the level of selectivity, for example, organ-specific,
cell-specific, and organelle-specific. Mechanistic targeting can be
distinguished based upon whether it is passive or active. Passive
targeting utilizes the natural tendency of liposomes to distribute
to cells of the reticulo-endothelial system (RES) in organs that
contain sinusoidal capillaries. Active targeting, on the other
hand, involves alteration of the liposome by coupling the liposome
to a specific ligand such as a monoclonal antibody, sugar,
glycolipid, or protein, or by changing the composition or size of
the liposome in order to achieve targeting to organs and cell types
other than the naturally occurring sites of localization.
[0136] The surface of the targeted delivery system may be modified
in a variety of ways. In the case of a liposomal targeted delivery
system, lipid groups can be incorporated into the lipid bilayer of
the liposome in order to maintain the targeting ligand in stable
association with the liposomal bilayer. Various linking groups can
be used for joining the lipid chains to the targeting ligand. In
general, the compounds bound to the surface of the targeted
delivery system will be ligands and receptors which will allow the
targeted delivery system to find and "home in" on the desired
cells. A ligand may be any compound of interest that will bind to
another compound, such as a receptor.
[0137] The agents useful in the method for the invention can be
administered, for in vivo application, parenterally by injection or
by gradual perfusion over time. Administration may be
intravenously, intraperitoneally, intramuscularly, subcutaneously,
intracavity, or transdermally. For in vitro studies the agents may
be added or dissolved in an appropriate biologically acceptable
buffer and added to a cell or tissue.
[0138] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's intravenous vehicles include fluid and nutrient
replenishers, electrolyte replenishers (such as those based on
Ringer's dextrose), and the like. Preservatives and other additives
may also be present such as, for example, antimicrobials,
anti-oxidants, chelating agents and inert gases and the like.
[0139] It is envisioned that the invention can be used to treat
pathologies associated with hyperalgesia and nociceptive pain
associated disorders. Therefore, the present invention encompasses
methods for ameliorating a disorder associated with nociception,
including treating a subject having the disorder, at the site of
the disorder, with an agent which modulates an invention
ANKTM1-related polypeptide, Generally, the terms "treating",
"treatment" and the like are used herein to mean affecting a
subject, tissue or cell to obtain a desired pharmacologic and/or
physiologic effect. The effect may be prophylactic in terms of
completely or partially preventing a disease or sign or symptom
thereof, and/or may be therapeutic in terms of a partial or
complete cure for hyperalgesia and nociceptive pain associated
disorders and/or adverse effect, such as pain, attributable to the
hyperalgesia and nociceptive pain associated disorders. "Treating"
as used herein covers any treatment of, or prevention of
hyperalgesia and nociceptive pain associated disorders in an
invertebrate, a vertebrate, a mammal, particularly a human, and
includes: (a) preventing the disorder from occurring in a subject
that may be predisposed to the disorder, but has not yet been
diagnosed as having it; (b) inhibiting the disorder, i.e.,
arresting its development; or (c) relieving or ameliorating the
disorder cause regression of one or more symptoms of the
hyperalgesia and/or nociceptive pain associated disorder.
[0140] The invention includes various pharmaceutical compositions
useful for ameliorating symptoms attributable to a nociceptive
pain-associated disorder. The pharmaceutical compositions according
to the invention are prepared by bringing an antibody against
ANKTM1-related polypeptide, a polypeptide or peptide derivative of
an invention ANKTM1-related polypeptide, an invention
ANKTM1-related polypeptide mimetic, a drug, chemical or combination
of chemicals or an invention ANKTM1-related polypeptide-modulating
agent into a form suitable for administration to a subject using
carriers, excipients and additives or auxiliaries. Frequently used
carriers or auxiliaries include magnesium carbonate, titanium
dioxide, lactose, mannitol and other sugars, talc, milk protein,
gelatin, starch, vitamins, cellulose and its derivatives, animal
and vegetable oils, polyethylene glycols and solvents, such as
sterile water, alcohols, glycerol and polyhydric alcohols.
Intravenous vehicles include fluid and nutrient replenishers.
Preservatives include antimicrobial, anti-oxidants, chelating
agents and inert gases. Other pharmaceutically acceptable carriers
include aqueous solutions, non-toxic excipients, including salts,
preservatives, buffers and the like, as described, for instance, in
Remington's Pharmaceutical Sciences, 15th ed. Easton: Mack
Publishing Co., 1405-1412, 1461-1487 (1975) and The National
Formulary XIV., 14th ed. Washington: American Pharmaceutical
Association (1975), the contents of which are hereby incorporated
by reference. The pH and exact concentration of the various
components of the pharmaceutical composition are adjusted according
to routine skills in the art. See Goodman and Oilman's The
Pharmacological Basis for Therapeutics (7th ed.).
[0141] The pharmaceutical compositions are preferably prepared and
administered in dose units. Solid dose units are tablets, capsules
and suppositories. For treatment of a subject, depending on
activity of the compound, manner of administration, nature and
severity of the disorder, age and body weight of the subject,
different daily doses are necessary. Under certain circumstances,
however, higher or lower daily doses may be appropriate. The
administration of the daily dose can be carried out both by single
administration in the form of an individual dose unit or else
several smaller dose units and also by multiple administrations of
subdivided doses at specific intervals.
[0142] The pharmaceutical compositions according to the invention
may be administered locally or systemically in a therapeutically
effective dose. Amounts effective for this use will, of course,
depend on the severity of the disease and the weight and general
state of the subject. Typically, dosages used in vitro may provide
useful guidance in the amounts useful for in situ administration of
the pharmaceutical composition, and animal models may be used to
determine effective dosages for treatment of particular disorders.
Various considerations are described, e.g., in Langer, Science,
249:1527. (1990); Gilman et al., (eds.) (1990), each of which is
herein incorporated by reference.
[0143] In one embodiment, the invention provides a pharmaceutical
composition useful for administering an invention ANKTM1-related
polypeptide, or nucleic acid encoding an invention ANKTM1-related
polypeptide, to a subject in need of such treatment.
"Administering" the pharmaceutical composition of the present
invention may be accomplished by any means known to the skilled
artisan. Preferably a "subject" refers to a mammal, most preferably
a human, but may be any organism.
[0144] An invention ANKTM1-related polypeptide or antibody can be
administered parenterally, enterically, by injection, rapid
infusion, nasopharyngeal absorption, dermal absorption, rectally
and orally. Pharmaceutically acceptable carrier preparations for
parenteral administration include sterile or aqueous or non-aqueous
solutions, suspensions, and emulsions. Examples of non-aqueous
solvents are propylene glycol, polyethylene glycol, vegetable oils
such as olive oil, and injectable organic esters such as ethyl
oleate. Carriers for occlusive dressings can be used to increase
skin permeability and enhance antigen absorption. liquid dosage
forms for oral administration may generally comprise a liposome
solution containing the liquid dosage form. Suitable solid or
liquid pharmaceutical preparation forms are, for example, granules,
powders, tablets, coated tablets, (micro)capsules, suppositories,
syrups, emulsions, suspensions, creams, aerosols, drops or
injectable solution in ampule form and also preparations with
protracted release of active compounds, in whose preparation
excipients and additives and/or auxiliaries such as disintegrants,
binders, coating agents, swelling agents, lubricants, flavorings,
sweeteners and elixirs containing inert diluents commonly used in
the art, such as purified water.
[0145] In another embodiment, the invention provides a method for
identifying a agent which modulates ANKTM1-related polypeptide
expression or activity including incubating components comprising
the agent and an invention ANKTM1-related polypeptide, or a
recombinant cell expressing an invention ANKTM1-related
polypeptide, under conditions sufficient to allow the agent to
interact and determining the affect of the agent on the expression
or activity of the gene or polypeptide, respectively. The term
"affect", as used herein, encompasses any means by which gene
expression or protein activity can be modulated. Such agents can
include, for example, polypeptides, peptidomimetics, chemical
compounds, small molecules and biologic agents as described
below.
[0146] Incubating includes conditions that allow contact between
the test agent and an invention ANKTM1-related polypeptide, a cell
expressing an invention ANKTM1-related polypeptide or nucleic acid
encoding an invention ANKTM1-related polypeptide, Contacting
includes in solution and in solid phase. The test agent may
optionally be a combinatorial library for screening a plurality of
agents. Agents identified in the method for the invention can be
further evaluated, detected, cloned, sequenced, and the like,
either in solution or after binding to a solid support, by any
method usually applied to the detection of a specific DNA sequence
such as PCR, oligomer restriction (Saiki, et al., Bio/Technology,
3:1008-1012, 1985), oligonucleotide ligation assays (OLAs)
(Landegren, et al., Science, 241:1077, 1988), and the like.
Molecular techniques for DNA analysis have been reviewed
(Landegren, et al., Science, 242:229-237, 1988).
[0147] Thus, the method for the invention includes combinatorial
chemistry methods for identifying chemical agents that bind to or
affect ANKTM1-related polypeptide expression or activity.
[0148] Areas of investigation are the development of therapeutic
treatments. The screening identifies agents that provide modulation
of ANKTM1-related polypeptide function in targeted organisms. Of
particular interest are screening assays for agents mat have a low
toxicity or a reduced number of side effects for humans.
[0149] The term "agent" as used herein describes any molecule, e.g.
protein or pharmaceutical, with the capability of altering or
mimicking the physiological function or expression of an invention
ANKTM1-related polypeptide. Generally, a plurality of assay
mixtures is run in parallel with different agent concentrations to
obtain a differential response to the various concentrations.
Typically, one of these concentrations serves as a negative
control, i.e. at zero concentration or below the level of
detection.
[0150] As used herein, an agent that acts, directly or indirectly
via a receptor or receptors responsible for mediating or involved
in peripheral hyperalgesia, by antagonizing the activity of
hyperalgesia mediating agents, such as a prostaglandin, is an agent
intended for use herein, if it also does not exhibit CNS effects as
defined herein. Such agent is a peripheral antihyperalgesic. The
activity of antihyperalgesic agents is distinct from the activity
of centrally acting analgesic agents (e.g., agents that act by
virtue of crossing the blood brain barrier). Anti-hyperalgesic
agents act to block hypersensitivity. The compositions and methods
for the invention are intended for prevention and/or the
amelioration of the symptoms of hyperalgesia by decreasing or
eliminating the hyperalgesia or by preventing its onset An
antihyperalgesic agent is distinct from a local anesthetic, which
is an agent that produces numbness by abolishing sensitivity to
touch, and other stimuli, including pain stimuli. Local anesthetics
abolish sensation, including pain, by blocking conduction in nerve
axons in the peripheral nervous system. Antihyperalgesics, on the
other hand, alleviate pain by elevating a patient's threshold to
pain. Thus, unlike anesthetics, antihyperalgesics reduce sensation
to pain during states of increased sensitivity (e.g., hyperalgesia)
without substantially affecting normal sensitivity to touch and/or
other stimuli.
[0151] Antihyperalgesics are agents that may reduce
hypersensitivity to touch and other stimuli that would not, under
normal circumstances, evoke a pain response. The hyperalgesic
response is an exaggerated response, such as excessive
sensitiveness or sensibility to pain from touch, slight exertion,
warmth and the like. Antihyperalgesics may be identified, for
example, by the Randall-Selitto method (see, e.g., Randall et al.,
Arch. Int. Pharmacodyn. 111:409-419, 1957), as well as die
formalin, carrageenan and yeast induced inflammation methods. In
addition to the antihyperalgesic effect, the antihyperalgesic
agents provided herein may concurrently provide an analgesic
effect.
[0152] Analgesics are agents that may reduce a patient's perception
of pain evoked by stimuli that are acutely painful under normal
circumstances. Thus, analgesics may be effective in reducing the
acute and immediate pain associated with trauma (e.g., pinpricks,
burns, or crushing wounds) as well as chronic pain, that is not
normally associated with peripheral sensitization, such as cancer
or headache pain.
[0153] In addition, cells or organisms which have a mutation in an
invention ANKTM1-related polypeptide sequence may be used as models
to screen for agents which modulate disorders associated with the
mutation. For example, if organisms are identified that lack normal
nociceptive response activity due to a mutation in an invention
nociception-related polypeptide sequence, administration of agents
to an organism having such a mutation, or cells derived or
recombinantly modified to have a reduced nociceptive activity, may
be used to determine the effect of the drug or agent on
nociception.
[0154] In a further embodiment, the invention provides a method for
detecting ANKTM1-related polypeptide or polynucleotide or
diagnosing a nociceptive-associated disorder in a subject including
contacting a cell component containing ANKTM1-related polypeptide
or polynucleotide with a reagent which binds to the cell
polypeptide or polynucleotide (herein after cell component). The
cell component can be or contain a nucleic acid, such as DNA or
RNA, or a protein. When the component is nucleic acid, the reagent
is a nucleic acid probe or PCR primer. When the cell component is
protein, the reagent is an antibody probe. The probes are
detectably labeled, for example, with a radioisotope, a fluorescent
compound, a bioluminescent compound, a chemiluminescent compound, a
metal chelator or an enzyme. Those of ordinary skill in the art
will know of other labels suitable for binding to an antibody or
nucleic acid probe, or will be able to ascertain such, using
routine experimentation. There are many different labels and
methods for labeling known to those of ordinary skill in the art.
Examples of the types of labels, which can be used in the present
invention, include enzymes, radioisotopes, colloidal metals,
fluorescent compounds, chemiluminescent compounds, and
bioluminescent compounds. In addition, the antibodies, polypeptides
and polynucleotide sequences of the invention can be used to
diagnosis a nociceptive disorder.
[0155] A monoclonal antibody of the invention, directed toward
ANKTM1-related polypeptide is useful for the in vivo and in vitro
detection of antigen. The detectably labeled monoclonal antibody is
given in a dose that is diagnostically effective. The term
"diagnostically effective" means that the amount of detectably
labeled monoclonal antibody is administered in sufficient quantity
to enable detection of ANKTM1-related polypeptide antigen for which
the monoclonal antibodies are specific.
[0156] The concentration of a detectably labeled monoclonal
antibody administered to a subject should be sufficient such that
the binding to those cells, body fluid, or tissue having
ANKTM1-related polypeptide that is detectable compared to the
background, Further, it is desirable that the detectably labeled
monoclonal antibody be rapidly cleared from the circulatory system
in order to give the best target-to-background signal ratio.
[0157] For in vivo diagnostic imaging, the type of detection
instrument available is a major factor in selecting a given
radioisotope. The radioisotope chosen must have a type of decay
that is detectable for a given type of instrument. Still another
important factor in selecting a radioisotope for in vivo diagnosis
is the half-life of the radioisotope which should be long enough so
that it is still detectable at the time of maximum uptake by the
target, but short enough so that deleterious radiation with respect
to the host is minimized. Ideally, a radioisotope used for in vivo
imaging will lack a particle emission, but produce a large number
of photons in the 140-250 nm key range, which may be readily
detected by conventional gamma cameras.
[0158] For in vivo diagnosis, radioisotopes may be bound to
immunoglobulin either directly or indirectly by using an
intermediate functional group. Intermediate functional groups,
which often are used to bind radioisotopes that exist as metallic
ions to immunoglobulins, are the bifunctional chelating agents such
as diethylenetriaminepentacetic acid (DTPA) and
ethylenediaminetetraacetic acid (EDTA) and similar molecules.
Typical examples of metallic ions that can be bound to the
monoclonal antibodies of the invention are .sup.111In, .sup.97Ru,
.sup.67Ga, .sup.68Ga, .sup.72As, .sup.89Zr, and .sup.201Tl.
[0159] The monoclonal antibodies of the invention can also be
labeled with a paramagnetic isotope for purposes of in vivo
diagnosis, as in magnetic resonance imaging (MRI) or electron spin
resonance (ESR). In general, any conventional method for
visualizing diagnostic imaging can be utilized. Usually gamma and
positron emitting radioisotopes are used for camera imaging and
paramagnetic isotopes for MRI. Elements that are particularly
useful in such techniques include .sup.157Gd, .sup.55M, .sup.162Dy,
.sup.52Cr, and .sup.56Fe.
[0160] In another embodiment, nucleic acid probes can be used to
identify a polynucleotide encoding an invention ANKTM1-related
polypeptide from a specimen obtained from a subject. Examples of
specimens from which nucleic acid sequence encoding an invention
ANKTM1-related polypeptide can be derived include insect, human,
swine, porcine, feline, canine, equine, murine, cervine, caprine,
lupine, leporidine and bovine species.
[0161] Oligonucleotide probes, which correspond to a part of the
sequence encoding the protein in question, can be synthesized
chemically. This requires that short, oligopeptide stretches of
amino acid sequence must be known. The DNA sequence encoding the
protein can be deduced from the genetic code, however, the
degeneracy of the code must be taken into account. It is possible
to perform a mixed addition reaction when the sequence is
degenerate. This includes a heterogeneous mixture of denatured
double-stranded DNA. For such screening, hybridization is
preferably performed on either single-stranded DNA or denatured
double-stranded DNA. Hybridization is particularly useful in the
detection of cDNA clones derived from sources where an extremely
low amount of mRN A sequences relating to the polypeptide of
interest are present. In other words, by using stringent
hybridization conditions directed to avoid non-specific binding, it
is possible, for example, to allow the autoradiographic
visualization of a specific cDNA clone by the hybridization of the
target DNA to that single probe in the mixture which is its
complete complement (Wallace, et al., Nucl. Acid Res. 9:879,
1981).
[0162] In an embodiment of the invention, purified nucleic acid
fragments containing intervening sequences or oligonucleotide
sequences of 10-50 base pairs are radioactively labeled. The
labeled preparations are used to probe nucleic acids from a
specimen by the Southern hybridization technique. Nucleotide
fragments from a specimen, before or after amplification, are
separated into fragments of different molecular masses by gel
electrophoresis and transferred to filters that bind nucleic acid.
After exposure to the labeled probe, which will hybridize to
nucleotide fragments containing target nucleic acid sequences,
binding of the radioactive probe to target nucleic acid fragments
is identified by autoradiography (see Genetic Engineering, 1, ed.
Robert Williamson, Academic Press, (1981), 72-81). Alternatively,
nucleic acid from the specimen can be bound directly to filters to
which the radioactive probe selectively attaches by binding nucleic
acids having the sequence of interest Specific sequences and the
degree of binding are quantitated by directly counting the
radioactive emissions,
[0163] Where the target nucleic acid is not amplified, detection
using an appropriate hybridization probe may be performed directly
on the separated nucleic acid. In those instances where the target
nucleic acid is amplified, detection with the appropriate
hybridization probe would be performed after amplification.
[0164] For the most part, the probe will be detectably labeled with
an atom or inorganic radical, most commonly using radionuclides,
but also heavy metals can be used. Conveniently, a radioactive
label may be employed. Radioactive labels include .sup.32P,
.sup.125I, .sup.3H, .sup.14C, .sup.111In, .sup.99Tc, or the like.
Any radioactive label may be employed which provides for an
adequate signal and has sufficient half-life. Other labels include
ligands, which can serve as a specific binding pair member for a
labeled ligand, and the like. A wide variety of labels routinely
employed in immunoassays can readily be employed in the present
assay. The choice of the label will be governed by the effect of
the label on the rate of hybridization and binding of the probe to
mutant nucleotide sequence. It will be necessary that the label
provide sufficient sensitivity to detect the amount of mutant
nucleotide sequence available for hybridization.
[0165] The manner in which the label is bound to the probe will
vary depending upon the nature of the label. For a radioactive
label, a wide variety of techniques can be employed. Commonly
employed is nick translation with a .sup.32P-dNTP or terminal
phosphate hydrolysis with alkaline phosphatase followed by labeling
with radioactive .sup.32P employing .sup.32P-NTP and T4
polynucleotide kinase. Alternatively, nucleotides can be
synthesized where one or more of the elements present are replaced
with a radioactive isotope, e.g., hydrogen with tritium. If
desired, complementary labeled strands can be used as probes to
enhance the concentration of hybridized label.
[0166] Where other radionucleotide labels are involved, various
linking groups can be employed. A terminal hydroxyl can be
esterified with inorganic acids, e.g., .sup.32P phosphate, or
.sup.14C organic acids, or else esterified to provide linking
groups to the label. Alternatively, intermediate bases may be
substituted with activatable linking groups that can then be linked
to a label.
[0167] Enzymes of interest as reporter groups will primarily be
hydrolases, particularly esterases and glycosidases, or
oxidoreductases, particularly peroxidases. Fluorescent compounds
include fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, umbelliferone, and so forth. Chemiluminescers
include luciferin, and 2,3-dihydrophthalazinediones (e.g.,
luminol).
[0168] Standard hybridization techniques for detecting a nucleic
acid sequence are known in the art. The particular hybridization
technique is not essential to the invention. Other hybridization
techniques are described by Gall and Pardue, Proc. Natl. Acad. Sci.
63:378, 1969); and John, et al., Nature, 223:582, 1969). As
improvements are made in hybridization techniques they can readily
be applied in the method for the invention.
[0169] The amount of labeled probe present in the hybridization
solution will vary widely, depending upon the nature of the label,
the amount of the labeled probe that can reasonably bind to the
filter, and the stringency of the hybridization. Generally,
substantial excess over stoichiometric concentrations of the probe
will be employed to enhance the rate of binding of the probe to the
fixed target nucleic acid.
[0170] The present invention also contemplates transgenic non-human
organisms, including invertebrates, vertebrates and mammals. For
purposes of the subject invention, these animals are referred to as
"transgenic" when such animal has had a heterologous DNA sequence,
or one or more additional DNA sequences normally endogenous to the
animal (collectively referred to herein as "transgenes")
chromosomally integrated into the germ cells of the animal. The
transgenic animal (including its progeny) will also have the
transgene integrated into the chromosomes of somatic cells.
[0171] Various methods to make the transgenic animals of the
subject invention can be employed. Generally speaking, three such
methods may be employed. In one such method, an embryo at the
pronuclear stage (a "one cell embryo") is harvested from a female
and the transgene is microinjected into the embryo, in which case
the transgene will be chromosomally integrated into both the germ
cells and somatic cells of the resulting mature animal. In another
such method, embryonic stem cells are isolated and the transgene
incorporated therein by electroporation, plasmid transfection or
microinjection, followed by reintroduction of the stem cells into
the embryo where they colonize and contribute to the germ line.
Methods for microinjection of mammalian species are described in
U.S. Pat. No. 4,873,191. In yet another such method, embryonic
cells are infected with a retrovirus containing the transgene
whereby the germ cells of the embryo have the transgene
chromosomally integrated therein. When the animals to be made
transgenic are avian, because avian fertilized ova generally go
through cell division for the first twenty hours in the oviduct,
microinjection into the pronucleus of the fertilized egg is
problematic due to the inaccessibility of the pronucleus.
Therefore, of the methods to make transgenic animals described
generally above, retrovirus infection is preferred for avian
species, for example as described in U.S. Pat. No. 5,162,215. If
microinjection is to be used with avian species, however, a
published procedure by Love et al., (Biotechnology, 12, January
1994) can be utilized whereby the embryo is obtained from a
sacrificed hen approximately two and one-half hours after the
laying of the previous laid egg, the transgene is microinjected
into the cytoplasm of the germinal disc and the embryo is cultured
in a host shell until maturity. When the animals to be made
transgenic are bovine or porcine, microinjection can be hampered by
the opacity of the ova thereby making the nuclei difficult to
identify by traditional differential interference-contrast
microscopy. To overcome this problem, the ova can first be
centrifuged to segregate the pronuclei for better
visualization.
[0172] The "non-human animals" of the invention include, for
example, bovine, porcine, ovine and avian animals (e.g., cow, pig,
sheep, chicken, turkey). The "transgenic non-human animals" of the
invention are produced by introducing "transgenes" into the germ
line of the non-human animal. Embryonal target cells at various
developmental stages can be used to introduce transgenes. Different
methods are used depending on the stage of development of the
embryonal target cell. The zygote is the best target for
micro-injection. The use of zygotes as a target for gene transfer
has a major advantage in that in most cases the injected DNA will
be incorporated into the host gene before the first cleavage
(Brinster et al., Proc. Natl. Acad. Sci, USA 82:4438-4442, 1985).
As a consequence, all cells of the transgenic non-human animal will
carry the incorporated transgene. This will in general also be
reflected in the efficient transmission of the transgene to
offspring of the founder since 50% of the germ cells will harbor
the transgene.
[0173] The term "transgenic" is used to describe an animal that
includes exogenous genetic material within all of its cells. A
"transgenic" animal can be produced by crossbreeding two chimeric
animals that include exogenous genetic material within cells used
in reproduction. Twenty-five percent of the resulting offspring
will be transgenic i.e., animals which include the exogenous
genetic material within all of their cells in both alleles. 50% of
the resulting animals will include the exogenous genetic material
within one allele and 25% will include no exogenous genetic
material.
[0174] In the microinjection method useful in the practice of the
invention, the transgene is digested and purified free from any
vector DNA e.g. by gel electrophoresis. It is preferred that the
transgene include an operatively associated promoter which
interacts with cellular proteins involved in transcription,
ultimately resulting in constitutive expression. Promoters useful
in this regard include those from cytomegalovirus (CMV), Moloney
leukemia virus (MLV), and herpes virus, as well as those from the
genes encoding metallothionein, skeletal actin, P-enolpyruvate
carboxylase (PEPCK), phosphoglycerate (PGK), DHFR, and thymidine
kinase. Promoters for viral long terminal repeats (LTRS) such as
Rous Sarcoma Virus can also be employed. When the animals to be
made transgenic are avian, preferred promoters include those for
the chicken-globin gene, chicken lysozyme gene, and avian leukosis
virus. Constructs useful in plasmid transfection of embryonic stem
cells will employ additional regulatory elements well known in the
art such as enhancer elements to stimulate transcription, splice
acceptors, termination and polyadenylation signals, and ribosome
binding sites to permit translation.
[0175] Retroviral infection can also be used to introduce transgene
into a non-human animal, as described above. The developing
non-human embryo can be cultured in vitro to the blastocyst stage.
During this time, the blastomeres can be targets for retro viral
infection (Jaenich, R., Proc. Natl. Acad. Sci. USA 71:1260-1264,
1976). Efficient infection of the blastomeres is obtained by
enzymatic treatment to remove the zona pellucida (Hogan, et al.
(1986) in Manipulating the Mouse Embryo, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.). The viral vector
system used to introduce the transgene is typically a
replication-defective retro virus carrying the transgene (Jahner,
et al., Proc. Natl. Acad. Sci. USA 82:6927-6931, 1985; Van der
Putten, et al., Proc. Natl. Acad. Sci. USA 82:6148-6152, 1985).
Transfection is easily and efficiently obtained by culturing the
blastomeres on a monolayer of virus-producing cells (Van der
Putten, supra; Stewart, et al., EMBO J. 6:383-388, 1987).
Alternatively, infection can be performed at a later stage. Virus
or virus-producing cells can be injected into the blastocoel (D.
Jahner et al., Nature 298:623-628, 1982). Most of the founders will
be mosaic for the transgene since incorporation occurs only in a
subset of the cells that formed the transgenic nonhuman animal.
Further, the founder may contain various retro viral insertions of
the transgene at different positions in the genome that generally
will segregate in the offspring. In addition, it is also possible
to introduce transgenes into the germ line, albeit with low
efficiency, by intrauterine retroviral infection of the
mid-gestation embryo (D. Jahner et al., supra).
[0176] A third type of target cell for transgene introduction is
the embryonal stem cell (ES). ES cells are obtained from
pre-implantation embryos cultured in vitro and fused with embryos
(M. J. Evans et al. Nature 292:154-156, 1981; M. O. Bradley et al.,
Nature 309:255-258, 1984; Gossler, et al., Proc. Natl. Acad. Sci.
USA 83:9065-9069, 1986; and Robertson et al., Nature 322:445-448,
1986). Transgenes can be efficiently introduced into the ES cells
by DNA transfection or by retro virus-mediated transduction. Such
transformed ES cells can thereafter be combined with blastocysts
from a nonhuman animal. The ES cells thereafter colonize the embryo
and contribute to the germ line of the resulting chimeric animal.
(For review see Jaenisch, R., Science 240:1468-1474, 1988).
[0177] "Transformed" means a cell into which (or into an ancestor
of which) has been introduced, by means of recombinant nucleic acid
techniques, a heterologous nucleic acid molecule. "Heterologous"
refers to a nucleic acid sequence that either originates from
another species or is modified from either its original form or the
form primarily expressed in the cell.
[0178] "Transgene" means any piece of DNA that is inserted by
artifice into a cell, and becomes part of the genome of the
organism (i.e., either stably integrated or as a stable
extrachromosomal element) that develops from that cell. Such a
transgene may include a gene that is partly or entirely
heterologous (i.e., foreign) to the transgenic organism, or may
represent a gene homologous to an endogenous gene of the organism.
Included within this definition is a transgene created by the
providing of an RNA sequence that is transcribed into DNA and then
incorporated into the genome. The transgenes of the invention
include DNA sequences which encode an invention ANKTM1-related
polypeptide, and include sense, antisense, dominant negative
encoding polynucleotides, which may be expressed in a transgenic
non-human animal. The term "transgenic" as used herein additionally
includes any organism whose genome has been altered by in vitro
manipulation of the early embryo or fertilized egg or by any
transgenic technology to induce a specific gene knockout (i.e.,
knockout of an invention ANKTM1-related polypeptide). The term
"gene knockout" as used herein, refers to the targeted disruption
of a gene in vivo with complete or partial loss of function that
has been achieved by any transgenic technology familiar to those in
the art (e.g., insertion of a P-element in Drosophila). In one
embodiment, transgenic animals having gene knockouts are those in
which the target gene has been rendered nonfunctional by an
insertion targeted to the gene to be rendered non-functional by
homologous recombination. As used herein, the term "transgenic"
includes any transgenic technology familiar to those in the art
which can produce an organism carrying an introduced transgene or
one in which an endogenous gene has been rendered non-functional or
"knocked out."
[0179] In one embodiment, the transgene comprises DNA antisense to
the coding sequence for an invention ANKTM1-related polypeptide. In
another embodiment, the transgene comprises DNA encoding an
antibody that is able to bind to an invention ANKTM1-related
polypeptide. Where appropriate, DNA sequences that encode proteins
having nociceptive activity but differ in nucleic acid sequence due
to the degeneracy of the genetic code may also be used herein, as
may truncated forms, allelic variants and interspecies
homologues.
[0180] The invention also includes animals having heterozygous
mutations in or partial inhibition of function or expression of an
invention ANKTM1-related polypeptide. One of skill in the art would
readily be able to determine if a particular mutation or if an
antisense molecule was able to partially inhibit ANKTM1-related
polypeptide. For example, in vitro testing may be desirable
initially by comparison with wild-type (e.g., comparison of
northern blots to examine a decrease in expression).
[0181] After an embryo has been microinjected, colonized with
transfected embryonic stem cells or infected with a retrovirus
containing the transgene (except for practice of the subject
invention in avian species which is addressed elsewhere herein) the
embryo is implanted into the oviduct of a pseudopregnant female.
The consequent progeny are tested for incorporation of the
transgene by Southern blot analysis of blood samples using
transgene specific probes. PCR is particularly useful in this
regard. Positive progeny (G.sub.0) are crossbred to produce
offspring (G.sub.1), which are analyzed for transgene expression by
Northern blot analysis of tissue samples. To be able to distinguish
expression of like-species transgenes from expression of the
animal's endogenous ANKTM1-related polypeptide gene(s), a marker
gene fragment can be included in the construct in the 3'
untranslated region of the transgene and the Northern probe
designed to probe for the marker gene fragment. The serum levels of
ANKTM1-related polypeptide can also be measured in the transgenic
animal to establish appropriate expression. Expression of the
nociceptive-related transgenes, thereby decreasing the
ANKTM1-related polypeptide in the tissue and serum levels of the
transgenic animals.
[0182] Transgenic organisms of the invention are highly useful in
the production of organisms for study of tumorgenesis and in
identifying agents or drugs with inhibit or modulate tumorgenesis
and inheritance.
[0183] It will be recognized that the method for creating a
transgenic organism include methods for inserting a transgene into,
for example, an embryo of an already created transgenic organism,
the organism being transgenic for a different unrelated gene or
gene product.
[0184] In one embodiment the transgenic organism is an insect. An
insect as used herein denotes all insect species. Typically the
insect is selected from the group consisting of bristletails,
springtails, mayflies, dragonflies, damselflies, grasshoppers,
crickets, walkingsticks, praying-mantises, cockroaches, earwigs,
termites, stoneflies, lice, thrips, bed bugs, plant bugs, damsel
bugs, flower bugs, assassin bugs, ambush bugs, lace bugs, stink
bugs, cicadas, treehoppers, leafhoppers, spittlebugs, planthoppers,
aphids, whiteflies, beetles, scropionflies, caddisflies, moths,
skippers, butterflies, crane flies, sand flies, mosquitoes, horse
flies, fruit flies louse flies, bees, wasps, and ants.
[0185] The transgenic insects of the invention can be produced by
introducing into single cell embryos DNA disrupting expression of a
nucleic acid encoding the wild type ANKTM1-related polypeptide
sequence. Transgenic insects can be generated by microinjection,
which can produce P-element mediated germ line transformation. For
transgenic insects, generally the transgene is introduced at an
embryonic stage. For example, transgenic insects of the present
invention can be produced by introducing into single cell embryos
invention polynucleotides, either naked or contained in an
appropriate vector, by microinjection, for example, which can
produce insects by P-element mediated germ line transformation (see
e.g., Rubin et al., Science 218:348-353 (1982)). Totipotent or
pluripotent stem cells transformed by microinjection, calcium
phosphate mediated precipitation, liposome fusion, retroviral
infection or other means are then introduced into the embryo, and
the polynucleotides are stably integrated into the genome. A
transgenic embryo so transformed then develops into a mature
transgenic insect in which the transgene is inherited in normal
Mendelian fashion. Additional methods for producing transgenic
insects can be found, for example, in O=Brochta et al., Insect
Biochem. Mol. Biol. 26:739-753 (1996) and in Louleris et al.,
Science 270:2002-2005 (1995).
[0186] In one method, developing insect embryos are infected with a
virus, such as a baculovirus (e.g., Autographa californica AcNPV),
containing a polynucleotide sequence of the invention, and
transgenic insects produced from the infected embryo. The virus can
be an occluded virus or a nonoccluded virus. A virus can be
occluded by coinfection of cells with a helper virus that supplies
polyhedrin gene function. The skilled artisan will understand how
to construct recombinant viruses in which the polynucleotide is
inserted into a nonessential region of the baculovirus genome. For
example, in the AcNPV genome, nonessential regions include the p10
region (Adan et al., Virology 444:782-793, 1982), the DA26 region
(O'Reily et al., J. Gen. Virol 71:1029-1037, 1990), the ETL region
(Crawford et al., Virology 62:2773-2781, 1988), the egt region
(O'Reily et al., J. Gen, Virol 64:1321-1328), amongst others.
Significant homology exists among particular genes of different
baculoviruses and therefore, one of skill in the art will
understand how to insert an invention polynucleotide into similar
nonessential regions of other baculoviruses. Thus, for example, a
sequence encoding an invention ANKTM1-related polypeptide as
described herein may be placed under control of an AcNPV promoter
(e.g., the polyhedrin promoter). Depending on the vector utilized,
any of a number of suitable transcription and translation elements,
including constitutive, inducible and conditional promoters,
enhancers, transcription terminators, etc. may be used in order to
transcribe invention polynucleotides or express invention
polypeptides. Alternatively, a transgene containing a nucleic acid
sequence disrupting expression of an invention ANKTM1-related
polypeptide may not contain a promoter as the nucleic acid sequence
need not be transcribed or translated to obtain a transgenic insect
having disrupted ANKTM1-related polypeptide.
[0187] Thus, the invention provides methods for producing
transgenic insects having a disrupted nucleic acid sequence
encoding an invention ANKTM1-related polypeptide. The methods
include introducing into the genome of an insect a nucleic acid
construct, including a disrupted or mutated ANKTM1-related
polynucleotide sequence, and obtaining a transgenic insect having a
disrupted nucleic acid sequence encoding ANKTM1-related
polypeptide. The invention further provides methods for producing
transgenic insects having a nucleic acid encoding ANKTM1-related
polypeptide or functional fragment thereof.
[0188] The results disclosed herein were also described in Cell,
Vol. 112, 819-829, Mar. 21, 2003, which publication is incorporated
herein by reference, in particular the Figures, Examples and
Results reported therein.
[0189] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following examples are
to be considered illustrative and thus are not limiting of the
remainder of the disclosure in any way whatsoever.
Example 1
Molecular Cloning of ANKTM1
[0190] Bioinformatic searches were done as previously described
(Peier et al., 2002a, supra; Peier et al., 2002b, supra). Sequence
analysis was performed using the Biology Workbench at the San Diego
Supercomputing Center. A 902-base pair fragment of the mouse
homologue of ANKTM1 was amplified from newborn mouse DRG cDNA using
the following primers:
TABLE-US-00001 mANK-like F2 (SEQ ID NO:9) (5'-AGTGGGGAGACTACCCTGTG)
and mANK-like R2 (SEQ ID NO:10) (5'-TTTATCATGCCCATTCTTTGC).
[0191] From this initial sequence and subsequent hits to DNA
databases, the following primers were designed to PCR-amplify
full-length ANKTM1 from adult mouse trigeminal ganglia cDNA:
TABLE-US-00002 mANK-like start (SEQ ID NO:11)
(5'TTTGGATCCGCCACCATGAAGCGCGGCTTGAGGAGG) and mANK-like stop (SEQ ID
NO 12) (5'TTTGCGGCCGCCTAAAAGTCCGGGTGGCTAATAGAAC).
Example 2
Expression Analysis
[0192] Overall tissue distribution of ANKTM1 was analyzed by
Northern blot analysis using a probe corresponding to nucleotides
590-1492 of mouse ANKTM1 (SEQ ID NO:13). To further determine the
expression pattern of ANKTM1, a rat tissue Northern blot was
prepared as follows: total RNA was purified from adult tissues
using Trizol (Invitrogen/Gibco life Technologies, San Diego,
Calif.), followed by polyA+ purification with OLIGOTEX.TM. (Qiagen,
Valencia, Calif.) according to manufacturer protocols.
Approximately 3 .mu.g polyA+ RNA was electrophoresed on a 1%
glyoxal gel, transferred to nylon membranes and hybridized with a
.sup.32P-labeled probe corresponding to nucleotides 590-1492 of
mouse full-length ANKTM1 (SEQ ID NO:13). Blots were hybridized 3
hours at 65.degree. C. in EXPRESSHYB.TM. solution (Clonetech, Palo
Alto, Calif.) and analyzed using a phosphorimager. A commercial
mouse tissue northern blot (Clonetech) was similarly treated.
[0193] The tissue distribution studies showed mat ANKTM1 expression
was not detected on a blot containing various mouse tissues,
including heart, lung, skeletal muscle, and kidney (FIG. 2A).
However, probe of a blot containing rat tissues relevant to sensory
neurons and their projections such as DRG, spinal cord, and skin
detected a single mRNA species of approximately 5 kb only in DRG
(FIG. 2B).
In Situ Hybridization Studies
[0194] More detailed expression analysis of ANKTM1 in DRG was
carried out via in situ hybridization and immunostaining. For in
situ hybridization and immunostaining of DRG's, adult mice were
perfused with 4% paraformaldehyde and DRG's rapidly dissected.
Following post-fixation and cryo-protection in 30% sucrose in PBS,
single DRG's were embedded in OCT compound, frozen in liquid
nitrogen and sectioned at 10-.mu.m thickness. Digoxigenin-labeled
ANKTM1 cRNA probes strongly hybridized to neurons in adult DRG and
trigeminal ganglia (FIG. 3). ANKTM1 expression was found to be
restricted to a small sub-population of DRG neurons (59 of 1608 or
3.6% of the DRG neurons). To further characterize its expression in
sensory neurons, tests were conducted to determine whether other
DRG markers co-localized with ANKTM1. These tests showed that
ANKTM1 is not expressed in heavily myelinated neurons marked by
NF-150 kd (FIGS. 3A-3C). This finding shows that ANKTM1 is most
likely expressed in the non-myelinated C-- or lightly myelinated
A.sub..delta.- fiber population of neurons that sense temperature
and/or noxious stimuli.
Example 3
Double In-Situ Hybridization Studies
[0195] The expression of ANKTM1 relative to known thermo-activated
TRP channels was also studied. TRPV1 (VR1) is a well-characterized
receptor for noxious heat, pH, and capsaicin. For double in-situ
hybridizations, sections were hybridized with in vitro transcribed
digoxigenin- or fluorescein-labeled cRNA probes (Roche, Basel,
Switzerland) corresponding to nucleotides 590-1492 of mANKTM1, and
nucleotides 1410-1980 of mTRPM8 (NM.sub.--029310) (SEQ ID NO:14).
We used two cRNA probes corresponding to bases 1516-2065 or
1516-2482 of TRPV1 sequence (AF029310). Both probes showed
consistent patterns of hybridization. Peroxidase-conjugated
anti-digoxigenin-POD (1:500) and alkaline phosphatase-conjugated
anti-fluorescein (1:2000) antibodies (Roche) were used to detect
hybridized cRNA probes and visualized using tyramide signal
amplification (TSA; NEN) and fast-red detection (Roche) systems,
respectively. The immunostaining experiments followed hybridization
of sections with digoxigenin-labeled cRNA probes and TSA
fluorescent detection. Anti-NF150 kd (1:1000; Chemicon, Temecula,
Calif.) and anti-CGRP (1:100; Biogenesis,) primary antibodies and
anti-rabbit Cy3 (1:200, Jackson Immunoresearch,) secondary
anybodies were used.
[0196] The results of these experiments utilizing ANKTM1 and TRPV1
probes (FIGS. 3G-3I) revealed that 97%, or 100 out of 103, of
ANKTM1-positive neurons also express TRPV1, while 30% (or 100 out
of 336) of TRPV1-positive neurons express ANKTM1. This expression
pattern differs from that of TRPM8, the cold and menthol receptor,
which is not coexpressed with CGRP and TRPV1 in DRG neurons. Since
ANKTM1 expression overlaps with these two markers, coexpression of
ANKTM1 and TRPM8 would not be expected. However, these double in
situ hybridizations revealed no overlap of expression between
ANKTM1 and TRPM8 (n=113 for ANKTM1 and 137 for TRPM8) (FIGS.
3J-3L). Taken together, these results indicate that ANKTM1 is
expressed in a sub-population of nociceptive/thermoceptive neurons
that coincides with noxious heat-activated TRPV1 receptor, but not
with the cool/cold-activated TRPM8 receptor.
[0197] In accordance with this observation, it was further
discovered that ANKTM1 is expressed in Calcitonin gene-related
peptide (CGRP)- and in Substance P(SP)-positive neurons (FIGS.
3D-3F). 97% or 69 out of 71 of these ANKTM1-positive neurons were
CGRP-positive. Both CGRP and SP are secreted inflammatory peptides
expressed in a subset of nociceptive neurons (Scott, 1992).
Example 4
CHO Cell Expression System
[0198] Nevertheless, since ANKTM1 bears similarities to TRP-like
channels expressed in sensory neurons, tests were conducted to
determine activation of ANKTM1 by various sensory stimuli. To this
end, a series of tests were conducted using full-length murine
ANKTM1 stably transfected in Chinese Hamster Ovary (CHO) cells
containing FRT sites (CHO-K1/FRT) under control of a tetracycline
(Tet)-inducible promoter via Flp recombinase mediated
recombination.
[0199] CHO-K1/FRT cells were stably transfected with full-length
murine ANKTM1 in the pcDNA5/FRT vector using Lipofectamine
(Invitrogen) according to the manufacturer's protocol. Transfected
cells were selected in growth medium containing Hygromycin (200
.mu.g/mL). Northern blot analysis identified stable clones
expressing ANKTM1 mRNA. The generation of stable murine
TRPM8-expressing CHO-K1/FRT cells has been previously described
(Peier et al., 2002). ANKTM1-expressing stable CHO-K1/FRT lines
appeared unhealthy (membrane blebbing, cytoplasmic granulations) in
culture, and after several passages, a loss of ANKTM1 expression
was observed. Previous investigators have also claimed difficulty
generating stable cell lines expressing human ANKTM1.
[0200] To circumvent this problem, cell lines were generated in
which ANKTM1 expression could be controlled. Tetracycline-inducible
CHO-K1/FRT cells lines were generated by transfecting the CHO
Flp-In host cell line with pcDNA6/TR according to manufacturer's
instructions (Invitrogen, San Diego, Calif.) and selected in 5
.mu.g/mL Blasticidin. Clones stably expressing the tetracycline
repressor identified by blasticidin resistance were transiently
transfected with a control plasmid containing the CAT gene and
selected based on high levels of CAT expression after treatment
with tetracycline. The gene expression vector, pcDNA5FRT/TO
containing full-length murine ANKTM1 and the Flp recombinase
expression plasmid pOG44 were co-transfected into the
tetracycline-inducible CHO-K1/FRT cell line and selected via
Hygromycin resistance (200 .mu.g/ml). Induction of ANKTM1 was
accomplished by treating CHO cells with 1-2 .mu.g/mL tetracycline
(Tet) 5-24 hours before experiments.
[0201] Stable clones expressing ANKTM1 were identified by Northern
blot analysis. Northern blot analysis of control and Tet-treated
ANKTM1-inducible CHO cells showed high levels of ANKTM1 expression.
A low but not absent level in the absence of Tet was also present
in these cells. Therefore, CHO cells that stably expressed the
Tet-repressor were used as controls in subsequent experiments. For
some of the experiments, cells were maintained in culture medium
supplemented with 5 .mu.m ruthenium red (RR, Fluka), which blocks
ANKTM1 activity. This was done to overcome the slight leaky
expression of ANKTM1 in the Tet-system. Cells cultured in RR looked
healthier and a higher percentage of cells responded to cold
compared to non-RR treated cells.
[0202] As shown in FIG. 4A, when buffer is cooled,
[Ca.sup.2+].sub.i increases rapidly in ANKTM1-expressing CHO cells,
but not in untransfected CHO cells. Average activation temperature
(measured at the inlet of the cell chamber by a miniature
thermocouple) is approximately 17.degree. C. Cooling in the
presence of 5 .mu.m ruthenium red and in the absence of
extracellular Ca.sup.2+ eliminates cold-evoked responses. The
threshold of activation was not affected.
Examples
Intracellular Calcium Imaging Experiments
[0203] Calcium imaging experiments were performed essentially as
described (Peier et al., 2002a, supra). Briefly, cells were plated
on glass coverslips 24-48 hours prior to imaging experiments. Cells
were washed in HEPES buffered saline solution (2 mM Ca.sup.2+),
loaded with Fura-2 acetoxymethyl ester (5 mM), 1.5 mM pluronic acid
(Molecular Probes, Eugene, Oreg.) and incubated for 1 hour at room
temperature in the dark. Coverslips were placed in laminar flow
chamber (Warner Instrument Corp., Hamden, Conn.) and perfused with
HEPES buffered saline (2 mM Ca.sup.2+) flowing through
small-diameter tubing at the inlet of the chamber. Perfusion rate
was approximately 2 ml/min and was stopped and started via a
solenoid switch (Warner Instrument Corp., Hamden, Conn. Perfusate
temperature was controlled by a Peltier device and monitored via
miniature thermocouple at the inlet of the chamber. Heated (up to
52.degree. C.), chilled (down to 9.degree. C., the limit of our
system), and room temperature buffer was delivered through the same
application tubing at the inlet of the chamber. Alternatively, in
experiments where the compounds menthol (Sigma, St. Louis, Mo.]),
capsaicin (Fluka, Buchs, Switzerland) and Icilin (Tocris Woodson,)
were applied, cells were plated on 24-well tissue culture plates
and loaded with Fura-2. Compounds were delivered with a 3 cc
syringe during a period of ten seconds.
[0204] Images of Fura-2 loaded cells with the excitation wavelength
alternating between 340 nm and 380 nm were captured with a cooled
LCD camera. The ratio of fluorescence intensity of the two
wavelengths of groups of 30-50 cells in each experiment was
analyzed using MetaFluor (Universal Imaging Corp). Experiments were
performed in triplicate. Unless otherwise indicated, graphs
represent averaged responses of 20-40 cells from representative
experiments. For direct comparison of results from different
experiments, values were normalized to baseline of the ratio
340/380. For ANKTM1 and TRPM8 threshold experiments, the threshold
temperature of activation of 60-100 cells each from three replicate
experiments was analyzed. Threshold of activation was defined as
20% above baseline. Hanks Balanced Salt Solution (HBSS) and HEPES
buffers were obtained from GibcoBRL. The results of these
experiments are shown in FIG. 5 herein.
Example 6
Electrophysiology
[0205] Cells were plated onto poly-D-lysine-coated coverslips for
recording purposes, and recordings were undertaken 18-24 hr later.
Experiments were carried out at room temperature using whole-cell
voltage clamp technique, with an Axopatch 2B amplifier filtered at
5 kHz and pClamp suite of software (Axon Instruments).
Series-resistance compensation was 80% for all experiments, using
2-5 M.OMEGA. fire-polished pipettes. Recording solutions were as
follows: pipette solution for all experiments was [(mM) CsCl, 140;
BAPTA, 10; HEPES, 10; MgATP, 2; titrated to pH7.4 with CsOH. The
external solution in the recording chamber was kept at 32.degree.
C. and consisted of [(mM): CholineCl, 100; NaCl, 40; HEPES, 10;
CaCl.sub.2, 2; MgCl.sub.2 1; titrated to pH 7.4 with NaOH]. In some
studies the above solution was used with 140 mM NaCl and no
Choline. In monovalent permeability studies, equimolar KCl or CsCl
replaced NaCl (40 mM). In divalent permeability studies, the
solutions either contained 1 mM of test ion and (mM) CholineCl,
100; NaCl, 40; Hepes, 10; sucrose, 80 or 30 mM test ion in the
above solution without sucrose. The current-voltage relationships
were determined using a 2 second ramp from -100 to +80 mV.
[0206] Permeability ratios relative to Na.sup.+, were calculated
for monovalent cations as follows:
P.sub.X/P.sub.Na=E.sub.shift={RT/F} log(P.sub.X/P.sub.Na
[X].sub.O/[Na].sub.O), where F is Faraday's constant, R is the
universal gas constant, and T is absolute temperature. Permeability
ratios relative to Na.sup.+, for divalent cations were calculated
as follows: Eshift={RT/F} log {[Na].sub.O+4B'
[Ca].sub.O(2)}/{[Na].sub.O4B' [Ca].sub.0(1)}, where
B'=P'.sub.Ca/P.sub.Na and P'.sub.Ca=P.sub.Ca/(1+e.sup.EF/RT) and
[Ca].sub.O(1) and [Ca].sub.O(2) refer to the two different
concentrations of the divalent ion tested.
[0207] ANKTM1 and TRPM8 were cloned into the pOX expression vector
for expression in Xenopus oocytes (Jegla and Salkoff, 1997). Capped
cRNAs were prepared by run-off transcription using the T3 mMessage
mMachine kit (Ambion) and cleaned prior to injection using
QIAQUICK.TM. columns (Qiagen). Mature oocytes were isolated
enzymatically using known methodology. Oocytes were injected with 5
ng to 25 ng of cRNA in a 50 nl volume and incubated 2-5 days prior
to recording at 18.degree. C. in ND96 (96 mM NaCl, 2 mM KCl, 1.8 mM
CaCl, 1 mM MgCl, 5 mM Hepes, 2.5 mM Na-pyruvate, 100 U/ml
penicillin, 100 .mu.g/ml streptomycin, pH7.5). Recordings were made
using standard two-electrode voltage clamp techniques with 3 M KCl
electrodes (0.3-0.5 M.OMEGA.). The recording solution consisted of
96 mM NaCl, 4 mM KCl, 1 mM MgCl, 100 .mu.M CaCl and 5 mM Hepes, pH
7.5. Temperature control was achieved with a combination of a
peltier-cooled stage and constant perfusion of cooled/heated
solution. Temperatures were monitored using a miniature
thermocouple placed adjacent to the oocyte in the recording
chamber. Currents and temperatures were recorded using a TEV-200
amplifier (Dagan Instruments, Minneapolis, Minn.), an HCC-100A
temperature controller (Dagan Instruments), and pCLAMP8.TM.
software suite (Axon Instruments, Union City, Calif.). The oocytes
were held at -70 mV during the recordings.
[0208] FIGS. 7A-D are a series of graphs summarizing the results of
these experiments. FIGS. 7A and 7C show, respectively, inward
currents recorded in response to first and second cold steps from
Xenopus oocytes expressing ANKTM1; while FIGS. 7B and 7D show,
respectively, inward currents recorded in response to cold steps
from Xenopus oocytes expressing TRPM8. By comparison to TRPM8,
ANKTM1 current responses were markedly reduced in the second cold
step.
[0209] It will be apparent to those skilled in the art that various
modifications and variations can be made to the compounds and
processes of this invention. Thus, it is intended that the present
invention cover such modifications and variations, provided they
come within the scope of the appended claims. Accordingly, the
invention is limited only by the following claims.
Sequence CWU 1
1
131320PRTMus musculus 1Leu Asn Val Met Val Gln His Asn Arg Ile Glu
Leu Leu Asn His Pro1 5 10 15Val Cys Arg Glu Tyr Leu Leu Met Lys Trp
Cys Ala Tyr Gly Phe Arg 20 25 30Ala His Met Met Asn Leu Gly Ser Tyr
Cys Leu Gly Leu Ile Pro Met 35 40 45Thr Leu Leu Val Val Lys Ile Gln
Pro Gly Met Ala Phe Asn Ser Thr 50 55 60Gly Ile Ile Asn Gly Thr Ser
Ser Thr His Glu Glu Arg Ile Asp Thr65 70 75 80Leu Asn Ser Phe Pro
Ile Lys Ile Cys Met Ile Leu Val Phe Leu Ser 85 90 95Ser Ile Phe Gly
Tyr Cys Lys Glu Val Ile Gln Ile Phe Gln Gln Lys 100 105 110Arg Asn
Tyr Phe Leu Asp Tyr Asn Asn Ala Leu Glu Trp Val Ile Tyr 115 120
125Thr Thr Ser Ile Ile Phe Val Leu Pro Leu Phe Leu Asn Ile Pro Ala
130 135 140Tyr Met Gln Trp Gln Cys Gly Ala Ile Ala Ile Phe Phe Tyr
Trp Met145 150 155 160Asn Phe Leu Leu Tyr Leu Gln Arg Phe Glu Asn
Cys Gly Ile Phe Ile 165 170 175Val Met Leu Glu Val Ile Phe Lys Thr
Leu Leu Arg Ser Thr Gly Val 180 185 190Phe Ile Phe Leu Leu Leu Ala
Phe Gly Leu Ser Phe Tyr Val Leu Leu 195 200 205Asn Phe Gln Asp Ala
Phe Ser Thr Pro Leu Leu Ser Leu Ile Gln Thr 210 215 220Phe Ser Met
Met Leu Gly Asp Ile Asn Tyr Arg Asp Ala Phe Leu Glu225 230 235
240Pro Leu Phe Arg Asn Glu Leu Ala Tyr Pro Val Leu Thr Phe Gly Gln
245 250 255Leu Ile Ala Phe Thr Met Phe Val Pro Ile Val Leu Met Asn
Leu Leu 260 265 270Ile Gly Leu Ala Val Gly Asp Ile Ala Glu Val Gln
Lys His Ala Ser 275 280 285Leu Lys Arg Ile Ala Met Gln Val Glu Leu
His Thr Asn Leu Glu Lys 290 295 300Lys Leu Pro Leu Trp Tyr Leu Arg
Lys Val Asp Gln Arg Ser Thr Ile305 310 315 3202319PRTHomo sapiens
2Leu Asn Ala Met Val Gln Asn Asn Arg Ile Glu Leu Leu Asn His Pro1 5
10 15Val Cys Lys Glu Tyr Leu Leu Met Lys Trp Leu Ala Tyr Gly Phe
Arg 20 25 30Ala His Met Met Asn Leu Gly Ser Tyr Cys Leu Gly Leu Ile
Pro Met 35 40 45Thr Ile Leu Val Val Asn Ile Lys Pro Gly Met Ala Phe
Asn Ser Thr 50 55 60Gly Ile Ile Asn Glu Thr Ser Asp His Ser Glu Ile
Leu Asp Thr Thr65 70 75 80Asn Ser Tyr Leu Ile Lys Thr Cys Met Ile
Leu Val Phe Leu Ser Ser 85 90 95Ile Phe Gly Tyr Cys Lys Glu Ala Gly
Gln Ile Phe Gln Gln Lys Arg 100 105 110Asn Tyr Phe Met Asp Ile Ser
Asn Val Leu Glu Trp Ile Ile Tyr Thr 115 120 125Thr Gly Ile Ile Phe
Val Leu Pro Leu Phe Val Glu Ile Pro Ala His 130 135 140Leu Gln Trp
Gln Cys Gly Ala Ile Ala Val Tyr Phe Tyr Trp Met Asn145 150 155
160Phe Leu Leu Tyr Leu Gln Arg Phe Glu Asn Cys Gly Ile Phe Ile Val
165 170 175Met Leu Glu Val Ile Leu Lys Thr Leu Leu Arg Ser Thr Val
Val Phe 180 185 190Ile Phe Leu Leu Leu Ala Phe Gly Leu Ser Phe Tyr
Ile Leu Leu Asn 195 200 205Leu Gln Asp Pro Phe Ser Ser Pro Leu Leu
Ser Ile Ile Gln Thr Phe 210 215 220Ser Met Met Leu Gly Asp Ile Asn
Tyr Arg Glu Ser Phe Leu Glu Pro225 230 235 240Tyr Leu Arg Asn Glu
Leu Ala His Pro Val Leu Ser Phe Ala Gln Leu 245 250 255Val Ser Phe
Thr Ile Phe Val Pro Ile Val Leu Met Asn Leu Leu Ile 260 265 270Gly
Leu Ala Val Gly Asp Ile Ala Glu Val Gln Lys His Ala Ser Leu 275 280
285Lys Arg Ile Ala Met Gln Val Glu Leu His Thr Ser Leu Glu Lys Lys
290 295 300Leu Pro Leu Trp Phe Leu Arg Lys Val Asp Gln Lys Ser Thr
Ile305 310 3153352PRTDrosophila melanogaster 3Leu Asn Thr Met Val
Thr His Gly Arg Val Glu Leu Leu Ala His Pro1 5 10 15Leu Ser Gln Lys
Tyr Leu Gln Met Lys Trp Asn Ser Tyr Gly Lys Tyr 20 25 30Phe His Leu
Ala Asn Leu Leu Ile Tyr Ser Ile Phe Leu Val Phe Val 35 40 45Thr Ile
Tyr Ser Ser Leu Met Met Asn Asn Ile Glu Leu Lys Ala Gly 50 55 60Asp
Asn Lys Thr Met Ser Gln Tyr Cys Asn Met Gly Trp Glu Gln Leu65 70 75
80Thr Met Asn Leu Ser Gln Asn Pro Ser Val Ala Ser Gln Ile Arg Leu
85 90 95Asp Ser Cys Glu Glu Arg Ile Asn Arg Thr Thr Ala Ile Leu Phe
Cys 100 105 110Ala Val Val Ile Val Val Tyr Ile Leu Leu Asn Ser Met
Arg Glu Leu 115 120 125Ile Gln Ile Tyr Gln Gln Lys Leu His Tyr Ile
Leu Glu Thr Val Asn 130 135 140Leu Ile Ser Trp Val Leu Tyr Ile Ser
Ala Leu Val Met Val Thr Pro145 150 155 160Ala Phe Gln Pro Asp Gly
Gly Ile Asn Thr Ile His Tyr Ser Ala Ala 165 170 175Ser Ile Ala Val
Phe Leu Ser Trp Phe Arg Leu Leu Leu Phe Leu Gln 180 185 190Arg Phe
Asp Gln Val Gly Ile Tyr Val Val Met Phe Leu Glu Ile Leu 195 200
205Gln Thr Leu Ile Lys Val Leu Met Val Phe Ser Ile Leu Ile Ile Ala
210 215 220Phe Gly Leu Ala Phe Tyr Ile Leu Leu Ser Lys Ile Ile Asp
Pro Gln225 230 235 240Pro Asn His Leu Ser Phe Ser Asn Ile Pro Met
Ser Leu Leu Arg Thr 245 250 255Phe Ser Met Met Leu Gly Glu Leu Asp
Phe Val Gly Thr Tyr Val Asn 260 265 270Thr Tyr Tyr Arg Asp Gln Leu
Lys Val Pro Met Thr Ser Phe Leu Ile 275 280 285Leu Ser Val Phe Met
Ile Leu Met Pro Ile Leu Leu Met Asn Leu Leu 290 295 300Ile Gly Leu
Ala Val Gly Asp Ile Glu Ser Val Arg Arg Asn Ala Gln305 310 315
320Leu Lys Arg Leu Ala Met Gln Val Val Leu His Thr Glu Leu Glu Arg
325 330 335Lys Leu Pro His Val Trp Leu Gln Arg Val Asp Lys Met Glu
Leu Ile 340 345 3504368PRTDrosophila melanogaster 4Leu Asp Val Leu
Ile Glu Asn Glu Gln Lys Glu Val Ile Ala His Thr1 5 10 15Val Val Gln
Arg Tyr Leu Gln Glu Leu Trp His Gly Ser Leu Thr Trp 20 25 30Ala Ser
Trp Lys Ile Leu Leu Leu Leu Val Ala Phe Ile Val Cys Pro 35 40 45Pro
Val Trp Ile Gly Phe Thr Phe Pro Met Gly His Lys Phe Asn Lys 50 55
60Val Pro Ile Ile Lys Phe Met Ser Tyr Leu Thr Ser His Ile Tyr Leu65
70 75 80Met Ile His Leu Ser Ile Val Gly Ile Thr Pro Ile Tyr Pro Val
Leu 85 90 95Arg Leu Ser Leu Val Pro Tyr Trp Tyr Glu Val Gly Leu Leu
Ile Trp 100 105 110Leu Ser Gly Leu Leu Leu Phe Glu Leu Thr Asn Pro
Ser Asp Lys Ser 115 120 125Gly Leu Gly Ser Ile Lys Val Leu Val Leu
Leu Leu Gly Met Ala Gly 130 135 140Val Gly Val His Val Ser Ala Phe
Leu Phe Val Ser Lys Glu Tyr Trp145 150 155 160Pro Thr Leu Val Tyr
Cys Arg Asn Gln Cys Phe Ala Leu Ala Phe Leu 165 170 175Leu Ala Cys
Val Gln Ile Leu Asp Phe Leu Ser Phe His His Leu Phe 180 185 190Gly
Pro Trp Ala Ile Ile Ile Gly Asp Leu Leu Lys Asp Leu Ala Arg 195 200
205Phe Leu Ala Val Leu Ala Ile Phe Val Phe Gly Phe Ser Met His Ile
210 215 220Val Ala Leu Asn Gln Ser Phe Ala Asn Phe Ser Pro Glu Asp
Leu Arg225 230 235 240Ser Phe Glu Lys Lys Asn Arg Asn Arg Gly Tyr
Phe Ser Asp Val Arg 245 250 255Met His Pro Ile Asn Ser Phe Glu Leu
Leu Phe Phe Ala Val Phe Gly 260 265 270Gln Thr Thr Thr Glu Gln Thr
Gln Val Asp Lys Ile Lys Asn Val Ala 275 280 285Thr Pro Thr Gln Pro
Tyr Trp Val Glu Tyr Leu Phe Lys Ile Val Phe 290 295 300Gly Ile Tyr
Met Leu Val Ser Val Val Val Leu Ile Asn Leu Leu Ile305 310 315
320Ala Met Met Ser Asp Thr Tyr Gln Arg Ile Gln Val Val Leu Leu Asn
325 330 335Ala Leu Leu Ser Asn Ser Thr Leu Phe Ile Asn Ser Tyr Phe
Asn His 340 345 350Lys Tyr Ile Asn Phe Ile Leu His Cys Val Leu Ile
Ile Leu Tyr Phe 355 360 3655365PRTCaenorhabditis elegans 5Leu Asp
Val Leu Ile Glu Asn Glu Gln Lys Glu Val Val Ser Tyr Ala1 5 10 15Ser
Val Gln Arg Tyr Leu Thr Glu Val Trp Thr Ala Arg Val Asp Trp 20 25
30Ser Phe Gly Lys Phe Val Ala Phe Ser Leu Phe Val Leu Ile Cys Pro
35 40 45Pro Ala Trp Phe Tyr Phe Ser Leu Pro Leu Asp Ser Arg Ile Gly
Arg 50 55 60Ala Pro Ile Ile Lys Phe Val Cys His Ile Val Ser His Val
Tyr Phe65 70 75 80Thr Ile Leu Leu Thr Ile Val Val Leu Asn Ile Thr
His Lys Met Tyr 85 90 95Glu Val Thr Ser Val Val Pro Asn Pro Val Glu
Trp Leu Leu Leu Leu 100 105 110Trp Leu Ser Gly Asn Leu Val Ser Glu
Leu Ser Thr Val Gly Gly Gly 115 120 125Ser Gly Leu Gly Ile Val Lys
Val Leu Ile Leu Val Leu Ser Ala Met 130 135 140Ala Ile Ala Val His
Val Leu Ala Phe Leu Leu Pro Ala Val Phe Leu145 150 155 160Thr His
Leu Asp Asn Asp Glu Lys Leu His Phe Ala Arg Thr Met Leu 165 170
175Tyr Leu Lys Asn Gln Leu Phe Ala Phe Ala Leu Leu Phe Ala Phe Val
180 185 190Glu Tyr Leu Asp Phe Leu Thr Val His His Leu Phe Gly Pro
Trp Ala 195 200 205Ile Ile Ile Arg Asp Leu Met Tyr Asp Leu Ala Arg
Phe Leu Val Ile 210 215 220Leu Met Leu Phe Val Ala Gly Phe Thr Leu
His Val Thr Ser Ile Phe225 230 235 240Gln Pro Ala Tyr Gln Pro Val
Asp Glu Asp Ser Ala Glu Leu Met Arg 245 250 255Leu Ala Ser Pro Ser
Gln Thr Leu Glu Met Leu Phe Phe Ser Leu Phe 260 265 270Gly Leu Val
Glu Pro Asp Ser Met Pro Pro Leu His Leu Val Pro Asp 275 280 285Phe
Ala Lys Ile Ile Leu Lys Leu Leu Phe Gly Ile Tyr Met Met Val 290 295
300Thr Leu Ile Val Leu Ile Asn Leu Leu Ile Ala Met Met Ser Asp
Thr305 310 315 320Tyr Gln Arg Ile Gln Ala Gln Ser Asp Lys Glu Trp
Lys Phe Gly Arg 325 330 335Ala Ile Leu Ile Arg Gln Met Asn Lys Lys
Ser Ala Thr Pro Ser Pro 340 345 350Ile Asn Met Leu Thr Lys Leu Ile
Ile Val Leu Arg Val 355 360 3656331PRTCaenorhabditis elegans 6Leu
Lys Leu Met Ala Asp Ala Glu Lys Leu His Leu Leu Asn His Pro1 5 10
15Leu Ser Lys Ala Leu Leu Lys Tyr Lys Trp Asn Arg Leu Gly Arg Pro
20 25 30Met Tyr Tyr Phe Ala Leu Phe Met Tyr Leu Val Phe Ile Val Ser
Leu 35 40 45Thr Gln Tyr Val Arg His Thr Lys Ala Pro Tyr Asn Val Trp
Asn Glu 50 55 60Glu Ser Tyr Tyr Asp Ser Glu Tyr Phe Asp Glu Asn Glu
Thr Cys Pro65 70 75 80Gln Ile Asn Thr Thr Lys Pro Asp Val Val Trp
Lys Ile Ile Ile Gln 85 90 95Thr Leu Ala Val Cys Gln Ile Leu Val Glu
Cys Phe Gln Leu Phe Gln 100 105 110Arg Lys Phe Ala Tyr Leu Val Asn
Trp Glu Asn Trp Ile Asp Cys Phe 115 120 125Ile Tyr Ser Thr Ala Leu
Ile Thr Val Tyr Asp Phe Ser Glu Cys Ser 130 135 140Ala Thr Ser Gly
Val Arg Gln Asn Trp Gln Trp Ile Leu Ala Ala Leu145 150 155 160Cys
Ile Phe Phe Gly Trp Ile Asn Leu Leu Phe Met Ile Arg Lys Met 165 170
175Pro Arg Phe Gly Ile Phe Val Val Met Phe Val Asp Ile Val Lys Thr
180 185 190Phe Phe Arg Phe Phe Pro Val Phe Val Leu Phe Ile Ile Ala
Phe Ser 195 200 205Ser Ser Phe Tyr Val Ile Leu Gln Asn Arg Pro Glu
Phe Ser Thr Ile 210 215 220Phe Met Ser Pro Leu Lys Thr Thr Val Met
Met Ile Gly Glu Phe Glu225 230 235 240Phe Thr Gly Ile Phe His Gly
Asp Glu Thr Thr His Ala Glu Lys Met 245 250 255Phe Gly Pro Ala His
Thr Ala Val Ala Cys Ala Leu Phe Phe Phe Phe 260 265 270Cys Ile Ile
Met Thr Ile Leu Leu Met Asn Leu Leu Val Gly Leu Ala 275 280 285Val
Asp Asp Ile Lys Gly Val Gln Glu Lys Ala Glu Leu Lys Arg Leu 290 295
300Ala Met Gln Val Asp Leu Val Leu Gln Ile Glu Ala Ser Leu His
Phe305 310 315 320Phe Ile Gln Arg Thr Lys Lys Tyr Ala Thr Cys 325
3307333PRTDrosophila melanogaster 7Leu Asn Thr Phe Val Asp Glu Gly
Gln Lys Glu Ile Leu Glu His Pro1 5 10 15Leu Cys Ser Ser Phe Leu Tyr
Ile Lys Trp Gly Lys Ile Arg Lys Tyr 20 25 30Tyr Ile Gly Arg Leu Ile
Phe Cys Phe Ser Phe Val Leu Phe Leu Thr 35 40 45Leu Tyr Val Leu Thr
Ala Leu Ala His Asn Cys Tyr Asn Gly Ser Lys 50 55 60Asn Asp Asn Thr
Thr Ile Pro Ala Gln Glu Leu Cys Gln Lys Gln Ser65 70 75 80Ile Leu
Gly Asp Met Leu Arg Asn Asn Pro Phe Val Met Glu Met Gln 85 90 95Trp
Trp Val Leu Val Ala Ile Thr Ile Val Glu Ile Phe Arg Lys Leu 100 105
110Tyr Gly Ile Thr Gly Tyr Ser Ser Phe Arg His Tyr Val Thr Gln Val
115 120 125Glu Asn Ile Met Glu Trp Phe Val Ile Thr Ser Val Phe Val
Ile Ser 130 135 140Tyr Ile Tyr Thr Asn Lys Thr Tyr Thr Phe Gln Asn
His Ile Gly Ala145 150 155 160Phe Ala Val Leu Leu Gly Trp Thr Asn
Leu Met Leu Met Ile Gly Gln 165 170 175Leu Pro Val Phe Asp Val Tyr
Val Ala Met Tyr Thr Arg Val Gln Gly 180 185 190Glu Phe Ala Lys Leu
Phe Met Ala Tyr Ser Cys Met Leu Ile Gly Phe 195 200 205Thr Ile Ser
Phe Cys Val Ile Phe Pro Ser Ser Ser Ser Phe Ala Asn 210 215 220Pro
Phe Met Gly Phe Ile Thr Val Leu Val Met Met Ile Gly Glu Gln225 230
235 240Asp Leu Ser Leu Leu Ile Asn Asp Pro Glu Gly Lys Asp Pro Pro
Phe 245 250 255Leu Leu Glu Val Ser Ala Gln Ile Thr Phe Val Leu Phe
Leu Leu Phe 260 265 270Val Thr Ile Ile Leu Met Asn Leu Leu Val Gly
Ile Ala Val His Asp 275 280 285Ile Gln Gly Leu Lys Lys Thr Ala Gly
Leu Ser Lys Leu Val Arg Gln 290 295 300Thr Lys Leu Ile Ser Tyr Ile
Glu Ser Ala Leu Phe Asn Gly Tyr Leu305 310 315 320Pro Thr Trp Leu
Arg Asn Leu Leu His Tyr Thr Ala Leu 325 3308314PRTDrosophila
melanogaster 8Leu Leu Ser Leu Ile Glu Val Gly Gln Lys Arg Ile Leu
Met His Pro1 5 10 15Leu Cys Glu Thr Phe Leu Phe Leu Lys Trp Arg Arg
Ile Arg Lys Phe 20 25 30Phe Leu Met Ser Leu Ala Tyr His Thr Leu Phe
Val Ile Leu Phe Thr 35 40 45Phe Tyr Val Ile Trp Val Tyr Val Arg Cys
Cys Lys Lys Glu Glu Leu 50 55
60Cys Val Ala Pro Gly Tyr Val Ser Thr Ile Gly Tyr Leu Val Ile Ile65
70 75 80Leu Asn Leu Ile Leu Leu Gly Lys Glu Val Phe Gln Met Ala His
Gly 85 90 95Leu Arg Gly Tyr Ala Lys Tyr Trp Glu Asn Trp Leu Gln Trp
Thr Ile 100 105 110Gly Thr Gly Val Leu Leu Cys Val Thr Pro Glu Thr
Val Arg Thr Asp 115 120 125Asp Leu Thr Ala Val Pro Val Trp Gln His
His Val Ala Ala Ile Val 130 135 140Ile Leu Leu Val Trp Leu Glu Leu
Met Met Leu Val Gly Arg Phe Pro145 150 155 160Ile Phe Gly Val Tyr
Val Gln Met Phe Thr Lys Val Ala Val Asn Phe 165 170 175Ala Lys Phe
Leu Leu Ala Tyr Ile Cys Leu Leu Val Ala Phe Gly Leu 180 185 190Ser
Phe Ala Val Leu Phe Asn Asp Tyr Pro Ala Phe Glu Asn Ile Thr 195 200
205Trp Ser Phe Leu Lys Ser Ile Thr Met Met Ser Gly Glu Leu Glu Phe
210 215 220Glu Asp Ile Phe Tyr Gly Asp Tyr Ala Val Lys Phe Pro Val
Thr Ala225 230 235 240His Ile Ile Phe Leu Ser Phe Val Leu Leu Val
Thr Val Ile Leu Thr 245 250 255Asn Leu Met Val Gly Leu Ala Val Ser
Asp Ile Gln Gly Leu Gln Val 260 265 270Ser Ala Thr Leu Asp Arg Leu
Val Arg Gln Ala Glu Leu Val Ser Arg 275 280 285Leu Glu Ser Leu Phe
Phe Ser Arg Leu Leu Arg Ser Ala Pro Thr Asn 290 295 300Leu Ile Gln
Leu Cys Lys Arg Ser Ala Leu305 310920DNAArtificial sequencePrimer
9agtggggaga ctaccctgtg 201021DNAArtificial sequencePrimer
10tttatcatgc ccattctttg c 211136DNAArtificial sequencePrimer
11tttggatccg ccaccatgaa gcgcggcttg aggagg 361237DNAArtificial
sequencePrimer 12tttgcggccg cctaaaagtc cgggtggcta atagaac
37133378DNAMus musculus 13atgaagcgcg gcttgaggag gattctgctc
ccggaggaaa ggaaggaggt ccagggcgtt 60gtctatcgcg gcgtcgggga agacatggac
tgctccaagg aatcctttaa ggtggacatt 120gaaggagata tgtgtagatt
agaagacttc atcaagaacc gaagaaaact aagcaaatat 180gaggatgaaa
atctctgtcc tctgcatcac gcagcagcag aaggtcaagt tgaactgatg
240gaactgatca tcaatggttc ttcgtgtgaa gtgctgaata taatggatgg
ttatggaaat 300accccactgc attgtgctgc agaaaaaaat caagttgaaa
gtgtaaagtt tcttctcagc 360caaggagcaa atccaaacct ccgaaataga
aacatgatgt caccccttca catagctgtg 420catggcatgt acaacgaagt
gatcaaggtg ttgactgagc acaaggccac taacatcaat 480ttagaaggag
agaatgggaa cacggctttg atgtccacgt gtgccaaaga caacagtgaa
540gctttgcaaa ttttgttaga aaaaggagct aagctgtgta aatcaaataa
gtggggagac 600taccctgtgc accaggcagc attttcaggt gccaaaaaat
gcatggaatt aatcttagca 660tatggtgaaa agaacggcta cagcagggag
actcacatta attttgtgaa tcacaagaaa 720gccagccctc tccacctagc
agttcaaagc ggagacttgg acatgattaa gatgtgcctg 780gacaacggtg
cacacatcga catgatggag aatgccaaat gcatggccct ccattttgct
840gcaacccagg gagccactga catcgttaag ctcatgatct catcctatac
cggaagtagt 900gatattgtga atgcagttga tggcaatcag gagaccctgc
ttcacagagc ctcgttattt 960gatcaccatg acctggcaga atacctaata
tcagtgggag cagacatcaa cagcactgat 1020tctgaaggac gctctccact
tattttagca acagcttctg catcctggaa cattgtgaat 1080ttgctcctct
gtaaaggtgc caaagtagac ataaaagatc atcttggacg taactttttg
1140catttgactg tgcagcagcc ttatggacta agaaatttgc ggcctgagtt
tatgcagatg 1200caacacatca aagagctggt gatggatgaa gacaatgacg
gatgcacacc tctccattat 1260gcctgtaggc agggggttcc tgtctctgta
aataacctcc ttggcttcaa tgtgtccatt 1320catagcaaaa gtaaagataa
gaagtcgccc ctgcattttg cagccagtta tgggcgcatc 1380aatacatgtc
agagacttct gcaagacata agtgatacga ggcttttgaa tgaaggggat
1440ctccatggga tgacccctct ccacctggca gcaaaaaatg ggcatgataa
agtcgttcaa 1500ctccttctga agaaaggggc cttatttctc agtgaccaca
atggctggac tgctttgcat 1560cacgcctcca tgggtgggta cactcagacc
atgaaggtca ttcttgatac taacttgaaa 1620tgcacagacc gactagatga
agaagggaac acagcactcc actttgcagc acgggaaggc 1680catgccaagg
ctgttgcaat gcttttgagc tacaatgctg acatcctcct gaacaagaag
1740caagcttcct ttctgcatat tgccctgcac aataagcgca aggaagtggt
tctcacaacc 1800atcagaaata aaagatggga tgagtgtctt caagttttca
ctcataattc tccaagcaat 1860cgatgtccaa tcatggagat ggtagaatac
ctccccgagt gcatgaaagt tcttttagat 1920ttctgcatga taccttccac
agaagacaag tcctgtcaag actaccatat tgagtataat 1980ttcaagtatc
tccaatgccc attatccatg accaaaaaag tagcacctac ccaggatgtg
2040gtatatgagc ctcttacaat cctcaatgtc atggtccaac ataaccgcat
agaactcctc 2100aaccaccctg tgtgtaggga gtacttactc atgaaatggt
gtgcctatgg attcagggcc 2160catatgatga acctaggatc ttattgtctt
ggtctcatac ccatgaccct tcttgttgtc 2220aaaatacagc ctggaatggc
cttcaattct actggaataa tcaatggaac tagtagtact 2280catgaggaaa
gaatagacac tctgaattca tttccaataa aaatatgtat gattctagtt
2340tttttatcaa gtatatttgg atattgcaaa gaagtgatcc aaattttcca
acagaaaagg 2400aattacttcc tggattacaa caatgctctg gaatgggtta
tctatacaac tagtatcatc 2460ttcgtgttgc ccttgttcct caacatccca
gcgtatatgc agtggcaatg tggagcaata 2520gcgatattct tctactggat
gaacttccta ctgtatcttc aaaggtttga gaactgtgga 2580attttcattg
ttatgttgga ggtgattttt aaaacattgc tgagatcgac cggagtgttt
2640atcttcctcc tactggcttt tggcctcagc ttttatgttc tcctgaattt
ccaagatgcc 2700ttcagcaccc cattgctttc cttaatccag acattcagta
tgatgctagg agacatcaat 2760tatcgagatg ccttcctaga accattgttt
agaaatgagt tggcataccc agtcctgacc 2820tttgggcagc ttattgcctt
cacaatgttt gtcccaattg ttctcatgaa cttactgatt 2880ggcttggcgg
ttggggacat tgctgaggtc cagaagcatg cgtcattgaa gaggattgct
2940atgcaggtgg aacttcatac caacttagaa aaaaagctgc cactctggta
cttacgcaaa 3000gtggatcaga ggtccaccat cgtgtatcca aatagaccca
ggcacggcag gatgctacgg 3060ttttttcatt actttcttaa tatgcaagaa
acacgacaag aagtaccaaa cattgacaca 3120tgcttggaaa tggaaatatt
gaaacagaaa tatcggctga aggacctcac ttccctcttg 3180gaaaagcagc
atgagctcat caaactcatc atccagaaga tggagatcat ctcagagaca
3240gaagatgaag ataaccattg ctctttccaa gacaggttca agaaggagag
gctggaacag 3300atgcacagca agtggaattt tgtcttaaac gcagttaaga
ctaaaacaca ttgttctatt 3360agccacccgg acttttag 3378
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