U.S. patent application number 12/767294 was filed with the patent office on 2010-09-02 for modulators of odorant receptors.
This patent application is currently assigned to Duke University. Invention is credited to Hiroaki Matsunami, Momoka Matsunami, Harumi Saito, Hanyi Zhuang.
Application Number | 20100222561 12/767294 |
Document ID | / |
Family ID | 36034502 |
Filed Date | 2010-09-02 |
United States Patent
Application |
20100222561 |
Kind Code |
A1 |
Matsunami; Hiroaki ; et
al. |
September 2, 2010 |
MODULATORS OF ODORANT RECEPTORS
Abstract
The present invention relates to polypeptides capable of
promoting odorant receptor cell surface localization and odorant
receptor functional expression. The present invention further
provides assays for the detection of ligands specific for various
odorant receptors. Additionally, the present invention provides
methods of screening for odorant receptor accessory protein
polymorphisms and mutations associated with disease states, as well
as methods of screening for therapeutic agents, ligands, and
modulators of such proteins.
Inventors: |
Matsunami; Hiroaki; (Durham,
SC) ; Matsunami; Momoka; (Durham, NC) ; Saito;
Harumi; (Durham, NC) ; Zhuang; Hanyi; (Durham,
NC) |
Correspondence
Address: |
Casimir Jones, S.C.
2275 DEMING WAY, SUITE 310
MIDDLETON
WI
53562
US
|
Assignee: |
Duke University
Durham
NC
|
Family ID: |
36034502 |
Appl. No.: |
12/767294 |
Filed: |
April 26, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12261778 |
Oct 30, 2008 |
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12767294 |
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11811050 |
Jun 8, 2007 |
7691592 |
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12261778 |
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11156516 |
Jun 20, 2005 |
7425445 |
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11811050 |
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60581087 |
Jun 18, 2004 |
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60582011 |
Jun 22, 2004 |
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Current U.S.
Class: |
536/23.5 |
Current CPC
Class: |
A61P 27/16 20180101;
G01N 33/74 20130101; G01N 33/5058 20130101; A61P 11/02 20180101;
C07K 14/4705 20130101; G01N 2333/726 20130101; C07K 14/47 20130101;
G01N 2500/00 20130101; A61P 43/00 20180101; C12N 2510/00 20130101;
G01N 2500/04 20130101; A61P 25/02 20180101; C12N 2503/02 20130101;
G01N 33/6893 20130101; G01N 33/5041 20130101 |
Class at
Publication: |
536/23.5 |
International
Class: |
C07H 21/04 20060101
C07H021/04 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under Grant
No. DC05782 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. An isolated and purified nucleic acid sequence encoding a
protein having an amino acid sequence recited in SEQ ID NO: 38 and
variants thereof that are at least 90% identical to an amino acid
sequence recited in SEQ ID NO: 38.
2. The nucleic acid sequence of claim 1, wherein said nucleic acid
sequence is operably linked to a heterologous promoter.
3. The nucleic acid sequence of claim 1, wherein said nucleic acid
sequence is contained within a vector.
4. The nucleic acid sequence of claim 3, wherein said vector is
expressed in a host cell.
5. A composition comprising the nucleic acid sequence of claim
1.
6. An isolated and purified nucleic acid sequence that hybridizes
under conditions of high stringency to a nucleic acid sequence
recited in SEQ ID NO: 18, and variants thereof that are at least
90% identical to a nucleic acid sequence recited in SEQ ID NO:
18.
7. The nucleic acid sequence of claim 6, wherein said sequence is
operably linked to a heterologous promoter.
8. The nucleic acid sequence of claim 6, wherein said sequence is
contained within a vector.
9. The nucleic acid sequence of claim 8, wherein said vector is
expressed in a host cell.
10. A composition comprising the nucleic acid sequence of claim 6.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of pending U.S. patent
application Ser. No. 12/261,778 filed Oct. 30, 2008, which is a
Continuation of pending U.S. patent application Ser. No. 11/811,050
filed Jun. 8, 2007, which is a Divisional of U.S. patent
application Ser. No. 11/156,516 filed Jun. 20, 2005 which issued on
Sep. 16, 2008 as U.S. Pat. No. 7,425,445, which claims priority to
expired U.S. Provisional Application Ser. No. 60/581,087, filed
Jun. 18, 2004, and expired U.S. Provisional Application Ser. No.
60/582,011, filed Jun. 22, 2004, each of which is herein
incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0003] The present invention relates to polypeptides capable of
promoting odorant receptor cell surface localization and odorant
receptor functional expression. The present invention further
provides assays for the detection of ligands specific for various
odorant receptors. Additionally, the present invention provides
methods of screening for odorant receptor accessory protein
polymorphisms and mutations associated with disease states, as well
as methods of screening for therapeutic agents, ligands, and
modulators of such proteins.
BACKGROUND OF THE INVENTION
[0004] Olfactory dysfunction arises from a variety of causes and
profoundly influences a patient's quality of life. Approximately 2
million Americans experience some type of olfactory dysfunction.
Studies show that olfactory dysfunction affects at least 1% of the
population under the age of 65 years, and well over 50% of the
population older than 65 years. The sense of smell determines the
flavor of foods and beverages and serves as an early warning system
for the detection of environmental hazards, such as spoiled food,
leaking natural gas, smoke, or airborne pollutants. The losses or
distortions of smell sensation can adversely influence food
preference, food intake and appetite.
[0005] Olfactory disorders are classified as follows: 1) anosmia:
inability to detect qualitative olfactory sensations (e.g., absence
of smell function), 2) partial anosmia: ability to perceive some,
but not all, odorants, 3) hyposmia or microsmia: decreased
sensitivity to odorants, 4) hyperosmia: abnormally acute smell
function, 5) dysosmia (cacosmia or parosmia): distorted or
perverted smell perception or odorant stimulation, 6) phantosmia:
dysosmic sensation perceived in the absence of an odor stimulus
(a.k.a. olfactory hallucination), and 7) olfactory agnosia:
inability to recognize an odor sensation.
[0006] Olfactory dysfunction is further classified as 1) conductive
or transport impairments from obstruction of nasal passages (e.g.,
chronic nasal inflammation, polyposis, etc.), 2) sensorineural
impairments from damage to neuroepithelium (e.g., viral infection,
airborne toxins, etc.), 3) central olfactory neural impairment from
central nervous system damage (e.g., tumors, masses impacting on
olfactory tract, neurodegenerative disorders, etc.). These
categories are not mutually exclusive. For example, viruses can
cause damage to the olfactory neuroepithelium and they may also be
transported into the central nervous system via the olfactory nerve
causing damage to the central elements of the olfactory system.
[0007] Smelling abilities are initially determined by neurons in
the olfactory epithelium, the olfactory sensory neurons
(hereinafter "olfactory neurons). In olfactory neurons, odorant
receptor (hereinafter "OR") proteins, members of the G-protein
coupled receptor (hereinafter "GPCR") superfamily, are synthesized
in the endoplasmic reticulum, transported, and eventually
concentrated at the cell surface membrane of the cilia at the tip
of the dendrite. Considering that ORs have roles in target
recognition of developing olfactory axons, OR proteins are also
present at axon terminals (see, e.g., Mombaerts, P., (1996) Cell
87, 675-686; Wang, F., et al. (1998) Cell 93, 47-60; each herein
incorporated by reference in their entireties). In rodents,
odorants are transduced by as many as 1000 different ORs encoded by
a multigene family (see, e.g., Axel, R. (1995) Sci Am 1273,
154-159; Buck, L., and Axel, R. (1991) Cell 65, 175-187; Firestein,
S. (2001) Nature 413, 211-218; Mombaerts, P. (1999) Annu Rev
Neurosci 22, 487-509; Young, J. M., et al., (2002) Hum Mol Genet
11, 535-546; Zhang, X., and Firestein, S. (2002) Nat Neurosci 5,
124-133; each herein incorporated by reference in their entirety).
Each olfactory neuron expresses only one type of the OR, forming
the cellular basis of odorant discrimination by olfactory neurons
(see, e.g., Lewcock, J. W., and Reed, R. R. (2004) Proc Natl Acad
Sci USA; Malnic, B., et al., (1999) Cell 96, 713-723; Serizawa, S.,
et al., (2003) Science 302, 2088-2094; each herein incorporated by
reference in their entirety).
[0008] What is needed is a better understanding of olfactory
sensation. What is further needed is a better understanding of
odorant receptor function.
SUMMARY OF THE INVENTION
[0009] The present invention relates to polypeptides capable of
promoting odorant receptor cell surface localization and odorant
receptor functional expression. The present invention further
provides assays for the detection of ligands specific for various
odorant receptors. Additionally, the present invention provides
methods of screening for odorant receptor accessory protein
polymorphisms and mutations associated with disease states, as well
as methods of screening for therapeutic agents, ligands, and
modulators of such proteins.
[0010] In preferred embodiments, the present invention provides a
method for identifying an odorant receptor ligand, comprising the
steps of a) providing i) a cell line or cell membranes thereof
comprising an odorant receptor and a reporting agent, and ii) a
test compound; b) exposing the test compound to the cell line; and
c) measuring the activity of the reporting agent. In some
embodiments, the cell line expresses REEP1, RTP1, RTP2, RTP1-A,
RTP1-B, RTP1-C, RTP1-D, and RTP1-E, RTP1-A1, RTP1-D1, RTP-D2, and
RTP1-D3. In some embodiments, the cell line is a heterologous cell
line or a natural cell line. In some embodiments, the cell line is
a 293T cell line. In preferred embodiments, the odorant receptor is
a human odorant receptor. In other preferred embodiments, the test
compound is an odiferous molecule. In even further embodiments, the
reporting agent is regulated by a cAMP responsive element. In
preferred embodiments, the cell line further comprises
G.sub..alpha.olf. In other embodiments, the odorant receptor is a
murine odorant receptor. In other embodiments, the odorant receptor
is a synthetic odorant receptor. In preferred embodiments, the
odorant receptor comprises S6/79, S18, S46, S50, MOR23-1, MOR31-4,
MOR31-6, MOR32-5 and/or MOR32-11. In other embodiments, the
reporting agent is an illuminating agent. In even other
embodiments, the illuminating agent is luciferase. In alternate
embodiments, the method further comprises the step of detecting the
presence or absence of an odorant receptor ligand based upon the
reporting agent activity.
[0011] In preferred embodiments, the present invention provides a
cell line expressing an odorant receptor, wherein the expression is
localized to the cell surface. In preferred embodiments, the cell
line comprises a heterologous gene. In preferred embodiments, the
heterologous gene comprises one or more of REEP1, RTP1, and RTP2.
In other preferred embodiments, the cell line is a 293T cell line.
In some embodiments, the odorant receptor is a human odorant
receptor. In preferred embodiments, the odorant receptor is tagged
with a reporting agent. In some embodiments, the reporting agent is
an illuminating reporting agent. In some embodiments, the
illuminating reporting agent comprises glutathione-S-transferase
(GST), c-myc, 6-histidine (6.times.-His), green fluorescent protein
(GFP), maltose binding protein (MBP), influenza A virus
haemagglutinin (HA), .beta.-galactosidase, or GAL4. In preferred
embodiments, the cell line further comprises G.sub..alpha.olf
expression. In preferred embodiments, the odorant receptor is a
murine odorant receptor. In some embodiments, the odorant receptor
is a synthetic odorant receptor. In preferred embodiments, the
odorant receptor comprises S6/79, S18, S46, S50, MOR23-1, MOR31-4,
MOR31-6, MOR32-5 and MOR32-11.
[0012] The present invention further provides an isolated nucleic
acid comprising a sequence encoding a protein comprising SEQ ID
NOs: 21, 27, 33, 34, 37, 38, and 41-50, and variants thereof that
are at least 80% identical to SEQ ID NOs: 21, 27, 33, 34, 37, 38,
and 41-50. In preferred embodiments, the sequence is operably
linked to a heterologous promoter. In preferred embodiments, the
sequence is contained within a vector. In preferred embodiments,
the vector is within a host cell.
[0013] The present invention also provides isolated and purified
nucleic acid sequences that hybridize under conditions of high
stringency to a nucleic acid comprising SEQ ID NOs: 1, 7, 13, 14,
17 and/or 18. In preferred embodiments, the sequence is operably
linked to a heterologous promoter. In preferred embodiments, the
sequence is contained within a vector. In some embodiments, the
host vector is within a host cell. In further preferred
embodiments, the host vector is expressed in a host cell. In
preferred embodiments, the host cell is located in an organism,
wherein the organism is a non-human animal. In preferred
embodiments, the present invention provides a polynucleotide
sequence comprising at least fifteen (e.g., 15, 18, 20, 21, 25, 50,
100, 1000, . . . ) nucleotides capable of hybridizing under
stringent conditions to the isolated nucleotide sequence.
[0014] In preferred embodiments, the present invention provides a
polypeptide encoded by a nucleic acid selected from the group
consisting of SEQ ID NOs: 1, 7, 13, 14, 17 and 18 and variants
thereof that are at least 80% identical to SEQ ID NOs: 1, 7, 13,
14, 17 and 18. In further embodiments, the protein is at least 90%
identical to SEQ ID NOs: 1, 7, 13, 14, 17 and 18. In even further
embodiments, the protein is at least 95% identical to SEQ ID NOs:
1, 7, 13, 14, 17 and 18.
[0015] In preferred embodiments, the present invention provides a
composition comprising a nucleic acid that inhibits the binding of
at least a portion of a nucleic acid selected from the group
consisting of SEQ ID NOs: 1, 7, 13, 14, 17 and 18 to their
complementary sequences.
[0016] In preferred embodiments, the present invention provides a
method for detection of a variant REEP polypeptide in a subject,
comprising providing a biological sample from a subject, wherein
the biological sample comprises a REEP polypeptide; and detecting
the presence or absence of a variant REEP polypeptide in the
biological sample.
[0017] In preferred embodiments, the biological sample is selected
from the group consisting of a blood sample, a tissue sample, a
urine sample, and an amniotic fluid sample. In further embodiments,
the subject is selected from the group consisting of an embryo, a
fetus, a newborn animal, and a young animal. In further
embodiments, the animal is a human. In preferred embodiments, the
detecting comprises differential antibody binding. In further
embodiments, the detection comprises a Western blot. In some
preferred embodiments, the variant REEP polypeptide is a variant
REEP1 polypeptide. In further embodiments, the detecting comprises
detecting a REEP1 nucleic acid sequence.
[0018] In preferred embodiments, the present invention provides a
method for detection of a variant RTP polypeptide in a subject,
comprising providing a biological sample from a subject, wherein
the biological sample comprises a RTP polypeptide; and detecting
the presence or absence of a variant RTP polypeptide in the
biological sample. In preferred embodiments, the biological sample
is selected from the group consisting of a blood sample, a tissue
sample, a urine sample, and an amniotic fluid sample. In further
embodiments, the subject is selected from the group consisting of
an embryo, a fetus, a newborn animal, and a young animal. In
further embodiments, the animal is a human. In preferred
embodiments, the detecting comprises differential antibody binding.
In further embodiments, the detection comprises a Western blot. In
some preferred embodiments, the variant RTP polypeptide is a
variant RTP1 and/or RTP2 polypeptide. In further embodiments, the
detecting comprises detecting a RTP1 and/or RTP2 nucleic acid
sequence. In preferred embodiments, the RTP1 variant is selected
from the group consisting of RTP1-A1, RTP1-D1, and RTP1-D3.
[0019] In preferred embodiments, the present invention provides a
kit comprising a reagent for detecting the presence or absence of a
variant REEP polypeptide in a biological sample. In some
embodiments, the kit further comprises instruction for using the
kit for the detecting the presence or absence of a variant REEP
polypeptide in a biological sample. In preferred embodiments, the
REEP polypeptide is a REEP1 polypeptide. In other embodiments, the
REEP polypeptide is selected from the group consisting of REEP1-6.
In preferred embodiments, the instructions comprise instructions
required by the U.S. Food and Drug Agency for in vitro diagnostic
kits. In preferred embodiments, the reagent is one or more
antibodies. In preferred embodiments, the biological sample is
selected from the group consisting of a blood sample, a tissue
sample, a urine sample, and an amniotic fluid sample. In preferred
embodiments, the reagents are configured to detect a REEP1 nucleic
acid sequence.
[0020] In preferred embodiments, the present invention provides a
kit comprising a reagent for detecting the presence or absence of a
variant RTP polypeptide in a biological sample. In some
embodiments, the kit further comprises instruction for using the
kit for the detecting the presence or absence of a variant RTP
polypeptide in a biological sample. In preferred embodiments, the
RTP polypeptide is a RTP1 and/or RTP2 polypeptide. In other
embodiments, the RTP polypeptide is selected from the group
consisting of RTP1-4. In preferred embodiments, the instructions
comprise instructions required by the U.S. Food and Drug Agency for
in vitro diagnostic kits. In preferred embodiments, the reagent is
one or more antibodies. In preferred embodiments, the biological
sample is selected from the group consisting of a blood sample, a
tissue sample, a urine sample, and an amniotic fluid sample. In
preferred embodiments, the reagents are configured to detect a RTP1
and/or RTP2 nucleic acid sequence. In preferred embodiments, the
RTP1 polypeptide is a variant RTP1 polypeptide selected from the
group consisting of RTP1-A1, RTP1-D1, and RTP1-D3.
[0021] In preferred embodiments, the present invention provides a
method for screening compounds, comprising providing a sample
expressing a heterologous REEP polypeptide and a test compound; and
exposing the sample to the test compound and detecting a biological
effect. In preferred embodiments, the REEP polypeptide is selected
from the group consisting of REEP1-6. In preferred embodiments, the
sample comprises a cell. In preferred embodiments, the sample
comprises a tissue. In preferred embodiments, the sample is found
in a subject. In some embodiments, the biological effect comprises
a change in activity of REEP. In some embodiments, the biological
effect comprises a change in expression of REEP.
[0022] In preferred embodiments, the present invention provides a
method for screening compounds, comprising providing a sample
expressing a heterologous RTP polypeptide and a test compound; and
exposing the sample to the test compound and detecting a biological
effect. In preferred embodiments, the RTP polypeptide is selected
from the group consisting of RTP1-4 and RTP1-A1, RTP1-D1, and
RTP1-D3. In preferred embodiments, the sample comprises a cell. In
preferred embodiments, the sample comprises a tissue. In preferred
embodiments, the sample is found in a subject. In some embodiments,
the biological effect comprises a change in activity of RTP. In
some embodiments, the biological effect comprises a change in
expression of RTP.
DESCRIPTION OF THE FIGURES
[0023] FIG. 1 shows a screening strategy for identifying molecules
that promote cell-surface expression of odorant receptors. REEP1
was obtained from Digital Differential Display analysis. RTP1 was
obtained from SAGE libraries.
[0024] FIG. 2 shows REEP and/or RTP promote cell-surface expression
of odorant receptors in 293T Cells. (A) cDNAs encoding diverse ORs
(MOR203-1, OREG, olfr62, OR-S46 and rat 17) were transfected with
or without REEP1, RTP1 and/or RTP2. Increased cell-surface staining
of ORs was seen in cells co-expressing the accessory proteins. In
contrast, no difference in cell-surface staining was seen in cells
expressing .beta.2 adrenergic receptors. Using living-cell staining
protocols, cell-surface fluorescent signals are seen as distinctive
punctate staining. Scale bar equals to 50 um.
(B) Normalized numbers of labelled cells is shown for each
transfection condition (N=4918-15526). After double
immunofluorescent staining against Rho-tagged receptors and the
HA-tagged .beta.2adrenergic receptor, FACS analysis was performed
to quantify immunopositive cells. The number of Rho-tagged receptor
positive cells was normalized to that of HA-tagged .beta.2
adrenergic receptor positive cells. In almost all cases, more
immunopositive cells were observed when different ORs were
expressed with REEP1, RTP1 and/or RTP2. In contrast, when VR4 and
mT2R5 receptor was used instead of ORs, no enhancement was
observed. (C) Normalized mean fluorescence of labelled cells is
shown. The mean fluorescence of .beta.2adrenergic receptor was used
as a control. Stronger fluorescence was observed when different ORs
were expressed with REEP1, RTP1 and/or RTP2. In contrast, when VR4
and mT2R5 receptor was used instead of ORs, no enhancement was
observed. (D) A summary of the FACS analysis is shown.
[0025] FIG. 3 shows that REEP and/or RTP do not promote
cell-surface expression of VR4 and mT2R5 in 293T Cells. cDNAs
encoding VR4 and mT2R5 were transfected with or without REEP1, RTP1
and/or RTP2. Unlike ORs, increased cell-surface staining was not
seen in cells expressing these proteins. BFP expression is shown to
demonstrate high (.about.70%) transfection efficiency of VR4
transfected cells. Using living-cell staining protocols,
cell-surface fluorescent signals are seen as distinctive punctate
staining. Scale bar equals to 50 um.
[0026] FIG. 4 presents fluorescent hisogram data for REEP1, RTP1,
and RTP2 expression with odorant receptor (A) olfr62 and (B)
mT2R5.
[0027] FIG. 5 shows the REEP and the RTP families. (A) Deduced
amino acid sequences of REEP1 (SEQ ID NO: 21). Solid bar indicates
putative transmembrane region (TM). The first TM region could
function as a signal peptide. (B) Unrooted phylogenetic tree of
REEP family members. At least 6 REEP family members (REEP1-6) were
identified on the mouse genome. Yeast YOP1P, barley HVA22, and
human DP1 are homologous to REEP proteins. (C) Deduced amino acid
sequences of RTP1 and RTP2. Solid bar indicates putative
transmembrane domain. Shaded amino acids are conserved between RTP1
(SEQ ID NO: 33)and RTP2 (SEQ ID NO: 34). There are two more members
(RTP3 and 4) on the mouse genome.
[0028] FIG. 6 shows expression of REEP1, RTP1 and RTP2. (A)
Northern blot analysis. Total RNA was used for northern blotting
analysis. Olfactory epithelium, vomeronasal organ, and brain showed
.about.3.6 kb bands corresponding to REEP1 mRNA. Only olfactory
epithelium and vomeronasal organ RNAs showed .about.3.5 kb and
.about.2.6 kb bands corresponding RTP1 and RTP2 mRNA, respectively.
Ethidium bromide staining for 18S rRNA is shown as a control. (B)
In situ hybridization analysis in the olfactory epithelium. Among
REEP members, only REEP1 was expressed specifically by the
olfactory neurons. REEP6 was expressed by supporting cells. Among
RTP members, RTP1 and RTP2 are strongly expressed by the olfactory
neurons. RTP4 was also expressed by the olfactory neurons but at
much lower level. OMP is a marker for mature olfactory neurons.
Higher magnification of REEP1, RTP1, and RTP2 suggests that all
olfactory neurons may express all three molecules. Scale bar: 200
um (70 um in high magnification pictures). (C) In situ analysis of
REEP1 in the brain. REEP1 was expressed by a subset of brain cells.
Scale bar: 200 um.
[0029] FIG. 7 shows association of odorant Receptors with REEP1 and
RTP1. (A) Control western blot analysis indicating expression of
HA-tagged MOR203-1, Flag-tagged REEP1, RTP1 and ICAP1 in 293T
cells. (B) When Flag-RTP1 or Flag-REEP1 was precipitated,
HA-MOR203-1 proteins were co-precipitated (Lanes 1 and 2). However,
when Flag-ICAP-1 (a negative control protein) was precipitated,
HA-MOR203-1 proteins were not detected (Lane 3). (C) When
HA-MOR203-1 was precipitated, Flag-REEP1 and Flag-RTP1 were
co-purified when co-expressed (Lanes 1 and 2). Negative control
protein (Flag-ICAP-1) was not co-precipitated (Lane 3). Asterisks
indicate nonspecific Ig proteins. (D) Little cell-surface
expression was observed when RTP1 was transfected in 293T cells.
However, when RTP1 and an odorant receptor (OREG) were
co-transfected, more RTP1 staining signal was observed. (E) A small
amount of cell-surface signal was observed when REEP1 was
transfected in 293T cells. Co expression of an OR (olfr62) did not
change the expression of REEP1. Scale bars equal to 50 um.
[0030] FIG. 8 shows that expression of REEP1, RTP1 or RTP 2
enhances odorant receptor activation. (A) Diagram showing cAMP
responsive element (CRE) and luciferase was used to monitor
activation of ORs. Activation of ORs increases cAMP, which enhances
the expression of luciferase reporter gene through the CRE. (B)
Normalized luciferase activities .+-.SEM (N=4). REEP1, RTP1 and
RTP2, expressed in various combination together with OREG, enhanced
luciferase activities compared to OR alone. (C) Relative luciferase
activities .+-.SEM (N=4). OREG or OR-S46 was used to ask if REEP1,
RTP1, or RTP2 could change ligand specificities of ORs. To obtain
relative activation to different odorants, luciferase activity to
300 uM of vanillin (OREG) or decanoic acid (OR-S46) was regarded as
1 in each expression condition. (D) Normalized luciferase
activities .+-.SEM (N=8). Enhanced response in Hana3A cells, a
stable cell line expressing REEP1, RTP1, RTP2 and Golf, when three
different ORs were expressed.
(E) cAMP assays. Enhanced cAMP production to various concentrations
of eugenol in Hana3A cells when OREG was transfected. In contrast,
cAMP production was not different between Hana3A cells and 293T
cells expressing G.sub..alpha.olf when .beta.2adrenergic receptor
was transfected and isoproterenol was used.
[0031] FIG. 9 shows RT-PCR analysis of Hana3A cells; + indicates
PCR products using cDNA samples from Hana3A cells as template DNA;
- indicates negative controls without reverse transcriptase; M
indicates DNA marker.
[0032] FIG. 10 shows cell-surface expression of odorant receptors
in Hana3A and 293T cells. cDNAs encoding three ORs (OREG, olfr62
and OR-S46) were transfected into Hana3A cells or 293T cells.
Increased cell-surface staining was seen in Hana3A cells. Scale bar
equals to 50 um.
[0033] FIG. 11 shows recognition profiles of odorant receptors to
odorants. (A) Test odorants are shown on the left. The color
indicate relative luciferase activities (N=4). Each OR responded to
different subset of odorants. (B) and (C) Normalized luciferase
activities (N=4). 139 chemicals were used for initial ligand
screening of MOR203-1 and olfr62. MOR203-1 responded to nonanoic
acid. Olfr62 responded to five related aromatic compounds.
[0034] FIG. 12 shows cell-surface expression of 8 odorant receptors
in Hana3A cells. Scale bar equals to 50 um.
[0035] FIG. 13 shows models for the roles of REEP and/or RTP in
odorant receptor expression.
[0036] FIG. 14 shows the nucleic acid (mRNA) sequence (SEQ ID NO:
1) and amino acid sequence (SEQ ID NO: 21) for murine REEP1.
[0037] FIG. 15 shows the nucleic acid (mRNA) sequence (SEQ ID NO:
2) and amino acid sequence (SEQ ID NO: 22) for murine REEP2.
[0038] FIG. 16 shows the nucleic acid (mRNA) sequence (SEQ ID NO:
3) and amino acid sequence (SEQ ID NO: 23) for murine REEP3.
[0039] FIG. 17 shows the nucleic acid (mRNA) sequence (SEQ ID NO:
4) and amino acid sequence (SEQ ID NO: 24) for murine REEP4.
[0040] FIG. 18 shows the nucleic acid (mRNA) sequence (SEQ ID NO:
5) and amino acid sequence (SEQ ID NO: 25) for murine REEP5.
[0041] FIG. 19 shows the nucleic acid (mRNA) sequence (SEQ ID NO:
6) and amino acid sequence (SEQ ID NO: 26) for murine REEP6.
[0042] FIG. 20 shows the nucleic acid (mRNA) sequence (SEQ ID NO:
7) and amino acid sequence (SEQ ID NO: 27) for human REEP1.
[0043] FIG. 21 shows the nucleic acid (mRNA) sequence (SEQ ID NO:
8) and amino acid sequence (SEQ ID NO: 28) for human REEP2.
[0044] FIG. 22 shows the nucleic acid (mRNA) sequence (SEQ ID NO:
9) and amino acid sequence (SEQ ID NO: 29) for human REEP3.
[0045] FIG. 23 shows the nucleic acid (mRNA) sequence (SEQ ID NO:
10) and amino acid sequence (SEQ ID NO: 30) for human REEP4.
[0046] FIG. 24 shows the nucleic acid (mRNA) sequence (SEQ ID NO:
11) and amino acid sequence (SEQ ID NO: 31) for human REEP5.
[0047] FIG. 25 shows the nucleic acid (mRNA) sequence (SEQ ID NO:
12) and amino acid sequence (SEQ ID NO: 32) for human REEP6.
[0048] FIG. 26 shows the nucleic acid (mRNA) sequence (SEQ ID NO:
13) and amino acid sequence (SEQ ID NO: 33) for murine RTP1.
[0049] FIG. 27 shows the nucleic acid (mRNA) sequence (SEQ ID NO:
14) and amino acid sequence (SEQ ID NO: 34) for murine RTP2.
[0050] FIG. 28 shows the nucleic acid (mRNA) sequence (SEQ ID NO:
15) and amino acid sequence (SEQ ID NO: 35) for murine RTP3.
[0051] FIG. 29 shows the nucleic acid (mRNA) sequence (SEQ ID NO:
16) and amino acid sequence (SEQ ID NO: 36) for murine RTP4.
[0052] FIG. 30 shows the nucleic acid (mRNA) sequence (SEQ ID NO:
17) for human RTP1-A1 and the amino acid sequence (SEQ ID NO: 37)
for human RTP1.
[0053] FIG. 31 shows the nucleic acid (mRNA) sequence (SEQ ID NO:
18) and amino acid sequence (SEQ ID NO: 38) for human RTP2.
[0054] FIG. 32 shows the nucleic acid (mRNA) sequence (SEQ ID NO:
19) and amino acid sequence (SEQ ID NO: 39) for human RTP3.
[0055] FIG. 33 shows the nucleic acid (mRNA) sequence (SEQ ID NO:
20) and amino acid sequence (SEQ ID NO: 40) for human RTP4.
[0056] FIG. 34 shows the activation patterns of human odorant
receptors in response to odiferous agent exposure.
[0057] FIG. 35 schematically shows amino acid segments of RTP1-A,
RTP1-B, RTP1-C, RTP1-D, and RTP1-E in comparison to RTP1.
[0058] FIG. 36 shows the murine amino acid sequence for RTP1-A (SEQ
ID NO: 41).
[0059] FIG. 37 shows the murine amino acid sequence for RTP1-B (SEQ
ID NO: 42).
[0060] FIG. 38 shows the murine amino acid sequence for RTP1-C (SEQ
ID NO: 43).
[0061] FIG. 39 shows the murine amino acid sequence for RTP1-D (SEQ
ID NO: 44).
[0062] FIG. 40 shows the murine amino acid sequence for RTP1-E (SEQ
ID NO: 45).
[0063] FIG. 41 shows cell-surface expression of OLFR62 in Hana3A
and 293T cells. cDNAs encoding RTP1, RTP1-A, RTP1-B, RTP1-C, RTP1-D
and RTP1-E were transfected into Hana3A cells or 293T cells.
Increased cell-surface staining was seen in Hana3A cells and 239T
cells expressing RTP1-D.
[0064] FIG. 42 schematically shows a luciferase assay used to
monitor the activity of OLFR62 activity. cAMP responsive element
(CRE) and luciferase was used to monitor activation of OLFR62.
Activation of OLFR62 increases cAMP, which enhances the expression
of luciferase reporter gene through the CRE.
[0065] FIG. 43 shows OLFR62 activity as indicated by luciferase
expression in Hana3A cells and 293T cells expressing RTP1, RTP1-A,
RTP1-B, RTP1-C, RTP1-D, RTP1-E, and control pCI.
[0066] FIG. 44 schematically shows the amino acid segments of
RTP1-A1, RTP1-D1, RTP1-D2, and RTP1-D3 in comparison to RTP1-A and
RTP1-D, respectively.
[0067] FIG. 45 shows the murine amino acid sequence for RTP1-A1
(SEQ ID NO: 46), and the human amino acid sequence for RTP1-A1 (SEQ
ID NO: 47).
[0068] FIG. 46 shows the murine amino acid sequence for RTP1-D1
(SEQ ID NO: 48).
[0069] FIG. 47 shows the murine amino acid sequence for RTP-D2 (SEQ
ID NO: 49).
[0070] FIG. 48 shows the murine amino acid sequence for RTP-D3 (SEQ
ID NO: 50).
[0071] FIG. 49 shows cell-surface expression of OLFR62 in 293T
cells. cDNAs encoding RTP1, RTP1-A1, RTP1-D1, RTP1-D2, and RTP1-D3,
and control pCI were transfected into 293T cells. Increased
cell-surface staining was seen in 239T cells expressing RTP1-A1,
RTP1-D1 and RTP1-D3.
[0072] FIG. 50 shows OLFR62, OREG, S6, and 23-1 activity as
indicated by luciferase expression in 293T cells expressing RTP1,
RTP1-A1, RTP1-D1, RTP1-D2, and RTP1-D3, and control pCI.
[0073] FIG. 51 shows OLFR62, OREG, S6, and 23-1 activity as
indicated by luciferase expression in Hana3A cells expressing RTP1,
RTP1-A1, RTP1-D1, RTP1-D2, and RTP1-D3, and control pCI.
[0074] FIG. 52 shows cell-surface expression of OLFR62, OREG,
MOR203-1, S6, and 23-1 in 293T cells co-transfected with either
RTP1, RTP1-A1 or control pCI. cDNAs encoding RTP1, RTP1-A1, and
control pCI were transfected into cells.
[0075] FIG. 53 schematically shows the amino acid segments of
RTP1-A1-A (Chimera 1), RTP1-A1-D2 (Chimera 2), RTP1-A1-D1 (Chimera
3), RTP4-A1-A (Chimera 4), RTP4-A1-D2 (Chimera 5), and RTP4-A1-D1
(Chimera 6).
[0076] FIG. 54 shows cell-surface expression of an OR in cells
expressing RTP1, RTP4, Chimera 1, Chimera 2, Chimera 3, Chimera 4,
Chimera 5, Chimera 6, and control pCI. cDNAs encoding RTP1, RTP4,
RTP1-A1, Chimera 1, Chimera 2, Chimera 3, Chimera 4, Chimera 5,
Chimera 6, and control pCI were transfected into 293T cells.
[0077] FIG. 55 shows OLFR62, OREG, S6, and 23-1 activity as
indicated by luciferase expression in 293T cells expressing RTP1,
RTP4, RTP1-A1, RTP1-D1, RTP1-D2, Chimera 1, Chimera 2, Chimera 3,
Chimera 4, Chimera 5, Chimera 6, and control pCI.
[0078] FIG. 56 shows detection of RTP1, RTP1-A, RTP1-B, RTP1-C,
RTP1-A1, RTP1-D, Chimera 4, Chimera 5, RTP1-D3, RTP1-D1, Chimera 6,
and RTP4 using anti-RTP1.
DEFINITIONS
[0079] To facilitate understanding of the invention, a number of
terms are defined below.
[0080] As used herein, the term "REEP" when used in reference to
proteins or nucleic acid refers to a REEP protein or nucleic acid
encoding a REEP protein of the present invention. The term REEP
encompasses both proteins that are identical to wild-type REEPs
(e.g., REEP1, REEP2, REEP3, REEP4, REEP5, and REEP6) and those that
are derived from wild-type REEP (e.g. variants of REEP polypeptides
of the present invention). In some embodiments, the "REEP" is a
wild type murine REEP nucleic acid (mRNA) (e.g., SEQ ID NOs: 1-6)
or a polypeptide encoded by the wild type murine REEP amino acid
sequence (e.g., SEQ ID NOs:21-26). In other embodiments, the "REEP"
is a wild type human REEP nucleic acid (mRNA) (e.g., SEQ ID NOs:
7-12) or a polypeptide encoded by a wild type human REEP amino acid
sequence (e.g., SEQ ID NOs: 27-32). Examples of REEP proteins or
nucleic acids include, but are not limited to, REEP1, REEP2, REEP3,
REEP4, REEP5 and REEP6.
[0081] As used herein, the term "RTP" when used in reference to
proteins or nucleic acid refers to a RTP protein or nucleic acid
encoding a RTP protein of the present invention. The term RTP
encompasses both proteins that are identical to wild-type RTPs
(e.g., RTP1, RTP2, RTP3, and RTP4) and those that are derived from
wild-type RTP (e.g. variants of RTP polypeptides of the present
invention including but not limited to RTP1-A, RTP1-B, RTP1-C,
RTP1-D, RTP1-E, RTP1-A1, RTP1-D1, RTP-D2, RTP-D3, or chimeric genes
constructed with portions of RTP1 coding regions (e.g., RTP1-A1-A
(Chimera 1), RTP1-A1-D2 (Chimera 2), RTP1-A1-D1 (Chimera 3),
RTP4-A1-A (Chimera 4), RTP4-A1-D2 (Chimera 5), and RTP4-A1-D1
(Chimera 6)). In some embodiments, the "RTP" is a wild type murine
RTP nucleic acid (mRNA) (e.g., SEQ ID NOs: 13-16) or a polypeptide
encoded by the wild type or variant murine RTP amino acid sequence
(e.g., SEQ ID NOs: 33-36, 41-50). In other embodiments, the "RTP"
is a wild type human RTP nucleic acid (mRNA) (e.g., SEQ ID NOs: 17
for RTP1-A1, and SEQ ID NOs: 18-20 for RTP2, RTP3, and RTP4) or a
polypeptide encoded by a wild type human RTP amino acid sequence
(e.g., SEQ ID NOs: 37-40). Examples of RTP proteins or nucleic
acids include, but are not limited to, RTP1, RTP2, RTP3, RTP4,
RTP1-A, RTP1-B, RTP1-C, RTP1-D, RTP1-E, RTP1-A1, RTP1-D1, RTP-D2,
RTP-D3, RTP1-A1-A (Chimera 1), RTP1-A1-D2 (Chimera 2), RTP1-A1-D1
(Chimera 3), RTP4-A1-A (Chimera 4), RTP4-A1-D2 (Chimera 5), and
RTP4-A1-D1 (Chimera 6).
[0082] As used herein, the term "odorant receptor" refers to
odorant receptors generated from olfactory sensory neurons.
Examples of odorant receptors include, but are not limited to,
S6/79, S18, S46, S50, MOR23-1, MOR31-4, MOR31-6, MOR32-5 and
MOR32-11.
[0083] As used herein, the term "odorant receptor cell surface
localization" or equivalent terms refer to the molecular transport
of an odorant receptor to a cell surface membrane. Examples of cell
surface localization includes, but is not limited to, localization
to cilia at the tip of a dendrite, and localization to an axon
terminal.
[0084] As used herein, the term "odorant receptor functional
expression" or equivalent terms, refer to an odorant receptor's
ability to interact with an odorant receptor ligand (e.g., an
odiferous molecule).
[0085] As used herein, the term "olfactory disorder," "olfactory
dysfunction," "olfactory disease" or similar term refers to a
disorder, dysfunction or disease resulting in a diminished
olfactory sensation (e.g., smell aberration). Examples of olfactory
disorders, dysfunctions and/or diseases include, but are not
limited to, head trauma, upper respiratory infections, tumors of
the anterior cranial fossa, Kallmann syndrome, Foster Kennedy
syndrome, Parkinson's disease, Alzheimer's disease, Huntington
chorea, and exposure to toxic chemicals or infections. Diminished
olfactory sensation is classified as anosmia--absence of smell
sensation; hyposmia--decreased smell sensation;
dysosmia--distortion of smell sensation; cacosmia--sensation of a
bad or foul smell; and parosmia--sensation of smell in the absence
of appropriate stimulus.
[0086] As used herein, the term "REEP1" when used in reference to a
protein or nucleic acid refers to a REEP1 protein or nucleic acid
encoding a REEP1 protein of the present invention. The term REEP1
encompasses both proteins that are identical to wild-type REEP1 and
those that are derived from wild type REEP1 (e.g., variants of
REEP1 polypeptides of the present invention) or chimeric genes
constructed with portions of REEP1 coding regions). In some
embodiments, the "REEP1" is a wild type murine REEP1 nucleic acid
(mRNA) (SEQ ID NO:1) or polypeptide encoded by the wild type murine
amino acid sequence (SEQ ID NO: 21). In other embodiments, the
"REEP1" is a wild type human REEP1 nucleic acid (mRNA) (SEQ ID NO:
7) or polypeptide encoded by the wild type human REEP1 amino acid
sequence (SEQ ID NO: 27). In other embodiments, the "REEP1" is a
variant or mutant nucleic acid or amino acid.
[0087] As used herein, the term "RTP1" when used in reference to a
protein or nucleic acid refers to a RTP1 protein or nucleic acid
encoding a RTP1 protein of the present invention. The term RTP1
encompasses both proteins that are identical to wild-type RTP1 and
those that are derived from wild type RTP1 (e.g., variants of RTP1
polypeptides of the present invention including but not limited to
RTP1-A, RTP1-B, RTP1-C, RTP1-D, RTP1-E, RTP1-A1, RTP1-D1, RTP-D2,
RTP-D3) or chimeric genes constructed with portions of RTP1 coding
regions (e.g., RTP1-A1-A (Chimera 1), RTP1-A1-D2 (Chimera 2),
RTP1-A1-D1 (Chimera 3), RTP4-A1-A (Chimera 4), RTP4-A1-D2 (Chimera
5), and RTP4-A1-D1 (Chimera 6)). In some embodiments, the "RTP1" is
a wild type murine RTP1 nucleic acid (mRNA) (SEQ ID NO:13) or
polypeptide encoded by the wild type murine amino acid sequence
(SEQ ID NO: 33). In other embodiments, the "RTP1" is a wild type
human RTP1 nucleic acid (mRNA) (SEQ ID NO: 17 for RTP1-A1) or
polypeptide encoded by the wild type human RTP1 amino acid sequence
(SEQ ID NO: 37). In other embodiments, the "RTP1" is a variant or
mutant nucleic acid or amino acid.
[0088] As used herein, the term "RTP2" when used in reference to a
protein or nucleic acid refers to a RTP2 protein or nucleic acid
encoding a RTP2 protein of the present invention. The term RTP2
encompasses both proteins that are identical to wild-type RTP2 and
those that are derived from wild type RTP2 (e.g., variants of RTP2
polypeptides of the present invention) or chimeric genes
constructed with portions of RTP2 coding regions). In some
embodiments, the "RTP2" is a wild type murine RTP2 nucleic acid
(mRNA) (SEQ ID NO:14) or polypeptide encoded by the wild type
murine amino acid sequence (SEQ ID NO: 34). In other embodiments,
the "RTP2" is a wild type human RTP2 nucleic acid (mRNA) (SEQ ID
NO: 18) or polypeptide encoded by the wild type human REEP1 amino
acid sequence (SEQ ID NO: 38). In other embodiments, the "RTP2" is
a variant or mutant nucleic acid or amino acid.
[0089] As used herein, the terms "subject" and "patient" refer to
any animal, such as a mammal like a dog, cat, bird, livestock, and
preferably a human. Specific examples of "subjects" and "patients"
include, but are not limited to, individuals with an olfactory
disorder, and individuals with olfactory disorder-related
characteristics or symptoms.
[0090] As used herein, the phrase "symptoms of an olfactory
disorder" and "characteristics of an olfactory disorder" include,
but are not limited to, a diminished olfactory sensation (e.g.,
smell aberration).
[0091] The phrase "under conditions such that the symptoms are
reduced" refers to any degree of qualitative or quantitative
reduction in detectable symptoms of olfactory disorders, including
but not limited to, a detectable impact on the rate of recovery
from disease, or the reduction of at least one symptom of an
olfactory disorder.
[0092] The term "siRNAs" refers to short interfering RNAs. Methods
for the use of siRNAs are described in U.S. Patent App. No.:
20030148519/A1 (herein incorporated by reference). In some
embodiments, siRNAs comprise a duplex, or double-stranded region,
of about 18-25 nucleotides long; often siRNAs contain from about
two to four unpaired nucleotides at the 3' end of each strand. At
least one strand of the duplex or double-stranded region of a siRNA
is substantially homologous to or substantially complementary to a
target RNA molecule. The strand complementary to a target RNA
molecule is the "antisense strand;" the strand homologous to the
target RNA molecule is the "sense strand," and is also
complementary to the siRNA antisense strand. siRNAs may also
contain additional sequences; non-limiting examples of such
sequences include linking sequences, or loops, as well as stem and
other folded structures. siRNAs appear to function as key
intermediaries in triggering RNA interference in invertebrates and
in vertebrates, and in triggering sequence-specific RNA degradation
during posttranscriptional gene silencing in plants.
[0093] The term "RNA interference" or "RNAi" refers to the
silencing or decreasing of gene expression by siRNAs. It is the
process of sequence-specific, post-transcriptional gene silencing
in animals and plants, initiated by siRNA that is homologous in its
duplex region to the sequence of the silenced gene. The gene may be
endogenous or exogenous to the organism, present integrated into a
chromosome or present in a transfection vector that is not
integrated into the genome. The expression of the gene is either
completely or partially inhibited. RNAi may also be considered to
inhibit the function of a target RNA; the function of the target
RNA may be complete or partial.
[0094] As used herein, the terms "instructions for using said kit
for said detecting the presence or absence of a variant REEP1
nucleic acid or polypeptide in said biological sample,"
"instructions for using said kit for said detecting the presence or
absence of a variant RTP1 nucleic acid or polypeptide in said
biological sample," "instructions for using said kit for said
detecting the presence or absence of a variant RTP2 nucleic acid or
polypeptide in said biological sample" include instructions for
using the reagents contained in the kit for the detection of
variant and wild type REEP and/or RTP nucleic acids or
polypeptides.
[0095] The term "gene" refers to a nucleic acid (e.g., DNA)
sequence that comprises coding sequences necessary for the
production of a polypeptide, RNA (e.g., including but not limited
to, mRNA, tRNA and rRNA) or precursor (e.g., REEP1, RTP1 or RTP2).
The polypeptide, RNA, or precursor can be encoded by a full length
coding sequence or by any portion of the coding sequence so long as
the desired activity or functional properties (e.g., enzymatic
activity, ligand binding, signal transduction, etc.) of the
full-length or fragment are retained. The term also encompasses the
coding region of a structural gene and the sequences located
adjacent to the coding region on both the 5' and 3' ends for a
distance of about 1 kb on either end such that the gene corresponds
to the length of the full-length mRNA. The sequences that are
located 5' of the coding region and which are present on the mRNA
are referred to as 5' untranslated sequences. The sequences that
are located 3' or downstream of the coding region and that are
present on the mRNA are referred to as 3' untranslated sequences.
The term "gene" encompasses both cDNA and genomic forms of a gene.
A genomic form or clone of a gene contains the coding region
interrupted with non-coding sequences termed "introns" or
"intervening regions" or "intervening sequences." Introns are
segments of a gene that are transcribed into nuclear RNA (hnRNA);
introns may contain regulatory elements such as enhancers. Introns
are removed or "spliced out" from the nuclear or primary
transcript; introns therefore are absent in the messenger RNA
(mRNA) transcript. The mRNA functions during translation to specify
the sequence or order of amino acids in a nascent polypeptide.
[0096] In particular, the term "REEP1 gene," "RTP1 gene," "RTP1
genes," "RTP2 gene," or "RTP2 genes" refer to the full-length
respective REEP and/or RTP nucleotide sequence (e.g., contained in
SEQ ID NOs:1, 2 and 3). However, it is also intended that the term
encompass fragments of the REEP and/or RTP sequences (e.g., RTP1-A,
RTP1-B, RTP1-C, RTP1-D, and RTP1-E, RTP1-A1, RTP1-D1, RTP-D2,
RTP-D3), chimeric genes constructed with portions of RTP1 coding
regions (e.g., RTP1-A1-A (Chimera 1), RTP1-A1-D2 (Chimera 2),
RTP1-A1-D1 (Chimera 3), RTP4-A1-A (Chimera 4), RTP4-A1-D2 (Chimera
5), and RTP4-A1-D1 (Chimera 6)), mutants of the REEP and/or RTP
sequences, as well as other domains within the full-length REEP
and/or RTP nucleotide sequences. Furthermore, the terms "REEP1
nucleotide sequence," "REEP1 polynucleotide sequence," "RTP1
nucleotide sequence," "RTP1 polynucleotide sequence," "RTP2
nucleotide sequence," or "RTP2 polynucleotide sequence" encompasses
DNA sequences, cDNA sequences, RNA (e.g., mRNA) sequences, and
associated regulatory sequences.
[0097] Where "amino acid sequence" is recited herein to refer to an
amino acid sequence of a naturally occurring protein molecule,
"amino acid sequence" and like terms, such as "polypeptide" or
"protein" are not meant to limit the amino acid sequence to the
complete, native amino acid sequence associated with the recited
protein molecule.
[0098] In addition to containing introns, genomic forms of a gene
may also include sequences located on both the 5' and 3' end of the
sequences that are present on the RNA transcript. These sequences
are referred to as "flanking" sequences or regions (these flanking
sequences are located 5' or 3' to the non-translated sequences
present on the mRNA transcript). The 5' flanking region may contain
regulatory sequences such as promoters and enhancers that control
or influence the transcription of the gene. The 3' flanking region
may contain sequences that direct the termination of transcription,
post-transcriptional cleavage and polyadenylation.
[0099] The term "wild-type" refers to a gene or gene product that
has the characteristics of that gene or gene product when isolated
from a naturally occurring source. A wild-type gene is that which
is most frequently observed in a population and is thus arbitrarily
designed the "normal" or "wild-type" form of the gene. In contrast,
the terms "modified," "mutant," "polymorphism," and "variant" refer
to a gene or gene product that displays modifications in sequence
and/or functional properties (i.e., altered characteristics) when
compared to the wild-type gene or gene product. It is noted that
naturally-occurring mutants can be isolated; these are identified
by the fact that they have altered characteristics when compared to
the wild-type gene or gene product.
[0100] As used herein, the terms "nucleic acid molecule encoding,"
"DNA sequence encoding," and "DNA encoding" refer to the order or
sequence of deoxyribonucleotides along a strand of deoxyribonucleic
acid. The order of these deoxyribonucleotides determines the order
of amino acids along the polypeptide (protein) chain. The DNA
sequence thus codes for the amino acid sequence.
[0101] DNA molecules are said to have "5' ends" and "3' ends"
because mononucleotides are reacted to make oligonucleotides or
polynucleotides in a manner such that the 5' phosphate of one
mononucleotide pentose ring is attached to the 3' oxygen of its
neighbor in one direction via a phosphodiester linkage. Therefore,
an end of an oligonucleotides or polynucleotide, referred to as the
"5' end" if its 5' phosphate is not linked to the 3' oxygen of a
mononucleotide pentose ring and as the "3' end" if its 3' oxygen is
not linked to a 5' phosphate of a subsequent mononucleotide pentose
ring. As used herein, a nucleic acid sequence, even if internal to
a larger oligonucleotide or polynucleotide, also may be said to
have 5' and 3' ends. In either a linear or circular DNA molecule,
discrete elements are referred to as being "upstream" or 5' of the
"downstream" or 3' elements. This terminology reflects the fact
that transcription proceeds in a 5' to 3' fashion along the DNA
strand. The promoter and enhancer elements that direct
transcription of a linked gene are generally located 5' or upstream
of the coding region. However, enhancer elements can exert their
effect even when located 3' of the promoter element and the coding
region. Transcription termination and polyadenylation signals are
located 3' or downstream of the coding region.
[0102] As used herein, the terms "an oligonucleotide having a
nucleotide sequence encoding a gene" and "polynucleotide having a
nucleotide sequence encoding a gene," means a nucleic acid sequence
comprising the coding region of a gene or, in other words, the
nucleic acid sequence that encodes a gene product. The coding
region may be present in a cDNA, genomic DNA, or RNA form. When
present in a DNA form, the oligonucleotide or polynucleotide may be
single-stranded (i.e., the sense strand) or double-stranded.
Suitable control elements such as enhancers/promoters, splice
junctions, polyadenylation signals, etc. may be placed in close
proximity to the coding region of the gene if needed to permit
proper initiation of transcription and/or correct processing of the
primary RNA transcript. Alternatively, the coding region utilized
in the expression vectors of the present invention may contain
endogenous enhancers/promoters, splice junctions, intervening
sequences, polyadenylation signals, etc. or a combination of both
endogenous and exogenous control elements.
[0103] As used herein, the term "regulatory element" refers to a
genetic element that controls some aspect of the expression of
nucleic acid sequences. For example, a promoter is a regulatory
element that facilitates the initiation of transcription of an
operably linked coding region. Other regulatory elements include
splicing signals, polyadenylation signals, termination signals,
etc.
[0104] As used herein, the terms "complementary" or
"complementarity" are used in reference to polynucleotides (i.e., a
sequence of nucleotides) related by the base-pairing rules. For
example, for the sequence 5'-"A-G-T-3'," is complementary to the
sequence 3'-"T-C-A-5'." Complementarity may be "partial," in which
only some of the nucleic acids' bases are matched according to the
base pairing rules. Or, there may be "complete" or "total"
complementarity between the nucleic acids. The degree of
complementarity between nucleic acid strands has significant
effects on the efficiency and strength of hybridization between
nucleic acid strands. This is of particular importance in
amplification reactions, as well as detection methods that depend
upon binding between nucleic acids. Complementarity can include the
formation of base pairs between any type of nucleotides, including
non-natural bases, modified bases, synthetic bases and the
like.
[0105] The term "homology" refers to a degree of complementarity.
There may be partial homology or complete homology (i.e.,
identity). A partially complementary sequence is one that at least
partially inhibits a completely complementary sequence from
hybridizing to a target nucleic acid and is referred to using the
functional term "substantially homologous." The term "inhibition of
binding," when used in reference to nucleic acid binding, refers to
inhibition of binding caused by competition of homologous sequences
for binding to a target sequence. The inhibition of hybridization
of the completely complementary sequence to the target sequence may
be examined using a hybridization assay (Southern or Northern blot,
solution hybridization and the like) under conditions of low
stringency. A substantially homologous sequence or probe will
compete for and inhibit the binding (i.e., the hybridization) of a
completely homologous to a target under conditions of low
stringency. This is not to say that conditions of low stringency
are such that non-specific binding is permitted; low stringency
conditions require that the binding of two sequences to one another
be a specific (i.e., selective) interaction. The absence of
non-specific binding may be tested by the use of a second target
that lacks even a partial degree of complementarity (e.g., less
than about 30% identity); in the absence of non-specific binding
the probe will not hybridize to the second non-complementary
target.
[0106] The art knows well that numerous equivalent conditions may
be employed to comprise low stringency conditions; factors such as
the length and nature (DNA, RNA, base composition) of the probe and
nature of the target (DNA, RNA, base composition, present in
solution or immobilized, etc.) and the concentration of the salts
and other components (e.g., the presence or absence of formamide,
dextran sulfate, polyethylene glycol) are considered and the
hybridization solution may be varied to generate conditions of low
stringency hybridization different from, but equivalent to, the
above listed conditions. In addition, the art knows conditions that
promote hybridization under conditions of high stringency (e.g.,
increasing the temperature of the hybridization and/or wash steps,
the use of formamide in the hybridization solution, etc.).
[0107] When used in reference to a double-stranded nucleic acid
sequence such as a cDNA or genomic clone, the term "substantially
homologous" refers to any probe that can hybridize to either or
both strands of the double-stranded nucleic acid sequence under
conditions of low stringency as described above.
[0108] A gene may produce multiple RNA species that are generated
by differential splicing of the primary RNA transcript. cDNAs that
are splice variants of the same gene will contain regions of
sequence identity or complete homology (representing the presence
of the same exon or portion of the same exon on both cDNAs) and
regions of complete non-identity (for example, representing the
presence of exon "A" on cDNA 1 wherein cDNA 2 contains exon "B"
instead). Because the two cDNAs contain regions of sequence
identity they will both hybridize to a probe derived from the
entire gene or portions of the gene containing sequences found on
both cDNAs; the two splice variants are therefore substantially
homologous to such a probe and to each other.
[0109] When used in reference to a single-stranded nucleic acid
sequence, the term "substantially homologous" refers to any probe
that can hybridize (i.e., it is the complement of) the
single-stranded nucleic acid sequence under conditions of low
stringency as described above.
[0110] As used herein, the term "competes for binding" is used in
reference to a first polypeptide with an activity which binds to
the same substrate as does a second polypeptide with an activity,
where the second polypeptide is a variant of the first polypeptide
or a related or dissimilar polypeptide. The efficiency (e.g.,
kinetics or thermodynamics) of binding by the first polypeptide may
be the same as or greater than or less than the efficiency
substrate binding by the second polypeptide. For example, the
equilibrium binding constant (K.sub.D) for binding to the substrate
may be different for the two polypeptides. The term "K.sub.m" as
used herein refers to the Michaelis-Menton constant for an enzyme
and is defined as the concentration of the specific substrate at
which a given enzyme yields one-half its maximum velocity in an
enzyme catalyzed reaction.
[0111] As used herein, the term "hybridization" is used in
reference to the pairing of complementary nucleic acids.
Hybridization and the strength of hybridization (i.e., the strength
of the association between the nucleic acids) is impacted by such
factors as the degree of complementary between the nucleic acids,
stringency of the conditions involved, the T.sub.m of the formed
hybrid, and the G:C ratio within the nucleic acids.
[0112] As used herein, the term "T.sub.m" is used in reference to
the "melting temperature." The melting temperature is the
temperature at which a population of double-stranded nucleic acid
molecules becomes half dissociated into single strands. The
equation for calculating the T.sub.m of nucleic acids is well known
in the art. As indicated by standard references, a simple estimate
of the T.sub.m value may be calculated by the equation:
T.sub.m=81.5+0.41(% G+C), when a nucleic acid is in aqueous
solution at 1 M NaCl (See e.g., Anderson and Young, Quantitative
Filter Hybridization, in Nucleic Acid Hybridization [1985]). Other
references include more sophisticated computations that take
structural as well as sequence characteristics into account for the
calculation of T.sub.m.
[0113] As used herein the term "stringency" is used in reference to
the conditions of temperature, ionic strength, and the presence of
other compounds such as organic solvents, under which nucleic acid
hybridizations are conducted. Those skilled in the art will
recognize that "stringency" conditions may be altered by varying
the parameters just described either individually or in concert.
With "high stringency" conditions, nucleic acid base pairing will
occur only between nucleic acid fragments that have a high
frequency of complementary base sequences (e.g., hybridization
under "high stringency" conditions may occur between homologs with
about 85-100% identity, preferably about 70-100% identity). With
medium stringency conditions, nucleic acid base pairing will occur
between nucleic acids with an intermediate frequency of
complementary base sequences (e.g., hybridization under "medium
stringency" conditions may occur between homologs with about 50-70%
identity). Thus, conditions of "weak" or "low" stringency are often
required with nucleic acids that are derived from organisms that
are genetically diverse, as the frequency of complementary
sequences is usually less.
[0114] "High stringency conditions" when used in reference to
nucleic acid hybridization comprise conditions equivalent to
binding or hybridization at 42.degree. C. in a solution consisting
of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4H.sub.2O
and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,
5.times.Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm
DNA followed by washing in a solution comprising 0.1.times.SSPE,
1.0% SDS at 42.degree. C. when a probe of about 500 nucleotides in
length is employed.
[0115] "Medium stringency conditions" when used in reference to
nucleic acid hybridization comprise conditions equivalent to
binding or hybridization at 42.degree. C. in a solution consisting
of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2PO.sub.4H.sub.2O
and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,
5.times.Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm
DNA followed by washing in a solution comprising 1.0.times.SSPE,
1.0% SDS at 42.degree. C. when a probe of about 500 nucleotides in
length is employed.
[0116] "Low stringency conditions" comprise conditions equivalent
to binding or hybridization at 42.degree. C. in a solution
consisting of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l
NaH.sub.2PO.sub.4H.sub.2O and 1.85 g/l EDTA, pH adjusted to 7.4
with NaOH), 0.1% SDS, 5.times.Denhardt's reagent
[50.times.Denhardt's contains per 500 ml: 5 g Ficoll (Type 400,
Pharamcia), 5 g BSA (Fraction V; Sigma)] and 100 .mu.g/ml denatured
salmon sperm DNA followed by washing in a solution comprising
5.times.SSPE, 0.1% SDS at 42.degree. C. when a probe of about 500
nucleotides in length is employed.
[0117] The present invention is not limited to the hybridization of
probes of about 500 nucleotides in length. The present invention
contemplates the use of probes between approximately 10 nucleotides
up to several thousand (e.g., at least 5000) nucleotides in length.
One skilled in the relevant understands that stringency conditions
may be altered for probes of other sizes (See e.g., Anderson and
Young, Quantitative Filter Hybridization, in Nucleic Acid
Hybridization [1985] and Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press, NY [1989]).
[0118] The following terms are used to describe the sequence
relationships between two or more polynucleotides: "reference
sequence", "sequence identity", "percentage of sequence identity",
and "substantial identity". A "reference sequence" is a defined
sequence used as a basis for a sequence comparison; a reference
sequence may be a subset of a larger sequence, for example, as a
segment of a full-length cDNA sequence given in a sequence listing
or may comprise a complete gene sequence. Generally, a reference
sequence is at least 20 nucleotides in length, frequently at least
25 nucleotides in length, and often at least 50 nucleotides in
length. Since two polynucleotides may each (1) comprise a sequence
(i.e., a portion of the complete polynucleotide sequence) that is
similar between the two polynucleotides, and (2) may further
comprise a sequence that is divergent between the two
polynucleotides, sequence comparisons between two (or more)
polynucleotides are typically performed by comparing sequences of
the two polynucleotides over a "comparison window" to identify and
compare local regions of sequence similarity. A "comparison
window", as used herein, refers to a conceptual segment of at least
20 contiguous nucleotide positions wherein a polynucleotide
sequence may be compared to a reference sequence of at least 20
contiguous nucleotides and wherein the portion of the
polynucleotide sequence in the comparison window may comprise
additions or deletions (i.e., gaps) of 20 percent or less as
compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
Optimal alignment of sequences for aligning a comparison window may
be conducted by the local homology algorithm of Smith and Waterman
[Smith and Waterman, Adv. Appl. Math. 2: 482 (1981)] by the
homology alignment algorithm of Needleman and Wunsch [Needleman and
Wunsch, J. Mol. Biol. 48:443 (1970)], by the search for similarity
method of Pearson and Lipman [Pearson and Lipman, Proc. Natl. Acad.
Sci. (U.S.A.) 85:2444 (1988)], by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package Release 7.0, Genetics Computer Group, 575
Science Dr., Madison, Wis.), or by inspection, and the best
alignment (i.e., resulting in the highest percentage of homology
over the comparison window) generated by the various methods is
selected. The term "sequence identity" means that two
polynucleotide sequences are identical (i.e., on a
nucleotide-by-nucleotide basis) over the window of comparison. The
term "percentage of sequence identity" is calculated by comparing
two optimally aligned sequences over the window of comparison,
determining the number of positions at which the identical nucleic
acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to
yield the number of matched positions, dividing the number of
matched positions by the total number of positions in the window of
comparison (i.e., the window size), and multiplying the result by
100 to yield the percentage of sequence identity. The terms
"substantial identity" as used herein denotes a characteristic of a
polynucleotide sequence, wherein the polynucleotide comprises a
sequence that has at least 85 percent sequence identity, preferably
at least 90 to 95 percent sequence identity, more usually at least
99 percent sequence identity as compared to a reference sequence
over a comparison window of at least 20 nucleotide positions,
frequently over a window of at least 25-50 nucleotides, wherein the
percentage of sequence identity is calculated by comparing the
reference sequence to the polynucleotide sequence which may include
deletions or additions which total 20 percent or less of the
reference sequence over the window of comparison. The reference
sequence may be a subset of a larger sequence, for example, as a
segment of the full-length sequences of the compositions claimed in
the present invention (e.g., REEP1, RTP1 or RTP2).
[0119] As applied to polypeptides, the term "substantial identity"
means that two peptide sequences, when optimally aligned, such as
by the programs GAP or BESTFIT using default gap weights, share at
least 80 percent sequence identity, preferably at least 90 percent
sequence identity, more preferably at least 95 percent sequence
identity or more (e.g., 99 percent sequence identity). Preferably,
residue positions that are not identical differ by conservative
amino acid substitutions. Conservative amino acid substitutions
refer to the interchangeability of residues having similar side
chains. For example, a group of amino acids having aliphatic side
chains is glycine, alanine, valine, leucine, and isoleucine; a
group of amino acids having aliphatic-hydroxyl side chains is
serine and threonine; a group of amino acids having
amide-containing side chains is asparagine and glutamine; a group
of amino acids having aromatic side chains is phenylalanine,
tyrosine, and tryptophan; a group of amino acids having basic side
chains is lysine, arginine, and histidine; and a group of amino
acids having sulfur-containing side chains is cysteine and
methionine. Preferred conservative amino acids substitution groups
are: valine-leucine-isoleucine, phenylalanine-tyrosine,
lysine-arginine, alanine-valine, and asparagine-glutamine.
[0120] The term "fragment" as used herein refers to a polypeptide
that has an amino-terminal and/or carboxy-terminal deletion as
compared to the native protein, but where the remaining amino acid
sequence is identical to the corresponding positions in the amino
acid sequence deduced from a full-length cDNA sequence. Fragments
typically are at least 4 amino acids long, preferably at least 20
amino acids long, usually at least 50 amino acids long or longer,
and span the portion of the polypeptide required for intermolecular
binding of the compositions (claimed in the present invention) with
its various ligands and/or substrates.
[0121] The term "polymorphic locus" is a locus present in a
population that shows variation between members of the population
(i.e., the most common allele has a frequency of less than 0.95).
In contrast, a "monomorphic locus" is a genetic locus at little or
no variations seen between members of the population (generally
taken to be a locus at which the most common allele exceeds a
frequency of 0.95 in the gene pool of the population).
[0122] As used herein, the term "genetic variation information" or
"genetic variant information" refers to the presence or absence of
one or more variant nucleic acid sequences (e.g., polymorphism or
mutations) in a given allele of a particular gene (e.g., a REEP
and/or RTP gene of the present invention).
[0123] As used herein, the term "detection assay" refers to an
assay for detecting the presence or absence of variant nucleic acid
sequences (e.g., polymorphisms or mutations) in a given allele of a
particular gene (e.g., a REEP and/or RTP gene).
[0124] The term "naturally-occurring" as used herein as applied to
an object refers to the fact that an object can be found in nature.
For example, a polypeptide or polynucleotide sequence that is
present in an organism (including viruses) that can be isolated
from a source in nature and which has not been intentionally
modified by man in the laboratory is naturally-occurring.
[0125] "Amplification" is a special case of nucleic acid
replication involving template specificity. It is to be contrasted
with non-specific template replication (i.e., replication that is
template-dependent but not dependent on a specific template).
Template specificity is here distinguished from fidelity of
replication (i.e., synthesis of the proper polynucleotide sequence)
and nucleotide (ribo- or deoxyribo-) specificity. Template
specificity is frequently described in terms of "target"
specificity. Target sequences are "targets" in the sense that they
are sought to be sorted out from other nucleic acid. Amplification
techniques have been designed primarily for this sorting out.
[0126] As used herein, the term "primer" refers to an
oligonucleotide, whether occurring naturally as in a purified
restriction digest or produced synthetically, which is capable of
acting as a point of initiation of synthesis when placed under
conditions in which synthesis of a primer extension product which
is complementary to a nucleic acid strand is induced, (i.e., in the
presence of nucleotides and an inducing agent such as DNA
polymerase and at a suitable temperature and pH). The primer is
preferably single stranded for maximum efficiency in amplification,
but may alternatively be double stranded. If double stranded, the
primer is first treated to separate its strands before being used
to prepare extension products. Preferably, the primer is an
oligodeoxyribonucleotide. The primer must be sufficiently long to
prime the synthesis of extension products in the presence of the
inducing agent. The exact lengths of the primers will depend on
many factors, including temperature, source of primer and the use
of the method.
[0127] As used herein, the term "probe" refers to an
oligonucleotide (i.e., a sequence of nucleotides), whether
occurring naturally as in a purified restriction digest or produced
synthetically, recombinantly or by PCR amplification, that is
capable of hybridizing to another oligonucleotide of interest. A
probe may be single-stranded or double-stranded. Probes are useful
in the detection, identification and isolation of particular gene
sequences. It is contemplated that any probe used in the present
invention will be labeled with any "reporter molecule," so that is
detectable in any detection system, including, but not limited to
enzyme (e.g., ELISA, as well as enzyme-based histochemical assays),
fluorescent, radioactive, and luminescent systems. It is not
intended that the present invention be limited to any particular
detection system or label.
[0128] As used herein, the term "target," refers to a nucleic acid
sequence or structure to be detected or characterized. Thus, the
"target" is sought to be sorted out from other nucleic acid
sequences. A "segment" is defined as a region of nucleic acid
within the target sequence.
[0129] As used herein, the term "amplification reagents" refers to
those reagents (deoxyribonucleotide triphosphates, buffer, etc.),
needed for amplification except for primers, nucleic acid template,
and the amplification enzyme. Typically, amplification reagents
along with other reaction components are placed and contained in a
reaction vessel (test tube, microwell, etc.).
[0130] As used herein, the terms "restriction endonucleases" and
"restriction enzymes" refer to bacterial enzymes, each of which cut
double-stranded DNA at or near a specific nucleotide sequence.
[0131] As used herein, the term "recombinant DNA molecule" as used
herein refers to a DNA molecule that is comprised of segments of
DNA joined together by means of molecular biological
techniques.
[0132] As used herein, the term "antisense" is used in reference to
RNA sequences that are complementary to a specific RNA sequence
(e.g., mRNA). Included within this definition are antisense RNA
("asRNA") molecules involved in gene regulation by bacteria.
Antisense RNA may be produced by any method, including synthesis by
splicing the gene(s) of interest in a reverse orientation to a
viral promoter that permits the synthesis of a coding strand. Once
introduced into an embryo, this transcribed strand combines with
natural mRNA produced by the embryo to form duplexes. These
duplexes then block either the further transcription of the mRNA or
its translation. In this manner, mutant phenotypes may be
generated. The term "antisense strand" is used in reference to a
nucleic acid strand that is complementary to the "sense" strand.
The designation (-) (i.e., "negative") is sometimes used in
reference to the antisense strand, with the designation (+)
sometimes used in reference to the sense (i.e., "positive")
strand.
[0133] The term "isolated" when used in relation to a nucleic acid,
as in "an isolated oligonucleotide" or "isolated polynucleotide"
refers to a nucleic acid sequence that is identified and separated
from at least one contaminant nucleic acid with which it is
ordinarily associated in its natural source. Isolated nucleic acid
is present in a form or setting that is different from that in
which it is found in nature. In contrast, non-isolated nucleic
acids are nucleic acids such as DNA and RNA found in the state they
exist in nature. For example, a given DNA sequence (e.g., a gene)
is found on the host cell chromosome in proximity to neighboring
genes; RNA sequences, such as a specific mRNA sequence encoding a
specific protein, are found in the cell as a mixture with numerous
other mRNAs that encode a multitude of proteins. However, isolated
nucleic acid encoding REEP and/or RTP includes, by way of example,
such nucleic acid in cells ordinarily expressing REEP and/or RTP
where the nucleic acid is in a chromosomal location different from
that of natural cells, or is otherwise flanked by a different
nucleic acid sequence than that found in nature. The isolated
nucleic acid, oligonucleotide, or polynucleotide may be present in
single-stranded or double-stranded form. When an isolated nucleic
acid, oligonucleotide or polynucleotide is to be utilized to
express a protein, the oligonucleotide or polynucleotide will
contain at a minimum the sense or coding strand (i.e., the
oligonucleotide or polynucleotide may single-stranded), but may
contain both the sense and anti-sense strands (i.e., the
oligonucleotide or polynucleotide may be double-stranded).
[0134] As used herein, a "portion of a chromosome" refers to a
discrete section of the chromosome. Chromosomes are divided into
sites or sections by cytogeneticists as follows: the short
(relative to the centromere) arm of a chromosome is termed the "p"
arm; the long arm is termed the "q" arm. Each arm is then divided
into 2 regions termed region 1 and region 2 (region 1 is closest to
the centromere). Each region is further divided into bands. The
bands may be further divided into sub-bands. For example, the
11p15.5 portion of human chromosome 11 is the portion located on
chromosome 11 (11) on the short arm (p) in the first region (1) in
the 5th band (5) in sub-band 5 (0.5). A portion of a chromosome may
be "altered;" for instance the entire portion may be absent due to
a deletion or may be rearranged (e.g., inversions, translocations,
expanded or contracted due to changes in repeat regions). In the
case of a deletion, an attempt to hybridize (i.e., specifically
bind) a probe homologous to a particular portion of a chromosome
could result in a negative result (i.e., the probe could not bind
to the sample containing genetic material suspected of containing
the missing portion of the chromosome). Thus, hybridization of a
probe homologous to a particular portion of a chromosome may be
used to detect alterations in a portion of a chromosome.
[0135] The term "sequences associated with a chromosome" means
preparations of chromosomes (e.g., spreads of metaphase
chromosomes), nucleic acid extracted from a sample containing
chromosomal DNA (e.g., preparations of genomic DNA); the RNA that
is produced by transcription of genes located on a chromosome
(e.g., hnRNA and mRNA), and cDNA copies of the RNA transcribed from
the DNA located on a chromosome. Sequences associated with a
chromosome may be detected by numerous techniques including probing
of Southern and Northern blots and in situ hybridization to RNA,
DNA, or metaphase chromosomes with probes containing sequences
homologous to the nucleic acids in the above listed
preparations.
[0136] As used herein the term "coding region" when used in
reference to structural gene refers to the nucleotide sequences
that encode the amino acids found in the nascent polypeptide as a
result of translation of a mRNA molecule. The coding region is
bounded, in eukaryotes, on the 5' side by the nucleotide triplet
"ATG" that encodes the initiator methionine and on the 3' side by
one of the three triplets, which specify stop codons (i.e., TAA,
TAG, TGA).
[0137] As used herein, the term "purified" or "to purify" refers to
the removal of contaminants from a sample. For example, REEP and/or
RTP antibodies are purified by removal of contaminating
non-immunoglobulin proteins; they are also purified by the removal
of immunoglobulin that does not bind a REEP and/or RTP polypeptide.
The removal of non-immunoglobulin proteins and/or the removal of
immunoglobulins that do not bind a REEP and/or RTP polypeptide
results in an increase in the percent of REEP1, RTP1 or
RTP2-reactive immunoglobulins in the sample. In another example,
recombinant REEP and/or RTP polypeptides are expressed in bacterial
host cells and the polypeptides are purified by the removal of host
cell proteins; the percent of recombinant REEP and/or RTP
polypeptides is thereby increased in the sample.
[0138] The term "recombinant DNA molecule" as used herein refers to
a DNA molecule that is comprised of segments of DNA joined together
by means of molecular biological techniques.
[0139] The term "recombinant protein" or "recombinant polypeptide"
as used herein refers to a protein molecule that is expressed from
a recombinant DNA molecule.
[0140] The term "native protein" as used herein, is used to
indicate a protein that does not contain amino acid residues
encoded by vector sequences; that is the native protein contains
only those amino acids found in the protein as it occurs in nature.
A native protein may be produced by recombinant means or may be
isolated from a naturally occurring source.
[0141] As used herein the term "portion" when in reference to a
protein (as in "a portion of a given protein") refers to fragments
of that protein. The fragments may range in size from four
consecutive amino acid residues to the entire amino acid sequence
minus one amino acid.
[0142] The term "Southern blot," refers to the analysis of DNA on
agarose or acrylamide gels to fractionate the DNA according to size
followed by transfer of the DNA from the gel to a solid support,
such as nitrocellulose or a nylon membrane. The immobilized DNA is
then probed with a labeled probe to detect DNA species
complementary to the probe used. The DNA may be cleaved with
restriction enzymes prior to electrophoresis. Following
electrophoresis, the DNA may be partially depurinated and denatured
prior to or during transfer to the solid support. Southern blots
are a standard tool of molecular biologists (J. Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,
NY, pp 9.31-9.58 [1989]).
[0143] The term "Northern blot," as used herein refers to the
analysis of RNA by electrophoresis of RNA on agarose gels to
fractionate the RNA according to size followed by transfer of the
RNA from the gel to a solid support, such as nitrocellulose or a
nylon membrane. The immobilized RNA is then probed with a labeled
probe to detect RNA species complementary to the probe used.
Northern blots are a standard tool of molecular biologists (J.
Sambrook, et al., supra, pp 7.39-7.52 [1989]).
[0144] The term "Western blot" refers to the analysis of protein(s)
(or polypeptides) immobilized onto a support such as nitrocellulose
or a membrane. The proteins are run on acrylamide gels to separate
the proteins, followed by transfer of the protein from the gel to a
solid support, such as nitrocellulose or a nylon membrane. The
immobilized proteins are then exposed to antibodies with reactivity
against an antigen of interest. The binding of the antibodies may
be detected by various methods, including the use of radiolabeled
antibodies.
[0145] The term "antigenic determinant" as used herein refers to
that portion of an antigen that makes contact with a particular
antibody (i.e., an epitope). When a protein or fragment of a
protein is used to immunize a host animal, numerous regions of the
protein may induce the production of antibodies that bind
specifically to a given region or three-dimensional structure on
the protein; these regions or structures are referred to as
antigenic determinants. An antigenic determinant may compete with
the intact antigen (i.e., the "immunogen" used to elicit the immune
response) for binding to an antibody.
[0146] The term "transgene" as used herein refers to a foreign,
heterologous, or autologous gene that is placed into an organism by
introducing the gene into newly fertilized eggs or early embryos.
The term "foreign gene" refers to any nucleic acid (e.g., gene
sequence) that is introduced into the genome of an animal by
experimental manipulations and may include gene sequences found in
that animal so long as the introduced gene does not reside in the
same location as does the naturally-occurring gene. The term
"autologous gene" is intended to encompass variants (e.g.,
polymorphisms or mutants) of the naturally occurring gene. The term
transgene thus encompasses the replacement of the naturally
occurring gene with a variant form of the gene.
[0147] As used herein, the term "vector" is used in reference to
nucleic acid molecules that transfer DNA segment(s) from one cell
to another. The term "vehicle" is sometimes used interchangeably
with "vector."
[0148] The term "expression vector" as used herein refers to a
recombinant DNA molecule containing a desired coding sequence and
appropriate nucleic acid sequences necessary for the expression of
the operably linked coding sequence in a particular host organism.
Nucleic acid sequences necessary for expression in prokaryotes
usually include a promoter, an operator (optional), and a ribosome
binding site, often along with other sequences. Eukaryotic cells
are known to utilize promoters, enhancers, and termination and
polyadenylation signals.
[0149] As used herein, the term "host cell" refers to any
eukaryotic or prokaryotic cell (e.g., bacterial cells such as E.
coli, yeast cells, mammalian cells, avian cells, amphibian cells,
plant cells, fish cells, and insect cells), whether located in
vitro or in vivo. For example, host cells may be located in a
transgenic animal.
[0150] The terms "overexpression" and "overexpressing" and
grammatical equivalents, are used in reference to levels of mRNA to
indicate a level of expression approximately 3-fold higher than
that typically observed in a given tissue in a control or
non-transgenic animal. Levels of mRNA are measured using any of a
number of techniques known to those skilled in the art including,
but not limited to Northern blot analysis (See, Example 10, for a
protocol for performing Northern blot analysis).
[0151] The term "transfection" as used herein refers to the
introduction of foreign DNA into eukaryotic cells. Transfection may
be accomplished by a variety of means known to the art including
calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated
transfection, polybrene-mediated transfection, electroporation,
microinjection, liposome fusion, lipofection, protoplast fusion,
retroviral infection, and biolistics.
[0152] The term "stable transfection" or "stably transfected"
refers to the introduction and integration of foreign DNA into the
genome of the transfected cell. The term "stable transfectant"
refers to a cell that has stably integrated foreign DNA into the
genomic DNA.
[0153] The term "transient transfection" or "transiently
transfected" refers to the introduction of foreign DNA into a cell
where the foreign DNA fails to integrate into the genome of the
transfected cell. The foreign DNA persists in the nucleus of the
transfected cell for several days. During this time the foreign DNA
is subject to the regulatory controls that govern the expression of
endogenous genes in the chromosomes. The term "transient
transfectant" refers to cells that have taken up foreign DNA but
have failed to integrate this DNA.
[0154] The term "calcium phosphate co-precipitation" refers to a
technique for the introduction of nucleic acids into a cell. The
uptake of nucleic acids by cells is enhanced when the nucleic acid
is presented as a calcium phosphate-nucleic acid co-precipitate.
The original technique of Graham and van der Eb (Graham and van der
Eb, Virol., 52:456 [1973]), has been modified by several groups to
optimize conditions for particular types of cells. The art is well
aware of these numerous modifications.
[0155] A "composition comprising a given polynucleotide sequence"
as used herein refers broadly to any composition containing the
given polynucleotide sequence. The composition may comprise an
aqueous solution. Compositions comprising polynucleotide sequences
encoding REEP1s, RTP1s or RTP2s (e.g., SEQ ID NOs:1, 2 and 3) or
fragments thereof may be employed as hybridization probes. In this
case, the REEP and/or RTP encoding polynucleotide sequences are
typically employed in an aqueous solution containing salts (e.g.,
NaCl), detergents (e.g., SDS), and other components (e.g.,
Denhardt's solution, dry milk, salmon sperm DNA, etc.).
[0156] The term "test compound" refers to any chemical entity,
pharmaceutical, drug, and the like that can be used to treat or
prevent a disease, illness, sickness, or disorder of bodily
function, or otherwise alter the physiological or cellular status
of a sample. Test compounds comprise both known and potential
therapeutic compounds. A test compound can be determined to be
therapeutic by screening using the screening methods of the present
invention. A "known therapeutic compound" refers to a therapeutic
compound that has been shown (e.g., through animal trials or prior
experience with administration to humans) to be effective in such
treatment or prevention.
[0157] The term "sample" as used herein is used in its broadest
sense. A sample suspected of containing a human chromosome or
sequences associated with a human chromosome may comprise a cell,
chromosomes isolated from a cell (e.g., a spread of metaphase
chromosomes), genomic DNA (in solution or bound to a solid support
such as for Southern blot analysis), RNA (in solution or bound to a
solid support such as for Northern blot analysis), cDNA (in
solution or bound to a solid support) and the like. A sample
suspected of containing a protein may comprise a cell, a portion of
a tissue, an extract containing one or more proteins and the
like.
[0158] As used herein, the term "response," when used in reference
to an assay, refers to the generation of a detectable signal (e.g.,
accumulation of reporter protein, increase in ion concentration,
accumulation of a detectable chemical product).
[0159] As used herein, the term "reporter gene" refers to a gene
encoding a protein that may be assayed. Examples of reporter genes
include, but are not limited to, luciferase (See, e.g., deWet et
al., Mol. Cell. Biol. 7:725 [1987] and U.S. Pat. Nos. 6,074,859;
5,976,796; 5,674,713; and 5,618,682; all of which are incorporated
herein by reference), green fluorescent protein (e.g., GenBank
Accession Number U43284; a number of GFP variants are commercially
available from CLONTECH Laboratories, Palo Alto, Calif.),
chloramphenicol acetyltransferase, .beta.-galactosidase, alkaline
phosphatase, and horse radish peroxidase.
GENERAL DESCRIPTION
[0160] Continued progress in understanding olfactory coding has
been significantly hampered by the inability to functionally
express ORs in heterologous cells in order to identify cognate
ligands. To overcome this problem, experiments conducted during the
course of the present invention searched for molecules that are
included in cell-surface expression of ORs. Three transmembrane
proteins, REEP1, RTP1, and RTP2, as well as variants thereof, were
identified that promote functional cell surface expression of ORs
in 293T cells. REEP and/or RTP are expressed specifically by
olfactory neurons in the olfactory epithelium. REEP1 and RTP1
interacts with OR proteins. Using cells expressing REEP1 and RTP1
and RTP2, new ORs that respond to aliphatic odorants were
identified. The present invention is not limited to a particular
mechanism. Indeed, an understanding of the mechanism is not
necessary to practice the present invention. Nonetheless,
experiments conducted during the course of the present invention
demonstrated the importance of the accessory proteins of ORs in
functional cell-surface expression and in decoding OR-ligand
specificities.
[0161] The identification and use of proteins involved in the
localization of ORs provides numerous research, diagnostic, drug
screening, and therapeutic applications. For example, the nucleic
acids and proteins of the present invention permit the selective
and controllable presentation of ORs on test cells to, among other
things, identify new ORs, characterize ORs, identify OR ligands,
correlate olfactory responses to the molecular interactions
underlying such response, identify and characterize groups of ORs
and ligands responsible for olfactory responses and health
conditions, and identify, select, and characterize regulators of OR
response to study and control olfactory responses. The present
invention, also, thus provides means for manipulating olfactory
responses and the molecular basis for such response in vitro and in
vivo. Numerous commercial applications are thus made possible,
including the production, characterization, and use of in vitro or
in vivo cell arrays expressing desired localized ORs for screening
(e.g., high-throughput screening) compounds or use as synthetic
olfactory systems. Any industry, including food industries, health
industries, cosmetic industries, militaries, sanitary agencies,
animal sniffers (e.g., for drugs, explosives, accident victims,
etc.), among many others will find use of the compositions and
methods of the present invention.
[0162] Inhibitors (e.g., antibodies, small molecules, aptamers,
etc.) of OR/ligand interactions that are identified by the methods
of the present invention find may uses. For example, the present
invention provides a systematic way to identify which receptors and
ligands are responsible for particular olfactory sensations (e.g.,
perceived scents). Thus, for example, by blocking particular
interactions (e.g., via a nasal spray having the inhibitors) or
enhancing particular interactions (e.g., via a nasal spray that
provides certain ligands or a coating on the surface of an object
that emits certain ligands) one can control perceived scents. Thus,
undesired scents can be blocked, covered, or altered (e.g., a
sniffer dog can be treated so as to only smell a target of
interested and no other distracting smells, a sanitary worked can
be made immune to the scent of waste, etc.) and desired scents can
be enhanced.
[0163] The present invention also provides novel gene and protein
sequences and methods of their use. A detailed description of
certain preferred embodiments and uses of the present invention is
described below. The present invention is not limited to these
particular illustrative embodiments.
DETAILED DESCRIPTION OF THE INVENTION
[0164] The present invention relates to peptides capable of
promoting odorant receptor cell-surface localization and odorant
receptor functional expression. The present invention further
provides assays for the detection of therapeutic agents, and for
the detection of odorant receptor accessory protein polymorphisms
and mutations associated with disease states. Exemplary embodiments
of the present invention are described below.
[0165] Exemplary compositions and methods of the present invention
are described in more detail in the following sections: I.
Olfactory Sensation; II. REEP and RTP Polynucleotides; III. REEP
and RTP Polypeptides; IV. Detection of REEP and RTP Alleles; V.
Generation of REEP and RTP Antibodies; VI. Gene Therapy Using REEP
and RTP; VII. Transgenic Animals Expressing Exogenous REEP and RTP
Genes and Homologs, Mutants, and Variants Thereof, VIII. Drug
Screening Using REEP and RTP; IX. Pharmaceutical Compositions
Containing REEP and RTP Nucleic Acid, Peptides, and Analogs; X. RNA
Interference (RNAi); XI. RNAi for REEP and RTP; and XII.
Identification of Odorant Receptor Ligands.
[0166] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of organic chemistry,
pharmacology, molecular biology (including recombinant techniques),
cell biology, biochemistry, and immunology, which are within the
skill of the art. Such techniques are explained fully in the
literature, such as, "Molecular cloning: a laboratory manual"
Second Edition (Sambrook et al., 1989); "Oligonucleotide synthesis"
(M. J. Gait, ed., 1984); "Animal cell culture" (R. I. Freshney,
ed., 1987); the series "Methods in enzymology" (Academic Press,
Inc.); "Handbook of experimental immunology" (D. M. Weir & C.
C. Blackwell, eds.); "Gene transfer vectors for mammalian cells"
(J. M. Miller & M. P. Calos, eds., 1987); "Current protocols in
molecular biology" (F. M. Ausubel et al., eds., 1987, and periodic
updates); "PCR: the polymerase chain reaction" (Mullis et al.,
eds., 1994); and "Current protocols in immunology" (J. E. Coligan
et al., eds., 1991), each of which is herein incorporated by
reference in its entirety.
I. Olfactory Sensation
[0167] The olfactory system represents one of the oldest sensory
modalities in the phylogenetic history of mammals. Olfaction is
less developed in humans than in other mammals such as rodents. As
a chemical sensor, the olfactory system detects food and influences
social and sexual behavior. The specialized olfactory epithelial
cells characterize the only group of neurons capable of
regeneration. Activation occurs when odiferous molecules come in
contact with specialized processes known as the olfactory vesicles.
Within the nasal cavity, the turbinates or nasal conchae serve to
direct the inspired air toward the olfactory epithelium in the
upper posterior region. This area (only a few centimeters wide)
contains more than 100 million olfactory receptor cells. These
specialized epithelial cells give rise to the olfactory vesicles
containing kinocilia, which serve as sites of stimulus
transduction.
[0168] There are three specialized neural systems are present
within the nasal cavities in humans: 1) the main olfactory system
(cranial nerve I), 2) trigeminal somatosensory system (cranial
nerve V), 3) the nervus terminalis (cranial nerve 0). CN I mediates
odor sensation. It is responsible for determining flavors. CN V
mediates somatosensory sensations, including burning, cooling,
irritation, and tickling. CN 0 is a ganglionated neural plexus. It
spans much of the nasal mucosa before coursing through the
cribriform plate to enter the forebrain medial to the olfactory
tract. The exact function of the nervus terminalis is unknown in
humans.
[0169] The olfactory neuroepithelium is a pseudostratified columnar
epithelium. The specialized olfactory epithelial cells are the only
group of neurons capable of regeneration. The olfactory epithelium
is situated in the superior aspect of each nostril, including
cribriform plate, superior turbinate, superior septum, and sections
of the middle turbinate. It harbors sensory receptors of the main
olfactory system and some CN V free nerve endings. The olfactory
epithelium loses its general homogeneity postnatally, and as early
as the first few weeks of life metaplastic islands of
respiratory-like epithelium appear. The metaplasia increases in
extent throughout life. It is presumed that this process is the
result of insults from the environment, such as viruses, bacteria,
and toxins.
[0170] There are 6 distinct cells types in the olfactory
neuroepithelium: 1) bipolar sensory receptor neurons, 2)
microvillar cells, 3) supporting cells, 4) globose basal cells, 5)
horizontal basal cells, 6) cells lining the Bowman's glands. There
are approximately 6,000,000 bipolar neurons in the adult olfactory
neuroepithelium. They are thin dendritic cells with rods containing
cilia at one end and long central processes at the other end
forming olfactory fila. The olfactory receptors are located on the
ciliated dendritic ends. The unmyelinated axons coalesce into 40
bundles, termed olfactory fila, which are ensheathed by
Schwann-like cells. The fila transverses the cribriform plate to
enter the anterior cranial fossa and constitute CN I. Microvillar
cells are near the surface of the neuroepithelium, but the exact
functions of these cells are unknown. Supporting cells are also at
the surface of the epithelium. They join tightly with neurons and
microvillar cells. They also project microvilli into the mucus.
Their functions include insulating receptor cells from one another,
regulating the composition of the mucus, deactivating odorants, and
protecting the epithelium from foreign agents. The basal cells are
located near the basement membrane, and are the progenitor cells
from which the other cell types arise. The Bowman's glands are a
major source of mucus within the region of the olfactory
epithelium.
[0171] The odorant receptors are located on the cilia of the
receptor cells. Each receptor cell expresses a single odorant
receptor gene. There are approximately 1,000 classes of receptors
at present. The olfactory receptors are linked to the stimulatory
guanine nucleotide binding protein Golf. When stimulated, it can
activate adenylate cyclase to produce the second messenger cAMP,
and subsequent events lead to depolarization of the cell membrane
and signal propagation. Although each receptor cell only expresses
one type of receptor, each cell is electrophysiologically
responsive to a wide but circumscribed range of stimuli. This
implies that a single receptor accepts a range of molecular
entities.
[0172] The olfactory bulb is located on top of the cribriform plate
at the base of the frontal lobe in the anterior cranial fossa. It
receives thousands of primary axons from olfactory receptor
neurons. Within the olfactory bulb, these axons synapse with a much
smaller number of second order neurons which form the olfactory
tract and project to olfactory cortex. The olfactory cortex
includes the frontal and temporal lobes, thalamus, and
hypothalamus.
[0173] Although mammalian ORs were identified over 10 years ago,
little is known about the selectivity of the different ORs for
chemical stimuli, mainly because it has been difficult to express
ORs on the cell surface of heterologous cells and assay their
ligand-binding specificity (see, e.g., Mombaerts, P. (2004) Nat Rev
Neurosci 5, 263-278; herein incorporated by reference in its
entirety). The reason is that OR proteins are retained in the ER
and subsequently degraded in the proteosome (see, e.g., Lu, M., et
al., (2003) Traffic 4, 416-433; McClintock, T. S., (1997) Brain Res
Mol Brain Res 48, 270-278; each herein incorporated by reference in
their entireties). Despite these difficulties, extensive efforts
have matched about 20 ORs with cognate ligands with various degrees
of certainty (see, e.g., Bozza, T., et al., (2002) J Neurosci 22,
3033-3043; Gaillard, I., et al., (2002) Eur J Neurosci 15, 409-418;
Hatt, H., et al., (1999) Cell Mol Biol 45, 285-291; Kajiya, K., et
al., (2001) J Neurosci 21, 6018-6025; Krautwurst, D., et al.,
(1998) Cell 95, 917-926; Malnic, B., et al., (1999) Cell 96,
713-723; Raming, K., et al., (1993) Nature 361, 353-356; Spehr, M.,
et al., (2003) Science 299, 2054-2058; Touhara, K., et al., (1999)
Proc Natl Acad Sci USA 96, 4040-4045; Zhao, H., et al., (1998)
Science 279, 237-242; each herein incorporated by reference in
their entirety). Adding the 20 N-terminal amino acids of rhodopsin
(e.g., Rho-tag) or a foreign signal peptide to the N-terminus
facilitates surface expression of some ORs in heterologous cells
(see, e.g., Hatt, H., et al., (1999) Cell Mol Biol 45, 285-291;
Krautwurst, D., et al., (1998) Cell 95, 917-926; each herein
incorporated in their entirety). However, for most ORs,
modifications do not reliably promote cell-surface expression. For
example, ODR-4, which is required for proper localization of
chemosensory receptors in C. elegans, has a small effect on
facilitating cell-surface expression of one rat OR, but not another
OR (see, e.g., Gimelbrant, A. A., et al., (2001) J Biol Chem 276,
7285-7290; herein incorporated by reference). These findings
indicate that olfactory neurons have a selective molecular
machinery that promotes proper targeting of OR proteins to the cell
surface, but no components of this machinery have been identified
(see, e.g., Gimelbrant, A. A., et al., (2001) J Biol Chem 276,
7285-7290; McClintock, T. S., and Sammeta, N. (2003) Neuroreport
14, 1547-1552; each herein incorporated by reference in their
entirety).
[0174] For some GPCRs, accessory proteins are required for correct
targeting to the cell surface membrane (see, e.g., Brady, A. E.,
and Limbird, L. E. (2002) Cell Signal 14, 297-309; herein
incorporated by reference in its entirety). These proteins include
NinaA for Drosophila Rhodopsin (see, e.g., Baker, E. K., et al.,
(1994) Embo J 13, 4886-4895; Shieh, B. H., et al., (1989) Nature
338, 67-70; each herein incorporated by reference in their
entirety), RanBP2 for mammalian cone opsin (see, e.g., Ferreira, P.
A., et al., (1996) Nature 383, 637-640; herein incorporated by
reference in its entirety), RAMPs for the mammalian calcitonin
receptor-like receptor (CRLR) (see, e.g., McLatchie, L. M., et al.,
(1998) Nature 393, 333-339; herein incorporated by reference in its
entirety) and finally the M10 family of MHC class I proteins and
beta 2 microglobulin for V2Rs, the putative mammalian pheromone
receptors (see, e.g., Loconto, J., et al., (2003) Cell 112,
607-618; herein incorporated by reference in its entirety). With
the exception of NinaA and RanBP2, none of these accessory proteins
share any sequence homology to with each other; their only common
feature is their association with the membrane.
[0175] The present invention provides novel proteins (e.g., REEP1,
RTP1, RTP2, RTP1-A, RTP1-B, RTP1-C, RTP1-D, RTP1-E, RTP1-A1,
RTP1-D1, RTP-D2, RTP-D3, RTP1-A1-A (Chimera 1), RTP1-A1-D2 (Chimera
2), RTP1-A1-D1 (Chimera 3), RTP4-A1-A (Chimera 4), RTP4-A1-D2
(Chimera 5), and RTP4-A1-D1 (Chimera 6)) promoting OR cell surface
localization and OR functional expression, and numerous
compositions and methods related to these findings.
II. REEP and RTP Polynucleotides
[0176] As described above, the present invention provides novel
proteins promoting odorant receptor cell surface localization and
odorant receptor functional expression. In particular, the present
invention provides REEP genes and polypeptides (e.g., REEP1, REEP2,
REEP3, REEP4, REEP5, and REEP6) and RTP genes and polypeptides
(e.g., RTP1, RTP2, RTP3, RTP4, RTP1-A, RTP1-B, RTP1-C, RTP1-D,
RTP1-E, RTP1-A1, RTP1-D1, RTP-D2, RTP-D3, RTP1-A1-A (Chimera 1),
RTP1-A1-D2 (Chimera 2), RTP1-A1-D1 (Chimera 3), RTP4-A1-A (Chimera
4), RTP4-A1-D2 (Chimera 5), and RTP4-A1-D1 (Chimera 6)). In
preferred embodiments, REEP1, RTP1, RTP2, and variants of RTP1
(e.g., RTP1-A, RTP1-B, RTP1-C, RTP1-D, and RTP1-E, RTP1-A1,
RTP1-D1, RTP-D2, RTP-D3, RTP1-A1-A (Chimera 1), RTP1-A1-D2 (Chimera
2), RTP1-A1-D1 (Chimera 3), RTP4-A1-A (Chimera 4), RTP4-A1-D2
(Chimera 5), and RTP4-A1-D1 (Chimera 6) promote odorant receptor
cell surface localization and odorant receptor functional
expression.
[0177] Accordingly, the present invention provides nucleic acids
encoding REEP genes, homologs, variants (e.g., polymorphisms and
mutants), including but not limited to, those described in SEQ ID
NOs:1-12. The present invention provides nucleic acids encoding RTP
genes, homologs, variants (e.g., polymorphisms and mutants),
including but not limited to, those described in SEQ ID NOs:13-20.
Table 1 describes exemplary REEP and RTP genes of the present
invention. In some embodiments, the present invention provides
polynucleotide sequences that are capable of hybridizing to SEQ ID
NOs: 1-20 under conditions of low to high stringency as long as the
polynucleotide sequence capable of hybridizing encodes a protein
that retains a biological activity of the naturally occurring REEP
and/or RTP protein. In some embodiments, the protein that retains a
biological activity of a naturally occurring REEP and/or RTP is 70%
homologous to the wild-type REEP and/or RTP, preferably 80%
homologous to the wild-type REEP and/or RTP, more preferably 90%
homologous to the wild-type REEP and/or RTP, and most preferably
95% homologous to wild-type the REEP and/or RTP. In preferred
embodiments, hybridization conditions are based on the melting
temperature (T.sub.m) of the nucleic acid binding complex and
confer a defined "stringency" as explained above (see e.g., Wahl,
et al., Meth. Enzymol., 152:399-407 (1987), incorporated herein by
reference).
[0178] In other embodiments of the present invention, additional
alleles of REEP and/or RTP genes are provided. In preferred
embodiments, alleles result from a polymorphism or mutation (i.e.,
a change in the nucleic acid sequence) and generally produce
altered mRNAs or polypeptides whose structure or function may or
may not be altered. Any given gene may have none, one or many
allelic forms. Common mutational changes that give rise to alleles
are generally ascribed to deletions, additions or substitutions of
nucleic acids. Each of these types of changes may occur alone, or
in combination with the others, and at the rate of one or more
times in a given sequence. Additional examples include truncation
mutations (e.g., such that the encoded mRNA does not produce a
complete protein).
[0179] In still other embodiments of the present invention, the
nucleotide sequences of the present invention may be engineered in
order to alter a REEP and/or RTP coding sequence for a variety of
reasons, including but not limited to, alterations which modify the
cloning, processing and/or expression of the gene product. For
example, mutations may be introduced using techniques that are well
known in the art (e.g., site-directed mutagenesis to insert new
restriction sites, to alter glycosylation patterns, to change codon
preference, etc.). Variants of RTP1 include but are not limited to
RTP1-A, RTP1-B, RTP1-C, RTP1-D, and RTP1-E, RTP1-A1, RTP1-D1,
RTP-D2, RTP-D3, RTP1-A1-A (Chimera 1), RTP1-A1-D2 (Chimera 2),
RTP1-A1-D1 (Chimera 3), RTP4-A1-A (Chimera 4), RTP4-A1-D2 (Chimera
5), and RTP4-A1-D1 (Chimera 6).
[0180] In some embodiments of the present invention, the
polynucleotide sequence of REEP and/or RTP may be extended
utilizing the nucleotide sequence in various methods known in the
art to detect upstream sequences such as promoters and regulatory
elements. For example, it is contemplated that restriction-site
polymerase chain reaction (PCR) will find use in the present
invention. This is a direct method that uses universal primers to
retrieve unknown sequence adjacent to a known locus (Gobinda et
al., PCR Methods Applic., 2:318-22 (1993); herein incorporated by
reference in its entirety). First, genomic DNA is amplified in the
presence of a primer to a linker sequence and a primer specific to
the known region. The amplified sequences are then subjected to a
second round of PCR with the same linker primer and another
specific primer internal to the first one. Products of each round
of PCR are transcribed with an appropriate RNA polymerase and
sequenced using reverse transcriptase.
[0181] In another embodiment, inverse PCR can be used to amplify or
extend sequences using divergent primers based on a known region
(Triglia et al., Nucleic Acids Res., 16:8186 [1988]). The primers
may be designed using Oligo 4.0 (National Biosciences Inc, Plymouth
Minn.), or another appropriate program, to be 22-30 nucleotides in
length, to have a GC content of 50% or more, and to anneal to the
target sequence at temperatures about 68-72.degree. C. The method
uses several restriction enzymes to generate a suitable fragment in
the known region of a gene. The fragment is then circularized by
intramolecular ligation and used as a PCR template. In still other
embodiments, walking PCR is utilized. Walking PCR is a method for
targeted gene walking that permits retrieval of unknown sequence
(Parker et al., Nucleic Acids Res., 19:3055-60 [1991]). The
PROMOTERFINDER kit (Clontech) uses PCR, nested primers and special
libraries to "walk in" genomic DNA. This process avoids the need to
screen libraries and is useful in finding intron/exon
junctions.
[0182] Preferred libraries for screening for full length cDNAs
include mammalian libraries that have been size-selected to include
larger cDNAs. Also, random primed libraries are preferred, in that
they will contain more sequences that contain the 5' and upstream
gene regions. A randomly primed library may be particularly useful
in case where an oligo d(T) library does not yield full-length
cDNA. Genomic mammalian libraries are useful for obtaining introns
and extending 5' sequence.
[0183] In other embodiments of the present invention, variants of
the disclosed REEP and/or RTP sequences are provided. In preferred
embodiments, variants result from polymorphisms or mutations (i.e.,
a change in the nucleic acid sequence) and generally produce
altered mRNAs or polypeptides whose structure or function may or
may not be altered. Any given gene may have none, one, or many
variant forms. Common mutational changes that give rise to variants
are generally ascribed to deletions, additions or substitutions of
nucleic acids. Each of these types of changes may occur alone, or
in combination with the others, and at the rate of one or more
times in a given sequence.
[0184] It is contemplated that it is possible to modify the
structure of a peptide having a function (e.g., REEP and/or RTP
function) for such purposes as altering the biological activity
(e.g., altered REEP and/or RTP function). Such modified peptides
are considered functional equivalents of peptides having an
activity of a REEP and/or RTP peptide as defined herein. A modified
peptide can be produced in which the nucleotide sequence encoding
the polypeptide has been altered, such as by substitution,
deletion, or addition. In particularly preferred embodiments, these
modifications do not significantly reduce the biological activity
of the modified REEP and/or RTP genes. In other words, construct
"X" can be evaluated in order to determine whether it is a member
of the genus of modified or variant REEP and/or RTP of the present
invention as defined functionally, rather than structurally. In
preferred embodiments, the activity of variant REEP and/or RTP
polypeptides is evaluated by methods described herein (e.g., the
generation of transgenic animals or the use of signaling
assays).
[0185] Moreover, as described above, variant forms of REEP and/or
RTP genes are also contemplated as being equivalent to those
peptides and DNA molecules that are set forth in more detail
herein. For example, it is contemplated that isolated replacement
of a leucine with an isoleucine or valine, an aspartate with a
glutamate, a threonine with a serine, or a similar replacement of
an amino acid with a structurally related amino acid (i.e.,
conservative mutations) will not have a major effect on the
biological activity of the resulting molecule. Accordingly, some
embodiments of the present invention provide variants of REEP
and/or RTP containing conservative replacements. Conservative
replacements are those that take place within a family of amino
acids that are related in their side chains. Genetically encoded
amino acids can be divided into four families: (1) acidic
(aspartate, glutamate); (2) basic (lysine, arginine, histidine);
(3) nonpolar (alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan); and (4) uncharged polar
(glycine, asparagine, glutamine, cysteine, serine, threonine,
tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes
classified jointly as aromatic amino acids. In similar fashion, the
amino acid repertoire can be grouped as (1) acidic (aspartate,
glutamate); (2) basic (lysine, arginine, histidine), (3) aliphatic
(glycine, alanine, valine, leucine, isoleucine, serine, threonine),
with serine and threonine optionally be grouped separately as
aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine,
tryptophan); (5) amide (asparagine, glutamine); and (6)
sulfur-containing (cysteine and methionine) (e.g., Stryer ed.,
Biochemistry, pg. 17-21, 2nd ed, WH Freeman and Co., 1981). Whether
a change in the amino acid sequence of a peptide results in a
functional polypeptide can be readily determined by assessing the
ability of the variant peptide to function in a fashion similar to
the wild-type protein. Peptides having more than one replacement
can readily be tested in the same manner.
[0186] More rarely, a variant includes "nonconservative" changes
(e.g., replacement of a glycine with a tryptophan). Analogous minor
variations can also include amino acid deletions or insertions, or
both. Guidance in determining which amino acid residues can be
substituted, inserted, or deleted without abolishing biological
activity can be found using computer programs (e.g., LASERGENE
software, DNASTAR Inc., Madison, Wis.).
[0187] As described in more detail below, variants may be produced
by methods such as directed evolution or other techniques for
producing combinatorial libraries of variants, described in more
detail below. In still other embodiments of the present invention,
the nucleotide sequences of the present invention may be engineered
in order to alter a REEP and/or RTP coding sequence including, but
not limited to, alterations that modify the cloning, processing,
localization, secretion, and/or expression of the gene product. For
example, mutations may be introduced using techniques that are well
known in the art (e.g., site-directed mutagenesis to insert new
restriction sites, alter glycosylation patterns, or change codon
preference, etc.).
[0188] Variants of RTP1 include but are not limited to RTP1-A,
RTP1-B, RTP1-C, RTP1-D, and RTP1-E, RTP1-A1, RTP1-D1, RTP-D2,
RTP-D3, RTP1-A1-A (Chimera 1), RTP1-A1-D2 (Chimera 2), RTP1-A1-D1
(Chimera 3), RTP4-A1-A (Chimera 4), RTP4-A1-D2 (Chimera 5), and
RTP4-A1-D1 (Chimera 6).
TABLE-US-00001 TABLE 1 OR Modulator Genes SEQ ID NO SEQ ID NO Gene
(Nucleic acid) (Polypeptide) Murine REEP1 1 21 Murine REEP2 2 22
Murine REEP3 3 23 Murine REEP4 4 24 Murine REEP5 5 25 Murine REEP6
6 26 Human REEP1 7 27 Human REEP2 8 28 Human REEP3 9 29 Human REEP4
10 30 Human REEP5 11 31 Human REEP6 12 32 Murine RTP1 13 33 Murine
RTP2 14 34 Murine RTP3 15 35 Murine RTP4 16 36 Human RTP1 17 (for
RTP1-A1) 37 Human RTP2 18 38 Human RTP3 19 39 Human RTP4 20 40
III. REEP and RTP Polypeptides
[0189] In other embodiments, the present invention provides REEP
and/or RTP polynucleotide sequences that encode REEP and/or RTP
polypeptide sequences (e.g., the polypeptides of SEQ ID NOs: 21-40,
41-50 respectively). In preferred embodiments, the present
invention provides a polypeptide encoded by a nucleic acid selected
from the group consisting of SEQ ID NOs: 1, 7, 13, 14, 17 and 18
and variants thereof that are at least 80% identical to SEQ ID NOs:
1, 7, 13, 14, 17 and 18. In further embodiments, the protein is at
least 90% identical to SEQ ID NOs: 1, 7, 13, 14, 17 and 18. In even
further embodiments, the protein is at least 95% identical to SEQ
ID NOs: 1, 7, 13, 14, 17 and 18. Other embodiments of the present
invention provide fragments, fusion proteins or functional
equivalents of REEP and/or RTP proteins (e.g., RTP1-A, RTP1-B,
RTP1-C, RTP1-D, and RTP1-E, RTP1-A1, RTP1-D1, RTP-D2, RTP-D3,
RTP1-A1-A (Chimera 1), RTP1-A1-D2 (Chimera 2), RTP1-A1-D1 (Chimera
3), RTP4-A1-A (Chimera 4), RTP4-A1-D2 (Chimera 5), and RTP4-A1-D1
(Chimera 6). In some embodiments, the present invention provides
mutants of REEP and/or RTP polypeptides. In still other embodiments
of the present invention, nucleic acid sequences corresponding to
REEP and/or RTP variants, homologs, and mutants may be used to
generate recombinant DNA molecules that direct the expression of
the REEP and/or RTP variants, homologs, and mutants in appropriate
host cells. In some embodiments of the present invention, the
polypeptide may be a naturally purified product, in other
embodiments it may be a product of chemical synthetic procedures,
and in still other embodiments it may be produced by recombinant
techniques using a prokaryotic or eukaryotic host (e.g., by
bacterial, yeast, higher plant, insect and mammalian cells in
culture). In some embodiments, depending upon the host employed in
a recombinant production procedure, the polypeptide of the present
invention may be glycosylated or may be non-glycosylated. In other
embodiments, the polypeptides of the invention may also include an
initial methionine amino acid residue.
[0190] In one embodiment of the present invention, due to the
inherent degeneracy of the genetic code, DNA sequences other than
the polynucleotide sequences of SEQ ID NOs: 21-50 that encode
substantially the same or a functionally equivalent amino acid
sequence, may be used to clone and express REEP and/or RTP
proteins. In general, such polynucleotide sequences hybridize to
one of SEQ ID NOs: 21-50 under conditions of high to medium
stringency as described above. As will be understood by those of
skill in the art, it may be advantageous to produce REEP and/or
RTP-encoding nucleotide sequences possessing non-naturally
occurring codons. Therefore, in some preferred embodiments, codons
preferred by a particular prokaryotic or eukaryotic host (Murray et
al., Nucl. Acids Res., 17 [1989]) are selected, for example, to
increase the rate of REEP and/or RTP expression or to produce
recombinant RNA transcripts having desirable properties, such as a
longer half-life, than transcripts produced from naturally
occurring sequence.
[0191] In preferred embodiments, REEP1, RTP1 and RTP2 polypeptides
promote odorant receptor cell surface localization and odorant
receptor functional expression.
[0192] 1. Vectors for Production of REEP and RTP
[0193] The polynucleotides of the present invention may be employed
for producing polypeptides by recombinant techniques. Thus, for
example, the polynucleotide may be included in any one of a variety
of expression vectors for expressing a polypeptide. In some
embodiments of the present invention, vectors include, but are not
limited to, chromosomal, nonchromosomal and synthetic DNA sequences
(e.g., derivatives of SV40, bacterial plasmids, phage DNA;
baculovirus, yeast plasmids, vectors derived from combinations of
plasmids and phage DNA, and viral DNA such as vaccinia, adenovirus,
fowl pox virus, and pseudorabies). It is contemplated that any
vector may be used as long as it is replicable and viable in the
host.
[0194] In particular, some embodiments of the present invention
provide recombinant constructs comprising one or more of the
sequences as broadly described above (e.g., SEQ ID NOs: 1-20). In
some embodiments of the present invention, the constructs comprise
a vector, such as a plasmid or viral vector, into which a sequence
of the invention has been inserted, in a forward or reverse
orientation. In still other embodiments, the heterologous
structural sequence (e.g., SEQ ID NOs: 1-20 is assembled in
appropriate phase with translation initiation and termination
sequences. In preferred embodiments of the present invention, the
appropriate DNA sequence is inserted into the vector using any of a
variety of procedures. In general, the DNA sequence is inserted
into an appropriate restriction endonuclease site(s) by procedures
known in the art.
[0195] Large numbers of suitable vectors are known to those of
skill in the art, and are commercially available. Such vectors
include, but are not limited to, the following vectors: 1)
Bacterial--pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript,
psiX174, pbluescript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A
(Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5
(Pharmacia); 2) Eukaryotic--pWLNEO, pSV2CAT, pOG44, PXT1, pSG
(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia); and 3)
Baculovirus--pPbac and pMbac (Stratagene). Any other plasmid or
vector may be used as long as they are replicable and viable in the
host. In some preferred embodiments of the present invention,
mammalian expression vectors comprise an origin of replication, a
suitable promoter and enhancer, and also any necessary ribosome
binding sites, polyadenylation sites, splice donor and acceptor
sites, transcriptional termination sequences, and 5' flanking
non-transcribed sequences. In other embodiments, DNA sequences
derived from the SV40 splice, and polyadenylation sites may be used
to provide the required non-transcribed genetic elements.
[0196] In certain embodiments of the present invention, the DNA
sequence in the expression vector is operatively linked to an
appropriate expression control sequence(s) (promoter) to direct
mRNA synthesis. Promoters useful in the present invention include,
but are not limited to, the LTR or SV40 promoter, the E. coli lac
or trp, the phage lambda P.sub.L and P.sub.R, T3 and T7 promoters,
and the cytomegalovirus (CMV) immediate early, herpes simplex virus
(HSV) thymidine kinase, and mouse metallothionein-I promoters and
other promoters known to control expression of genes in prokaryotic
or eukaryotic cells or their viruses. In other embodiments of the
present invention, recombinant expression vectors include origins
of replication and selectable markers permitting transformation of
the host cell (e.g., dihydrofolate reductase or neomycin resistance
for eukaryotic cell culture, or tetracycline or ampicillin
resistance in E. coli).
[0197] In some embodiments of the present invention, transcription
of the DNA encoding the polypeptides of the present invention by
higher eukaryotes is increased by inserting an enhancer sequence
into the vector. Enhancers are cis-acting elements of DNA, usually
about from 10 to 300 bp that act on a promoter to increase its
transcription. Enhancers useful in the present invention include,
but are not limited to, the SV40 enhancer on the late side of the
replication origin bp 100 to 270, a cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication
origin, and adenovirus enhancers.
[0198] In other embodiments, the expression vector also contains a
ribosome binding site for translation initiation and a
transcription terminator. In still other embodiments of the present
invention, the vector may also include appropriate sequences for
amplifying expression.
[0199] 2. Host Cells for Production of REEP and RTP
Polypeptides
[0200] In a further embodiment, the present invention provides host
cells containing the above-described constructs. In some
embodiments of the present invention, the host cell is a higher
eukaryotic cell (e.g., a mammalian or insect cell). In other
embodiments of the present invention, the host cell is a lower
eukaryotic cell (e.g., a yeast cell). In still other embodiments of
the present invention, the host cell can be a prokaryotic cell
(e.g., a bacterial cell). Specific examples of host cells include,
but are not limited to, Escherichia coli, Salmonella typhimurium,
Bacillus subtilis, and various species within the genera
Pseudomonas, Streptomyces, and Staphylococcus, as well as
Saccharomycees cerivisiae, Schizosaccharomycees pombe, Drosophila
S2 cells, Spodoptera Sf9 cells, Chinese hamster ovary (CHO) cells,
COS-7 lines of monkey kidney fibroblasts, (Gluzman, Cell 23:175
[1981]), C127, 3T3, 293, 293T, HeLa and BHK cell lines.
[0201] The constructs in host cells can be used in a conventional
manner to produce the gene product encoded by the recombinant
sequence. In some embodiments, introduction of the construct into
the host cell can be accomplished by calcium phosphate
transfection, DEAE-Dextran mediated transfection, or
electroporation (See e.g., Davis et al., Basic Methods in Molecular
Biology, [1986]). Alternatively, in some embodiments of the present
invention, the polypeptides of the invention can be synthetically
produced by conventional peptide synthesizers.
[0202] Proteins can be expressed in mammalian cells, yeast,
bacteria, or other cells under the control of appropriate
promoters. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA constructs of
the present invention. Appropriate cloning and expression vectors
for use with prokaryotic and eukaryotic hosts are described by
Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., [1989].
[0203] In some embodiments of the present invention, following
transformation of a suitable host strain and growth of the host
strain to an appropriate cell density, the selected promoter is
induced by appropriate means (e.g., temperature shift or chemical
induction) and cells are cultured for an additional period. In
other embodiments of the present invention, cells are typically
harvested by centrifugation, disrupted by physical or chemical
means, and the resulting crude extract retained for further
purification. In still other embodiments of the present invention,
microbial cells employed in expression of proteins can be disrupted
by any convenient method, including freeze-thaw cycling,
sonication, mechanical disruption, or use of cell lysing
agents.
[0204] In preferred embodiments, the present invention provides a
cell line (e.g., heterologous 293T cell line) comprising expression
of an odorant receptor (e.g., human odorant receptor, murine
odorant receptor, synthetic odorant receptor) localized to the cell
surface, REEP1, RTP1, RTP2, and G.sub..alpha.olf. In some
embodiments, the odorant receptor is tagged with a reporting agent
(e.g., glutathione-S-transferase (GST), c-myc, 6-histidine
(6.times.-His), green fluorescent protein (GFP), maltose binding
protein (MBP), influenza A virus haemagglutinin (HA),
b-galactosidase, and GAL4). The cell line described in this
embodiment is not limited to particular odorant receptors. In some
embodiments, the odorant receptors expressed in the cell line
include, but are not limited to, S6/79, S18, S46, S50, MOR23-1,
MOR31-4, MOR31-6, MOR32-5 and MOR32-11. In preferred embodiments,
cell lines expressing odorant receptors are used in the
classification of an odorant receptor's functional expression
(e.g., ligand specificity). In even further embodiments, cell lines
expressing odorant receptors are used in the classification of an
animal's olfactory sensation.
[0205] 3. Purification of REEP and RTP Polypeptides
[0206] The present invention also provides methods for recovering
and purifying REEP and/or RTP polypeptides from recombinant cell
cultures including, but not limited to, ammonium sulfate or ethanol
precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. In other
embodiments of the present invention, protein-refolding steps can
be used as necessary, in completing configuration of the mature
protein. In still other embodiments of the present invention, high
performance liquid chromatography (HPLC) can be employed for final
purification steps.
[0207] The present invention further provides polynucleotides
having a coding sequence of a REEP and/or RTP gene (e.g., SEQ ID
NOs: 1-20) fused in frame to a marker sequence that allows for
purification of the polypeptide of the present invention. A
non-limiting example of a marker sequence is a hexahistidine tag
which may be supplied by a vector, preferably a pQE-9 vector, which
provides for purification of the polypeptide fused to the marker in
the case of a bacterial host, or, for example, the marker sequence
may be a hemagglutinin (HA) tag when a mammalian host (e.g., COS-7
cells) is used. The HA tag corresponds to an epitope derived from
the influenza hemagglutinin protein (Wilson et al., Cell, 37:767
[1984]).
[0208] 4. Truncation Mutants of REEP and RTP Polypeptides
[0209] In addition, the present invention provides fragments of
REEP and/or RTP polypeptides (i.e., truncation mutants). In some
embodiments of the present invention, when expression of a portion
of the REEP and/or RTP protein is desired, it may be necessary to
add a start codon (ATG) to the oligonucleotide fragment containing
the desired sequence to be expressed. It is well known in the art
that a methionine at the N-terminal position can be enzymatically
cleaved by the use of the enzyme methionine aminopeptidase (MAP).
MAP has been cloned from E. coli (Ben-Bassat et al, J. Bacteriol.,
169:751 [1987]) and Salmonella typhimurium and its in vitro
activity has been demonstrated on recombinant proteins (Miller et
al., Proc. Natl. Acad. Sci. USA 84:2718 [1990]). Therefore, removal
of an N-terminal methionine, if desired, can be achieved either in
vivo by expressing such recombinant polypeptides in a host which
produces MAP (e.g., E. coli or CM89 or S. cerivisiae), or in vitro
by use of purified MAP.
[0210] 5. Fusion Proteins Containing REEP and RTP
[0211] The present invention also provides fusion proteins
incorporating all or part of the REEP and/or RTP polypeptides of
the present invention. Accordingly, in some embodiments of the
present invention, the coding sequences for the polypeptide can be
incorporated as a part of a fusion gene including a nucleotide
sequence encoding a different polypeptide. It is contemplated that
this type of expression system will find use under conditions where
it is desirable to produce an immunogenic fragment of a REEP and/or
RTP protein. In some embodiments of the present invention, the VP6
capsid protein of rotavirus is used as an immunologic carrier
protein for portions of a REEP and/or RTP polypeptide, either in
the monomeric form or in the form of a viral particle. In other
embodiments of the present invention, the nucleic acid sequences
corresponding to the portion of a REEP and/or RTP polypeptide
against which antibodies are to be raised can be incorporated into
a fusion gene construct which includes coding sequences for a late
vaccinia virus structural protein to produce a set of recombinant
viruses expressing fusion proteins comprising a portion of REEP
and/or RTP as part of the virion. It has been demonstrated with the
use of immunogenic fusion proteins utilizing the hepatitis B
surface antigen fusion proteins that recombinant hepatitis B
virions can be utilized in this role as well. Similarly, in other
embodiments of the present invention, chimeric constructs coding
for fusion proteins containing a portion of a REEP and/or RTP
polypeptide and the poliovirus capsid protein are created to
enhance immunogenicity of the set of polypeptide antigens (See
e.g., EP Publication No. 025949; and Evans et al., Nature 339:385
[1989]; Huang et al, J. Virol., 62:3855 [1988]; and Schlienger et
al., J. Virol., 66:2 [1992]).
[0212] In still other embodiments of the present invention, the
multiple antigen peptide system for peptide-based immunization can
be utilized. In this system, a desired portion of REEP and/or RTP
is obtained directly from organo-chemical synthesis of the peptide
onto an oligomeric branching lysine core (see e.g., Posnett et al.,
J. Biol. Chem., 263:1719 [1988]; and Nardelli et al., J. Immunol.,
148:914 [1992]). In other embodiments of the present invention,
antigenic determinants of the REEP and/or RTP proteins can also be
expressed and presented by bacterial cells.
[0213] In addition to utilizing fusion proteins to enhance
immunogenicity, it is widely appreciated that fusion proteins can
also facilitate the expression of proteins, such as a REEP and/or
RTP protein of the present invention. Accordingly, in some
embodiments of the present invention, REEP and/or RTP polypeptides
can be generated as glutathione-S-transferase (i.e., GST fusion
proteins). It is contemplated that such GST fusion proteins will
enable easy purification of REEP and/or RTP polypeptides, such as
by the use of glutathione-derivatized matrices (See e.g., Ausabel
et al. (eds.), Current Protocols in Molecular Biology, John Wiley
& Sons, NY [1991]). In another embodiment of the present
invention, a fusion gene coding for a purification leader sequence,
such as a poly-(His)/enterokinase cleavage site sequence at the
N-terminus of the desired portion of a REEP and/or RTP polypeptide,
can allow purification of the expressed REEP and/or RTP fusion
protein by affinity chromatography using a Ni.sup.2+ metal resin.
In still another embodiment of the present invention, the
purification leader sequence can then be subsequently removed by
treatment with enterokinase (See e.g., Hochuli et al., J.
Chromatogr., 411:177 [1987]; and Janknecht et al., Proc. Natl.
Acad. Sci. USA 88:8972).
[0214] Techniques for making fusion genes are well known.
Essentially, the joining of various DNA fragments coding for
different polypeptide sequences is performed in accordance with
conventional techniques, employing blunt-ended or stagger-ended
termini for ligation, restriction enzyme digestion to provide for
appropriate termini, filling-in of cohesive ends as appropriate,
alkaline phosphatase treatment to avoid undesirable joining, and
enzymatic ligation. In another embodiment of the present invention,
the fusion gene can be synthesized by conventional techniques
including automated DNA synthesizers. Alternatively, in other
embodiments of the present invention, PCR amplification of gene
fragments can be carried out using anchor primers which give rise
to complementary overhangs between two consecutive gene fragments
which can subsequently be annealed to generate a chimeric gene
sequence (See e.g., Current Protocols in Molecular Biology,
supra).
[0215] 6. Variants of REEP and RTP
[0216] Still other embodiments of the present invention provide
mutant or variant forms of REEP and/or RTP polypeptides (i.e.,
muteins). It is possible to modify the structure of a peptide
having an activity of a REEP and/or RTP polypeptide of the present
invention for such purposes as enhancing therapeutic or
prophylactic efficacy, disabling the protein, or stability (e.g.,
ex vivo shelf life, and/or resistance to proteolytic degradation in
vivo). Such modified peptides are considered functional equivalents
of peptides having an activity of the subject REEP and/or RTP
proteins as defined herein. A modified peptide can be produced in
which the amino acid sequence has been altered, such as by amino
acid substitution, deletion, or addition.
[0217] Variant forms of RTP1 include but are not limited to RTP1-A,
RTP1-B, RTP1-C, RTP1-D, and RTP1-E, RTP1-A1, RTP1-D1, RTP-D2,
RTP-D3, RTP1-A1-A (Chimera 1), RTP1-A1-D2 (Chimera 2), RTP1-A1-D1
(Chimera 3), RTP4-A1-A (Chimera 4), RTP4-A1-D2 (Chimera 5), and
RTP4-A1-D1 (Chimera 6).
[0218] Moreover, as described above, variant forms (e.g., mutants
or polymorphic sequences) of the subject REEP and/or RTP proteins
are also contemplated as being equivalent to those peptides and DNA
molecules that are set forth in more detail. For example, as
described above, the present invention encompasses mutant and
variant proteins that contain conservative or non-conservative
amino acid substitutions.
[0219] This invention further contemplates a method of generating
sets of combinatorial mutants of the present REEP and/or RTP
proteins, as well as truncation mutants, and is especially useful
for identifying potential variant sequences (i.e., mutants or
polymorphic sequences) that are involved in neurological disorders
(e.g., olfactory disorders) or resistance to neurological
disorders. The purpose of screening such combinatorial libraries is
to generate, for example, novel REEP and/or RTP variants that can
act as either agonists or antagonists, or alternatively, possess
novel activities all together.
[0220] Therefore, in some embodiments of the present invention,
REEP and/or RTP variants are engineered by the present method to
provide altered (e.g., increased or decreased) biological activity.
In other embodiments of the present invention,
combinatorially-derived variants are generated which have a
selective potency relative to a naturally occurring REEP and/or
RTP. Such proteins, when expressed from recombinant DNA constructs,
can be used in gene therapy protocols.
[0221] Still other embodiments of the present invention provide
REEP and/or RTP variants that have intracellular half-lives
dramatically different than the corresponding wild-type protein.
For example, the altered protein can be rendered either more stable
or less stable to proteolytic degradation or other cellular process
that result in destruction of, or otherwise inactivate REEP and/or
RTP polypeptides. Such variants, and the genes which encode them,
can be utilized to alter the location of REEP and/or RTP expression
by modulating the half-life of the protein. For instance, a short
half-life can give rise to more transient REEP and/or RTP
biological effects and, when part of an inducible expression
system, can allow tighter control of REEP and/or RTP levels within
the cell. As above, such proteins, and particularly their
recombinant nucleic acid constructs, can be used in gene therapy
protocols.
[0222] In still other embodiments of the present invention, REEP
and/or RTP variants are generated by the combinatorial approach to
act as antagonists, in that they are able to interfere with the
ability of the corresponding wild-type protein to regulate cell
function.
[0223] In some embodiments of the combinatorial mutagenesis
approach of the present invention, the amino acid sequences for a
population of REEP and/or RTP homologs, variants or other related
proteins are aligned, preferably to promote the highest homology
possible. Such a population of variants can include, for example,
REEP and/or RTP homologs from one or more species, or REEP and/or
RTP variants from the same species but which differ due to mutation
or polymorphisms. Amino acids that appear at each position of the
aligned sequences are selected to create a degenerate set of
combinatorial sequences.
[0224] In a preferred embodiment of the present invention, the
combinatorial REEP and/or RTP library is produced by way of a
degenerate library of genes encoding a library of polypeptides
which each include at least a portion of potential REEP and/or RTP
protein sequences. For example, a mixture of synthetic
oligonucleotides can be enzymatically ligated into gene sequences
such that the degenerate set of potential REEP and/or RTP sequences
are expressible as individual polypeptides, or alternatively, as a
set of larger fusion proteins (e.g., for phage display) containing
the set of REEP and/or RTP sequences therein.
[0225] There are many ways by which the library of potential REEP
and/or RTP homologs and variants can be generated from a degenerate
oligonucleotide sequence. In some embodiments, chemical synthesis
of a degenerate gene sequence is carried out in an automatic DNA
synthesizer, and the synthetic genes are ligated into an
appropriate gene for expression. The purpose of a degenerate set of
genes is to provide, in one mixture, all of the sequences encoding
the desired set of potential REEP and/or RTP sequences. The
synthesis of degenerate oligonucleotides is well known in the art
(See e.g., Narang, Tetrahedron Lett., 39:39 [1983]; Itakura et al.,
Recombinant DNA, in Walton (ed.), Proceedings of the 3rd Cleveland
Symposium on Macromolecules, Elsevier, Amsterdam, pp 273-289
[1981]; Itakura et al., Annu. Rev. Biochem., 53:323 [1984]; Itakura
et al., Science 198:1056 [1984]; Ike et al., Nucl. Acid Res.,
11:477 [1983]). Such techniques have been employed in the directed
evolution of other proteins (See e.g., Scott et al., Science
249:386 [1980]; Roberts et al., Proc. Natl. Acad. Sci. USA 89:2429
[1992]; Devlin et al., Science 249: 404 [1990]; Cwirla et al.,
Proc. Natl. Acad. Sci. USA 87: 6378 [1990]; each of which is herein
incorporated by reference; as well as U.S. Pat. Nos. 5,223,409,
5,198,346, and 5,096,815; each of which is incorporated herein by
reference).
[0226] It is contemplated that the REEP and/or RTP nucleic acids of
the present invention (e.g., SEQ ID NOs: 1-20, and fragments and
variants thereof) can be utilized as starting nucleic acids for
directed evolution. These techniques can be utilized to develop
REEP and/or RTP variants having desirable properties such as
increased or decreased biological activity.
[0227] In some embodiments, artificial evolution is performed by
random mutagenesis (e.g., by utilizing error-prone PCR to introduce
random mutations into a given coding sequence). This method
requires that the frequency of mutation be finely tuned. As a
general rule, beneficial mutations are rare, while deleterious
mutations are common. This is because the combination of a
deleterious mutation and a beneficial mutation often results in an
inactive enzyme. The ideal number of base substitutions for
targeted gene is usually between 1.5 and 5 (Moore and Arnold, Nat.
Biotech., 14, 458 [1996]; Leung et al., Technique, 1:11 [1989];
Eckert and Kunkel, PCR Methods Appl., 1: 17-24 [1991]; Caldwell and
Joyce, PCR Methods Appl., 2:28 [1992]; and Zhao and Arnold, Nuc.
Acids. Res., 25:1307 [1997]). After mutagenesis, the resulting
clones are selected for desirable activity (e.g., screened for REEP
and/or RTP activity). Successive rounds of mutagenesis and
selection are often necessary to develop enzymes with desirable
properties. It should be noted that only the useful mutations are
carried over to the next round of mutagenesis.
[0228] In other embodiments of the present invention, the
polynucleotides of the present invention are used in gene shuffling
or sexual PCR procedures (e.g., Smith, Nature, 370:324 [1994]; U.S.
Pat. Nos. 5,837,458; 5,830,721; 5,811,238; 5,733,731; all of which
are herein incorporated by reference). Gene shuffling involves
random fragmentation of several mutant DNAs followed by their
reassembly by PCR into full length molecules. Examples of various
gene shuffling procedures include, but are not limited to, assembly
following DNase treatment, the staggered extension process (STEP),
and random priming in vitro recombination. In the DNase mediated
method, DNA segments isolated from a pool of positive mutants are
cleaved into random fragments with DNaseI and subjected to multiple
rounds of PCR with no added primer. The lengths of random fragments
approach that of the uncleaved segment as the PCR cycles proceed,
resulting in mutations in present in different clones becoming
mixed and accumulating in some of the resulting sequences. Multiple
cycles of selection and shuffling have led to the functional
enhancement of several enzymes (Stemmer, Nature, 370:398 [1994];
Stemmer, Proc. Natl. Acad. Sci. USA, 91:10747 [1994]; Crameri et
al., Nat. Biotech., 14:315 [1996]; Zhang et al., Proc. Natl. Acad.
Sci. USA, 94:4504 [1997]; and Crameri et al., Nat. Biotech., 15:436
[1997]). Variants produced by directed evolution can be screened
for REEP and/or RTP activity by the methods described herein.
[0229] A wide range of techniques are known in the art for
screening gene products of combinatorial libraries made by point
mutations, and for screening cDNA libraries for gene products
having a certain property. Such techniques will be generally
adaptable for rapid screening of the gene libraries generated by
the combinatorial mutagenesis or recombination of REEP and/or RTP
homologs or variants. The most widely used techniques for screening
large gene libraries typically comprises cloning the gene library
into replicable expression vectors, transforming appropriate cells
with the resulting library of vectors, and expressing the
combinatorial genes under conditions in which detection of a
desired activity facilitates relatively easy isolation of the
vector encoding the gene whose product was detected.
[0230] 7. Chemical Synthesis of REEP and/or RTP Polypeptides
[0231] In an alternate embodiment of the invention, the coding
sequence of REEP and/or RTP is synthesized, whole or in part, using
chemical methods well known in the art (See e.g., Caruthers et al.,
Nucl. Acids Res. Symp. Ser., 7:215 [1980]; Crea and Horn, Nucl.
Acids Res., 9:2331 [1980]; Matteucci and Caruthers, Tetrahedron
Lett., 21:719 [1980]; and Chow and Kempe, Nucl. Acids Res., 9:2807
[1981]). In other embodiments of the present invention, the protein
itself is produced using chemical methods to synthesize either an
entire REEP and/or RTP amino acid sequence or a portion thereof.
For example, peptides can be synthesized by solid phase techniques,
cleaved from the resin, and purified by preparative high
performance liquid chromatography (See e.g., Creighton, Proteins
Structures And Molecular Principles, W H Freeman and Co, New York
N.Y. [1983]). In other embodiments of the present invention, the
composition of the synthetic peptides is confirmed by amino acid
analysis or sequencing (See e.g., Creighton, supra).
[0232] Direct peptide synthesis can be performed using various
solid-phase techniques (Roberge et al., Science 269:202 [1995]) and
automated synthesis may be achieved, for example, using ABI 431A
Peptide Synthesizer (Perkin Elmer) in accordance with the
instructions provided by the manufacturer. Additionally, the amino
acid sequence of a REEP and/or RTP polypeptide, or any part
thereof, may be altered during direct synthesis and/or combined
using chemical methods with other sequences to produce a variant
polypeptide.
IV. Detection of REEP and RTP Alleles
[0233] In some embodiments, the present invention provides methods
of detecting the presence of wild type or variant (e.g., mutant or
polymorphic) REEP and/or RTP nucleic acids or polypeptides. The
detection of mutant REEP and/or RTP polypeptides finds use in the
diagnosis of disease (e.g., olfactory disorder).
[0234] A. Detection of Variant REEP and/or RTP Alleles
[0235] In some embodiments, the present invention provides alleles
of REEP and/or RTP that increase a patient's susceptibility to
olfactory disorders (e.g., upper respiratory infections, tumors of
the anterior cranial fossa, Kallmann syndrome, Foster Kennedy
syndrome, Parkinson's disease, Alzheimer's disease, and Huntington
chorea). Any mutation that results in an altered phenotype (e.g.,
diminished olfactory sensing abilility) is within the scope of the
present invention.
[0236] Accordingly, the present invention provides methods for
determining whether a patient has an increased susceptibility to
olfactory disorders (e.g., upper respiratory infections, tumors of
the anterior cranial fossa, and Kallmann syndrome, Foster Kennedy
syndrome, Parkinson's disease, Alzheimer's disease, Huntington
chorea) by determining, directly or indirectly, whether the
individual has a variant REEP and/or RTP allele. In other
embodiments, the present invention provides methods for providing a
prognosis of increased risk for olfactory disorder to an individual
based on the presence or absence of one or more variant REEP and/or
RTP alleles.
[0237] A number of methods are available for analysis of variant
(e.g., mutant or polymorphic) nucleic acid or polypeptide
sequences. Assays for detection variants (e.g., polymorphisms or
mutations) via nucleic acid analysis fall into several categories
including, but not limited to, direct sequencing assays, fragment
polymorphism assays, hybridization assays, and computer based data
analysis. Protocols and commercially available kits or services for
performing multiple variations of these assays are available. In
some embodiments, assays are performed in combination or in hybrid
(e.g., different reagents or technologies from several assays are
combined to yield one assay). The following exemplary assays are
useful in the present invention: directs sequencing assays, PCR
assays, mutational analysis by dHPLC (e.g., available from
Transgenomic, Omaha, Nebr. or Varian, Palo Alto, Calif.), fragment
length polymorphism assays (e.g., RFLP or CFLP (See e.g. U.S. Pat.
Nos. 5,843,654; 5,843,669; 5,719,208; and 5,888,780; each of which
is herein incorporated by reference)), hybridization assays (e.g.,
direct detection of hybridization, detection of hybridization using
DNA chip assays (See e.g., U.S. Pat. Nos. 6,045,996; 5,925,525;
5,858,659; 6,017,696; 6,068,818; 6,051,380; 6,001,311; 5,985,551;
5,474,796; PCT Publications WO 99/67641 and WO 00/39587, each of
which is herein incorporated by reference), enzymatic detection of
hybridization (See e.g., U.S. Pat. Nos. 5,846,717, 6,090,543;
6,001,567; 5,985,557; 5,994,069; 5,962,233; 5,538,848; 5,952,174
and 5,919,626, each of which is herein incorporated by reference)),
polymorphisms detected directly or indirectly (e.g., detecting
sequences (other polymorphisms) that are in linkage disequilibrium
with the polymorphism to be identified; for example, other
sequences in the SPG-6 locus may be used; this method is described
in U.S. Pat. No. 5,612,179 (herein incorporated by reference)) and
mass spectrometry assays.
[0238] In addition, assays for the detection of variant REEP and/or
RTP proteins find use in the present invention (e.g., cell free
translation methods, See e.g., U.S. Pat. No. 6,303,337, herein
incorporated by reference) and antibody binding assays. The
generation of antibodies that specifically recognize mutant versus
wild type proteins are discussed below.
[0239] B. Kits for Analyzing Risk of Olfactory Disorders
[0240] The present invention also provides kits for determining
whether an individual contains a wild-type or variant (e.g., mutant
or polymorphic) allele or polypeptide of REEP and/or RTP. In some
embodiments, the kits are useful determining whether the subject is
at risk of developing an olfactory disorder (e.g., upper
respiratory infections, tumors of the anterior cranial fossa, and
Kallmann syndrome, Foster Kennedy syndrome, Parkinson's disease,
Alzheimer's disease, Huntington chorea). The diagnostic kits are
produced in a variety of ways. In some embodiments, the kits
contain at least one reagent for specifically detecting a mutant
REEP and/or RTP allele or protein. In preferred embodiments, the
reagent is a nucleic acid that hybridizes to nucleic acids
containing the mutation and that does not bind to nucleic acids
that do not contain the mutation. In other embodiments, the
reagents are primers for amplifying the region of DNA containing
the mutation. In still other embodiments, the reagents are
antibodies that preferentially bind either the wild-type or mutant
REEP and/or RTP proteins.
[0241] In some embodiments, the kit contains instructions for
determining whether the subject is at risk for an olfactory
disorder (e.g, upper respiratory infections, tumors of the anterior
cranial fossa, and Kallmann syndrome, Foster Kennedy syndrome,
Parkinson's disease, Alzheimer's disease, Huntington chorea). In
preferred embodiments, the instructions specify that risk for
developing an olfactory disorder is determined by detecting the
presence or absence of a mutant REEP and/or RTP allele in the
subject, wherein subjects having an mutant allele are at greater
risk for developing an olfactory disorder.
[0242] The presence or absence of a disease-associated mutation in
a REEP and/or RTP gene can be used to make therapeutic or other
medical decisions. For example, couples with a family history of
odorant receptor related diseases may choose to conceive a child
via in vitro fertilization and pre-implantation genetic screening.
In this case, fertilized embryos are screened for mutant (e.g.,
disease associated) alleles of a REEP and/or RTP gene and only
embryos with wild type alleles are implanted in the uterus.
[0243] In other embodiments, in utero screening is performed on a
developing fetus (e.g., amniocentesis or chorionic villi
screening). In still other embodiments, genetic screening of
newborn babies or very young children is performed. The early
detection of a REEP and/or RTP allele known to be associated with
an olfactory disorder allows for early intervention (e.g., genetic
or pharmaceutical therapies).
[0244] In some embodiments, the kits include ancillary reagents
such as buffering agents, nucleic acid stabilizing reagents,
protein stabilizing reagents, and signal producing systems (e.g.,
florescence generating systems as Fret systems). The test kit may
be packaged in any suitable manner, typically with the elements in
a single container or various containers as necessary along with a
sheet of instructions for carrying out the test. In some
embodiments, the kits also preferably include a positive control
sample.
[0245] C. Bioinformatics
[0246] In some embodiments, the present invention provides methods
of determining an individual's risk of developing an olfactory
disorder (e.g., upper respiratory infections, tumors of the
anterior cranial fossa, and Kallmann syndrome, Foster Kennedy
syndrome, Parkinson's disease, Alzheimer's disease, Huntington
chorea) based on the presence of one or more variant alleles of a
REEP and/or RTP gene. In some embodiments, the analysis of variant
data is processed by a computer using information stored on a
computer (e.g., in a database). For example, in some embodiments,
the present invention provides a bioinformatics research system
comprising a plurality of computers running a multi-platform object
oriented programming language (See e.g., U.S. Pat. No. 6,125,383;
herein incorporated by reference). In some embodiments, one of the
computers stores genetics data (e.g., the risk of contacting an
REEP and/or RTP related olfactory disorder associated with a given
polymorphism, as well as the sequences). In some embodiments, one
of the computers stores application programs (e.g., for analyzing
the results of detection assays). Results are then delivered to the
user (e.g., via one of the computers or via the internet).
[0247] For example, in some embodiments, a computer-based analysis
program is used to translate the raw data generated by the
detection assay (e.g., the presence, absence, or amount of a given
REEP and/or RTP allele or polypeptide) into data of predictive
value for a clinician. The clinician can access the predictive data
using any suitable means. Thus, in some preferred embodiments, the
present invention provides the further benefit that the clinician,
who is not likely to be trained in genetics or molecular biology,
need not understand the raw data. The data is presented directly to
the clinician in its most useful form. The clinician is then able
to immediately utilize the information in order to optimize the
care of the subject.
[0248] The present invention contemplates any method capable of
receiving, processing, and transmitting the information to and from
laboratories conducting the assays, information providers, medical
personal, and subjects. For example, in some embodiments of the
present invention, a sample (e.g., a biopsy or a serum or urine
sample) is obtained from a subject and submitted to a profiling
service (e.g., clinical lab at a medical facility, genomic
profiling business, etc.), located in any part of the world (e.g.,
in a country different than the country where the subject resides
or where the information is ultimately used) to generate raw data.
Where the sample comprises a tissue or other biological sample, the
subject may visit a medical center to have the sample obtained and
sent to the profiling center, or subjects may collect the sample
themselves (e.g., a urine sample) and directly send it to a
profiling center. Where the sample comprises previously determined
biological information, the information may be directly sent to the
profiling service by the subject (e.g., an information card
containing the information may be scanned by a computer and the
data transmitted to a computer of the profiling center using an
electronic communication systems). Once received by the profiling
service, the sample is processed and a profile is produced (i.e.,
presence of wild type or mutant REEP and/or RTP genes or
polypeptides), specific for the diagnostic or prognostic
information desired for the subject.
[0249] The profile data is then prepared in a format suitable for
interpretation by a treating clinician. For example, rather than
providing raw data, the prepared format may represent a diagnosis
or risk assessment (e.g., likelihood of developing an REEP and/or
RTP related olfactory disorder) for the subject, along with
recommendations for particular treatment options. The data may be
displayed to the clinician by any suitable method. For example, in
some embodiments, the profiling service generates a report that can
be printed for the clinician (e.g., at the point of care) or
displayed to the clinician on a computer monitor.
[0250] In some embodiments, the information is first analyzed at
the point of care or at a regional facility. The raw data is then
sent to a central processing facility for further analysis and/or
to convert the raw data to information useful for a clinician or
patient. The central processing facility provides the advantage of
privacy (all data is stored in a central facility with uniform
security protocols), speed, and uniformity of data analysis. The
central processing facility can then control the fate of the data
following treatment of the subject. For example, using an
electronic communication system, the central facility can provide
data to the clinician, the subject, or researchers.
[0251] In some embodiments, the subject is able to directly access
the data using the electronic communication system. The subject may
chose further intervention or counseling based on the results. In
some embodiments, the data is used for research use. For example,
the data may be used to further optimize the association of a given
REEP and/or RTP allele with olfactory disorders.
V. Generation of REEP and RTP Antibodies
[0252] The present invention provides isolated antibodies or
antibody fragments (e.g., FAB fragments). Antibodies can be
generated to allow for the detection of REEP and/or RTP proteins
(e.g., wild type or mutant) of the present invention. The
antibodies may be prepared using various immunogens. In one
embodiment, the immunogen is a human REEP and/or RTP peptide to
generate antibodies that recognize human REEP and/or RTP. Such
antibodies include, but are not limited to polyclonal, monoclonal,
chimeric, single chain, Fab fragments, Fab expression libraries, or
recombinant (e.g., chimeric, humanized, etc.) antibodies, as long
as it can recognize the protein. Antibodies can be produced by
using a protein of the present invention as the antigen according
to a conventional antibody or antiserum preparation process.
[0253] Various procedures known in the art may be used for the
production of polyclonal antibodies directed against a REEP and/or
RTP polypeptide. For the production of antibody, various host
animals can be immunized by injection with the peptide
corresponding to the REEP and/or RTP epitope including but not
limited to rabbits, mice, rats, sheep, goats, etc. In a preferred
embodiment, the peptide is conjugated to an immunogenic carrier
(e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyhole
limpet hemocyanin (KLH)). Various adjuvants may be used to increase
the immunological response, depending on the host species,
including but not limited to Freund's (complete and incomplete),
mineral gels (e.g., aluminum hydroxide), surface active substances
(e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanins, dinitrophenol, and
potentially useful human adjuvants such as BCG (Bacille
Calmette-Guerin) and Corynebacterium parvum).
[0254] For preparation of monoclonal antibodies directed toward
REEP and/or RTP, it is contemplated that any technique that
provides for the production of antibody molecules by continuous
cell lines in culture will find use with the present invention (See
e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.). These include
but are not limited to the hybridoma technique originally developed
by Kohler and Milstein (Kohler and Milstein, Nature 256:495-497
[1975]), as well as the trioma technique, the human B-cell
hybridoma technique (See e.g., Kozbor et al., Immunol. Tod., 4:72
[1983]), and the EBV-hybridoma technique to produce human
monoclonal antibodies (Cole et al., in Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 [1985]).
[0255] In an additional embodiment of the invention, monoclonal
antibodies are produced in germ-free animals utilizing technology
such as that described in PCT/US90/02545). Furthermore, it is
contemplated that human antibodies will be generated by human
hybridomas (Cote et al., Proc. Natl. Acad. Sci. USA 80:2026-2030
[1983]) or by transforming human B cells with EBV virus in vitro
(Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, pp. 77-96 [1985]).
[0256] In addition, it is contemplated that techniques described
for the production of single chain antibodies (U.S. Pat. No.
4,946,778; herein incorporated by reference) will find use in
producing REEP and/or RTP specific single chain antibodies. An
additional embodiment of the invention utilizes the techniques
described for the construction of Fab expression libraries (Huse et
al., Science 246:1275-1281 [1989]) to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity for a REEP and/or RTP polypeptide.
[0257] In other embodiments, the present invention contemplated
recombinant antibodies or fragments thereof to the proteins of the
present invention. Recombinant antibodies include, but are not
limited to, humanized and chimeric antibodies. Methods for
generating recombinant antibodies are known in the art (See e.g.,
U.S. Pat. Nos. 6,180,370 and 6,277,969 and "Monoclonal Antibodies"
H. Zola, BIOS Scientific Publishers Limited 2000. Springer-Verlay
New York, Inc., New York; each of which is herein incorporated by
reference).
[0258] It is contemplated that any technique suitable for producing
antibody fragments will find use in generating antibody fragments
that contain the idiotype (antigen binding region) of the antibody
molecule. For example, such fragments include but are not limited
to: F(ab')2 fragment that can be produced by pepsin digestion of
the antibody molecule; Fab' fragments that can be generated by
reducing the disulfide bridges of the F(ab')2 fragment, and Fab
fragments that can be generated by treating the antibody molecule
with papain and a reducing agent.
[0259] In the production of antibodies, it is contemplated that
screening for the desired antibody will be accomplished by
techniques known in the art (e.g., radioimmunoassay, ELISA
(enzyme-linked immunosorbant assay), "sandwich" immunoassays,
immunoradiometric assays, gel diffusion precipitation reactions,
immudiffusion assays, in situ immunoassays (e.g., using colloidal
gold, enzyme or radioisotope labels), Western blots, precipitation
reactions, agglutination assays (e.g., gel agglutination assays,
hemagglutination assays, etc.), complement fixation assays,
immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc.
[0260] In one embodiment, antibody binding is detected by detecting
a label on the primary antibody. In another embodiment, the primary
antibody is detected by detecting binding of a secondary antibody
or reagent to the primary antibody. In a further embodiment, the
secondary antibody is labeled. Many means are known in the art for
detecting binding in an immunoassay and are within the scope of the
present invention. As is well known in the art, the immunogenic
peptide should be provided free of the carrier molecule used in any
immunization protocol. For example, if the peptide was conjugated
to KLH, it may be conjugated to BSA, or used directly, in a
screening assay.)
[0261] The foregoing antibodies can be used in methods known in the
art relating to the localization and structure of REEP and/or RTP
(e.g., for Western blotting), measuring levels thereof in
appropriate biological samples, etc. The antibodies can be used to
detect a REEP and/or RTP in a biological sample from an individual.
The biological sample can be a biological fluid, such as, but not
limited to, blood, serum, plasma, interstitial fluid, urine,
cerebrospinal fluid, and the like, containing cells.
[0262] The biological samples can then be tested directly for the
presence of a human REEP and/or RTP using an appropriate strategy
(e.g., ELISA or radioimmunoassay) and format (e.g., microwells,
dipstick (e.g., as described in International Patent Publication WO
93/03367), etc. Alternatively, proteins in the sample can be size
separated (e.g., by polyacrylamide gel electrophoresis (PAGE), in
the presence or not of sodium dodecyl sulfate (SDS), and the
presence of REEP and/or RTP detected by immunoblotting (Western
blotting). Immunoblotting techniques are generally more effective
with antibodies generated against a peptide corresponding to an
epitope of a protein, and hence, are particularly suited to the
present invention.
[0263] Another method uses antibodies as agents to alter signal
transduction. Specific antibodies that bind to the binding domains
of REEP and/or RTP or other proteins involved in intracellular
signaling can be used to inhibit the interaction between the
various proteins and their interaction with other ligands.
Antibodies that bind to the complex can also be used
therapeutically to inhibit interactions of the protein complex in
the signal transduction pathways leading to the various
physiological and cellular effects of REEP and/or RTP. Such
antibodies can also be used diagnostically to measure abnormal
expression of REEP1 and/or RTP, or the aberrant formation of
protein complexes, which may be indicative of a disease state.
VI. Gene Therapy Using REEP and RTP
[0264] The present invention also provides methods and compositions
suitable for gene therapy to alter REEP and/or RTP expression,
production, or function for research, generation of transgenic
animals, and/or therapeutic applications. As described above, the
present invention provides human REEP and/or RTP genes and provides
methods of obtaining REEP and/or RTP genes from other species.
Thus, the methods described below are generally applicable across
many species. In some embodiments, it is contemplated that the gene
therapy is performed by providing a subject with a wild-type allele
of a REEP and/or RTP gene (i.e., an allele that does not contain a
REEP and/or RTP disease allele (e.g., free of disease causing
polymorphisms or mutations)). Subjects in need of such therapy are
identified by the methods described above. In some embodiments,
transient or stable therapeutic nucleic acids are used (e.g.,
antisense oligonucleotides, siRNAs) to reduce or prevent expression
of mutant proteins. In other embodiments, genes are deleted to
reduce or block desired olfactory senses.
[0265] Viral vectors commonly used for in vivo or ex vivo targeting
and therapy procedures are DNA-based vectors and retroviral
vectors. Methods for constructing and using viral vectors are known
in the art (See e.g., Miller and Rosman, BioTech., 7:980-990
[1992]). Preferably, the viral vectors are replication defective,
that is, they are unable to replicate autonomously in the target
cell. In general, the genome of the replication defective viral
vectors that are used within the scope of the present invention
lack at least one region that is necessary for the replication of
the virus in the infected cell. These regions can either be
eliminated (in whole or in part), or be rendered non-functional by
any technique known to a person skilled in the art. These
techniques include the total removal, substitution (by other
sequences, in particular by the inserted nucleic acid), partial
deletion or addition of one or more bases to an essential (for
replication) region. Such techniques may be performed in vitro
(i.e., on the isolated DNA) or in situ, using the techniques of
genetic manipulation or by treatment with mutagenic agents.
[0266] Preferably, the replication defective virus retains the
sequences of its genome that are necessary for encapsidating the
viral particles. DNA viral vectors include an attenuated or
defective DNA viruses, including, but not limited to, herpes
simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV),
adenovirus, adeno-associated virus (AAV), and the like. Defective
viruses, that entirely or almost entirely lack viral genes, are
preferred, as defective virus is not infective after introduction
into a cell. Use of defective viral vectors allows for
administration to cells in a specific, localized area, without
concern that the vector can infect other cells. Thus, a specific
tissue can be specifically targeted. Examples of particular vectors
include, but are not limited to, a defective herpes virus 1 (HSV1)
vector (Kaplitt et al., Mol. Cell. Neurosci., 2:320-330 [1991]),
defective herpes virus vector lacking a glycoprotein L gene (See
e.g., Patent Publication RD 371005 A), or other defective herpes
virus vectors (See e.g., WO 94/21807; and WO 92/05263); an
attenuated adenovirus vector, such as the vector described by
Stratford-Perricaudet et al. (J. Clin. Invest., 90:626-630 [1992];
See also, La Salle et al., Science 259:988-990 [1993]); and a
defective adeno-associated virus vector (Samulski et al., J.
Virol., 61:3096-3101 [1987]; Samulski et al., J. Virol.,
63:3822-3828 [1989]; and Lebkowski et al., Mol. Cell. Biol.,
8:3988-3996 [1988]).
[0267] Preferably, for in vivo administration, an appropriate
immunosuppressive treatment is employed in conjunction with the
viral vector (e.g., adenovirus vector), to avoid
immuno-deactivation of the viral vector and transfected cells. For
example, immunosuppressive cytokines, such as interleukin-12
(IL-12), interferon-gamma (IFN-.gamma.), or anti-CD4 antibody, can
be administered to block humoral or cellular immune responses to
the viral vectors. In addition, it is advantageous to employ a
viral vector that is engineered to express a minimal number of
antigens.
[0268] In a preferred embodiment, the vector is an adenovirus
vector. Adenoviruses are eukaryotic DNA viruses that can be
modified to efficiently deliver a nucleic acid of the invention to
a variety of cell types. Various serotypes of adenovirus exist. Of
these serotypes, preference is given, within the scope of the
present invention, to type 2 or type 5 human adenoviruses (Ad 2 or
Ad 5), or adenoviruses of animal origin (See e.g., WO 94/26914).
Those adenoviruses of animal origin that can be used within the
scope of the present invention include adenoviruses of canine,
bovine, murine (e.g., Mav1, Beard et al., Virol., 75-81 [1990]),
ovine, porcine, avian, and simian (e.g., SAV) origin. Preferably,
the adenovirus of animal origin is a canine adenovirus, more
preferably a CAV2 adenovirus (e.g. Manhattan or A26/61 strain (ATCC
VR-800)).
[0269] Preferably, the replication defective adenoviral vectors of
the invention comprise the ITRs, an encapsidation sequence and the
nucleic acid of interest. Still more preferably, at least the E1
region of the adenoviral vector is non-functional. The deletion in
the E1 region preferably extends from nucleotides 455 to 3329 in
the sequence of the Ad5 adenovirus (PvuII-BglII fragment) or 382 to
3446 (HinfII-Sau3A fragment). Other regions may also be modified,
in particular the E3 region (e.g., WO 95/02697), the E2 region
(e.g., WO 94/28938), the E4 region (e.g., WO 94/28152, WO 94/12649
and WO 95/02697), or in any of the late genes L1-L5.
[0270] In a preferred embodiment, the adenoviral vector has a
deletion in the E1 region (Ad 1.0). Examples of E1-deleted
adenoviruses are disclosed in EP 185,573, the contents of which are
incorporated herein by reference. In another preferred embodiment,
the adenoviral vector has a deletion in the E1 and E4 regions (Ad
3.0). Examples of E1/E4-deleted adenoviruses are disclosed in WO
95/02697 and WO 96/22378. In still another preferred embodiment,
the adenoviral vector has a deletion in the E1 region into which
the E4 region and the nucleic acid sequence are inserted.
[0271] The replication defective recombinant adenoviruses according
to the invention can be prepared by any technique known to the
person skilled in the art (See e.g., Levrero et al., Gene 101:195
[1991]; EP 185 573; and Graham, EMBO J., 3:2917 [1984]). In
particular, they can be prepared by homologous recombination
between an adenovirus and a plasmid that carries, inter alia, the
DNA sequence of interest. The homologous recombination is
accomplished following co-transfection of the adenovirus and
plasmid into an appropriate cell line. The cell line that is
employed should preferably (i) be transformable by the elements to
be used, and (ii) contain the sequences that are able to complement
the part of the genome of the replication defective adenovirus,
preferably in integrated form in order to avoid the risks of
recombination. Examples of cell lines that may be used are the
human embryonic kidney cell line 293 (Graham et al, J. Gen. Virol.,
36:59 [1977]), which contains the left-hand portion of the genome
of an Ad5 adenovirus (12%) integrated into its genome, and cell
lines that are able to complement the E1 and E4 functions, as
described in applications WO 94/26914 and WO 95/02697. Recombinant
adenoviruses are recovered and purified using standard molecular
biological techniques that are well known to one of ordinary skill
in the art.
[0272] The adeno-associated viruses (AAV) are DNA viruses of
relatively small size that can integrate, in a stable and
site-specific manner, into the genome of the cells that they
infect. They are able to infect a wide spectrum of cells without
inducing any effects on cellular growth, morphology or
differentiation, and they do not appear to be involved in human
pathologies. The AAV genome has been cloned, sequenced and
characterized. It encompasses approximately 4700 bases and contains
an inverted terminal repeat (ITR) region of approximately 145 bases
at each end, which serves as an origin of replication for the
virus. The remainder of the genome is divided into two essential
regions that carry the encapsidation functions: the left-hand part
of the genome, that contains the rep gene involved in viral
replication and expression of the viral genes; and the right-hand
part of the genome, that contains the cap gene encoding the capsid
proteins of the virus.
[0273] The use of vectors derived from the AAVs for transferring
genes in vitro and in vivo has been described (See e.g., WO
91/18088; WO 93/09239; U.S. Pat. No. 4,797,368; U.S. Pat. No.
5,139,941; and EP 488 528, all of which are herein incorporated by
reference). These publications describe various AAV-derived
constructs in which the rep and/or cap genes are deleted and
replaced by a gene of interest, and the use of these constructs for
transferring the gene of interest in vitro (into cultured cells) or
in vivo (directly into an organism). The replication defective
recombinant AAVs according to the invention can be prepared by
co-transfecting a plasmid containing the nucleic acid sequence of
interest flanked by two AAV inverted terminal repeat (ITR) regions,
and a plasmid carrying the AAV encapsidation genes (rep and cap
genes), into a cell line that is infected with a human helper virus
(for example an adenovirus). The AAV recombinants that are produced
are then purified by standard techniques.
[0274] In another embodiment, the gene can be introduced in a
retroviral vector (e.g., as described in U.S. Pat. Nos. 5,399,346,
4,650,764, 4,980,289 and 5,124,263; all of which are herein
incorporated by reference; Mann et al., Cell 33:153 [1983];
Markowitz et al., J. Virol., 62:1120 [1988]; PCT/US95/14575; EP
453242; EP178220; Bernstein et al. Genet. Eng., 7:235 [1985];
McCormick, BioTechnol., 3:689 [1985]; WO 95/07358; and Kuo et al.,
Blood 82:845 [1993]). The retroviruses are integrating viruses that
infect dividing cells. The retrovirus genome includes two LTRs, an
encapsidation sequence and three coding regions (gag, pol and env).
In recombinant retroviral vectors, the gag, pol and env genes are
generally deleted, in whole or in part, and replaced with a
heterologous nucleic acid sequence of interest. These vectors can
be constructed from different types of retrovirus, such as, HIV,
MoMuLV ("murine Moloney leukemia virus" MSV ("murine Moloney
sarcoma virus"), HaSV ("Harvey sarcoma virus"); SNV ("spleen
necrosis virus"); RSV ("Rous sarcoma virus") and Friend virus.
Defective retroviral vectors are also disclosed in WO 95/02697.
[0275] In general, in order to construct recombinant retroviruses
containing a nucleic acid sequence, a plasmid is constructed that
contains the LTRs, the encapsidation sequence and the coding
sequence. This construct is used to transfect a packaging cell
line, which cell line is able to supply in trans the retroviral
functions that are deficient in the plasmid. In general, the
packaging cell lines are thus able to express the gag, pol and env
genes. Such packaging cell lines have been described in the prior
art, in particular the cell line PA317 (U.S. Pat. No. 4,861,719,
herein incorporated by reference), the PsiCRIP cell line (See,
WO90/02806), and the GP+envAm-12 cell line (See, WO89/07150). In
addition, the recombinant retroviral vectors can contain
modifications within the LTRs for suppressing transcriptional
activity as well as extensive encapsidation sequences that may
include a part of the gag gene (Bender et al., J. Virol., 61:1639
[1987]). Recombinant retroviral vectors are purified by standard
techniques known to those having ordinary skill in the art.
[0276] Alternatively, the vector can be introduced in vivo by
lipofection. For the past decade, there has been increasing use of
liposomes for encapsulation and transfection of nucleic acids in
vitro. Synthetic cationic lipids designed to limit the difficulties
and dangers encountered with liposome mediated transfection can be
used to prepare liposomes for in vivo transfection of a gene
encoding a marker (Felgner et. al., Proc. Natl. Acad. Sci. USA
84:7413-7417 [1987]; See also, Mackey, et al., Proc. Natl. Acad.
Sci. USA 85:8027-8031 [1988]; Ulmer et al., Science 259:1745-1748
[1993]). The use of cationic lipids may promote encapsulation of
negatively charged nucleic acids, and also promote fusion with
negatively charged cell membranes (Felgner and Ringold, Science
337:387-388 [1989]). Particularly useful lipid compounds and
compositions for transfer of nucleic acids are described in
WO95/18863 and WO96/17823, and in U.S. Pat. No. 5,459,127, herein
incorporated by reference.
[0277] Other molecules are also useful for facilitating
transfection of a nucleic acid in vivo, such as a cationic
oligopeptide (e.g., WO95/21931), peptides derived from DNA binding
proteins (e.g., WO96/25508), or a cationic polymer (e.g.,
WO95/21931).
[0278] It is also possible to introduce the vector in vivo as a
naked DNA plasmid. Methods for formulating and administering naked
DNA to mammalian muscle tissue are disclosed in U.S. Pat. Nos.
5,580,859 and 5,589,466, both of which are herein incorporated by
reference.
[0279] DNA vectors for gene therapy can be introduced into the
desired host cells by methods known in the art, including but not
limited to transfection, electroporation, microinjection,
transduction, cell fusion, DEAE dextran, calcium phosphate
precipitation, use of a gene gun, or use of a DNA vector
transporter (See e.g., Wu et al., J. Biol. Chem., 267:963 [1992];
Wu and Wu, J. Biol. Chem., 263:14621 [1988]; and Williams et al.,
Proc. Natl. Acad. Sci. USA 88:2726 [1991]). Receptor-mediated DNA
delivery approaches can also be used (Curiel et al., Hum. Gene
Ther., 3:147 [1992]; and Wu and Wu, J. Biol. Chem., 262:4429
[1987]).
VII. Transgenic Animals Expressing Exogenous REEP and RTP Genes and
Homologs, Mutants, and Variants Thereof
[0280] The present invention contemplates the generation of
transgenic animals comprising an exogenous REEP and/or RTP gene or
homologs, mutants, or variants thereof. In preferred embodiments,
the transgenic animal displays an altered phenotype as compared to
wild-type animals. In some embodiments, the altered phenotype is
the overexpression of mRNA for a REEP and/or RTP gene as compared
to wild-type levels of REEP and/or RTP expression. In other
embodiments, the altered phenotype is the decreased expression of
mRNA for an endogenous REEP and/or RTP gene as compared to
wild-type levels of endogenous REEP and/or RTP expression. In some
preferred embodiments, the transgenic animals comprise mutant
alleles of REEP and/or RTP. Methods for analyzing the presence or
absence of such phenotypes include Northern blotting, mRNA
protection assays, and RT-PCR. In other embodiments, the transgenic
mice have a knock out mutation of a REEP and/or RTP gene. In
preferred embodiments, the transgenic animals display an altered
susceptibility to olfactory disorders (e.g., upper respiratory
infections, tumors of the anterior cranial fossa, and Kallmann
syndrome, Foster Kennedy syndrome, Parkinson's disease, Alzheimer's
disease, Huntington chorea).
[0281] Such animals find use in research applications (e.g.,
identifying signaling pathways that a REEP and/or RTP protein is
involved in), as well as drug screening applications (e.g., to
screen for drugs that prevent or treat olfactory disorders). For
example, in some embodiments, test compounds (e.g., a drug that is
suspected of being useful to treat an olfactory disorder) are
administered to the transgenic animals and control animals with a
wild type REEP and/or RTP allele and the effects evaluated. The
effects of the test and control compounds on disease symptoms are
then assessed.
[0282] The transgenic animals can be generated via a variety of
methods. In some embodiments, embryonal cells at various
developmental stages are used to introduce transgenes for the
production of transgenic animals. Different methods are used
depending on the stage of development of the embryonal cell. The
zygote is the best target for micro-injection. In the mouse, the
male pronucleus reaches the size of approximately 20 micrometers in
diameter, which allows reproducible injection of 1-2 picoliters
(pl) of DNA solution. 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 genome 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. U.S. Pat. No. 4,873,191 describes a method for the
micro-injection of zygotes; the disclosure of this patent is
incorporated herein in its entirety.
[0283] In other embodiments, retroviral infection is used to
introduce transgenes into a non-human animal. In some embodiments,
the retroviral vector is utilized to transfect oocytes by injecting
the retroviral vector into the perivitelline space of the oocyte
(U.S. Pat. No. 6,080,912, incorporated herein by reference). In
other embodiments, the developing non-human embryo can be cultured
in vitro to the blastocyst stage. During this time, the blastomeres
can be targets for retroviral infection (Janenich, Proc. Natl.
Acad. Sci. USA 73:1260 [1976]). Efficient infection of the
blastomeres is obtained by enzymatic treatment to remove the zona
pellucida (Hogan et al., in Manipulating the Mouse Embryo, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. [1986]).
The viral vector system used to introduce the transgene is
typically a replication-defective retrovirus carrying the transgene
(Jahner et al., Proc. Natl. Acad. Sci. USA 82:6927 [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 [1987]).
Alternatively, infection can be performed at a later stage. Virus
or virus-producing cells can be injected into the blastocoele
(Jahner et al., Nature 298:623 [1982]). Most of the founders will
be mosaic for the transgene since incorporation occurs only in a
subset of cells that form the transgenic animal. Further, the
founder may contain various retroviral 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 germline, albeit with low efficiency, by
intrauterine retroviral infection of the midgestation embryo
(Jahner et al., supra [1982]). Additional means of using
retroviruses or retroviral vectors to create transgenic animals
known to the art involves the micro-injection of retroviral
particles or mitomycin C-treated cells producing retrovirus into
the perivitelline space of fertilized eggs or early embryos (PCT
International Application WO 90/08832 [1990], and Haskell and
Bowen, Mol. Reprod. Dev., 40:386 [1995]).
[0284] In other embodiments, the transgene is introduced into
embryonic stem cells and the transfected stem cells are utilized to
form an embryo. ES cells are obtained by culturing pre-implantation
embryos in vitro under appropriate conditions (Evans et al., Nature
292:154 [1981]; Bradley et al., Nature 309:255 [1984]; Gossler et
al., Proc. Acad. Sci. USA 83:9065 [1986]; and Robertson et al.,
Nature 322:445 [1986]). Transgenes can be efficiently introduced
into the ES cells by DNA transfection by a variety of methods known
to the art including calcium phosphate co-precipitation, protoplast
or spheroplast fusion, lipofection and DEAE-dextran-mediated
transfection. Transgenes may also be introduced into ES cells by
retrovirus-mediated transduction or by micro-injection. Such
transfected ES cells can thereafter colonize an embryo following
their introduction into the blastocoel of a blastocyst-stage embryo
and contribute to the germ line of the resulting chimeric animal
(for review, See, Jaenisch, Science 240:1468 [1988]). Prior to the
introduction of transfected ES cells into the blastocoel, the
transfected ES cells may be subjected to various selection
protocols to enrich for ES cells which have integrated the
transgene assuming that the transgene provides a means for such
selection. Alternatively, the polymerase chain reaction may be used
to screen for ES cells that have integrated the transgene. This
technique obviates the need for growth of the transfected ES cells
under appropriate selective conditions prior to transfer into the
blastocoel.
[0285] In still other embodiments, homologous recombination is
utilized to knock-out gene function or create deletion mutants
(e.g., mutants in which a particular domain of REEP and/or RTP is
deleted). Methods for homologous recombination are described in
U.S. Pat. No. 5,614,396, incorporated herein by reference.
VIII. Compound Screening Using REEP and RTP
[0286] In some embodiments, the isolated nucleic acid and
polypeptides of REEP and/or RTP genes of the present invention
(e.g., SEQ ID NOS: 1-50) and related proteins and nucleic acids are
used in drug screening applications for compounds that alter (e.g.,
enhance or inhibit) REEP and/or RTP activity and signaling. The
present invention further provides methods of identifying ligands
and signaling pathways of the REEP and/or RTP proteins of the
present invention.
[0287] The present invention is not limited to a particular
mechanism. Indeed, an understanding of the mechanism is not
necessary to practice the present invention. Nonetheless, based
upon OR expression analysis experiments conducted during the course
of the present invention, it is contemplated that REEP and/or RTP
family proteins function in promoting odorant receptor cell surface
localization and functional expression.
[0288] In some embodiments, the present invention provides methods
of screening compounds for the ability to alter REEP and/or RTP
activity mediated by natural ligands (e.g., identified using the
methods described above). Such compounds find use in the treatment
of disease mediated by REEP and/or RTP (e.g., olfactory disorders),
the alteration of olfactory sensory responses, and the like.
[0289] In some embodiments, the present invention provides methods
of screening compounds for an ability to interact with mutant REEP
and/or RTP nucleic acid and/or mutant REEP and/or RTP polypeptides,
while simultaneously not interacting with wild type REEP and/or RTP
nucleic acid (e.g., SEQ ID NOS:1-20) and/or wild type REEP and/or
RTP polypeptides (e.g., SEQ ID NOS:21-50). Such compounds find use
in the treatment of olfactory disorders facilitated by the presence
of mutant forms of REEP and/or RTP nucleic acids and/or
proteins.
[0290] In some embodiments, the activity of cell surface localized
ORs in cells expressing exogenous REEP or RTP polypeptides is
assessed in response to compounds (e.g., candidate or ligands or
inhibitors).
[0291] One technique uses REEP, RTP, or OR antibodies, generated as
discussed above. Such antibodies are capable of specifically
binding to REEP, RTP, or OR peptides and compete with a test
compound for binding to REEP, RTP, or OR peptides. Similar screens
can be carried out with small molecule libraries, aptamers,
etc.
[0292] The present invention contemplates the use of cell lines
transfected with REEP and/or RTP genes and variants thereof for
screening compounds for activity, and in particular to high
throughput screening of compounds from combinatorial libraries
(e.g., libraries containing greater than 10.sup.4 compounds). The
cell lines of the present invention can be used in a variety of
screening methods. In some embodiments, the cells can be used in
second messenger assays that monitor signal transduction following
activation of cell-surface receptors. In other embodiments, the
cells can be used in reporter gene assays that monitor cellular
responses at the transcription/translation level.
[0293] In second messenger assays, the host cells are preferably
transfected as described above with vectors encoding REEP and/or
RTP or variants or mutants thereof. The host cells are then treated
with a compound or plurality of compounds (e.g., from a
combinatorial library) and assayed for the presence or absence of a
response. It is contemplated that at least some of the compounds in
the combinatorial library can serve as agonists, antagonists,
activators, or inhibitors of the protein or proteins encoded by the
vectors or of ORs localized at the cell membrane. It is also
contemplated that at least some of the compounds in the
combinatorial library can serve as agonists, antagonists,
activators, or inhibitors of protein acting upstream or downstream
of the protein encoded by the vector in a signal transduction
pathway.
[0294] In some embodiments, the second messenger assays measure
fluorescent signals from reporter molecules that respond to
intracellular changes (e.g., Ca.sup.2+ concentration, membrane
potential, pH, IP.sub.3, cAMP, arachidonic acid release) due to
stimulation of membrane receptors and ion channels (e.g., ligand
gated ion channels; see Denyer et al., Drug Discov. Today 3:323
[1998]; and Gonzales et al., Drug. Discov. Today 4:431-39 [1999]).
Examples of reporter molecules include, but are not limited to,
FRET (florescence resonance energy transfer) systems (e.g.,
Cuo-lipids and oxonols, EDAN/DABCYL), calcium sensitive indicators
(e.g., Fluo-3, FURA 2, INDO 1, and FLUO3/AM, BAPTA AM),
chloride-sensitive indicators (e.g., SPQ, SPA), potassium-sensitive
indicators (e.g., PBFI), sodium-sensitive indicators (e.g., SBFI),
and pH sensitive indicators (e.g., BCECF).
[0295] In general, the host cells are loaded with the indicator
prior to exposure to the compound. Responses of the host cells to
treatment with the compounds can be detected by methods known in
the art, including, but not limited to, fluorescence microscopy,
confocal microscopy (e.g., FCS systems), flow cytometry,
microfluidic devices, FLIPR systems (See, e.g., Schroeder and
Neagle, J. Biomol. Screening 1:75 [1996]), and plate-reading
systems. In some preferred embodiments, the response (e.g.,
increase in fluorescent intensity) caused by compound of unknown
activity is compared to the response generated by a known agonist
and expressed as a percentage of the maximal response of the known
agonist. The maximum response caused by a known agonist is defined
as a 100% response. Likewise, the maximal response recorded after
addition of an agonist to a sample containing a known or test
antagonist is detectably lower than the 100% response.
[0296] The cells are also useful in reporter gene assays. Reporter
gene assays involve the use of host cells transfected with vectors
encoding a nucleic acid comprising transcriptional control elements
of a target gene (i.e., a gene that controls the biological
expression and function of a disease target) spliced to a coding
sequence for a reporter gene. Therefore, activation of the target
gene results in activation of the reporter gene product.
[0297] The test compounds of the present invention can be obtained
using any of the numerous approaches in combinatorial library
methods known in the art, including biological libraries; peptoid
libraries (libraries of molecules having the functionalities of
peptides, but with a novel, non-peptide backbone, which are
resistant to enzymatic degradation but which nevertheless remain
bioactive; see, e.g., Zuckennann et al., J. Med. Chem. 37: 2678-85
[1994]); spatially addressable parallel solid phase or solution
phase libraries; synthetic library methods requiring deconvolution;
the `one-bead one-compound` library method; and synthetic library
methods using affinity chromatography selection. The biological
library and peptoid library approaches are preferred for use with
peptide libraries, while the other four approaches are applicable
to peptide, non-peptide oligomer or small molecule libraries of
compounds (Lam (1997) Anticancer Drug Des. 12:145).
[0298] The ability of the test compound to modulate REEP and/or RTP
binding to a compound, e.g., an odorant receptor, can also be
evaluated. This can be accomplished, for example, by coupling the
compound, e.g., the substrate, with a radioisotope or enzymatic
label such that binding of the compound, e.g., the substrate, to
REEP and/or RTP can be determined by detecting the labeled
compound, e.g., substrate, in a complex.
[0299] Alternatively, REEP and/or RTP is coupled with a
radioisotope or enzymatic label to monitor the ability of a test
compound to modulate REEP and/or RTP binding to a REEP and/or RTP
substrate in a complex. For example, compounds (e.g., substrates)
can be labeled with .sup.125I, .sup.35S .sup.14C or .sup.3H, either
directly or indirectly, and the radioisotope detected by direct
counting of radioemmission or by scintillation counting.
Alternatively, compounds can be enzymatically labeled with, for
example, horseradish peroxidase, alkaline phosphatase, or
luciferase, and the enzymatic label detected by determination of
conversion of an appropriate substrate to product.
[0300] The ability of a compound (e.g., an odorant receptor) to
interact with REEP and/or RTP with or without the labeling of any
of the interactants can be evaluated. For example, a
microphysiorneter can be used to detect the interaction of a
compound with REEP and/or RTP without the labeling of either the
compound or the REEP and/or RTP (McConnell et al. Science
257:1906-1912 [1992]). As used herein, a "microphysiometer" (e.g.,
Cytosensor) is an analytical instrument that measures the rate at
which a cell acidifies its environment using a light-addressable
potentiometric sensor (LAPS). Changes in this acidification rate
can be used as an indicator of the interaction between a compound
and a REEP and/or RTP polypeptide.
[0301] In yet another embodiment, a cell-free assay is provided in
which REEP and/or RTP protein or biologically active portion
thereof is contacted with a test compound and the ability of the
test compound to bind to the REEP and/or RTP protein or
biologically active portion thereof is evaluated. Preferred
biologically active portions of REEP and/or RTP proteins to be used
in assays of the present invention include fragments that
participate in interactions with substrates or other proteins,
e.g., fragments with high surface probability scores.
[0302] Cell-free assays involve preparing a reaction mixture of the
target gene protein and the test compound under conditions and for
a time sufficient to allow the two components to interact and bind,
thus forming a complex that can be removed and/or detected.
[0303] The interaction between two molecules can also be detected,
e.g., using fluorescence energy transfer (FRET) (see, for example,
Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos et al.,
U.S. Pat. No. 4,968,103; each of which is herein incorporated by
reference). A fluorophore label is selected such that a first donor
molecule's emitted fluorescent energy will be absorbed by a
fluorescent label on a second, `acceptor` molecule, which in turn
is able to fluoresce due to the absorbed energy.
[0304] Alternately, the `donor` protein molecule may simply utilize
the natural fluorescent energy of tryptophan residues. Labels are
chosen that emit different wavelengths of light, such that the
`acceptor` molecule label may be differentiated from that of the
`donor`. Since the efficiency of energy transfer between the labels
is related to the distance separating the molecules, the spatial
relationship between the molecules can be assessed. In a situation
in which binding occurs between the molecules, the fluorescent
emission of the `acceptor` molecule label in 1 5 the assay should
be maximal. An FRET binding event can be conveniently measured
through standard fluorometric detection means well known in the art
(e.g., using a fluorimeter).
[0305] Modulators of REEP and/or RTP expression can also be
identified. For example, a cell or cell free mixture is contacted
with a candidate compound and the expression of REEP and/or RTP
mRNA or protein evaluated relative to the level of expression of
the REEP and/or RTP mRNA or protein in the absence of the candidate
compound. When expression of the REEP and/or RTP mRNA or protein is
greater in the presence of the candidate compound than in its
absence, the candidate compound is identified as a stimulator of a
REEP and/or RTP mRNA or protein expression. Alternatively, when
expression of REEP and/or RTP mRNA or protein is less (i.e.,
statistically significantly less) in the presence of the candidate
compound than in its absence, the candidate compound is identified
as an inhibitor of REEP and/or RTP mRNA or protein expression. The
level of REEP and/or RTP mRNA or protein expression can be
determined by methods described herein for detecting REEP and/or
RTP mRNA or protein.
[0306] A modulating agent can be identified using a cell-based or a
cell free assay, and the ability of the agent to modulate the
activity of a REEP and/or RTP protein can be confirmed in vivo,
e.g., in an animal such as an animal model for a disease (e.g., an
animal with an REEP and/or RTP related olfactory disorder).
[0307] B. Therapeutic Agents
[0308] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein (e.g., a REEP and/or RTP modulating agent or
mimetic, a REEP and/or RTP specific antibody, a REEP and/or
RTP--binding partner, or an OR agonist or inhibitor) in an
appropriate animal model (such as those described herein) to
determine the efficacy, toxicity, side effects, or mechanism of
action, of treatment with such an agent. Furthermore, as described
above, novel agents identified by the above-described screening
assays can be, e.g., used for treatments of olfactory disorders
(e.g., including, but not limited to, olfactory disorders).
IX. Pharmaceutical Compositions Containing REEP and RTP Nucleic
Acid, Peptides, and Analogs
[0309] The present invention further provides pharmaceutical
compositions which may comprise all or portions of REEP and/or RTP
polynucleotide sequences, REEP and/or RTP polypeptides, inhibitors
or antagonists of REEP and/or RTP bioactivity, including
antibodies, alone or in combination with at least one other agent,
such as a stabilizing compound, and may be administered in any
sterile, biocompatible pharmaceutical carrier, including, but not
limited to, saline, buffered saline, dextrose, and water.
[0310] The methods of the present invention find use in treating
diseases or altering physiological states characterized by mutant
REEP and/or RTP alleles (e.g., upper respiratory infections, tumors
of the anterior cranial fossa, and Kallmann syndrome, Foster
Kennedy syndrome, Parkinson's disease, Alzheimer's disease,
Huntington chorea). Peptides can be administered to the patient
intravenously in a pharmaceutically acceptable carrier such as
physiological saline. Standard methods for intracellular delivery
of peptides can be used (e.g., delivery via liposome). Such methods
are well known to those of ordinary skill in the art. The
formulations of this invention are useful for parenteral
administration, such as intravenous, subcutaneous, intramuscular,
and intraperitoneal. Therapeutic administration of a polypeptide
intracellularly can also be accomplished using gene therapy as
described above.
[0311] As is well known in the medical arts, dosages for any one
patient depends upon many factors, including the patient's size,
body surface area, age, the particular compound to be administered,
sex, time and route of administration, general health, and
interaction with other drugs being concurrently administered.
[0312] Accordingly, in some embodiments of the present invention,
REEP and/or RTP nucleotide and REEP and/or RTP amino acid sequences
can be administered to a patient alone, or in combination with
other nucleotide sequences, drugs or hormones or in pharmaceutical
compositions where it is mixed with excipient(s) or other
pharmaceutically acceptable carriers. In one embodiment of the
present invention, the pharmaceutically acceptable carrier is
pharmaceutically inert. In another embodiment of the present
invention, REEP and/or RTP polynucleotide sequences or REEP and/or
RTP amino acid sequences may be administered alone to individuals
subject to or suffering from a disease.
[0313] Depending on the condition being treated, these
pharmaceutical compositions may be formulated and administered
systemically or locally. Techniques for formulation and
administration may be found in the latest edition of "Remington's
Pharmaceutical Sciences" (Mack Publishing Co, Easton Pa.). Suitable
routes may, for example, include oral or transmucosal
administration; as well as parenteral delivery, including
intramuscular, subcutaneous, intramedullary, intrathecal,
intraventricular, intravenous, intraperitoneal, or intranasal
administration.
[0314] For injection, the pharmaceutical compositions of the
invention may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiologically buffered saline. For tissue
or cellular administration, penetrants appropriate to the
particular barrier to be permeated are used in the formulation.
Such penetrants are generally known in the art.
[0315] In other embodiments, the pharmaceutical compositions of the
present invention can be formulated using pharmaceutically
acceptable carriers well known in the art in dosages suitable for
oral administration. Such carriers enable the pharmaceutical
compositions to be formulated as tablets, pills, capsules, liquids,
gels, syrups, slurries, suspensions and the like, for oral or nasal
ingestion by a patient to be treated.
[0316] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose.
For example, an effective amount of REEP and/or RTP may be that
amount that suppresses olfactory disorder related symptoms.
Determination of effective amounts is well within the capability of
those skilled in the art, especially in light of the disclosure
provided herein.
[0317] In addition to the active ingredients these pharmaceutical
compositions may contain suitable pharmaceutically acceptable
carriers comprising excipients and auxiliaries that facilitate
processing of the active compounds into preparations that can be
used pharmaceutically. The preparations formulated for oral
administration may be in the form of tablets, dragees, capsules, or
solutions.
[0318] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is itself known (e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes).
[0319] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances that increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents that increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0320] Pharmaceutical preparations for oral use can be obtained by
combining the active compounds with solid excipient, optionally
grinding a resulting mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee cores. Suitable excipients are carbohydrate or
protein fillers such as sugars, including lactose, sucrose,
mannitol, or sorbitol; starch from corn, wheat, rice, potato, etc;
cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose,
or sodium carboxymethylcellulose; and gums including arabic and
tragacanth; and proteins such as gelatin and collagen. If desired,
disintegrating or solubilizing agents may be added, such as the
cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt
thereof such as sodium alginate.
[0321] Dragee cores are provided with suitable coatings such as
concentrated sugar solutions, which may also contain gum arabic,
talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol,
and/or titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures. Dyestuffs or pigments may be added to
the tablets or dragee coatings for product identification or to
characterize the quantity of active compound, (i.e., dosage).
[0322] Pharmaceutical preparations that can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating such as glycerol or sorbitol. The
push-fit capsules can contain the active ingredients mixed with a
filler or binders such as lactose or starches, lubricants such as
talc or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid paraffin, or liquid
polyethylene glycol with or without stabilizers.
[0323] Compositions comprising a compound of the invention
formulated in a pharmaceutical acceptable carrier may be prepared,
placed in an appropriate container, and labeled for treatment of an
indicated condition. For polynucleotide or amino acid sequences of
REEP and/or RTP, conditions indicated on the label may include
treatment of condition related to olfactory disorders.
[0324] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic,
etc. Salts tend to be more soluble in aqueous or other protonic
solvents that are the corresponding free base forms. In other
cases, the preferred preparation may be a lyophilized powder in 1
mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range
of 4.5 to 5.5 that is combined with buffer prior to use.
[0325] For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. Then, preferably, dosage can be formulated in
animal models (particularly murine models) to achieve a desirable
circulating concentration range that adjusts REEP and/or RTP
levels.
[0326] A therapeutically effective dose refers to that amount of
REEP and/or RTP that ameliorates symptoms of the disease state.
Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index, and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds
that exhibit large therapeutic indices are preferred. The data
obtained from these cell culture assays and additional animal
studies can be used in formulating a range of dosage for human use.
The dosage of such compounds lies preferably within a range of
circulating concentrations that include the ED.sub.50 with little
or no toxicity. The dosage varies within this range depending upon
the dosage form employed, sensitivity of the patient, and the route
of administration.
[0327] The exact dosage is chosen by the individual physician in
view of the patient to be treated. Dosage and administration are
adjusted to provide sufficient levels of the active moiety or to
maintain the desired effect. Additional factors which may be taken
into account include the severity of the disease state; age,
weight, and gender of the patient; diet, time and frequency of
administration, drug combination(s), reaction sensitivities, and
tolerance/response to therapy. Long acting pharmaceutical
compositions might be administered every 3 to 4 days, every week,
or once every two weeks depending on half-life and clearance rate
of the particular formulation.
[0328] Normal dosage amounts may vary from 0.01 to 100,000
micrograms, up to a total dose of about 1 g, depending upon the
route of administration. Guidance as to particular dosages and
methods of delivery is provided in the literature (See, U.S. Pat.
Nos. 4,657,760; 5,206,344; or 5,225,212, all of which are herein
incorporated by reference). Those skilled in the art will employ
different formulations for REEP and/or RTP than for the inhibitors
of REEP and/or RTP. Administration to the bone marrow may
necessitate delivery in a manner different from intravenous
injections.
X. RNA Interference (RNAi)
[0329] RNAi represents an evolutionary conserved cellular defense
for controlling the expression of foreign genes in most eukaryotes,
including humans. RNAi is triggered by double-stranded RNA (dsRNA)
and causes sequence-specific mRNA degradation of single-stranded
target RNAs homologous in response to dsRNA. The mediators of mRNA
degradation are small interfering RNA duplexes (siRNAs), which are
normally produced from long dsRNA by enzymatic cleavage in the
cell. siRNAs are generally approximately twenty-one nucleotides in
length (e.g. 21-23 nucleotides in length), and have a base-paired
structure characterized by two nucleotide 3'-overhangs. Following
the introduction of a small RNA, or RNAi, into the cell, it is
believed the sequence is delivered to an enzyme complex called
RISC(RNA-induced silencing complex). RISC recognizes the target and
cleaves it with an endonuclease. It is noted that if larger RNA
sequences are delivered to a cell, RNase III enzyme (Dicer)
converts longer dsRNA into 21-23 nt ds siRNA fragments.
[0330] Chemically synthesized siRNAs have become powerful reagents
for genome-wide analysis of mammalian gene function in cultured
somatic cells. Beyond their value for validation of gene function,
siRNAs also hold great potential as gene-specific therapeutic
agents (Tuschl and Borkhardt, Molecular Intervent. 2002;
2(3):158-67, herein incorporated by reference).
[0331] The transfection of siRNAs into animal cells results in the
potent, long-lasting post-transcriptional silencing of specific
genes (Caplen et al, Proc Natl Acad Sci U.S.A. 2001; 98: 9742-7;
Elbashir et al., Nature. 2001; 411:494-8; Elbashir et al., Genes
Dev. 2001; 15: 188-200; and Elbashir et al., EMBO J. 2001; 20:
6877-88, all of which are herein incorporated by reference).
Methods and compositions for performing RNAi with siRNAs are
described, for example, in U.S. Pat. No. 6,506,559, herein
incorporated by reference.
[0332] siRNAs are extraordinarily effective at lowering the amounts
of targeted RNA, and by extension proteins, frequently to
undetectable levels. The silencing effect can last several months,
and is extraordinarily specific, because one nucleotide mismatch
between the target RNA and the central region of the siRNA is
frequently sufficient to prevent silencing Brummelkamp et al,
Science 2002; 296:550-3; and Holen et al, Nucleic Acids Res. 2002;
30:1757-66, both of which are herein incorporated by reference.
XI. RNAi for REEP and RTP
[0333] As discussed above, the present invention provides RNAi for
inhibiting the expression of the REEP and/or RTP polypeptide in
cells, ORs, or pathway components involved in the expression or
activity of such components.
[0334] A. Designing and Testing RNAi for REEP and/or RTP
[0335] In order to design siRNAs for REEP and/or RTP (e.g. that
target REEP and/or RTP mRNA) software design tools are available in
the art (e.g. on the Internet). For example, Oligoengine's web page
has one such design tool that finds RNAi candidates based on
Elbashir's (Elbashir et al, Methods 2002; 26: 199-213, herein
incorporated by reference) criteria. Other design tools may also be
used, such as the Cenix Bioscience design tool offered by Ambion.
In addition, there is also the Si2 silencing duplex offered by
Oligoengine.
[0336] There are also RNA folding software programs available that
allow one to determine if the mRNA has a tendency to fold on its
own and form a "hair-pin" (which in the case of dsRNAi is not as
desirable since one goal is to have the RNAi attach to the mRNA and
not itself). One preferred configuration is an open configuration
with three or less bonds. Generally, a positive delta G is
desirable to show that it would not tend to fold on itself
spontaneously.
[0337] siRNA candidate molecules that are generated can be, for
example, screened in an animal model of an olfactory disorder for
the quantitative evaluation of REEP and/or RTP expression in vivo
using similar techniques as described above.
[0338] B. Expression Cassettes
[0339] REEP and/or RTP specific siRNAs of the present invention may
be synthesized chemically. Chemical synthesis can be achieved by
any method known or discovered in the art. Alternatively, REEP
and/or RTP specific siRNAs of the present invention may be
synthesized by methods which comprise synthesis by transcription.
In some embodiments, transcription is in vitro, as from a DNA
template and bacteriophage RNA polymerase promoter, in other
embodiments, synthesis is in vivo, as from a gene and a promoter.
Separate-stranded duplex siRNA, where the two strands are
synthesized separately and annealed, can also be synthesized
chemically by any method known or discovered in the art.
Alternatively, ds siRNA are synthesized by methods that comprise
synthesis by transcription. In some embodiments, the two strands of
the double-stranded region of a siRNA are expressed separately by
two different expression cassettes, either in vitro (e.g., in a
transcription system) or in vivo in a host cell, and then brought
together to form a duplex.
[0340] Thus, in another aspect, the present invention provides a
composition comprising an expression cassette comprising a promoter
and a gene that encodes a siRNA specific for REEP and/or RTP. In
some embodiments, the transcribed siRNA forms a single strand of a
separate-stranded duplex (or double-stranded, or ds) siRNA of about
18 to 25 base pairs long; thus, formation of ds siRNA requires
transcription of each of the two different strands of a ds siRNA.
The term "gene" in the expression cassette refers to a nucleic acid
sequence that comprises coding sequences necessary for the
production of a siRNA. Thus, a gene includes but is not limited to
coding sequences for a strand of a ds siRNA.
[0341] Generally, a DNA expression cassette comprises a chemically
synthesized or recombinant DNA molecule containing at least one
gene, or desired coding sequence for a single strand of a ds siRNA,
and appropriate nucleic acid sequences necessary for the expression
of the operably linked coding sequence, either in vitro or in vivo.
Expression in vitro may include expression in transcription systems
and in transcription/translation systems. Expression in vivo may
include expression in a particular host cell and/or organism.
Nucleic acid sequences necessary for expression in a prokaryotic
cell or in a prokaryotic in vitro expression system are well known
and usually include a promoter, an operator, and a ribosome binding
site, often along with other sequences. Eukaryotic in vitro
transcription systems and cells are known to utilize promoters,
enhancers, and termination and polyadenylation signals. Nucleic
acid sequences necessary for expression via bacterial RNA
polymerases (such as T3, T7, and SP6), referred to as a
transcription template in the art, include a template DNA strand
which has a polymerase promoter region followed by the complement
of the RNA sequence desired (or the coding sequence or gene for the
siRNA). In order to create a transcription template, a
complementary strand is annealed to the promoter portion of the
template strand.
[0342] In any of the expression cassettes described above, the gene
may encode a transcript that contains at least one cleavage site,
such that when cleaved results in at least two cleavage products.
Such products can include the two opposite strands of a ds siRNA.
In an expression system for expression in a eukaryotic cell, the
promoter may be constitutive or inducible; the promoter may also be
tissue or organ specific (e.g. specific to the eye), or specific to
a developmental phase. Preferably, the promoter is positioned 5' to
the transcribed region. Other promoters are also contemplated; such
promoters include other polymerase III promoters and microRNA
promoters.
[0343] Preferably, a eukaryotic expression cassette further
comprises a transcription termination signal suitable for use with
the promoter; for example, when the promoter is recognized by RNA
polymerase III, the termination signal is an RNA polymerase III
termination signal. The cassette may also include sites for stable
integration into a host cell genome.
[0344] C. Vectors
[0345] In other aspects of the present invention, the compositions
comprise a vector comprising a gene encoding an siRNA specific for
REEP and/or RTP or preferably at least one expression cassette
comprising a promoter and a gene which encodes a sequence necessary
for the production of a siRNA specific for REEP and/or RTP (an
siRNA gene). The vectors may further comprise marker genes,
reporter genes, selection genes, or genes of interest, such as
experimental genes. Vectors of the present invention include
cloning vectors and expression vectors. Expression vectors may be
used in in vitro transcription/translation systems, as well as in
in vivo in a host cell. Expression vectors used in vivo in a host
cell may be transfected into a host cell, either transiently, or
stably. Thus, a vector may also include sites for stable
integration into a host cell genome.
[0346] In some embodiments, it is useful to clone a siRNA gene
downstream of a bacteriophage RNA polymerase promoter into a
multicopy plasmid. A variety of transcription vectors containing
bacteriophage RNA polymerase promoters (such as T7 promoters) are
available. Alternatively, DNA synthesis can be used to add a
bacteriophage RNA polymerase promoter upstream of a siRNA coding
sequence. The cloned plasmid DNA, linearized with a restriction
enzyme, can then be used as a transcription template (See for
example Milligan, J F and Uhlenbeck, O C (1989) Methods in
Enzymology 180: 51-64).
[0347] In other embodiments of the present invention, vectors
include, but are not limited to, chromosomal, nonchromosomal and
synthetic DNA sequences (e.g., derivatives of viral DNA such as
vaccinia, adenovirus, fowl pox virus, and pseudorabies). It is
contemplated that any vector may be used as long as it is expressed
in the appropriate system (either in vitro or in vivo) and viable
in the host when used in vivo; these two criteria are sufficient
for transient transfection. For stable transfection, the vector is
also replicable in the host.
[0348] Large numbers of suitable vectors are known to those of
skill in the art, and are commercially available. In some
embodiments of the present invention, mammalian expression vectors
comprise an origin of replication, suitable promoters and
enhancers, and also any necessary ribosome binding sites,
polyadenylation sites, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
non-transcribed sequences. In other embodiments, DNA sequences
derived from the SV40 splice, and polyadenylation sites may be used
to provide the required non-transcribed genetic elements.
[0349] In certain embodiments of the present invention, a gene
sequence in an expression vector which is not part of an expression
cassette comprising a siRNA gene (specific for REEP1, RTP1, RTP2,
RTP1-A, RTP1-B, RTP1-C, RTP1-D, and RTP1-E, RTP1-A1, RTP1-D1,
RTP-D2, RTP-D3, RTP1-A1-A (Chimera 1), RTP1-A1-D2 (Chimera 2),
RTP1-A1-D1 (Chimera 3), RTP4-A1-A (Chimera 4), RTP4-A1-D2 (Chimera
5), and RTP4-A1-D1 (Chimera 6)) is operatively linked to an
appropriate expression control sequence(s) (promoter) to direct
mRNA synthesis. In some embodiments, the gene sequence is a marker
gene or a selection gene. Promoters useful in the present invention
include, but are not limited to, the cytomegalovirus (CMV)
immediate early, herpes simplex virus (HSV) thymidine kinase, and
mouse metallothionein promoters and other promoters known to
control expression of gene in mammalian cells or their viruses. In
other embodiments of the present invention, recombinant expression
vectors include origins of replication and selectable markers
permitting transformation of the host cell (e.g., dihydrofolate
reductase or neomycin resistance for eukaryotic cell culture).
[0350] In some embodiments of the present invention, transcription
of DNA encoding a gene is increased by inserting an enhancer
sequence into the vector. Enhancers are cis-acting elements of DNA,
usually about from 10 to 300 bp that act on a promoter to increase
its transcription. Enhancers useful in the present invention
include, but are not limited to, a cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication
origin, and adenovirus enhancers.
[0351] Preferably the design of a vector is configured to deliver
the RNAi for more permanent inhibition. For example the pSilencer
siRNA expression vector offered by Ambion, the pSuper RNAi system
offered by Oligoengine, and the GneSilencer System offered by
IMGENEX. These are all plasmid vector based RNAis. BD Biosciences
offer the RNAi-Ready pSIREN Vectors, that allow both a
Plasmid-based vectors and an Adenoviral or a Retroviral delivery
formats. Ambion is expected to release an adenoviral vector for
siRNA shortly. For the design of a vector there is no limitation
regarding the folding pattern since there is no concern regarding
the formation of a hairpin or at least there are no studies that
found any difference in performance related to the mRNA folding
pattern. Therefore, SEQ ID NOS: 1-20, for example, may be used with
in a Vector (both Plasmid and Viral) delivery systems.
[0352] It is noted that Ambion offers a design tool for a vector on
their web page, and BD Biosciences offers a manual for the design
of a vector, both of which are useful for designing vectors for
siRNA.
[0353] D. Transfecting Cells
[0354] In yet other aspects, the present invention provides
compositions comprising cells transfected by an expression cassette
of the present invention as described above, or by a vector of the
present invention, where the vector comprises an expression
cassette (or simply the siRNA gene) of the present invention, as
described above. In some embodiments of the present invention, the
host cell is a mammalian cell. A transfected cell may be a cultured
cell or a tissue, organ, or organismal cell. Specific examples of
cultured host cells include, but are not limited to, Chinese
hamster ovary (CHO) cells, COS-7 lines of monkey kidney
fibroblasts, 293T, C127, 3T3, HeLa, and BHK cell lines. Specific
examples of host cells in vivo include tumor tissue and eye
tissue.
[0355] The cells may be transfected transiently or stably (e.g. DNA
expressing the siRNA is stably integrated and expressed by the host
cell's genome). The cells may also be transfected with an
expression cassette of the present invention, or they are
transfected with an expression vector of the present invention. In
some embodiments, transfected cells are cultured mammalian cells,
preferably human cells. In other embodiments, they are tissue,
organ, or organismal cells.
[0356] In the present invention, cells to be transfected in vitro
are typically cultured prior to transfection according to methods
which are well known in the art, as for example by the preferred
methods as defined by the American Tissue Culture Collection. In
certain embodiments of the present invention, cells are transfected
with siRNAs that are synthesized exogenously (or in vitro, as by
chemical methods or in vitro transcription methods), or they are
transfected with expression cassettes or vectors, which express
siRNAs within the transfected cell.
[0357] In some embodiments, cells are transfected with siRNAs by
any method known or discovered in the art which allows a cell to
take up exogenous RNA and remain viable. Non-limiting examples
include electroporation, microinjection, transduction, cell fusion,
DEAE dextran, calcium phosphate precipitation, use of a gene gun,
osmotic shock, temperature shock, and electroporation, and pressure
treatment. In alternative, embodiments, the siRNAs are introduced
in vivo by lipofection, as has been reported (as, for example, by
Elbashir et al. (2001) Nature 411: 494-498, herein incorporated by
reference).
[0358] In other embodiments expression cassettes or vectors
comprising at least one expression cassette are introduced into the
desired host cells by methods known in the art, including but not
limited to transfection, electroporation, microinjection,
transduction, cell fusion, DEAE dextran, calcium phosphate
precipitation, use of a gene gun, or use of a DNA vector
transporter (See e.g., Wu et al. (1992) J. Biol. Chem., 267:963; Wu
and Wu (1988) J. Biol. Chem., 263:14621; and Williams et al. (1991)
Proc. Natl. Acad. Sci. USA 88:272). Receptor-mediated DNA delivery
approaches are also used (Curiel et al. (1992) Hum. Gene Ther.,
3:147; and Wu and Wu (1987) J. Biol. Chem., 262:4429). In some
embodiments, various methods are used to enhance transfection of
the cells. These methods include but are not limited to osmotic
shock, temperature shock, and electroporation, and pressure
treatment.
[0359] Alternatively, the vector can be introduced in vivo by
lipofection. For the past decade, there has been increasing use of
liposomes for encapsulation and transfection of nucleic acids in
vitro. Synthetic cationic lipids designed to limit the difficulties
and dangers encountered with liposome mediated transfection can be
used to prepare liposomes for in vivo transfection of a gene
encoding a marker. The use of cationic lipids may promote
encapsulation of negatively charged nucleic acids, and also promote
fusion with negatively charged cell membranes. Particularly useful
lipid compounds and compositions for transfer of nucleic acids are
described in WO95/18863 and WO96/17823, and in U.S. Pat. No.
5,459,127, herein incorporated by reference. Other molecules are
also useful for facilitating transfection of a nucleic acid in
vivo, such as a cationic oligopeptide (e.g., WO95/21931), peptides
derived from DNA binding proteins (e.g., WO96/25508), or a cationic
polymer (e.g., WO95/21931).
[0360] It is also possible to introduce a sequence encoding a siRNA
in vivo as a naked DNA, either as an expression cassette or as a
vector. Methods for formulating and administering naked DNA to
mammalian muscle tissue are disclosed in U.S. Pat. Nos. 5,580,859
and 5,589,466, both of which are herein incorporated by
reference.
[0361] Stable transfection typically requires the presence of a
selectable marker in the vector used for transfection. Transfected
cells are then subjected to a selection procedure. Generally,
selection involves growing the cells in a toxic substance, such as
G418 or Hygromycin B, such that only those cells expressing a
transfected marker gene conferring resistance to the toxic
substance upon the transfected cell survive and grow. Such
selection techniques are well known in the art. Typical selectable
markers are well known, and include genes encoding resistance to
G418 or hygromycin B.
[0362] In preferred embodiments, the transfecting agent is
OLIGOFECTAMINE. OLIGOFECTAMINE is a lipid based transfection
reagent. Additional example of lipid based transfection reagents
that were designed for the transfection of dsRNAis are the
Transit-TKO reagent which is provided by Mirus (Madison, Wis.) and
the jetSI which was introduced by Polyplus-trasfection SAS. In
addition, the Silencer siRNA Transfection Kit provided by Ambion's
includes siPORT Amine and siPORT Lipid transfection agents. Roche
offers the Fugene 6 transfection reagents that are also lipid
based. There is an option to use electroporation in cell culture.
Preferably a plasmid vector delivery system is transfected into the
cell with OLIGOFECTAMINE provided by Invitrogen or with siPORT XP-1
transfection agent provided by Ambion.
[0363] In certain embodiments, certain chemical modifications of
the dsRNAis such as changing the lipophilicity of the molecule may
be employed (e.g., attachment of lipophilic residues at the 3'
termini of the dsRNA). Delivery of dsRNAs into organisms may also
be achieved with methods previously developed for the application
of antisense oligonucleotides such as injection of
liposomes-encapsulated molecules.
[0364] E. Kits
[0365] The present invention also provides kits comprising at least
one expression cassette comprising a siRNA gene specific for REEP
and/or RTP. In some aspects, a transcript from the expression
cassette forms a double stranded siRNA of about 18 to 25 base pairs
long. In other embodiments, the expression cassette is contained
within a vector, as described above, where the vector can be used
in in vitro transcription or transcription/translation systems, or
used in vivo to transfect cells, either transiently or stably.
[0366] In other aspects, the kit comprises at least two expression
cassettes, each of which comprises a siRNA gene, such that at least
one gene encodes one strand of a siRNA that combines with a strand
encoded by a second cassette to form a ds siRNA; the ds siRNA so
produced is any of the embodiments described above. These cassettes
may comprise a promoter and a sequence encoding one strand of a ds
siRNA. In some further embodiments, the two expression cassettes
are present in a single vector; in other embodiments, the two
expression cassettes are present in two different vectors. A vector
with at least one expression cassette, or two different vectors,
each comprising a single expression cassette, can be used in in
vitro transcription or transcription/translation systems, or used
in vivo to transfect cells, either transiently or stably.
[0367] In yet other aspects, the kit comprises at least one
expression cassettes which comprises a gene which encodes two
separate strands of a ds siRNA and a processing site between the
sequences encoding each strand such that, when the gene is
transcribed, the transcript is processed, such as by cleavage, to
result in two separate strands which can combine to form a ds
siRNA, as described above.
[0368] In some embodiments, the present invention provides kits
comprising; a) a composition comprising small interfering RNA
duplexes (siRNAs) configured to inhibit expression of the REEP
and/or RTP protein, and b) printed material with instructions for
employing the composition for treating a target cell expressing
REEP and/or RTP protein via expression of REEP and/or RTP mRNA
under conditions such that the REEP and/or RTP mRNA is cleaved or
otherwise disabled. In certain embodiments, the printed material
comprises instructions for employing the composition for treating
eye disease.
[0369] F. Generating REEP and/or RTP Specific siRNA
[0370] The present invention also provides methods of synthesizing
siRNAs specific for REEP and/or RTP (e.g. human REEP and/or RTP) or
specific for mutant or wild type forms of REEP and/or RTP. The
siRNAs may be synthesized in vitro or in vivo. In vitro synthesis
includes chemical synthesis and synthesis by in vitro
transcription. In vitro transcription is achieved in a
transcription system, as from a bacteriophage RNA polymerase, or in
a transcription/translation system, as from a eukaryotic RNA
polymerase. In vivo synthesis occurs in a transfected host
cell.
[0371] The siRNAs synthesized in vitro, either chemically or by
transcription, are used to transfect cells. Therefore, the present
invention also provides methods of transfecting host cells with
siRNAs synthesized in vitro; in particular embodiments, the siRNAs
are synthesized by in vitro transcription. The present invention
further provides methods of silencing the REEP and/or RTP gene in
vivo by transfecting cells with siRNAs synthesized in vitro. In
other methods, the siRNAs is expressed in vitro in a
transcription/translation system from an expression cassette or
expression vector, along with an expression vector encoding and
expressing a reporter gene.
[0372] The present invention also provides methods of expressing
siRNAs in vivo by transfecting cells with expression cassettes or
vectors which direct synthesis of siRNAs in vivo. The present
invention also provides methods of silencing genes in vivo by
transfecting cells with expression cassettes or vectors that direct
synthesis of siRNAs in vivo.
XII. Identification of Odorant Receptor Ligands
[0373] The present invention provides methods for identifying
ligands specific for odorant receptors. The present invention is
not limited to a particular method for identifying ligands specific
for odorant receptors. In preferred embodiments, the present
invention provides a cell line (e.g., heterologous 293T cell line)
expressing an odorant receptor of interest (e.g., any human odorant
receptor) localized to the cell surface, REEP1, RTP1 or variant
thereof, RTP2, and G.sub..alpha.olf. Activation of an odorant
receptor results in an increase in cAMP. As such, in some
embodiments, the cell line further comprises a cAMP responsive
element linked with a reporting agent (e.g., luciferase) for
detecting odorant receptor activation. An odiferous molecule (e.g.,
eugenol) is exposed to the cell line. If the odiferous molecule is
a ligand specific for the odorant receptor, luciferase expression
or a change in luciferase expression is detectable (see, e.g.,
Example 7).
EXAMPLES
[0374] To identify accessory proteins that are involved in
targeting ORs to the cell surface, genes were screened for inducing
functional cell surface expression of ORs in HEK293T (293T) cells.
It was discovered REEP1, RTP1, RTP2, RTP1-A, RTP1-B, RTP1-C,
RTP1-D, and RTP1-E, RTP1-A1, RTP1-D1, RTP-D2, RTP-D3, RTP1-A1-A
(Chimera 1), RTP1-A1-D2 (Chimera 2), RTP1-A1-D1 (Chimera 3),
RTP4-A1-A (Chimera 4), RTP4-A1-D2 (Chimera 5), and RTP4-A1-D1
(Chimera 6), were discovered that promote cell surface expression
of ORs. These proteins are expressed by olfactory neurons, interact
with OR proteins, and enhance responses to odorants when
co-expressed with ORs in 293T cells. Furthermore, this has allowed
construction of a heterologous expression system to identify new
ORs that respond to aliphatic odorants.
Example 1
Identification of Odorant Receptor Accessory Proteins
[0375] After hypothesizing that mammalian ORs require accessory
protein(s) for functional cell surface expression, a search was
instituted for detecting such molecule(s). Long-SAGE (serial
analysis of gene expression) libraries (see, e.g., Saha, S., et
al., (2002) Nat Biotechnol 20, 508-512; herein incorporated by
reference in its entirety) were constructed from single olfactory
neurons as well as neurons from the vomeronasal organ and genes
were collected that are expressed by these neurons. To identify
candidate genes expressed by the olfactory neurons Digital
Differential Display (see, e.g., http://www.ncbi.nlm.nih.gov
UniGene/info_ddd.shtml) was also used. Candidate genes were
investigated for ORFs that encode membrane associated proteins.
Genes were selected with similarities to known chaperones and
cloned the cDNAs from olfactory epithelium cDNAs. The mRNA
expression of each gene was verified by in situ hybridization.
After isolating and subcloning into mammalian expression vectors,
each cDNA together with a mouse OR (MOR203-1) tagged with a 20
N-terminal amino acids of rhodopsin (Rho-tag), was transfected into
293T cells. Measurements were made assessing whether these clones
had any effect on the cell-surface expression of ORs by staining
living cells using antibodies against the Rho-tag (see, e.g.,
Laird, D. W., and Molday, R. S. (1988) Invest Opthalmol V is Sci
29, 419-428; herein incorporated by reference in its entirety).
When MOR203-1 was transfected alone, antibody staining detected
only faint cell-surface expression in less than 1% of the cells. A
schematic diagram outlining the screening procedure utilized with
the present invention is provided at FIG. 1.
Example 2
REEP and/or RTP Enhance Cell Surface Expression of ORs
[0376] Two unrelated clones (of 61 tested) enhanced both the number
and staining intensity of cell surface expression of MOR203-1 (see
FIG. 2A). The proteins encoded by these clones were named REEP1,
for Receptor Expression Enhancing Protein 1 and RTP1, for Receptor
Transporting Protein 1. Subsequently, RTP2 was found, a close
relative of RTP1. RTP2 also enhanced cell surface expression of
MOR203-1. Next, REEP1, RTP1, and RTP2 were tested to detect a
similar effect in promoting cell-surface expression of other ORs.
Four different ORs (mouse OREG, mouse olfr62, mouse OR--S46 and rat
17) were expressed in 293T cells with or without REEP1, RTP1, or
RTP2. Co-transfection of BFP or GFP demonstrated that transfection
efficiency was consistent (.about.70%). Additionally, ORs
transfected with REEP and/or RTP generated more immunofluorescent
cells and stronger signals in positive cells compared with ORs
without REEP and/or RTP (see FIG. 2A). The signal intensity and the
number of immunopositive cells varied when using different ORs at
each condition. For example, in the case of rat 17, the surface
expression was significantly lower than that of other ORs tested.
Nonetheless, occasional immunopositive cells were observed only
when the accessory proteins were co-expressed. The effects of RTP1
or RTP2 were consistently more robust than that of REEP1. The
enhancement of cell-surface expression was specific for ORs and not
for other GPCRs: neither REEP and/or RTP enhanced expression of the
.beta.2 adrenergic receptor, mT2R5 (a mouse bitter taste receptor)
(see, e.g., Chandrashekar, J., et al. (2000) Cell 100, 703-711;
herein incorporated by reference in its entirety), or a V2R
pheromone receptor (VR4) (see, e.g., Matsunami, H., and Buck, L. B.
(1997) Cell 90, 775-784; herein incorporated by reference in its
entirety) (see FIG. 2A and FIG. 3). Finally, enhancement of
cell-surface expression of MOR203-1 was not observed when other
members of the REEP and RTP families (REEP2 and RTP4) were
co-expressed.
[0377] In order to quantify the numbers and intensity of
immunopositive cells, Fluorescence-activated cell sorting (FACS)
analysis was performed. To monitor transfection and staining
efficiency, HA-tagged .beta.2 adrenergic receptor was used as a
control. More cells were labelled and the fluorescent signal was
higher when ORs were expressed with the accessory proteins (see
FIGS. 2B and 2C and FIG. 4).
Example 3
REEP and/or RTP Genes Encode Transmembrane Proteins
[0378] The REEP1 gene encodes a protein of 201 amino acids,
containing two putative transmembrane domains (see FIG. 5A).
Immunostaining of C-terminal tagged REEP1 protein indicate that the
C-terminal end is extracellular. BLAST searches identified
homologous genes in diverse eukaryotic species. REEP1 showed
limited similarities with yeast YOP1, barley HVA22, and human
DP1/TB2 (see FIG. 5B). YOP1 is implicated in vesicular transport
(see, e.g., Brands, A., and Ho, T. H. (2002) Plant Physiol 130,
1121-1131; herein incorporated by reference in its entirety).
Expression of HVA22 is induced by abscisic acid and regulated by
various environmental stresses such as extreme temperatures or
dehydration (see, e.g., Chen, C. N., et al. (2002) Plant Mol Biol
49, 633-644; Shen, Q., et al. (1993) J Biol Chem 268, 23652-23660;
each herein incorporated by reference in their entireties). DP1/TB2
is encoded by a gene deleted in colon cancers (see, e.g., Kinzler,
K. W., et al. (1991) Science 253, 661-665; herein incorporated by
reference in its entirety) and a mouse homolog of DP1 (REEP5) is
downregulated when mast cells are triggered by IgE plus antigen
(see, e.g., Prieschl, E., et al. (1996) Gene 169, 215-218; herein
incorporated by reference in its entirety). In the mouse genome,
REEP1 has at least 5 additional homologous genes (designated
REEP2-6) (see FIG. 5B).
[0379] RTP1 and RTP2 genes encode proteins with 263 and 223 amino
acids, respectively and share a 73% sequence identity in amino acid
level (see FIG. 5C). Neither protein appears to have a signal
sequence but both have a single putative transmembrane domain
located near the C-terminal end. Immunostaining of the C-terminal
tagged RTP1 suggest that C-terminal end is extracellular. BLAST
searches of the mouse genome identified two additional members,
RTP3 and RTP4. There were no obvious RTP homologs outside
vertebrate species. Nevertheless, C. elegans ODR-4 (see, e.g.,
Dwyer, N. D., et al. (1998) Cell 93, 455-466; herein incorporated
by reference in its entirety) appears to have the same membrane
topology as the RTPs.
Example 4
REEP and/or RTP are Specifically Expressed in Olfactory Neurons
[0380] Northern blot analysis with RNAs extracted from various
mouse tissues revealed that REEP1 and especially RTP1 and RTP2 are
most prominently expressed in olfactory and vomeronasal organs.
REEP1 RNA was also detected at significant levels in the brain (see
FIG. 6A). Long exposure revealed faint signals for RTP1 and RTP2 in
the brain. Expression in testis was not observed, where a subset of
ORs are expressed (see, e.g., Parmentier, M., et al. (1992) Nature
355, 453-455; Spehr, M., et al. (2003) Science 299, 2054-2058; each
herein incorporated by reference in their entireties).
[0381] In the olfactory epithelium, REEP and/or RTP were expressed
specifically in olfactory neurons, which is evident from comparison
with OMP expression, a marker for mature olfactory sensory neurons
(see FIG. 6B). To avoid cross hybridization between RTP1 and RTP2
RNA, which are 87% identical at nucleotide level across the coding
sequence, non-homologous 3'UTR regions as probes were used in
addition to probes corresponding to the open reading frames. The
signals were identical. No expression of other REEP or RTP genes
was detected in olfactory neurons with the exception of RTP4 which
was expressed at lower levels (see FIG. 6B). Finally, REEP1 was
expressed by a subset of brain cells (see FIG. 6C).
Example 5
REEP1 and RTP1 can Interact with ORs
[0382] Given the ability of REEP and/or RTP to promote cell-surface
expression of ORs, it was hypothesized that they may also interact
with OR proteins. This was assessed using co-immunoprecipitation
assays. HA-tagged MOR203-1 and Flag-tagged REEP1, RTP1 or ICAP-1, a
negative control (see, e.g., Zawistowski, J. S., et al. (2002) Hum
Mol Genet. 11, 389-396; herein incorporated by reference in its
entirety) were transfected in 293T cells. After the cell extracts
were precipitated with anti-Flag antibodies, proteins were eluted
in SDS-sample buffer at room temperature whereupon western blotting
analysis was performed to detect the OR proteins. OR proteins were
detected as high molecular weight bands after precipitation of
REEP1 or RTP1 (see FIG. 7B, lanes 1 and 2). The majority of a
control GPCR, .beta.2 adrenergic receptor, did not form
high-molecular weight oligomers using these elution conditions.
Similarly, when the HA-MOR203-1 proteins were precipitated, REEP1
or RTP1 proteins were co-precipitated whereas ICAP-1 was not
detectable (see FIG. 7C, lanes 1, 2 and 3). The present invention
is not limited to a particular mechanism. Indeed, an understanding
of the mechanism is not necessary to practice the present
invention. Nonetheless, these results indicate that REEP1 and RTP1
complex with ORs.
[0383] Based on the protein interaction, it was hypothesized that
the functional expression of the accessory proteins might be
regulated by the OR proteins. When only C-terminal Flag-tagged RTP1
was transfected into 293T cells, little cell-surface signal was
detected, indicating that the majority of RTP proteins was inside
the cells. In contrast, co-transfection of RTP1 and OR greatly
enhanced cell-surface RTP1 (see FIG. 7D). The present invention is
not limited to a particular mechanism. Indeed, an understanding of
the mechanism is not necessary to practice the present invention.
Nonetheless, these results demonstrated mutual dependence of ORs
and RTP1 for cell-surface expression and indicated that effective
cell surface expression of the both ORs and RTP1 requires the
formation of a relatively stable receptor complex between the two.
When the C-terminal tagged REEP1 was expressed, a small amount of
cell-surface REEP1 was observed. Unlike RTP1, co-expression of the
OR proteins did not facilitate cell-surface expression of REEP1
(see FIG. 7E).
Example 6
REEP and/or RTP Enhance OR Function
[0384] Poor odorant evoked signaling activity in heterologous cell
culture systems expressing ORs has been attributed to the poor cell
surface expression of ORs. The identification of REEP and/or RTP
allowed a direct assessment of this issue. A luciferase reporter
gene assay was employed in which a cAMP responsive element (CRE)
mediated luciferase gene expression (see FIG. 8A). Because OR
activation leads to an increase in cAMP, activation of the mouse
odorant receptor OREG by its ligand eugenol was measured in the
presence and absence of REEP and/or RTP (see, e.g., Kajiya, K., et
al. (2001) J Neurosci 21, 6018-6025; Touhara, K., et al., (1999)
Proc Natl Acad Sci USA 96, 4040-4045; each herein incorporated by
reference in their entireties). As reported previously, eugenol
increased levels of OREG dependent luciferase activity (see, e.g.,
Katada, S., et al. (2003) Biochem Biophys Res Commun 305, 964-969;
herein incorporated by reference in its entirety). Co-expression of
OREG with REEP and/or RTP markedly enhanced odorant-dependent
luciferase activity (FIG. 8B). Similar results were obtained when
vanillin or ethyl vanillin, two other OREG ligands were applied.
Since RTP4 is also expressed at low levels in olfactory epithelium,
this protein was co-expressed with OREG, but this did not produce a
significant increase in luciferase reporter gene activity.
[0385] Other GPCRs can exhibit change in ligand specificity
depending on accessory proteins (see, e.g., McLatchie, L. M., et
al. (1998) Nature 393, 333-339; herein incorporated by reference in
its entirety). (McLatchie et al., 1998). To investigate whether
REEP1, RTP1, or RTP2 alter the ligand selectivity of ORs, OREG and
OR-S46 with their agonists and related chemicals were tested. No
substantial changes in relative chemical selectivity were observed
when the receptors were co-expressed with the accessory proteins
(see FIG. 8C).
Example 7
Constructing a Functional Assay to Identify Odorant-Receptor
Interactions
[0386] To facilitate analysis of odorant-OR interactions, 293T cell
lines were established which stably express REEP1, RTP1, RTP2 and
G.sub..alpha.olf, the G protein alpha subunit that couples to OR
(see, e.g., Belluscio, L., et al. (1998) Neuron 20, 69-81; Jones,
D. T., and Reed, R. R. (1989). Science 244, 790-795; each herein
incorporated by reference in their entireties). To establish such
cells, linearized expression vectors containing mouse REEP1, RTP1,
RTP2 and G.sub..alpha.olf ORFs were transfected into 293T cells
with PGK-Pac (puromycin resistant gene) (see, e.g., Watanabe, S.,
et al. (1995) Biochem Biophys Res Commun 213, 130-137; herein
incorporated by reference in its entirety). Among the puromycin
resistant clones, clone 3A showed a large response to eugenol when
OREG was transfected, and was named Hana3A. RT-PCR analysis
indicated that Hana3A cells express exogenous REEP1, RTP1, RTP2 and
G.sub..alpha.olf (see FIG. 9). Enhanced cell-surface expression was
observed when OREG or other ORs were transfected in Hana3A cells
and immunostained (see FIG. 10). To test whether Hana3A cells also
increased the ligand response in the luciferase assay, the
CRE-luciferase reporter gene along with either OREG (HA-tagged),
OR-S46 or OR-S50 were co-transfected and stimulated the cells with
their ligands, eugenol, nonanoic acid, and nonanedioic acid,
respectively (see, e.g., Malnic, B., et al. (1999) Cell 96,
713-723; Touhara, K., et al. (1999) Proc Natl Acad Sci USA 96,
4040-4045; each herein incorporated by reference in their
entireties). Little luciferase induction was observed when HA-OREG
was expressed in 293T cells. In contrast, when Hana3A cells were
used, an enhancement in luciferase activity was observed following
eugenol stimulation (see FIG. 8D). Similar results were obtained
using two additional ORs, OR-S46 and OR-S50. The OR-S50 gene did
not produce a luciferase response in 293T cells, whereas the same
receptor transfected into the Hana3A cells produced robust
luciferase activity (see FIG. 8D). Expression of G.sub..alpha.olf
alone in 293T cells had little or no effect on OR activation using
this assay.
[0387] In order to confirm the increased OR function in the
presence of REEP1, RTP1 and RTP2, the amount of cAMP upon ligand
stimulation was measured using 293T cells expressing
G.sub..alpha.olf and Hana3A cells. When OREG was transfected and
eugenol was added to stimulate the OR, more cAMP was produced in
Hana3A cells. In contrast, when the .beta.2 adrenergic receptor was
expressed and isoproterenol was used, no significant differences in
cAMP production were observed (see FIG. 8E). The present invention
is not limited to a particular mechanism. Indeed, an understanding
of the mechanism is not necessary to practice the present
invention. Nonetheless, this further supports a specific role of
the accessory proteins in functional OR expression.
[0388] Previous studies demonstrated that single olfactory neurons
that are activated by aliphatic alcohols and acids express specific
ORs, primarily class I (fish-like) ORs (see, e.g., Malnic, B., et
al. (1999) Cell 96, 713-723; Zhang, X., and Firestein, S. (2002)
Nat Neurosci 5, 124-133; each herein incorporated by reference in
their entireties). Four ORs (S6/79, S18, S46 and S50) previously
assayed using other techniques (see, e.g., Malnic, B., et al.
(1999) Cell 96, 713-723; herein incorporated by reference in its
entirety) were tested against an assay panel of aliphatic alcohols,
aldehydes and acids and some other odorants. Additionally, five
"orphan" class I ORs (MOR23-1, MOR31-4, MOR31-6, MOR32-5 and
MOR32-11) whose cognate ligands were unknown were tested. At a
suprathreshold concentration of 100 uM, all these ORs were odorant
selective, responding to only a small subset of the odorants tested
(see FIG. 11A). This specificity was retained at lower, more
physiologically relevant concentrations. Many of these ORs
responded to odorants present in micromolar concentrations. The
cell-surface expression of these ORs by living-cell
immunofluorescence were evaluated. Some ORs (S18, MOR31-4, MOR31-6
and MOR32-5) were strongly expressed while other ORs (S6, S50,
MOR23-1, MOR32-11) were weakly expressed (see FIG. 12). The present
invention is not limited to a particular mechanism. Indeed, an
understanding of the mechanism is not necessary to practice the
present invention. Nonetheless, these results suggest that weak
expression was sufficient to produce a significant response to
odorants at physiologically relevant concentrations. Finally, two
additional orphan class II ORs, MOR203-1 and olfr62, were tested
against a panel of 139 odorants. MOR203-1 responded to high
concentrations of nonanoic acid (see FIG. 11B). Olfr62 responded to
coumarine and piperonal (see FIG. 11C). Several related aromatic
compounds were next tested and 2-coumaranone was identified as a
preferred ligand for olfr62 (see FIG. 11C). When parental 293T
cells for these ORs were used in this luciferase assay, little or
no response to the odorants was observed. The present invention is
not limited to a particular mechanism. Indeed, an understanding of
the mechanism is not necessary to practice the present invention.
Nonetheless, these results further demonstrate the importance of
REEP and/or RTP in functional OR expression.
Example 8
REEP and/or RTP Function during Receptor Folding, Transport, and/or
Odorant Recognition
[0389] Expression of GPCRs is a complex process that includes
protein folding, post-translational modifications and transport
through cellular compartments including the ER and Golgi apparatus.
Additionally, evidence indicates that the proper targeting of GPCRs
to the plasma membrane may involve homo or heterodimerization (see,
e.g., Angers, S., et al. (2002) Annu Rev Pharmacol Toxicol 42,
409-435; herein incorporated by reference in its entirety). The
present invention is not limited to a particular mechanism. Indeed,
an understanding of the mechanism is not necessary to practice the
present invention. Nonetheless, though REEP and/or RTP can function
at any of these steps of OR expression, three possibilities are
presented in FIG. 13 regarding their possible interaction.
[0390] First, REEP and/or RTP promote correct folding of ORs in the
ER. NinaA, a cyclophilin homolog of Drosophila, was identified as a
chaperone protein for rhodopsin and thought to facilitate the
correct folding (see, e.g., Baker, E. K., et al. (1994) Embo J 13,
4886-4895; Shieh, B. H., et al. (1989) Nature 338, 67-70; each
herein incorporated by reference in their entireties). The plant
homologs of REEP1, HVA22s, are stress-induced genes and may allow
plants to tolerate adverse conditions (see, e.g., Chen, C. N., et
al. (2002) Plant Mol Biol 49, 633-644; Shen, Q., et al. (1993) J
Biol Chem 268, 23652-23660; each herein incorporated by reference
in their entireties). The present invention is not limited to a
particular mechanism. Indeed, an understanding of the mechanism is
not necessary to practice the present invention. Nonetheless, while
the precise roles of HVA22s are not known, since a number of stress
induced proteins, such as heat shock proteins, function as
chaperones, it is conceivable that HVA22s and, by analogy, perhaps
REEP1 act as chaperones to promote folding.
[0391] Second, REEP1, RTP1, and RTP2 facilitate the transport of
specific vesicles/cargos that include ORs. Consistent with this
idea, a REEP1 homolog in yeast, YOP1P, has been implicated in
Rab-mediated vesicle transport (see, e.g., Brands, A., and Ho, T.
H. (2002) Plant Physiol 130, 1121-1131; Calero, M. (2001) J Biol
Chem 276, 12100-12112; each herein incorporated by reference in
their entireties). In C. elegans, a clathrin adaptor subunit,
UNC-101, mediates trafficking of chemosensory receptors to
olfactory cilia (see, e.g., Dwyer, N. D., et al., (2001) Neuron 31,
277-287; herein incorporated by reference in its entirety).
[0392] Third, REEP1, RTP1, and RTP2 act as a co-receptor with ORs.
As shown in FIG. 7D, RTP1 cell surface expression is enhanced by
co-expression of ORs. ORs may contain ER retention signal(s) that
are masked by the association with RTPs (or REEP1), a mechanism
similar to the regulation of cell-surface expression of GABA(B)R1
receptor by the association of GABA(B)R2 (see, e.g., Jones, K. A.,
et al. (1998) Nature 396, 674-679; Kaupmann, K., et al. (1998)
Nature 396, 683-687; White, J. H., et al. (1998) Nature 396,
679-682; each herein incorporated by reference in their
entireties). The REEPs and RTPs may have different or complementary
roles, a hypothesis that is consistent with the absence of any
amino acid sequence similarity or specific sequence motifs.
[0393] The present invention is not limited to a particular
mechanism. Indeed, an understanding of the mechanism is not
necessary to practice the present invention. Nonetheless, the three
roles outlined above are reasonable for REEP1, RTP1 and RTP2;
however, other possible functions are not excluded. Even though
changes in ligand specificity of OREG or OR-S46 was not observed
when expressed with REEP1, RTP1 or RTP2, it is possible that they
do play a role in modulating recognition profiles of some ORs. For
example, different RAMP members change the ligand specificity of
calcitonin receptor like receptor (CRLR), a member of GPCRs. CRLR
expressed with RAMP1 function as a CGRP receptor, whereas CRLR
expressed with RAMP2 functions as adrenomedullin receptor (see,
e.g., McLatchie, L. M., et al. (1998) Nature 393, 333-339; herein
incorporated by reference in its entirety).
[0394] The present invention is not limited to a particular
mechanism. Indeed, an understanding of the mechanism is not
necessary to practice the present invention. Nonetheless, many
GPCRs, including V1R pheromone receptors (see, e.g., Dulac, C., and
Axel, R. (1995) Cell 83, 195-206; herein incorporated by reference
in its entirety), T2R taste receptors (see, e.g., Adler et al.
(2000) Cell 100, 693-702; Matsunami, H., Montmayeur, J. P., and
Buck, L. B. (2000) Nature 404, 601-604; each herein incorporated by
reference in their entireties), the .alpha.2C adrenergic receptor
(see, e.g., Hurt, C. M., et al. (2000) J Biol Chem 275,
35424-35431; herein incorporated by reference in its entirety), and
the thyrotropin-releasing hormone receptor (see, e.g., Yu, R., and
Hinkle, P. M. (1997) Mol Pharmacol 51, 785-793; herein incorporated
by reference in its entirety), appear to require cofactor(s) for
their cell surface expression. Thus, REEP and RTP members may
regulate trafficking of such GPCRs. In situ hybridization analysis
has shown that REEP3, REEP5, RTP1 and RTP2 are all expressed by the
VNO neurons. In addition, REEP members are differentially expressed
in subset of brain cells (M.M. and H.M., unpublished observations).
The strategy to create a list of genes expressed in specific cell
types using SAGE and/or Digital Differential Display and screen
genes that promote cell-surface expression of the receptors could
be applied in such cases.
Example 9
REEP and/or RTP Enable Investigations of Odorant Receptor-Odorant
Interactions
[0395] An expression system has been established that permits rapid
identification of ligands for ORs. This system was tested with
twelve ORs. Four of the tested ORs (S6/S79, S18, S46, and S50) were
expressed in single olfactory neurons responding to aliphatic
odorants (see, e.g., Malnic, B., et al. (1999) Cell 96, 713-723;
herein incorporated by reference in its entirety). The response
profiles of OR-S50, but not that of OR-S18 agreed with the previous
report (see, e.g., Malnic, B., et al. (1999) Cell 96, 713-723;
herein incorporated by reference in its entirety). In previous
studies, olfactory neurons S6 and S79 expressed the same
OR(OR-S6/S79) and both responded to nonanedioic acid, although only
the olfactory neuron S79 responded to two odorants, heptanoic acid
and octanoic acid (see, e.g., Malnic, B., et al. (1999) Cell 96,
713-723; herein incorporated by reference in its entirety). In
experiments conducted during the course of the present invention,
OR-S6/S79 responded to nonanedioic acid but not to heptanoic acid
or octanoic acid. The present invention is not limited to a
particular mechanism. Indeed, an understanding of the mechanism is
not necessary to practice the present invention. Nonetheless, these
results support the olfactory neuron S6 response profile.
Differences may be due to the variation of responses when recording
from single olfactory neurons. When multiple single olfactory
neurons that expressed the same OR were recorded against the same
set of odorants using calcium imaging, their response profiles were
similar but different (see, e.g., Bozza, T., et al. (2002) J
Neurosci 22, 3033-3043; herein incorporated by reference in its
entirety).
[0396] Seven new ORs were identified that responded to different
odorants in the test panels. The present invention is not limited
to a particular mechanism. Indeed, an understanding of the
mechanism is not necessary to practice the present invention.
Nonetheless, these results demonstrate the applicability of this
system to decode the ligand specificity of ORs. The profiles of the
ORs in response to various odorants are consistent with the idea of
"combinatorial receptor code" where one OR responds to multiple
related odorants and one odorant activates multiple receptors (see,
e.g., Kajiya, K., et al. (2001) J Neurosci 21, 6018-6025; Malnic,
B., et al. Cell 96, 713-723; each herein incorporated by reference
in their entireties).
[0397] In experiments conducted during the course of the present
invention, not only three class I ORs (S46, MOR23-1, MOR31-4) but
also MOR203-1, a class II OR, responded to nonanoic acid. The
present invention is not limited to a particular mechanism. Indeed,
an understanding of the mechanism is not necessary to practice the
present invention. Nonetheless, these results indicate that very
different ORs can respond to the same chemical, as MOR203-1 and
other nonanoic acid ORs (MOR23-1, MOR31-4 and S46) are only 29-32%
identical. Olfr62 is one of the closely related ORs located near or
at the IVA locus, implicated in isovaleric acid sensation (see,
e.g., Griff, I. C., and Reed, R. R. (1995) Cell 83, 407-414; Zhang,
X., and Firestein, S. (2002) Nat Neurosci 5, 124-133; each herein
incorporated by reference in their entireties). In experiments
conducted during the course of the present invention, olfr62 did
not respond to isovaleric acid but responded to coumarin and other
related aromatic compounds (see FIG. 11C). Eight other ORs located
near the IVA locus were also tested, but none of them responded to
isovaleric acid. The present invention is not limited to a
particular mechanism. Indeed, an understanding of the mechanism is
not necessary to practice the present invention. Nonetheless, these
results suggest that these ORs are not involved in isovaleric acid
detection.
[0398] The present invention is not limited to a particular
mechanism. Indeed, an understanding of the mechanism is not
necessary to practice the present invention. Nonetheless, the
functional OR expression system together with the annotation of
virtually all the ORs in the mouse and human genomes (see, e.g.,
Glusman, G., et al. (2001) Genome Res 11, 685-702; Young, J. M., et
al. (2002) Hum Mol Genet. 11, 535-546; Zhang, X., and Firestein, S.
(2002) Nat Neurosci 5, 124-133; Zozulya, S., et al. (2001) Genome
Biol 2, 18; each herein incorporated by reference in their
entireties), provide a platform to investigate mammalian OR-odorant
interaction in a comprehensive manner.
Example 10
Single-Cell Long SAGE Analysis
[0399] Single-cell RT-PCR was conducted as described (see, e.g.,
Brady, G., and Iscove, N. N. (1993) Methods Enzymol 225, 611-623;
Dulac, C., and Axel, R. (1995) Cell 83, 195-206; Matsunami, H., and
Buck, L. B. (1997) Cell 90, 775-784; each herein incorporated by
reference in their entireties) with modifications. Briefly, adult
mouse olfactory tissues were dissociated with dispase (Invitrogen)
and collagenase (Invitrogen). Single cells were picked under
inverted microscope using micromanipulator and transferred into
4.75 ul of lysis mix (1.times.PCR buffer (Roche), 1.5 mM
MgCl.sub.2, 50 uM dNTPs, 200 ng/mg anchor primer
(biotin-TATAGAATTCGCGGCCGCTCGCGA (T) 24), 0.3 U/ul Prime RNase
Inhibitor (Eppendorf), and 0.4 U/ul rRNasin (Promega). PCR tubes
containing lysed cells were heated to 65 degrees C. for 1 min,
cooled at 4 degrees C. and 0.25 ul of RT mix (170 U/ul Superscript
II (Invitrogen), 35 U/ul Prime RNase Inhibitor and 45 U/ul
rRNasin.) was added and incubated at 37 degrees C. for 10 min then
65 degrees C. for 10 min. 5 ul of TdT mix (1.times.PCR buffer
(Roche), 1.5 mM MgCl.sub.2, 3 mM dATP, 1.25 U/ul TdT (Roche), 0.05
U RNase H (Roche)) was added to each tube and incubated at 37
degrees C. for 20 min then at 65 degrees C. for 10 min. 5 ul of the
product was added to 50 ul of PCR mix (1.times.EX Taq buffer
(Takara), 0.25 mM dNTPs, 20 ng/ul anchor primer, 2.5 U EX Taq HS
polymerase (Takara)) and incubated at 95 degrees C. for 2 min, 37
degrees C. 5 min, 72 degrees C. 20 min, then 28 cycles of 95
degrees C. 30 sec, 67 degrees C. 1 min, 72 degrees C. 6 min plus 6
sec extension for each cycle, then 72 degrees C. for 10 min.
Contents of amplified PCR products were analyzed using long SAGE
protocols (see, e.g., Saha, S., et al. (2002) Nat Biotechnol 20,
508-512; herein incorporated by reference in its entirety).
Briefly, single-cell PCR products were cut with NlaIII (NEB). After
biotinylated DNA was bound to streptavidin magnetic beads (Dynal),
linkers were ligated. The ligated DNA was cut by MmeI (NEB). The
cleaved tags were ligated to form ditags and amplified by PCR. The
PCR product was cut with NlaIII and the ditags were ligated to form
concatemers. They were ligated into pZero-1 vector (Invitrogen) and
transformed. Single colonies were picked and sequenced. Tag
sequences were analyzed using SAGE2002 software and NCBI Blast
searches.
Example 11
Vector Construction
[0400] cDNAs were amplified from olfactory epithelium cDNA using
HotstarTaq DNA polymerase (Qiagen) or KOD DNA polymerase
(Toyobo/Novagen) and subcloned into pCI expression vectors
(Promega). OR open reading frames were amplified from genomic DNA
of C57BL6 (MOR203-1 and S46), 129 (S18) or DBA2 (olfr62, S6/S79,
S50, MOR23-1, MOR31-4, MOR31-6, MOR32-5 and MOR32-11) and subcloned
into pCI containing Rho-tag.
Example 12
Cell Culture and Immunocytochemistry
[0401] 293T cells were maintained in minimal essential medium
containing 10% fetal bovine serum (M10). Lipofectamine 2000
(Invitrogen) was used for transfection. In live-cell staining, 16
hours after transfection, cells were incubated in M10 containing
anti rhodopsin antibody, 4D2 (see, e.g., Laird, D. W., and Molday,
R. S. (1988) Invest Opthalmol V is Sci 29, 419-428; herein
incorporated by reference in its entirety) and 15 mM NaN.sub.3 at 4
degrees C. for 1 hour. After washing, cells were incubated with
Cy3-conjugated anti mouse IgG (Jackson Immunologicals), washed and
mounted. For FACS analysis, 4D2 and PE-conjugated anti mouse IgG
(Jackson Immunologicals) were used to monitor the Rho tagged
receptor expression. Anti HA rabbit antibodies (Sigma) and Alexa
488 conjugated anti rabbit IgG (Molecular Probes) was used to stain
the HA-.beta.2 adrenergic receptor. To establish the Hana3a cells,
1 ug/ml of puromycin was used for selection. 96 colonies were
picked and assayed using luciferase assay using OREG.
Example 13
Analysis of REEPs and RTPs
[0402] For prediction of signal peptide and transmembrane regions
of REEPs and RTPs, SignalP (see, e.g., Nielsen, H., et al. (1997)
Protein Eng 10, 1-6; herein incorporated by reference in its
entirety) was used and TMHMM, respectively. In order to create a
phylogenetic tree, ClustalW was used.
Example 14
Northern and In Situ Hybridization
[0403] Total RNAs from various tissues were extracted using Trizol
reagent (Invitrogen) or Aurum Total RNA (Biorad). RNAs were
electrophoresed on formaldehyde-agarose gel and transferred onto
HybondN membrane (Amersham). Dig-labeled probes were hybridized to
the membrane in Dig easyhyb solution (Roche) at 65 degrees C. After
washing, anti-Dig AP (Roche) were applied and the membranes were
washed. The signals were detected with CDP-Star (Roche). The same
membrane for all three probes was used. In situ hybridization was
performed as described (see, e.g., Matsunami, H., and Buck, L. B.
(1997) Cell 90, 775-784; Matsunami, H., et al. (2000) Nature 404,
601-604; Schaeren-Wiemers, N., and Gerfin-Moser, A. (1993)
Histochemistry 100, 431-440; each herein incorporated by reference
in their entireties). Briefly, Dig-labeled RNA probes were
hybridized with fresh frozen sections of three weeks old CD-1 mice.
After washing, Dig probes were reacted with anti-Dig AP and signals
were detected using NBT-BCIP.
Example 15
Immunoprecipitation
[0404] 293T cells in 100 mm dishes were transfected with ORs,
REEP1, and/or RTP1 cDNAs. 16 hours after transfection, cells were
lysed in lysis buffer (50 mM Tris (7.4), 150 mM NaCl, 1% NP-40, 0.5
mM PMSF, 2 mM Benzamidene, 0.5 ug/ml Leupeptin, 1.4 ug/ml pepstatin
A, 2.4 ug/ml chymostatin, 15 ug/ml aprotinin, 1 mM sodium
orthovanadate). The lysis were incubated with anti-Flag M2 affinity
gel (Sigma) or anti-HA affinity matrix (Roche) for 2 hours at 4
degrees C. and washed with lysis buffer. Subsequently, the bound
proteins were eluted by incubation with SDS sample buffer at room
temperature for 2 hours. SDS-PAGE and western blotting were
performed according to Mini-Protean 3 Cell (Bio-Rad) instruction
manual. ECL (Amersham) was used for detecting proteins on
membranes.
Example 16
Luciferase Assay
[0405] Dual-Glo system (Promega) for luciferase assay was used.
CRE-Luciferase (Stratagene) was used to measure the receptor
activities. Renilla luciferase driven by constitutively active SV40
promoter (pRL-SV40: Promega) was used as an internal control. Cells
were plated on poly-D-lysine coated 96 well plates (BIOCOAT,
Beckton Dickinson). After 8 hours (for experiments shown in FIGS.
8B and 8C) or 12 hours (for experiments shown in FIG. 8D and FIG.
11) after transfection, the medium was replaced with CD293
chemically defined medium (Invitrogen) and the plates were
incubated for one hour at 37 degrees C. The medium was replaced
with 50 ul of odorant solutions dissolved in CD293 and incubated
for 10 hours (for experiments shown in FIGS. 8B and 8C) or 4 hours
(for experiments shown in FIG. 8D and FIG. 11) at 37 degrees C. The
manufacture's protocol for measuring luciferase and Renilla
luciferase activities was followed. Luminescence was measured using
Wallac Victor 1420 (Perkin-Elmer). Normalized luciferase activity
was calculated as [Luc (N)-Luc (0)]/RL (N), where Luc
(N)=Luminescent count of a certain well, Luc (0)=Luminescent count
without odorant for each OR, and RL (N)=Luminescent count of
Renilla Luciferase of each well. For cAMP assays, cells were plated
onto 24-well plate. OREG or OREG/Golf cDNA was transfected into
Hana3a or HEK293-Tcells, respectively. 14 hours after transfection,
the cells were incubated in CD293 for 2 hrs, and exposed to eugenol
or isoproterenol in MEM containing 10 mM Hepes and 500 uM IBMX for
5 min. cAMP-Screen Direct System (Applied Biosystems) was used to
measure the cAMP levels. Prism software (Graphpad) was used for
data analysis.
Example 17
Chemicals
[0406] All odorants were purchased from Sigma except octanoic acid
from Calbiochem. The chemicals used in finding cognate ligands for
MOR203-1 and olfr62 are provided in Example 19.
Example 18
Genbank Accession Numbers
[0407] The genbank accession numbers of mouse and human REEP1-6 and
RTP1-4: AY562225-AY562244.
Example 19
Supplemental Materials
[0408] Chemicals that are used for initial ligand screening for
MOR203-1 and olfr62 are the following: 1 (+)-Carvone, 2 L-Canvone,
3 (-)-Fenchone, 4 Citral, 5 (1R)-(-)-Fenchone, 6 (+)-Fenchone, 7
Rosemary oil, 8 (-)-Rose oxide, 9 (+)-Rose oxide, 10 (-)-Camphor,
11 (S)-(-)-Limonene, 12 (R)-(+)-1-Phenylethanol, 13
(S)-(+)-2-Phenylbutyric acid, 14 (R)-(-)-2-Phenylbutyric acid, 15
2-Hexanone, 16 1-Pentanol, 17 1-Heptanol, 18 (+)-2-Butanol, 19
1-propanol, 20 1-Hexanol, 21 (-)-Menthol, 22 (R)-(-)-2-Heptanol, 23
(-)-.alpha.-Terpineol, 24 (+)-Menthol, 25 2-methyl-2-heptanol, 26
(S)-(+)-2-Octanol, 27 (S)-(+)-2-Butanol, 28 (S)-(+)-2-Heptanol, 29
(R)-(-)-2-Octanolor P(+)-2 Octanol, 30 1-Decanol, 31
(-)-.beta.-Citronellol, 32 (S)-(-)-1-Phenylethanol, 33
Propionaldehyde, 34 Undecanal, 35 Octanal(Caprylic aldehyde), 36
trans-Cinnamaldehyde, 37 Nonanal (Pelargonaldehyde), 38
Heptaldehyde, 39 Decanal, 40 Hexanoic acid, 41 Hexanoic acid, 42
Heptanoic acid(Oenanthic acid), 43 Pentanoic acid, 44 Propionic
acid, 45 Butyric acid, 46 Nonanoic acid, 47 Methyl propionate, 48
Ethyl butyrate, 49 Butyl butyrate, 50 tert-Butyl propionate, 51
Methyl butyrate, 52 Propyl butyrate, 53 Pentyl acetate, 54
Dimethylpyrazine, 55 Isobutylamine, 56 Geraniol, 57 2-Pentanone, 58
2-Butanone, 59 (1S)-(-)-.alpha.-Pinene, 60 1,4-Cineole, 61
Phenetole, 62 Butyl methyl ether, 63 (R)-(+)-Pulegone, 64 Benzene,
65 Benzyl alcohol, 66 Guaiacol, 67 Isopentylamine, 68
g-Caprolactone, 69 g-Caprolactone, 70 octen, 71 Allyl heptanoate,
72 a-Amylcinnamaldehyde, 73 Amyl hexanoate, 74 amylbutyrate, 75
Anethole, 76 Anisaldehyde, 77 Benzophenone, 78 Benzyl acetate, 79
Benzyl salicylate, 80 Butyl heptanoate, 81 camphor ((+)-Camphor),
82 Cedryl acetate, 83 Cinnamyl alcohol, 84 Cinnamaldehyde, 85
(R)-(+)-Citronellal, 86 (S)-(-)-Citronellal, 87 citronellol, 88
Coumarin, 89 Cyclohexanone, 90 p-cymene, 91
5,5-Dimethyl-1,3-cyclohexanedione (Dimedone), 92 ethylamylketone
(3-Octanone), 93 Eucalyptol, 94 Heptyl isobutyrate, 95 Hexyl
acetate, 96 a-Hexylcinnamaldehyde, 97 Isobornyl acetate, 98
Linalool, 99 Lyral (a-Amylcinnamaldehyde dimethyl acetal), 100
Hydroxycitronellal, 101 p-Tolyl isobutyrate, 102 o-Tolyl
isobutyrate, 103 p-Tolyl phenylacetate, 104
2-Methoxy-3-Methyl-pyrazine, 105 2-Methoxypyrazine, 106 Methyl
salicylate, 107 Myrcene, 108 w-Pentadecalactone, 109 prenylacetate,
110 2-Phenylethanol, 111 2-Phenethyl acetate, 112 Piperonal, 113
Pyrazine, 114 Sassafras oil, 115 thymol, 116 Triethylamine, 117
2-Heptanone, 118 Methyl eugenol, 119 eugenol, 120 Eugenol methyl
ether, 121 Butyraldehyde, 122 Hexanal, 123 1-Pentanol, 124
valeraldehyde, 125 Azelaic acid dichloride, 126 Azelaic acid, 127
Isovaleric acid, 128 Decanoic acid, 129 Vanillic acid, 130
1-Octanol, 131 4-Ethylphenol, 132 Heptaldehyde, 133 1-Nonanol, 134
Nonanal, 135 Ethyl vanillin, 136 Vanillin, 137 Acetophenone, 138
2-Ethylphenol, 139 Octanal.
[0409] Chemicals related to coumarin and piperonal (used for
olfr62) are the following: 140 Benzaldehyde, 141 Piperonyl alcohol,
142 4-Hydroxycoumarin, 143 4-Chromanone, 144 2-Coumaranone.
Example 20
Activation Patterns of Human Odorant Receptors
[0410] Hana3A cells (293T cells expressing mouse REEP1, RTP1, RTP2,
and G.sub..alpha.olf) were used. CRE-Luciferase (Stratagene) was
used to measure odorant receptor activities. The following human
odorant receptors were tested for expression patterns in response
to various odiferous agents: 36, 35, 11, 57, 58, 9, 3, 42, 81, 82,
66, 13, 87, 33, 44, 43, 77, 75, 64, 59, 12, 62, 60, 120, 90, 95,
160, and 106. The following odiferous agents were used to test
human odorant receptor expression patterns: Pyridine,
2,2'-(Dithiodimethylene)difuran, 1-Decanol, 1-Hexanol,
(-)-Fenchone, (+)-Fenchone, Geraniol, 2-Pentanone, Benzyl
salicylate, (+)-Menthol, (-)-Menthol, Benzene, Undecanal, Methyl
butylate, Heptyl isobutyrate, p-Tolyl isobutyrate, amylbutyrate,
Ethyl butyrate, Hexyl acetate, Pentyl Acetate, Piperonyl acetate,
(-)-b-Citronellol, citronellol, Eugenol methyl ether, Methyl
Eugenol, a-Amylcinnamaldehyde, a-Amylcinnamaldehyde dimethyl
acetal, Cinnamaldehyde, a-Hexylcinnamaldehyde, Hydroxycitronnellal,
Citral, (R)-(+)-Citronellal, (S)-(-)-Citronellal, p-Toly
phenylacetate, Allyl phenylacetate, Propionic acid, Azelaic acid
dichloride, isovaleric acid, (R)-(-)-2-Phenylbutyric acid,
(S)-(+)-2-Phenylbutyric acid, Heptanoic Acid, Octanoic Acid,
Valeric Acid, Hexanoic Acid, and Butyl butyrate. Renilla luciferase
driven by constitutively active SV40 promoter (pRL-SV40: Promega)
was used as an internal control. Dual-Glo system (Promega) was used
for the luciferase assay. Cells were plated on poly-D-lysine coated
96 well plates (BIOCOAT, Beckton Dickinson). 12-16 hours after
transfection, the medium was replaced with CD293 chemically defined
medium (Invitrogen) and the plates were incubated for one hour at
37 degrees C. The medium was replaced with 50 .mu.l of odorant
solutions dissolved in CD293 and incubated for 4 hours at 37
degrees C. The manufacture's protocol was followed for measuring
luciferase and Renilla luciferase activities. Luminescence was
measured using Wallac Victor 1420 (Perkin-Elmer). Normalized
luciferase activity was calculated as [Luc (N)-Luc (O)]/RL (N),
where Luc (N)=Luminescent count of a certain well, Luc
(O)=Luminescent count without odorant for each OR, and RL
(N)=Luminescent count of Renilla Luciferase of each well. FIG. 34
shows the activation patterns of the human odorant receptors in
response to odiferous agent exposure.
Example 21
Cell-Surface Expression of V1RE11 in Hana3A and 293T Cells
[0411] cDNAs encoding a putative pheromone receptor (V1RE11) were
transfected into Hana3A cells (HEK293T cells expressing REEP1,
RTP1, RTP2 and G.sub..alpha.olf) or 293T cells. V1RE11 is a
putative pheromone receptor in the mouse and is completely
different from odorant receptors in amino acid sequences. Hana3A
cells supported cell-surface expression of V1RE11. The present
invention is not limited to a particular mechanism. Indeed, an
understanding of the mechanism is not necessary to practice the
present invention. Nonetheless, these results indicate that REEP1,
RTP1, and RTP2 can support functional expression of receptors other
than odorant receptors.
Example 22
The Ability of RTP1-A, RTP1-B, RTP1-C, RTP1-D and RTP1-E to Enhance
OLFR62 Cell-Surface Expression and Activity
[0412] This example describes the generation of the RTP1 variants
RTP1-A, RTP1-B, RTP1-C, RTP1-D and RTP1-E and their ability to
enhance OLFR62 cell-surface expression and activity. Variants of
RTP1 were generated by deleting portions of RTP1. FIG. 35
schematically shows the amino acid segments of RTP1-A, RTP1-B,
RTP1-C, RTP1-D, and RTP1-E in comparison to RTP1. pCI was a control
vector. FIG. 36 shows the murine amino acid sequence for RTP1-A
(SEQ ID NO: 41), FIG. 37 shows the murine amino acid sequence for
RTP1-B (SEQ ID NO: 42), FIG. 38 shows the murine amino acid
sequence for RTP1-C (SEQ ID NO: 43), FIG. 39 shows the murine amino
acid sequence for RTP1-D (SEQ ID NO: 44), and FIG. 40 shows the
murine amino acid sequence for RTP1-E (SEQ ID NO: 45).
[0413] FIG. 41 shows cell-surface expression of OLFR62 in Hana3A
and 293T cells. cDNAs encoding RTP1, RTP1-A, RTP1-B, RTP1-C,
RTP1-D, RTP1-E, and control pCI were transfected into Hana3A cells
or 293T cells. Increased cell-surface staining was seen in Hana3A
cells and 239T cells expressing RTP1-D.
[0414] FIG. 42 schematically shows a luciferase assay used to
monitor the activity of OLFR62 activity. cAMP responsive element
(CRE) and luciferase was used to monitor activation of OLFR62.
Activation of OLFR62 increases cAMP, which enhances the expression
of luciferase reporter gene through the CRE.
[0415] FIG. 43 shows OLFR62 activity as indicated by luciferase
expression in Hana3A cells and 293T cells expressing RTP1, RTP1-A,
RTP1-B, RTP1-C, RTP1-D, RTP1-E, and control pCI. Increased
enhancement of OLFR62 activity was seen in 293T cells and Hana3A
cells expressing RTP1-D.
Example 23
The Ability of RTP1-A1, RTP1-D1, RTP1-D2, and RTP1-D3 to Enhance
OLFR62Cell-Surface Expression and Activity
[0416] This example describes the generation of the RTP1 variants
RTP1-A1, RTP1-D1, RTP1-D2, and RTP1-D3 and their ability to enhance
OLFR62 cell-surface expression and activity. Variants of RTP1 were
generated by deleting portions of RTP1-A and RTP1-D. In particular,
primer pairs were laid down at specific locations corresponding to
desired deletion segments and were amplified by PCR with KOD
polymerase. FIG. 44 schematically shows the amino acid segments of
RTP1-A1, RTP1-D1, RTP1-D2, and RTP1-D3 in comparison to RTP1-A and
RTP1-D, respectively. FIG. 45 shows the murine amino acid sequence
for RTP1-A1 (SEQ ID NO: 46) and the human amino acid sequence for
RTP1-A1 (SEQ ID NO: 47). FIG. 46 shows the murine amino acid
sequence for RTP1-D1 (SEQ ID NO: 48). FIG. 47 shows the murine
amino acid sequence for RTP-D2 (SEQ ID NO: 49). FIG. 48 shows the
murine amino acid sequence for RTP-D3 (SEQ ID NO: 50).
[0417] FIG. 49 shows cell-surface expression of OLFR62 in 293T
cells. cDNAs encoding RTP1, RTP1-A1, RTP1-D1, RTP1-D2, and RTP1-D3,
and control pCI were transfected into 293T cells. Increased
cell-surface staining was seen in 239T cells expressing RTP1-A1,
RTP1-D1 and RTP1-D3.
[0418] FIG. 50 shows OLFR62, OREG, S6, and 23-1 activity as
indicated by luciferase expression in 293T cells expressing RTP1,
RTP1-A1, RTP1-D1, RTP1-D2, and RTP1-D3, and control pCI. Increased
enhancement of OLFR62, OREG, S6, and 23-1 activity was seen in 293T
cells expressing RTP1-A1.
[0419] FIG. 51 shows OLFR62, OREG, S6, and 23-1 activity as
indicated by luciferase expression in Hana3A cells expressing RTP1,
RTP1-A1, RTP1-D1, RTP1-D2, and RTP1-D3, and control pCI.
[0420] FIG. 52 shows cell-surface expression of OLFR62, OREG,
MOR203-1, S6, and 23-1 in 293T cells co-transfected with either
RTP1, RTP1-A1 or control pCI. cDNAs encoding RTP1, RTP1-A1, and
control pCI were transfected into cells. Increased cell-surface
staining was seen in cells expressing RTP1-A1.
Example 24
RTP1 and RTP4 Chimeras
[0421] This example describes RTP1 and RTP4 chimeras generated with
chimeric PCR. In particular, complex chimera primers were designed
at the connection points of the RTP1 and RTP4 sequences. For each
chimera, two pairs of primers were first amplified (e.g., forward
primer and complex primer, complex primer and reverse primer).
Next, the two PCR products were used as templates in a subsequent
megaprimer PCR, with the original forward and reverse primers, to
obtain a desired chimera. FIG. 53 schematically shows the amino
acid segments of RTP1-A1-A (Chimera 1), RTP1-A1-D2 (Chimera 2),
RTP1-A1-D1 (Chimera 3), RTP4-A1-A (Chimera 4), RTP14-A1-D2 (Chimera
5), and RTP4-A1-D1 (Chimera 6).
[0422] FIG. 54 shows cell-surface expression of an OR in cells
expressing RTP1, RTP4, Chimera 1, Chimera 2, Chimera 3, Chimera 4,
Chimera 5, Chimera 6, and control pCI. cDNAs encoding RTP1, RTP4,
RTP1-A1, Chimera 1, Chimera 2, Chimera 3, Chimera 4, Chimera 5,
Chimera 6, and control pCI were transfected into 293T cells.
[0423] FIG. 55 shows OLFR62, OREG, S6, and 23-1 activity as
indicated by luciferase expression in 293T cells expressing RTP1,
RTP4, RTP1-A1, RTP1-D1, RTP1-D2, Chimera 1, Chimera 2, Chimera 3,
Chimera 4, Chimera 5, Chimera 6, and control pCI.
[0424] FIG. 56 shows detection of RTP1, RTP1-A, RTP1-B, RTP1-C,
RTP1-A1, RTP1-D, Chimera 4, Chimera 5, RTP1-D3, RTP1-D1, Chimera 6,
and RTP4 using anti-RTP1.
[0425] All publications and patents mentioned in the above
specification are herein incorporated by reference. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention that are obvious to those skilled in the relevant fields
are intended to be within the scope of the following claims.
Sequence CWU 1
1
511606DNAMus musculus 1atggtgtcgt ggatcatctc caggctggtg gtgcttatat
ttggcaccct ttatcctgca 60tattattcat acaaggctgt gaagtccaag gacattaaag
aatatgtcaa atggatgatg 120tattggatta tatttgccct cttcaccacg
gcagagacgt tcacagacat cttcctttgc 180tggtttccat tctattatga
actaaaaata gcgtttgtag cctggctgct gtctccctat 240acaaaaggat
ccagcctcct gtacaggaag tttgttcatc ccacattgtc ttcaaaagaa
300aaggaaatcg atgactgcct ggtccaagca aaagatcgaa gctatgacgc
ccttgtgcac 360tttgggaagc ggggcttgaa tgtggcagcc actgcagctg
tgatggctgc ctccaaggga 420cagggtgcct tgtcagagag actccggagc
ttcagcatgc aggacctcac caccatcagg 480ggtgatggtg ctcctgctcc
ctcgggccct cctccaccag ggactgggcg gtccagcggc 540aaacacagcc
agcccaagat gtccaggagt gcttctgaga gtgccggcag ctcgggcacc 600gcctag
6062765DNAMus musculus 2atggtgtcct ggatcatctc tcgcctggtg gtgctcatct
ttggcaccct gtacccagcc 60tattcttcct acaaggccgt gaagaccaaa aacgtgaagg
aatacgtaaa atggatgatg 120tattggatag tcttcgcctt cttcaccaca
gctgagacac ttacagatat aatactgtcc 180tggttcccct tctactttga
gctcaagatt gcctttgtga tatggctgtt gtccccttac 240accaagggct
ccagtgtcct ctaccgcaag ttcgtgcacc caacactgtc caacaaggaa
300aaggagatcg acgaatacat cacacaagct cgagacaaga gctatgagac
gatgatgagg 360gtgggcaaga ggggcctgaa cctggctgcc aatgctgcag
tcacagctgc tgccaagggc 420cagggggtgc tgtcggaaaa gctgcggagc
ttcagcatgc aggacctgac tctcattcga 480gatgaggatg cgttaccgct
gcaggggcca gatggccgcc tccaacccgg ccccgtgggt 540ctcctggaca
ctattgagga cttaggagat gagcctgccc taagtctaag gtctagcaca
600agccagccag atccccggac agagacctca gaagatgacc tgggagacaa
ggcacccaag 660aggaccaaac ctatcaaaaa agtacccaga gctgagccgc
cggcttccaa gacactgaag 720acccggccca agaagaagag ttctggaggg
ggcgactcag catga 7653765DNAMus musculus 3atggtgtcct ggatgatctc
ccgagccgtg gtgctggtgt ttggaatgct ctatccagcg 60tactattcct acaaagccgt
gaagacgaaa aacgtcaagg aatacgttcg ctggatgatg 120tattggatcg
tctttgccct ctacactgtc attgaaacgg tggccgatca gacacttgca
180tggtttcccc tgtactatga gctgaagatt gccttcgtca tttggctgct
gtcgccctac 240actagagggg cgagtttaat ctatagaaag ttccttcatc
ccctgctgtc atcaaaggaa 300agggaaattg atgattatat tgtccaagcc
aaagaaagag gctatgagac aatggtgaat 360tttggacggc aaggtttgaa
tttagcagct gcagccgccg tcactgcagc agtgaagagc 420caaggagcaa
taacggagcg tctgcgaagt ttcagcatgc atgatctgac agctatccaa
480ggggatgagc ccgtgggaca cagaccctac cagactttgc cagaagcaaa
gaggaaaggc 540aaacaagcca ccgagtcacc agcctatgga attccactga
aagatggaag tgagcagaca 600gacgaagaag cggaggggcc attctccgat
gacgagatgg tgactcacaa ggcgctgagg 660cgatcccaga gcatgaaatc
tgtcaagacc atcaaaggcc gcaaagaggt gcggtatggc 720tcactaaaat
ataaagtgaa gaagagaccg caagtgtatt tttag 7654774DNAMus musculus
4atggtgtcct ggatgatctg tcgcctggta gtgctcatat ttggcatgct gtatcctgcg
60tatgcttcct acaaggccgt gaagagcaag aacattcgag aatatgtacg gtggatgatg
120tattggattg tctttgcgat cttcatggca gcagaaacct tcacagacat
cttcatttcc 180tggttcccgt tttattacga gttcaagatg gcttttgtgc
tgtggctgct ctcaccttac 240accaaggggg ccagcctgct ttaccgaaag
tttgtccacc catccctatc ccgccatgag 300aaggagatcg acgcgtgtat
cgtgcaggca aaggagcgca gctatgaaac catgctcagt 360tttgggaagc
ggagcctcaa catcgctgcc tcagctgctg tgcaggctgc taccaagagt
420caaggcgctc tagctggaag gctgcggagt ttctctatgc aagacctgcg
ctctatccct 480gacacccctg tccccaccta ccaagatccc ctctacctgg
aagaccaggt accccgacgt 540agacccccta ttggataccg gccaggcggc
ctgcagggca gtgacacaga ggatgagtgt 600tggtcagaca atgagatcgt
cccccagcca cctgttcggc cccgagagaa gcctctaggc 660cgcagccaga
gccttcgggt ggtcaagagg aagccattga ctcgagaggg cacctcacgc
720tccctgaagg tccgaacccg gaaaaaggcc atgccctcag acatggacag ctag
7745570DNAMus musculus 5atgtccgcag ccatgaggga gaggttcgac cggttcctgc
acgagaagaa ctgcatgact 60gatctcctcg ccaagctcga ggccaagacc ggagtgaacc
ggagcttcat cgcgctcggt 120gtcatcggac tggtggcttt gtatctggtg
ttcggttatg gagcctctct cctctgcaac 180ctgataggtt tcggataccc
agcctacatc tcaatgaaag ccatcgagag tcccaacaaa 240gatgatgaca
cccagtggct gacgtactgg gtggtatatg gtgtgttcag cattgccgaa
300ttcttctccg atctcttcct gtcctggttc cccttctact acatgctgaa
gtgtggcttc 360ctgctgtggt gcatggcccc cagcccggct aatggggctg
agatgcgcta caggcgaatc 420atccgtccta tcttcctcaa gcacgagtcc
caggtagaca gtgtggtgaa ggacgtgaag 480gacaaagcca aagagactgc
agatgccatc agcaaagaag tcaagaaagc tacagtgaac 540ttgctgggcg
atgagaagaa gagcacctga 5706606DNAMus musculus 6atggacggtc tgcgccagcg
cttcgaacgt tttctggaac agaagaacgt ggccaccgaa 60gcgctcgggg cgctcgaagc
aaggaccggt gtagagaagc ggtatctcgc cgcgggagcc 120ctcgcccttc
taggcctgta tcttctgttc ggttacgggg cctctctact gtgcaatgtc
180atcggatttg tataccccgc atatgcttca gtcaaagcta tcgagagccc
aagcaaggaa 240gacgacactg tgtggctaac ctactgggtg gtgtacgccc
tgttcggtct ggtcgaattc 300ttcagcgatc tactcctgtt ctggttccct
ttctactacg cgggcaagtg cgccttcctg 360ttattttgca tgacgcccgg
accctggaac ggggcattac tactatacca tcgcgtcata 420agaccactct
ttctaaagca ccacatggct ctagacagcg ccgcgagcca gctaagcgga
480agagcattgg acctagcagc tgggataacc cgggacgtac ttcaggcctt
ggctcggggc 540cgggctctcg tcaccccagc atcaacatcg gaacccccag
ccgctctgga actggacccc 600aagtaa 6067606DNAHomo sapiens 7atggtgtcat
ggatcatctc caggctggtg gtgcttatat ttggcaccct ttaccctgcg 60tattattcct
acaaggctgt gaaatcaaag gacattaagg aatatgtcaa atggatgatg
120tactggatta tatttgcact tttcaccaca gcagagacat tcacagacat
cttcctttgt 180tggtttccat tctattatga actaaaaata gcatttgtag
cctggctgct gtctccctac 240acaaaaggct ccagcctcct gtacaggaag
tttgtacatc ccacactatc ttcaaaagaa 300aaggaaatcg atgattgtct
ggtccaagca aaagaccgaa gttacgatgc ccttgtgcac 360ttcgggaagc
ggggcttgaa cgtggccgcc acagcggctg tgatggctgc ttccaaggga
420cagggtgcct tatcggagag actgcggagc ttcagcatgc aggacctcac
caccatcagg 480ggagacggcg cccctgctcc ctcgggcccc ccaccaccgg
ggtctgggcg ggccagcggc 540aaacacggcc agcctaagat gtccaggagt
gcttctgaga gcgctagcag ctcaggcacc 600gcctag 6068759DNAHomo sapiens
8atggtgtcct ggatcatctc tcgcctggtg gtgctcatct ttggcaccct gtacccagcc
60tattcttcct acaaggccgt gaagacaaaa aacgtgaagg aatatgtgaa atggatgatg
120tactggatcg tctttgcctt cttcaccacg gccgagacgc tcacggatat
agtgctctcc 180tggttcccct tctactttga actgaagatc gccttcgtga
tatggctgct gtccccttac 240accaagggct ccagcgtgct ctaccgcaag
ttcgtgcacc caacgctgtc caacaaggag 300aaggagatcg acgagtacat
cacgcaggcc cgagacaaga gctatgagac catgatgagg 360gtgggcaaga
ggggcctgaa ccttgccgcc aatgctgcag tcacagctgc cgccaagggg
420gtgctgtcag agaagctccg cagcttcagc atgcaggacc tgaccctgat
ccgggacgag 480gacgcactgc ccctgcagag gcctgacggc cgcctccgac
ccagccctgg cagcctcctg 540gacaccatcg aggacttagg agatgaccct
gccctgagtc taaggtccag cacaaacccg 600gcagattccc ggacagaggc
ttctgaggat gacatgggag acaaagctcc caagagggcc 660aaacccatca
aaaaagcgcc caaagctgag ccactggctt ccaagacact gaagacccgg
720cccaagaaga agacctctgg cgggggcgac tcagcttga 7599441DNAHomo
sapiens 9atggtgtcct ggatgatctc cagagccgtg gtgctggtgt ttggaatgct
ttatcctgca 60tattattcat acaaagctgt gaaaacaaaa aacgtgaagg aatatgttcg
atggatgatg 120tactggattg tttttgctct ctatactgtg attgaaacag
tagccgatca aacagttgct 180tggtttcccc tgtactatga gctgaagatt
gcttttgtca tatggctgct ttctccctat 240accaaaggag caagtttaat
atatagaaaa ttccttcatc cacttctttc ttcaaaggaa 300agggagattg
atgattatat tgtacaagca aaggaacgag gctatgaaac catggtaaac
360tttggacggc aaggtttaaa ccttgcagct actgctgctg ttactgcagc
agtaaaggta 420attgttcatt taccttttta a 44110774DNAHomo sapiens
10atggtgtcct ggatgatctg tcgcctggtg gtgctggtgt ttgggatgct gtgtccagct
60tatgcttcct ataaggctgt gaagaccaag aacattcgtg aatatgtgcg gtggatgatg
120tactggattg tttttgcact cttcatggca gcagagatcg ttacagacat
ttttatctcc 180tggttccctt tctactatga gatcaagatg gccttcgtgc
tgtggctgct ctcaccctac 240accaagggcg ccagcctgct ttaccgcaag
tttgtccacc cgtccctgtc ccgccatgag 300aaggagatcg acgcgtacat
cgtgcaggcc aaggagcgca gctacgagac cgtgctcagc 360ttcgggaagc
ggggcctcaa cattgccgcc tccgctgctg tgcaggctgc caccaagagt
420cagggggcgc tggccggcag gctgcggagc ttctccatgc aggacctgcg
ctccatctct 480gacgcacctg cccctgccta ccatgacccc ctctacctgg
aggaccaggt gtcccaccgg 540aggccaccca ttgggtaccg ggccgggggc
ctgcaggaca gcgacaccga ggatgagtgt 600tggtcagata ctgaggcagt
cccccgggcg ccagcccggc cccgagagaa gcccctaatc 660cgcagccaga
gcctgcgtgt ggtcaagagg aagccaccgg tgcgggaggg cacctcgcgc
720tccctgaagg ttcggacgag gaaaaagact gtgccctcag acgtggacag ctag
77411570DNAHomo sapiens 11atgtctgcgg ccatgaggga gaggttcgac
cggttcctgc acgagaagaa ctgcatgact 60gaccttctgg ccaagctcga ggccaaaacc
ggcgtgaaca ggagcttcat cgctcttggt 120gtcatcggac tggtggcctt
gtacctggtg ttcggttatg gagcctctct cctctgcaac 180ctgataggat
ttggctaccc agcctacatc tcaattaaag ctatagagag tcccaacaaa
240gaagatgata cccagtggct gacctactgg gtagtgtatg gtgtgttcag
cattgctgaa 300ttcttctctg atatcttcct gtcatggttc cccttctact
acatgctgaa gtgtggcttc 360ctgttgtggt gcatggcccc gagcccttct
aatggggctg aactgctcta caagcgcatc 420atccgtcctt tcttcctgaa
gcacgagtcc cagatggaca gtgtggtcaa ggaccttaaa 480gacaaggcca
aagagactgc agatgccatc actaaagaag cgaagaaagc taccgtgaat
540ttactgggtg aagaaaagaa gagcacctaa 57012555DNAHomo sapiens
12atggacggcc tgaggcagcg cgtggagcac ttcctggagc aaaggaacct ggtcaccgaa
60gtgctggggg cgctggaggc caagaccggg gtggagaagc ggtatctggc tgcaggagcc
120gtcactctgc taagcctgta tctgctgttc ggctacggag cgtctctgct
gtgcaatctc 180atcggatttg tgtaccccgc atatgcctca atcaaagcta
tcgagagccc aagcaaggac 240gacgacactg tgtggctcac ctactgggtg
gtgtacgccc tgtttgggct ggccgagttc 300ttcagcgatc tactcctgtc
ctggttccct ttctactacg tgggcaagtg cgccttcctg 360ttgttctgca
tggctcccag gccctggaac ggggctctca tgctgtatca gcgcgtcgtg
420cgtccgctgt tcctaaggca ccacggggcc gtagacagaa tcatgaacga
cctcagcggg 480cgagccctgg acgcggcggc cggaataacc aggaacgtca
agccaagcca gaccccgcag 540ccgaaggaca agtga 55513792DNAMus musculus
13atgaggattt ttagaccgtg gagactgcgc tgccctgcct tacacttacc ctctttcccc
60acgttctcta taaagtgtag tttgcctcct cttcccactg acgaagacat gtgtaagagt
120gtgaccacag gtgagtggaa gaaggtcttc tacgagaaga tggaggaggt
gaagccagcg 180gacagctggg acttcatcat agaccccaac ctcaagcaca
atgtgttggc ccctggctgg 240aagcagtacc tggaacttca tgcctcaggc
aggttccact gttcctggtg ctggcacacc 300tggcagtcac cccatgtagt
catcctcttc cacatgtacc tggacaaggc tcagcgcgct 360ggttcggtgc
gcatgcgtgt gttcaagcag ctctgctacg agtgcggtac agcacggctg
420gatgagtcca gcatgctgga ggagaacatc gaaagcctgg tggacaacct
catcaccagt 480ttgcgagagc agtgctacgg ggagcgtggt ggccactacc
gcatccatgt ggccagccgg 540caggacaacc ggcgacaccg cggagagttc
tgcgaggcct gccaggaagg catcgtgcac 600tggaagccca gtgagaagct
gctggaggag gaggcgacca cctacacctt ctcccgtgct 660cccagcccca
ccaaaccgca ggctgaaaca ggctcaggct gcaacttctg ctccattccc
720tggtgcttat tttgggccac ggttttgatg ctcatcatct acctgcaatt
ctccttccgt 780acttctgtct aa 79214672DNAMus musculus 14atgtccacca
gcctgaccac ttgtgagtgg aagaaggtct tctacgagaa gatggaggtg 60gccaagccag
cggacagctg ggagctcatc atagacccca ccctcaagcc caatgagctg
120ggccctggct ggaagcagta cctggagcaa catgcctcag gcaggttcca
ctgttcctgg 180tgttggcaca catggcaatc tgccaatgtc gtcattctct
tccacatgca cctggaccgt 240gcccagcgtg ttggctcagt gcgcatgcgc
gtgttcaagc agctgtgcta tcagtgcggc 300acgtcgcggc tggacgagtc
cagcatgctg gaggagaata tcgagggcct ggtggacaac 360ctcatcacca
gtctgcgcga gcagtgttac gatgaggatg gtggccagta ccgcatccac
420gtagccagcc ggccagacag cggattgcac cgcagtgagt tctgcgaggc
ctgccaggaa 480ggcatcgtgc actggaagcc cagcgaaaag ctgctggagg
aggatgccgc ctataccgat 540gcctccaaga agaagggcca ggctggtttt
atctccagct tcttctcatt tcgttggtgc 600ctgttctggg gcaccctctg
cctggtcatt gtctacctgc agttcttccg aggccgctct 660ggcttccttt ag
672151425DNAMus musculus 15atgatggaag aagacatagg agacacagag
caatggcgac atgtgttcca ggagctaatg 60caagaggtga aaccctggca caaatggacc
ctcataccag acaagaacct tcttcccaac 120gttttgaagc caggatggac
gcaataccag caaaagacct ttgctaggtt ccactgtcct 180tcctgctctc
gaagttgggc atctggccga gttctgatag tcttccacat gcggtggtgt
240gagaagaagg ccaaggggtg ggtgaagatg agggtgtttg ctcagagatg
taatcagtgc 300cccgagcctc catttgcaac tccagaagtc acttgggaca
acatctcaag gatcttgaac 360aacctgctct tccaaattct gaagaagtgc
tataaagaag gatttaagca aatgggtgag 420attcctttgc tagggaacac
cagtctcgaa gggccacatg acagcagcaa ctgtgaggcc 480tgtctcctgg
gcttttgtgc tcagaatgac ttaggccaag cctcaaaacc accagcaccc
540ccattatctc ctacctcctc aaagtcagcc agggagccca aggtcactgc
cacctgtagc 600aacatttcct cctcacagcc ctcctctaaa gtacagatgc
cccaagcatc aaaagcgaac 660ccccaagcca gtaaccctac caaaaatgac
cccaaagtta gctgcacctc aaaaccacca 720gcacccccat tatctcctac
ctccttaaag tcagccaggg agcctaaggt cactgtcacc 780tgtagcaaca
tttcctcctc gcggtcctcc tctaaagtac agatgcccca agcatcaaaa
840gtgaaccccc aaaccagtaa tcctaccaaa aatgacccca agattagctg
tacctcaaaa 900ccatcaacta ctccaagact gacaatacaa cagctgtcag
tagtaagccc acctgcccct 960gcccctacat gtgtcattca aatgccttct
cccactccca tcgacggcag cagagcagca 1020gatgtagcaa aggagaacac
cagatccaag accccaaagg cattgctctc atccccttta 1080tacgtcccac
ccacttcctc ctatgtccca cccacttcct cctatgtccc acccacttcc
1140tcctatgtcc cgcccacttc ctcttatgtc ccacccactt cctcctcagt
tattgtgccc 1200atttcctcct cgtggagact accagaaaac actatttgcc
aagtagagag aaacagtcat 1260atccacccgc aaagccagtc ttcctgctgt
ggggcctgct gcgagtcctg gtgtgagatc 1320ttcaggtact catgctgtga
ggccgcctgt aattgcatgt cacagagtcc actgtgttgc 1380ttggcctttc
taatcttgtt cttattgctg tggtatttat tataa 142516750DNAMus musculus
16atgctgttcc ccgatgactt cagtacttgg gagcagacat ttcaagaact gatgcaggag
60gagaagcccg gggccaagtg gagcctgcat ttggataaga acattgtacc agatggtgca
120gccctgggat ggaggcagca ccagcagaca gtgcttggca ggttccagtg
ttccagatgc 180tgcagaagtt ggacctctgc tcaggtgatg atcttgtgcc
acatgtaccc ggacactttg 240aaatcgcagg gccaggcacg catgaggatc
tttggtcaga agtgccagaa gtgttttgga 300tgtcaatttg agactcccaa
gttctccaca gagatcatca aaagaattct gaataaccta 360gttaattata
ttctgcagag atactatgga cacaggaaga tagcattgac ctcgaatgca
420tctttgggtg agaaggtgac tttggatggg ccccacgaca cacgcaattg
tgaggcatgc 480agtctaaact ctcatggaag atgtgccctt gcacacaaag
taaaaccacc cagatctcca 540tctccattac caaatagttc ctccccatca
aagagctgcc ctcctccgcc tcagacccgg 600aatacggatt ttgggaataa
aactcttcag gattttggga atagaacttt tcagggatgc 660agagagcccc
cccaacgtga aatagagcca ccactatttc tgtttttgtc tattgctgca
720tttgcccttt ttagtctttt cactagataa 75017684DNAHomo sapiens
17atgtgtaaaa gcgtgaccac agatgagtgg aagaaagtct tctatgagaa gatggaggag
60gcaaagccgg ctgacagctg ggacctcatc atagacccca acctcaagca caatgtgctg
120agccctggtt ggaagcagta cctggaattg catgcttcag gcaggttcca
ctgctcctgg 180tgctggcaca cctggcagtc gccctacgtg gtcatcctct
tccacatgtt cctggaccgc 240gcccagcggg cgggctcggt gcgcatgcgc
gtcttcaagc agctgtgcta tgagtgcggc 300acggcgcggc tggacgagtc
cagcatgctg gaggagaaca tcgagggcct ggtggacaac 360ctcatcacca
gcctgcgcga gcagtgctac ggcgagcgtg gcggccagta ccgcatccac
420gtggccagcc gccaggacaa ccggcggcac cgcggagagt tctgcgaggc
ctgccaggag 480ggcatcgtgc actggaagcc cagcgagaag ctgctggagg
aggaggcgac cacctacacc 540ttctcccggg cgcccagccc caccaagtcg
caggaccaga cgggctcagg ctggaacttc 600tgctctatcc cctggtgctt
gttttgggcc acggtcctgc tgctgatcat ctacctgcag 660ttctctttcc
gtagctccgt ataa 68418678DNAHomo sapiens 18atgtgtacca gcttgaccac
ttgtgagtgg aagaaagtct tctatgagaa gatggaggtg 60gcaaagccag cggacagctg
ggagctcatc atagacccca acctcaagcc cagtgagctg 120gcccctggct
ggaagcagta cctggagcag cacgcctcag gcaggttcca ctgctcctgg
180tgctggcaca cctggcagtc tgcccatgtg gtcatcctct tccacatgtt
cctggaccgc 240gcccagcggg cgggctcggt gcgcatgcgc gtcttcaagc
agctgtgcta tgagtgcggc 300acggcgcggc tggacgagtc cagcatgctg
gaggagaaca tcgagggcct ggtggacaac 360ctcatcacca gcctgcgcga
gcagtgctac gaggaggatg gtggccagta ccgcatccac 420gtggccagcc
gcccggacag cgggccgcat cgtgcagagt tctgtgaggc ctgccaggag
480ggcatcgttc actggaagcc cagcgagaag ctgctggagg aggaggtgac
cacctacacc 540tctgaagcct ccaagccgag ggcccaggcg ggatccggct
acaacttctt gtctcttcgc 600tggtgcctct tctgggcctc tctctgcctg
ctcgttgttt acctgcagtt ctccttcctc 660agtcctgcct tcttttag
67819699DNAHomo sapiens 19atggctgggg acacagaagt gtggaagcaa
atgtttcagg agttaatgcg ggaggtgaag 60ccatggcaca ggtggaccct gagaccagac
aagggccttc ttcccaacgt cctgaagcca 120ggctggatgc aataccagca
gtggaccttc gccaggttcc agtgctcctc ctgctctcgt 180aactgggcct
ctgcccaagt tctggtcctt ttccacatga actggagtga ggagaagtcc
240aggggccagg tgaagatgag ggtgtttacc cagagatgta agaagtgccc
ccaacctctg 300tttgaggacc ctgagttcac acaagagaac atctcaagga
tcctgaaaaa cctggtgttc 360cgaattctga agaaatgcta tagaggaaga
tttcagttga tagaggaggt tcctatgatc 420aaggacatct ctcttgaagg
gccacacaat agtgacaact gtgaggcatg tctgcagggc 480ttctgtgctg
ggcccataca ggttacaagc ctccccccat ctcagacccc aagagtacac
540tccatttaca aggtggagga ggtagttaag ccctgggcct caggagagaa
tgtctattcc 600tacgcatgcc aaaaccacat ctgtaggaac ttaagcattt
tctgctgttg tgtcattctc 660attgttatcg tggtgattgt tgtaaaaact gctatatga
69920741DNAHomo sapiens 20atggttgtag atttctggac ttgggagcag
acatttcaag aactaatcca agaggcaaaa 60ccccgggcca catggacgct gaagttggat
ggcaaccttc agctagactg cctggctcaa 120gggtggaagc aataccaaca
gagagcattt ggctggttcc ggtgttcctc ctgccagcga 180agttgggctt
ccgccaagtt gcagattctg tgccacacgt actgggagca ctggacatcc
240cagggtcagg tgcgtatgag gctctttggc caaaggtgcc agaagtgctc
ctggtcccaa 300tatgagatgc ctgagttctc ctcggatagc accatgagga
ttctgagcaa cctggtgcag 360catatactga agaaatacta tggaaatggc
atgaggaagt ctccagaaat gccagtaatc 420ctggaagtgt ccctggaagg
atcccatgac acagccaatt gtgaggcatg cactttgggc 480atatgtggac
agggcttaaa aagctacatg acaaagccgt ccaaatccct actcccccac
540ctaaagactg ggaattcctc
acctggaatt ggtgctgtgt acctcgcaaa ccaagccaag 600aaccagtcag
atgaggcaaa agaggctaag gggagtgggt atgagaaatt agggcccagt
660cgagacccag atccactgaa catctgtgtc tttattttgc tgcttgtatt
tattgtagtc 720aaatgcttta catcagaatg a 74121201PRTMus musculus 21Met
Val Ser Trp Ile Ile Ser Arg Leu Val Val Leu Ile Phe Gly Thr1 5 10
15Leu Tyr Pro Ala Tyr Tyr Ser Tyr Lys Ala Val Lys Ser Lys Asp Ile
20 25 30Lys Glu Tyr Val Lys Trp Met Met Tyr Trp Ile Ile Phe Ala Leu
Phe 35 40 45Thr Thr Ala Glu Thr Phe Thr Asp Ile Phe Leu Cys Trp Phe
Pro Phe 50 55 60Tyr Tyr Glu Leu Lys Ile Ala Phe Val Ala Trp Leu Leu
Ser Pro Tyr65 70 75 80Thr Lys Gly Ser Ser Leu Leu Tyr Arg Lys Phe
Val His Pro Thr Leu 85 90 95Ser Ser Lys Glu Lys Glu Ile Asp Asp Cys
Leu Val Gln Ala Lys Asp 100 105 110Arg Ser Tyr Asp Ala Leu Val His
Phe Gly Lys Arg Gly Leu Asn Val 115 120 125Ala Ala Thr Ala Ala Val
Met Ala Ala Ser Lys Gly Gln Gly Ala Leu 130 135 140Ser Glu Arg Leu
Arg Ser Phe Ser Met Gln Asp Leu Thr Thr Ile Arg145 150 155 160Gly
Asp Gly Ala Pro Ala Pro Ser Gly Pro Pro Pro Pro Gly Thr Gly 165 170
175Arg Ser Ser Gly Lys His Ser Gln Pro Lys Met Ser Arg Ser Ala Ser
180 185 190Glu Ser Ala Gly Ser Ser Gly Thr Ala 195 20022254PRTMus
musculus 22Met Val Ser Trp Ile Ile Ser Arg Leu Val Val Leu Ile Phe
Gly Thr1 5 10 15Leu Tyr Pro Ala Tyr Ser Ser Tyr Lys Ala Val Lys Thr
Lys Asn Val 20 25 30Lys Glu Tyr Val Lys Trp Met Met Tyr Trp Ile Val
Phe Ala Phe Phe 35 40 45Thr Thr Ala Glu Thr Leu Thr Asp Ile Ile Leu
Ser Trp Phe Pro Phe 50 55 60Tyr Phe Glu Leu Lys Ile Ala Phe Val Ile
Trp Leu Leu Ser Pro Tyr65 70 75 80Thr Lys Gly Ser Ser Val Leu Tyr
Arg Lys Phe Val His Pro Thr Leu 85 90 95Ser Asn Lys Glu Lys Glu Ile
Asp Glu Tyr Ile Thr Gln Ala Arg Asp 100 105 110Lys Ser Tyr Glu Thr
Met Met Arg Val Gly Lys Arg Gly Leu Asn Leu 115 120 125Ala Ala Asn
Ala Ala Val Thr Ala Ala Ala Lys Gly Gln Gly Val Leu 130 135 140Ser
Glu Lys Leu Arg Ser Phe Ser Met Gln Asp Leu Thr Leu Ile Arg145 150
155 160Asp Glu Asp Ala Leu Pro Leu Gln Gly Pro Asp Gly Arg Leu Gln
Pro 165 170 175Gly Pro Val Gly Leu Leu Asp Thr Ile Glu Asp Leu Gly
Asp Glu Pro 180 185 190Ala Leu Ser Leu Arg Ser Ser Thr Ser Gln Pro
Asp Pro Arg Thr Glu 195 200 205Thr Ser Glu Asp Asp Leu Gly Asp Lys
Ala Pro Lys Arg Thr Lys Pro 210 215 220Ile Lys Lys Val Pro Arg Ala
Glu Pro Pro Ala Ser Lys Thr Leu Lys225 230 235 240Thr Arg Pro Lys
Lys Lys Ser Ser Gly Gly Gly Asp Ser Ala 245 25023254PRTMus musculus
23Met Val Ser Trp Met Ile Ser Arg Ala Val Val Leu Val Phe Gly Met1
5 10 15Leu Tyr Pro Ala Tyr Tyr Ser Tyr Lys Ala Val Lys Thr Lys Asn
Val 20 25 30Lys Glu Tyr Val Arg Trp Met Met Tyr Trp Ile Val Phe Ala
Leu Tyr 35 40 45Thr Val Ile Glu Thr Val Ala Asp Gln Thr Leu Ala Trp
Phe Pro Leu 50 55 60Tyr Tyr Glu Leu Lys Ile Ala Phe Val Ile Trp Leu
Leu Ser Pro Tyr65 70 75 80Thr Arg Gly Ala Ser Leu Ile Tyr Arg Lys
Phe Leu His Pro Leu Leu 85 90 95Ser Ser Lys Glu Arg Glu Ile Asp Asp
Tyr Ile Val Gln Ala Lys Glu 100 105 110Arg Gly Tyr Glu Thr Met Val
Asn Phe Gly Arg Gln Gly Leu Asn Leu 115 120 125Ala Ala Ala Ala Ala
Val Thr Ala Ala Val Lys Ser Gln Gly Ala Ile 130 135 140Thr Glu Arg
Leu Arg Ser Phe Ser Met His Asp Leu Thr Ala Ile Gln145 150 155
160Gly Asp Glu Pro Val Gly His Arg Pro Tyr Gln Thr Leu Pro Glu Ala
165 170 175Lys Arg Lys Gly Lys Gln Ala Thr Glu Ser Pro Ala Tyr Gly
Ile Pro 180 185 190Leu Lys Asp Gly Ser Glu Gln Thr Asp Glu Glu Ala
Glu Gly Pro Phe 195 200 205Ser Asp Asp Glu Met Val Thr His Lys Ala
Leu Arg Arg Ser Gln Ser 210 215 220Met Lys Ser Val Lys Thr Ile Lys
Gly Arg Lys Glu Val Arg Tyr Gly225 230 235 240Ser Leu Lys Tyr Lys
Val Lys Lys Arg Pro Gln Val Tyr Phe 245 25024257PRTMus musculus
24Met Val Ser Trp Met Ile Cys Arg Leu Val Val Leu Ile Phe Gly Met1
5 10 15Leu Tyr Pro Ala Tyr Ala Ser Tyr Lys Ala Val Lys Ser Lys Asn
Ile 20 25 30Arg Glu Tyr Val Arg Trp Met Met Tyr Trp Ile Val Phe Ala
Ile Phe 35 40 45Met Ala Ala Glu Thr Phe Thr Asp Ile Phe Ile Ser Trp
Phe Pro Phe 50 55 60Tyr Tyr Glu Phe Lys Met Ala Phe Val Leu Trp Leu
Leu Ser Pro Tyr65 70 75 80Thr Lys Gly Ala Ser Leu Leu Tyr Arg Lys
Phe Val His Pro Ser Leu 85 90 95Ser Arg His Glu Lys Glu Ile Asp Ala
Cys Ile Val Gln Ala Lys Glu 100 105 110Arg Ser Tyr Glu Thr Met Leu
Ser Phe Gly Lys Arg Ser Leu Asn Ile 115 120 125Ala Ala Ser Ala Ala
Val Gln Ala Ala Thr Lys Ser Gln Gly Ala Leu 130 135 140Ala Gly Arg
Leu Arg Ser Phe Ser Met Gln Asp Leu Arg Ser Ile Pro145 150 155
160Asp Thr Pro Val Pro Thr Tyr Gln Asp Pro Leu Tyr Leu Glu Asp Gln
165 170 175Val Pro Arg Arg Arg Pro Pro Ile Gly Tyr Arg Pro Gly Gly
Leu Gln 180 185 190Gly Ser Asp Thr Glu Asp Glu Cys Trp Ser Asp Asn
Glu Ile Val Pro 195 200 205Gln Pro Pro Val Arg Pro Arg Glu Lys Pro
Leu Gly Arg Ser Gln Ser 210 215 220Leu Arg Val Val Lys Arg Lys Pro
Leu Thr Arg Glu Gly Thr Ser Arg225 230 235 240Ser Leu Lys Val Arg
Thr Arg Lys Lys Ala Met Pro Ser Asp Met Asp 245 250
255Ser25185PRTMus musculus 25Met Arg Glu Arg Phe Asp Arg Phe Leu
His Glu Lys Asn Cys Met Thr1 5 10 15Asp Leu Leu Ala Lys Leu Glu Ala
Lys Thr Gly Val Asn Arg Ser Phe 20 25 30Ile Ala Leu Gly Val Ile Gly
Leu Val Ala Leu Tyr Leu Val Phe Gly 35 40 45Tyr Gly Ala Ser Leu Leu
Cys Asn Leu Ile Gly Phe Gly Tyr Pro Ala 50 55 60Tyr Ile Ser Met Lys
Ala Ile Glu Ser Pro Asn Lys Asp Asp Asp Thr65 70 75 80Gln Trp Leu
Thr Tyr Trp Val Val Tyr Gly Val Phe Ser Ile Ala Glu 85 90 95Phe Phe
Ser Asp Leu Phe Leu Ser Trp Leu Pro Phe Tyr Tyr Met Leu 100 105
110Lys Cys Gly Phe Leu Leu Trp Cys Met Ala Pro Ser Pro Ala Asn Gly
115 120 125Ala Glu Met Leu Tyr Arg Arg Ile Ile Arg Pro Ile Phe Leu
Arg His 130 135 140Glu Ser Gln Val Asp Ser Val Val Lys Asp Val Lys
Asp Lys Ala Lys145 150 155 160Glu Thr Ala Asp Ala Ile Ser Lys Glu
Val Lys Lys Ala Thr Val Asn 165 170 175Leu Leu Gly Asp Val Lys Lys
Ser Thr 180 18526201PRTMus musculus 26Met Asp Gly Leu Arg Gln Arg
Phe Glu Arg Phe Leu Glu Gln Lys Asn1 5 10 15Val Ala Thr Glu Ala Leu
Gly Ala Leu Glu Ala Arg Thr Gly Val Glu 20 25 30Lys Arg Tyr Leu Ala
Ala Gly Ala Leu Ala Leu Leu Gly Leu Tyr Leu 35 40 45Leu Phe Gly Tyr
Gly Ala Ser Leu Leu Cys Asn Val Ile Gly Phe Val 50 55 60Tyr Pro Ala
Tyr Ala Ser Val Lys Ala Ile Glu Ser Pro Ser Lys Glu65 70 75 80Asp
Asp Thr Val Trp Leu Thr Tyr Trp Val Val Tyr Ala Leu Phe Gly 85 90
95Leu Val Glu Phe Phe Ser Asp Leu Leu Leu Phe Trp Phe Pro Phe Tyr
100 105 110Tyr Ala Gly Lys Cys Ala Phe Leu Leu Phe Cys Met Thr Pro
Gly Pro 115 120 125Trp Asn Gly Ala Leu Leu Leu Tyr His Arg Val Ile
Arg Pro Leu Phe 130 135 140Leu Lys His His Met Ala Leu Asp Ser Ala
Ala Ser Gln Leu Ser Gly145 150 155 160Arg Ala Leu Asp Leu Ala Ala
Gly Ile Thr Arg Asp Val Leu Gln Ala 165 170 175Leu Ala Arg Gly Arg
Ala Leu Val Thr Pro Ala Ser Thr Ser Glu Pro 180 185 190Pro Ala Ala
Leu Glu Leu Asp Pro Lys 195 20027201PRTHomo sapiens 27Met Val Ser
Trp Ile Ile Ser Arg Leu Val Val Leu Ile Phe Gly Thr1 5 10 15Leu Tyr
Pro Ala Tyr Tyr Ser Tyr Lys Ala Val Lys Ser Lys Asp Ile 20 25 30Lys
Glu Tyr Val Lys Trp Met Met Tyr Trp Ile Ile Phe Ala Leu Phe 35 40
45Thr Thr Ala Glu Thr Phe Thr Asp Ile Phe Leu Cys Trp Phe Pro Phe
50 55 60Tyr Tyr Glu Leu Lys Ile Ala Phe Val Ala Trp Leu Leu Ser Pro
Tyr65 70 75 80Thr Lys Gly Ser Ser Leu Leu Tyr Arg Lys Phe Val His
Pro Thr Leu 85 90 95Ser Ser Lys Glu Lys Glu Ile Asp Asp Cys Leu Val
Gln Ala Lys Asp 100 105 110Arg Ser Tyr Asp Ala Leu Val His Phe Gly
Lys Arg Gly Leu Asn Val 115 120 125Ala Ala Thr Ala Ala Val Met Ala
Ala Ser Lys Gly Gln Gly Ala Leu 130 135 140Ser Glu Arg Leu Arg Ser
Phe Ser Met Gln Asp Leu Thr Thr Ile Arg145 150 155 160Gly Asp Gly
Ala Pro Ala Pro Ser Gly Pro Pro Pro Pro Gly Ser Gly 165 170 175Arg
Ala Ser Gly Lys His Gly Gln Pro Lys Met Ser Arg Ser Ala Ser 180 185
190Glu Ser Ala Ser Ser Ser Gly Thr Ala 195 20028252PRTHomo sapiens
28Met Val Ser Trp Ile Ile Ser Arg Leu Val Val Leu Ile Phe Gly Thr1
5 10 15Leu Tyr Pro Ala Tyr Ser Ser Tyr Lys Ala Val Lys Thr Lys Asn
Val 20 25 30Lys Glu Tyr Val Lys Trp Met Met Tyr Trp Ile Val Phe Ala
Phe Phe 35 40 45Thr Thr Ala Glu Thr Leu Thr Asp Ile Val Leu Ser Trp
Phe Pro Phe 50 55 60Tyr Phe Glu Leu Lys Ile Ala Phe Val Ile Trp Leu
Leu Ser Pro Tyr65 70 75 80Thr Lys Gly Ser Ser Val Leu Tyr Arg Lys
Phe Val His Pro Thr Leu 85 90 95Ser Asn Lys Glu Lys Glu Ile Asp Glu
Tyr Ile Thr Gln Ala Arg Asp 100 105 110Lys Ser Tyr Glu Thr Met Met
Arg Val Gly Lys Arg Gly Leu Asn Leu 115 120 125Ala Ala Asn Ala Ala
Val Thr Ala Ala Ala Lys Gly Val Leu Ser Glu 130 135 140Lys Leu Arg
Ser Phe Ser Met Gln Asp Leu Thr Leu Ile Arg Asp Glu145 150 155
160Asp Ala Leu Pro Leu Gln Arg Pro Asp Gly Arg Leu Arg Pro Ser Pro
165 170 175Gly Ser Leu Leu Asp Thr Ile Glu Asp Leu Gly Asp Asp Pro
Ala Leu 180 185 190Ser Leu Arg Ser Ser Thr Asn Pro Ala Asp Ser Arg
Thr Glu Ala Ser 195 200 205Glu Asp Asp Met Gly Asp Lys Ala Pro Lys
Arg Ala Lys Pro Ile Lys 210 215 220Lys Ala Pro Lys Ala Glu Pro Leu
Ala Ser Lys Thr Leu Lys Thr Arg225 230 235 240Pro Lys Lys Lys Thr
Ser Gly Gly Gly Asp Ser Ala 245 25029146PRTHomo sapiens 29Met Val
Ser Trp Met Ile Ser Arg Ala Val Val Leu Val Phe Gly Met1 5 10 15Leu
Tyr Pro Ala Tyr Tyr Ser Tyr Lys Ala Val Lys Thr Lys Asn Val 20 25
30Lys Glu Tyr Val Arg Trp Met Met Tyr Trp Ile Val Phe Ala Leu Tyr
35 40 45Thr Val Ile Glu Thr Val Ala Asp Gln Thr Val Ala Trp Phe Pro
Leu 50 55 60Tyr Tyr Glu Leu Lys Ile Ala Phe Val Ile Trp Leu Leu Ser
Pro Tyr65 70 75 80Thr Lys Gly Ala Ser Leu Ile Tyr Arg Lys Phe Leu
His Pro Leu Leu 85 90 95Ser Ser Lys Glu Arg Glu Ile Asp Asp Tyr Ile
Val Gln Ala Lys Glu 100 105 110Arg Gly Tyr Glu Thr Met Val Asn Phe
Gly Arg Gln Gly Leu Asn Leu 115 120 125Ala Ala Thr Ala Ala Val Thr
Ala Ala Val Lys Val Ile Val His Leu 130 135 140Pro
Phe14530257PRTHomo sapiens 30Met Val Ser Trp Met Ile Cys Arg Leu
Val Val Leu Val Phe Gly Met1 5 10 15Leu Cys Pro Ala Tyr Ala Ser Tyr
Lys Ala Val Lys Thr Lys Asn Ile 20 25 30Arg Glu Tyr Val Arg Trp Met
Met Tyr Trp Ile Val Phe Ala Leu Phe 35 40 45Met Ala Ala Glu Ile Val
Thr Asp Ile Phe Ile Ser Trp Phe Pro Phe 50 55 60Tyr Tyr Glu Ile Lys
Met Ala Phe Val Leu Trp Leu Leu Ser Pro Tyr65 70 75 80Thr Lys Gly
Ala Ser Leu Leu Tyr Arg Lys Phe Val His Pro Ser Leu 85 90 95Ser Arg
His Glu Lys Glu Ile Asp Ala Tyr Ile Val Gln Ala Lys Glu 100 105
110Arg Ser Tyr Glu Thr Val Leu Ser Phe Gly Lys Arg Gly Leu Asn Ile
115 120 125Ala Ala Ser Ala Ala Val Gln Ala Ala Thr Lys Ser Gln Gly
Ala Leu 130 135 140Ala Gly Arg Leu Arg Ser Phe Ser Met Gln Asp Leu
Arg Ser Ile Ser145 150 155 160Asp Ala Pro Ala Pro Ala Tyr His Asp
Pro Leu Tyr Leu Glu Asp Gln 165 170 175Val Ser His Arg Arg Pro Pro
Ile Gly Tyr Arg Ala Gly Gly Leu Gln 180 185 190Asp Ser Asp Thr Glu
Asp Glu Cys Trp Ser Asp Thr Glu Ala Val Pro 195 200 205Arg Ala Pro
Ala Arg Pro Arg Glu Lys Pro Leu Ile Arg Ser Gln Ser 210 215 220Leu
Arg Val Val Lys Arg Lys Pro Pro Val Arg Glu Gly Thr Ser Arg225 230
235 240Ser Leu Lys Val Arg Thr Arg Lys Lys Thr Val Pro Ser Asp Val
Asp 245 250 255Ser31189PRTHomo sapiens 31Met Ser Ala Ala Met Arg
Glu Arg Phe Asp Arg Phe Leu His Glu Lys1 5 10 15Asn Cys Met Thr Asp
Leu Leu Ala Lys Leu Glu Ala Lys Thr Gly Val 20 25 30Asn Arg Ser Phe
Ile Ala Leu Gly Val Ile Gly Leu Val Ala Leu Tyr 35 40 45Leu Val Phe
Gly Tyr Gly Ala Ser Leu Leu Cys Asn Leu Ile Gly Phe 50 55 60Gly Tyr
Pro Ala Tyr Ile Ser Ile Lys Ala Ile Glu Ser Pro Asn Lys65 70 75
80Glu Asp Asp Thr Gln Trp Leu Thr Tyr Trp Val Val Tyr Gly Val Phe
85 90 95Ser Ile Ala Glu Phe Phe Ser Asp Ile Phe Leu Ser Trp Phe Pro
Phe 100 105 110Tyr Tyr Met Leu Lys Cys Gly Phe Leu Leu Trp Cys Met
Ala Pro Ser 115 120 125Pro Ser Asn Gly Ala Glu Leu Leu Tyr Lys Arg
Ile Ile Arg Pro Phe 130 135 140Phe Leu Lys His Glu Ser Gln Met Asp
Ser Val Val Lys Asp Leu Lys145 150 155 160Asp Lys Ala Lys Glu Thr
Ala Asp Ala Ile Thr Lys Glu Ala Lys Lys 165 170 175Ala Thr Val Asn
Leu Leu Gly Glu Glu Lys Lys Ser Thr 180 18532184PRTHomo sapiens
32Met Asp Gly Leu Arg Gln Arg Val Glu His Phe Leu Glu Gln Arg Asn1
5 10
15Leu Val Thr Glu Val Leu Gly Ala Leu Glu Ala Lys Thr Gly Val Glu
20 25 30Lys Arg Tyr Leu Ala Ala Gly Ala Val Thr Leu Leu Ser Leu Tyr
Leu 35 40 45Leu Phe Gly Tyr Gly Ala Ser Leu Leu Cys Asn Leu Ile Gly
Phe Val 50 55 60Tyr Pro Ala Tyr Ala Ser Ile Lys Ala Ile Glu Ser Pro
Ser Lys Asp65 70 75 80Asp Asp Thr Val Trp Leu Thr Tyr Trp Val Val
Tyr Ala Leu Phe Gly 85 90 95Leu Ala Glu Phe Phe Ser Asp Leu Leu Leu
Ser Trp Phe Pro Phe Tyr 100 105 110Tyr Val Gly Lys Cys Ala Phe Leu
Leu Phe Cys Met Ala Pro Arg Pro 115 120 125Trp Asn Gly Ala Leu Met
Leu Tyr Gln Arg Val Val Arg Pro Leu Phe 130 135 140Leu Arg His His
Gly Ala Val Asp Arg Ile Met Asn Asp Leu Ser Gly145 150 155 160Arg
Ala Leu Asp Ala Ala Ala Gly Ile Thr Arg Asn Val Lys Pro Ser 165 170
175Gln Thr Pro Gln Pro Lys Asp Lys 18033263PRTMus musculus 33Met
Arg Ile Phe Arg Pro Trp Arg Leu Arg Cys Pro Ala Leu His Leu1 5 10
15Pro Ser Phe Pro Thr Phe Ser Ile Lys Cys Ser Leu Pro Pro Leu Pro
20 25 30Thr Asp Glu Asp Met Cys Lys Ser Val Thr Thr Gly Glu Trp Lys
Lys 35 40 45Val Phe Tyr Glu Lys Met Glu Glu Val Lys Pro Ala Asp Ser
Trp Asp 50 55 60Phe Ile Ile Asp Pro Asn Leu Lys His Asn Val Leu Ala
Pro Gly Trp65 70 75 80Lys Gln Tyr Leu Glu Leu His Ala Ser Gly Arg
Phe His Cys Ser Trp 85 90 95Cys Trp His Thr Trp Gln Ser Pro His Val
Val Ile Leu Phe His Met 100 105 110Tyr Leu Asp Lys Ala Gln Arg Ala
Gly Ser Val Arg Met Arg Val Phe 115 120 125Lys Gln Leu Cys Tyr Glu
Cys Gly Thr Ala Arg Leu Asp Glu Ser Ser 130 135 140Met Leu Glu Glu
Asn Ile Glu Ser Leu Val Asp Asn Leu Ile Thr Ser145 150 155 160Leu
Arg Glu Gln Cys Tyr Gly Glu Arg Gly Gly His Tyr Arg Ile His 165 170
175Val Ala Ser Arg Gln Asp Asn Arg Arg His Arg Gly Glu Phe Cys Glu
180 185 190Ala Cys Gln Glu Gly Ile Val His Trp Lys Pro Ser Glu Lys
Leu Leu 195 200 205Glu Glu Glu Ala Thr Thr Tyr Thr Phe Ser Arg Ala
Pro Ser Pro Thr 210 215 220Lys Pro Gln Ala Glu Thr Gly Ser Gly Cys
Asn Phe Cys Ser Ile Pro225 230 235 240Trp Cys Leu Phe Trp Ala Thr
Val Leu Met Leu Ile Ile Tyr Leu Gln 245 250 255Phe Ser Phe Arg Thr
Ser Val 26034223PRTMus musculus 34Met Ser Thr Ser Leu Thr Thr Cys
Glu Trp Lys Lys Val Phe Tyr Glu1 5 10 15Lys Met Glu Val Ala Lys Pro
Ala Asp Ser Trp Glu Leu Ile Ile Asp 20 25 30Pro Thr Leu Lys Pro Asn
Glu Leu Gly Pro Gly Trp Lys Gln Tyr Leu 35 40 45Glu Gln His Ala Ser
Gly Arg Phe His Cys Ser Trp Cys Trp His Thr 50 55 60Trp Gln Ser Ala
Asn Val Val Ile Leu Phe His Met His Leu Asp Arg65 70 75 80Ala Gln
Arg Val Gly Ser Val Arg Met Arg Val Phe Lys Gln Leu Cys 85 90 95Tyr
Gln Cys Gly Thr Ser Arg Leu Asp Glu Ser Ser Met Leu Glu Glu 100 105
110Asn Ile Glu Gly Leu Val Asp Asn Leu Ile Thr Ser Leu Arg Glu Gln
115 120 125Cys Tyr Asp Glu Asp Gly Gly Gln Tyr Arg Ile His Val Ala
Ser Arg 130 135 140Pro Asp Ser Gly Leu His Arg Ser Glu Phe Cys Glu
Ala Cys Gln Glu145 150 155 160Gly Ile Val His Trp Lys Pro Ser Glu
Lys Leu Leu Glu Glu Asp Ala 165 170 175Ala Tyr Thr Asp Ala Ser Lys
Lys Lys Gly Gln Ala Gly Phe Ile Ser 180 185 190Ser Phe Phe Ser Phe
Arg Trp Cys Leu Phe Trp Gly Thr Leu Cys Leu 195 200 205Val Ile Val
Tyr Leu Gln Phe Phe Arg Gly Arg Ser Gly Phe Leu 210 215
22035281PRTMus musculus 35Met Met Glu Glu Asp Ile Gly Asp Thr Glu
Gln Trp Arg His Val Phe1 5 10 15Gln Glu Leu Met Gln Glu Val Lys Pro
Trp His Lys Trp Thr Leu Ile 20 25 30Pro Asp Lys Asn Leu Leu Pro Asn
Val Leu Lys Pro Gly Trp Thr Gln 35 40 45Tyr Gln Gln Lys Thr Phe Ala
Arg Phe His Cys Pro Ser Cys Ser Arg 50 55 60Ser Trp Ala Ser Gly Arg
Val Leu Ile Val Phe His Met Arg Trp Cys65 70 75 80Glu Lys Lys Ala
Lys Gly Trp Val Lys Met Arg Val Phe Ala Gln Arg 85 90 95Cys Asn Gln
Cys Pro Glu Pro Pro Phe Ala Thr Pro Glu Val Thr Trp 100 105 110Asp
Asn Ile Ser Arg Ile Leu Asn Asn Leu Leu Phe Gln Ile Leu Lys 115 120
125Lys Cys Tyr Lys Glu Gly Phe Lys Gln Met Gly Glu Ile Pro Leu Leu
130 135 140Gly Asn Thr Ser Leu Glu Gly Pro His Asp Ser Ser Asn Cys
Glu Ala145 150 155 160Cys Leu Leu Gly Phe Cys Ala Gln Asn Asp Leu
Gly Gln Ala Ser Lys 165 170 175Pro Pro Ala Pro Pro Leu Ser Pro Thr
Ser Ser Lys Ser Ala Arg Glu 180 185 190Pro Lys Val Thr Val Thr Cys
Ser Asn Ile Ser Ser Ser Arg Pro Ser 195 200 205Ser Lys Val Gln Met
Pro Gln Ala Ser Lys Val Asn Pro Gln Ala Ser 210 215 220Asn Pro Thr
Lys Asn Asp Pro Lys Val Ser Cys Thr Ser Lys Pro Pro225 230 235
240Ala Pro Pro Leu Ser Pro Thr Ser Leu Lys Ser Ala Arg Glu Pro Lys
245 250 255Val Thr Val Thr Cys Ser Asn Ile Ser Ser Ser Arg Pro Ser
Ser Lys 260 265 270Val Gln Met Pro Gln Ala Ser Lys Val 275
28036248PRTMus musculus 36Met Leu Phe Pro Asp Asp Phe Ser Thr Trp
Glu Gln Thr Phe Gln Glu1 5 10 15Leu Met Gln Glu Glu Lys Pro Gly Ala
Lys Trp Ser Leu His Leu Asp 20 25 30Lys Asn Ile Val Pro Asp Gly Ala
Ala Leu Gly Trp Arg Gln His Gln 35 40 45Gln Thr Val Gly Arg Phe Gln
Cys Ser Arg Cys Cys Arg Ser Trp Thr 50 55 60Ser Ala Gln Val Met Ile
Leu Cys His Met Tyr Pro Asp Thr Leu Lys65 70 75 80Ser Gln Gly Gln
Ala Arg Met Arg Ile Phe Gly Gln Lys Cys Gln Lys 85 90 95Cys Phe Gly
Cys Gln Phe Glu Thr Pro Lys Phe Ser Thr Glu Ile Ile 100 105 110Lys
Arg Ile Leu Asn Asn Leu Val Asn Tyr Ile Leu Gln Arg Tyr Tyr 115 120
125Gly His Arg Lys Ile Ala Leu Thr Ser Asn Ala Ser Leu Gly Glu Lys
130 135 140Val Thr Leu Asp Gly Pro His Asp Thr Arg Asn Cys Glu Ala
Cys Ser145 150 155 160Leu Asn Ser His Gly Arg Cys Ala Leu Ala His
Lys Val Lys Pro Pro 165 170 175Arg Ser Pro Ser Pro Leu Pro Asn Ser
Ser Ser Pro Ser Lys Ser Cys 180 185 190Pro Pro Pro Pro Gln Thr Arg
Asn Thr Asp Phe Gly Asn Lys Thr Leu 195 200 205Gln Asp Phe Gly Asn
Arg Thr Phe Gln Gly Cys Arg Glu Pro Pro Gln 210 215 220Arg Glu Ile
Glu Pro Pro Leu Phe Leu Phe Leu Ser Ile Ala Ala Phe225 230 235
240Ala Leu Phe Ser Leu Phe Thr Arg 24537263PRTHomo sapiens 37Met
Arg Ile Phe Arg Pro Trp Arg Leu Arg Cys Pro Ala Leu His Leu1 5 10
15Pro Ser Leu Ser Val Phe Ser Leu Arg Trp Lys Leu Pro Ser Leu Thr
20 25 30Thr Asp Glu Thr Met Cys Lys Ser Val Thr Thr Asp Glu Trp Lys
Lys 35 40 45Val Phe Tyr Glu Lys Met Glu Glu Ala Lys Pro Ala Asp Ser
Trp Asp 50 55 60Leu Ile Ile Asp Pro Asn Leu Lys His Asn Val Leu Ser
Pro Gly Trp65 70 75 80Lys Gln Tyr Leu Glu Leu His Ala Ser Gly Arg
Phe His Cys Ser Trp 85 90 95Cys Trp His Thr Trp Gln Ser Pro Tyr Val
Val Ile Leu Phe His Met 100 105 110Phe Leu Asp Arg Ala Gln Arg Ala
Gly Ser Val Arg Met Arg Val Phe 115 120 125Lys Gln Leu Cys Tyr Glu
Cys Gly Thr Ala Arg Leu Asp Glu Ser Ser 130 135 140Met Leu Glu Glu
Asn Ile Glu Gly Leu Val Asp Asn Leu Ile Thr Ser145 150 155 160Leu
Arg Glu Gln Cys Tyr Gly Glu Arg Gly Gly Gln Tyr Arg Ile His 165 170
175Val Ala Ser Arg Gln Asp Asn Arg Arg His Arg Gly Glu Phe Cys Glu
180 185 190Ala Cys Gln Glu Gly Ile Val His Trp Lys Pro Ser Glu Lys
Leu Leu 195 200 205Glu Glu Glu Ala Thr Thr Tyr Thr Phe Ser Arg Ala
Pro Ser Pro Thr 210 215 220Lys Ser Gln Asp Gln Thr Gly Ser Gly Trp
Asn Phe Cys Ser Ile Pro225 230 235 240Trp Cys Leu Phe Trp Ala Thr
Val Leu Leu Leu Ile Ile Tyr Leu Gln 245 250 255Phe Ser Phe Arg Ser
Ser Val 26038225PRTHomo sapiens 38Met Cys Thr Ser Leu Thr Thr Cys
Glu Trp Lys Lys Val Phe Tyr Glu1 5 10 15Lys Met Glu Val Ala Lys Pro
Ala Asp Ser Trp Glu Leu Ile Ile Asp 20 25 30Pro Asn Leu Lys Pro Ser
Glu Leu Ala Pro Gly Trp Lys Gln Tyr Leu 35 40 45Glu Gln His Ala Ser
Gly Arg Phe His Cys Ser Trp Cys Trp His Thr 50 55 60Trp Gln Ser Ala
His Val Val Ile Leu Phe His Met Phe Leu Asp Arg65 70 75 80Ala Gln
Arg Ala Gly Ser Val Arg Met Arg Val Phe Lys Gln Leu Cys 85 90 95Tyr
Glu Cys Gly Thr Ala Arg Leu Asp Glu Ser Ser Met Leu Glu Glu 100 105
110Asn Ile Glu Gly Leu Val Asp Asn Leu Ile Thr Ser Leu Arg Glu Gln
115 120 125Cys Tyr Glu Glu Asp Gly Gly Gln Tyr Arg Ile His Val Ala
Ser Arg 130 135 140Pro Asp Ser Gly Pro His Arg Ala Glu Phe Cys Glu
Ala Cys Gln Glu145 150 155 160Gly Ile Val His Trp Lys Pro Ser Glu
Lys Leu Leu Glu Glu Glu Val 165 170 175Thr Thr Tyr Thr Ser Glu Ala
Ser Lys Pro Arg Ala Gln Ala Gly Ser 180 185 190Gly Tyr Asn Phe Leu
Ser Leu Arg Trp Cys Leu Phe Trp Ala Ser Leu 195 200 205Cys Leu Leu
Val Val Tyr Leu Gln Phe Ser Phe Leu Ser Pro Ala Phe 210 215
220Phe22539232PRTHomo sapiens 39Met Ala Gly Asp Thr Glu Val Trp Lys
Gln Met Phe Gln Glu Leu Met1 5 10 15Arg Glu Val Lys Pro Trp His Arg
Trp Thr Leu Arg Pro Asp Lys Gly 20 25 30Leu Leu Pro Asn Val Leu Lys
Pro Gly Trp Met Gln Tyr Gln Gln Trp 35 40 45Thr Phe Ala Arg Phe Gln
Cys Ser Ser Cys Ser Arg Asn Trp Ala Ser 50 55 60Ala Gln Val Leu Val
Leu Phe His Met Asn Trp Ser Glu Glu Lys Ser65 70 75 80Arg Gly Gln
Val Lys Met Arg Val Phe Thr Gln Arg Cys Lys Lys Cys 85 90 95Pro Gln
Pro Leu Phe Glu Asp Pro Glu Phe Thr Gln Glu Asn Ile Ser 100 105
110Arg Ile Leu Lys Asn Leu Val Phe Arg Ile Leu Lys Lys Cys Tyr Arg
115 120 125Gly Arg Phe Gln Leu Ile Glu Glu Val Pro Met Ile Lys Asp
Ile Ser 130 135 140Leu Glu Gly Pro His Asn Ser Asp Asn Cys Glu Ala
Cys Leu Gln Gly145 150 155 160Phe Cys Ala Gly Pro Ile Gln Val Thr
Ser Leu Pro Pro Ser Gln Thr 165 170 175Pro Arg Val His Ser Ile Tyr
Lys Val Glu Glu Val Val Lys Pro Trp 180 185 190Ala Ser Gly Glu Asn
Val Tyr Ser Tyr Ala Cys Gln Asn His Ile Cys 195 200 205Arg Asn Leu
Ser Ile Phe Cys Cys Cys Val Ile Leu Ile Val Ile Val 210 215 220Val
Ile Val Val Lys Thr Ala Ile225 23040246PRTHomo sapiens 40Met Val
Val Asp Phe Trp Thr Trp Glu Gln Thr Phe Gln Glu Leu Ile1 5 10 15Gln
Glu Ala Lys Pro Arg Ala Thr Trp Thr Leu Lys Leu Asp Gly Asn 20 25
30Leu Gln Leu Asp Cys Leu Ala Gln Gly Trp Lys Gln Tyr Gln Gln Arg
35 40 45Ala Phe Gly Trp Phe Arg Cys Ser Ser Cys Gln Arg Ser Trp Ala
Ser 50 55 60Ala Lys Leu Gln Ile Leu Cys His Thr Tyr Trp Glu His Trp
Thr Ser65 70 75 80Gln Gly Gln Val Arg Met Arg Leu Phe Gly Gln Arg
Cys Gln Lys Cys 85 90 95Ser Trp Ser Gln Tyr Glu Met Pro Glu Phe Ser
Ser Asp Ser Thr Met 100 105 110Arg Ile Leu Ser Asn Leu Val Gln His
Ile Leu Lys Lys Tyr Tyr Gly 115 120 125Asn Gly Met Arg Lys Ser Pro
Glu Met Pro Val Ile Leu Glu Val Ser 130 135 140Leu Glu Gly Ser His
Asp Thr Ala Asn Cys Glu Ala Cys Thr Leu Gly145 150 155 160Ile Cys
Gly Gln Gly Leu Lys Ser Tyr Met Thr Lys Pro Ser Lys Ser 165 170
175Leu Leu Pro His Leu Lys Thr Gly Asn Ser Ser Pro Gly Ile Gly Ala
180 185 190Val Tyr Leu Ala Asn Gln Ala Lys Asn Gln Ser Asp Glu Ala
Lys Glu 195 200 205Ala Lys Gly Ser Gly Tyr Glu Lys Leu Gly Pro Ser
Arg Asp Pro Asp 210 215 220Pro Leu Asn Ile Cys Val Phe Ile Leu Leu
Leu Val Phe Ile Val Val225 230 235 240Lys Cys Phe Thr Ser Glu
24541210PRTMus musculus 41Met Glu Glu Val Lys Pro Ala Asp Ser Trp
Asp Phe Ile Ile Asp Pro1 5 10 15Asn Leu Lys His Asn Val Leu Ala Pro
Gly Trp Lys Gln Tyr Leu Glu 20 25 30Leu His Ala Ser Gly Arg Phe His
Cys Ser Trp Cys Trp His Thr Trp 35 40 45Gln Ser Pro His Val Val Ile
Leu Phe His Met Tyr Leu Asp Lys Ala 50 55 60Gln Arg Ala Gly Ser Val
Arg Met Arg Val Phe Lys Gln Leu Cys Tyr65 70 75 80Glu Cys Gly Thr
Ala Arg Leu Asp Glu Ser Ser Met Leu Glu Glu Asn 85 90 95Ile Glu Ser
Leu Val Asp Asn Leu Ile Thr Ser Leu Arg Glu Gln Cys 100 105 110Tyr
Gly Glu Arg Gly Gly His Tyr Arg Ile His Val Ala Ser Arg Gln 115 120
125Asp Asn Arg Arg His Arg Gly Glu Phe Cys Glu Ala Cys Gln Glu Gly
130 135 140Ile Val His Trp Lys Pro Ser Glu Lys Leu Leu Glu Glu Glu
Ala Thr145 150 155 160Thr Tyr Thr Phe Ser Arg Ala Pro Ser Pro Thr
Lys Pro Gln Ala Glu 165 170 175Thr Gly Ser Gly Cys Asn Phe Cys Ser
Ile Pro Trp Cys Leu Phe Trp 180 185 190Ala Thr Val Leu Met Leu Ile
Ile Tyr Leu Gln Phe Ser Phe Arg Thr 195 200 205Ser Val
21042152PRTMus musculus 42Met Tyr Leu Asp Lys Ala Gln Arg Ala Gly
Ser Val Arg Met Arg Val1 5 10 15Phe Lys Gln Leu Cys Tyr Glu Cys Gly
Thr Ala Arg Leu Asp Glu Ser 20 25 30Ser Met Leu Glu Glu Asn Ile Glu
Ser Leu Val Asp Asn Leu Ile Thr 35 40 45Ser Leu Arg Glu Gln Cys Tyr
Gly Glu Arg Gly Gly His Tyr Arg Ile 50 55 60His Val Ala Ser Arg Gln
Asp Asn Arg Arg His Arg Gly Glu Phe Cys65 70 75 80Glu Ala Cys Gln
Glu Gly Ile Val His Trp Lys Pro Ser Glu Lys Leu 85
90 95Leu Glu Glu Glu Ala Thr Thr Tyr Thr Phe Ser Arg Ala Pro Ser
Pro 100 105 110Thr Lys Pro Gln Ala Glu Thr Gly Ser Gly Cys Asn Phe
Cys Ser Ile 115 120 125Pro Trp Cys Leu Phe Trp Ala Thr Val Leu Met
Leu Ile Ile Tyr Leu 130 135 140Gln Phe Ser Phe Arg Thr Ser Val145
15043119PRTMus musculus 43Met Leu Glu Glu Asn Ile Glu Ser Leu Val
Asp Asn Leu Ile Thr Ser1 5 10 15Leu Arg Glu Gln Cys Tyr Gly Glu Arg
Gly Gly His Tyr Arg Ile His 20 25 30Val Ala Ser Arg Gln Asp Asn Arg
Arg His Arg Gly Glu Phe Cys Glu 35 40 45Ala Cys Gln Glu Gly Ile Val
His Trp Lys Pro Ser Glu Lys Leu Leu 50 55 60Glu Glu Glu Ala Thr Thr
Tyr Thr Phe Ser Arg Ala Pro Ser Pro Thr65 70 75 80Lys Pro Gln Ala
Glu Thr Gly Ser Gly Cys Asn Phe Cys Ser Ile Pro 85 90 95Trp Cys Leu
Phe Trp Ala Thr Val Leu Met Leu Ile Ile Tyr Leu Gln 100 105 110Phe
Ser Phe Arg Thr Ser Val 11544234PRTMus musculus 44Met Arg Ile Phe
Arg Pro Trp Arg Leu Arg Cys Pro Ala Leu His Leu1 5 10 15Pro Ser Phe
Pro Thr Phe Ser Ile Lys Cys Ser Leu Pro Pro Leu Pro 20 25 30Thr Asp
Glu Asp Met Cys Lys Ser Val Thr Thr Gly Glu Trp Lys Lys 35 40 45Val
Phe Tyr Glu Lys Met Glu Glu Val Lys Pro Ala Asp Ser Trp Asp 50 55
60Phe Ile Ile Asp Pro Asn Leu Lys His Asn Val Leu Ala Pro Gly Trp65
70 75 80Lys Gln Tyr Leu Glu Leu His Ala Ser Gly Arg Phe His Cys Ser
Trp 85 90 95Cys Trp His Thr Trp Gln Ser Pro His Val Val Ile Leu Phe
His Met 100 105 110Tyr Leu Asp Lys Ala Gln Arg Ala Gly Ser Val Arg
Met Arg Val Phe 115 120 125Lys Gln Leu Cys Tyr Glu Cys Gly Thr Ala
Arg Leu Asp Glu Ser Ser 130 135 140Met Leu Glu Glu Asn Ile Glu Ser
Leu Val Asp Asn Leu Ile Thr Ser145 150 155 160Leu Arg Glu Gln Cys
Tyr Gly Glu Arg Gly Gly His Tyr Arg Ile His 165 170 175Val Ala Ser
Arg Gln Asp Asn Arg Arg His Arg Gly Glu Phe Cys Glu 180 185 190Ala
Cys Gln Glu Gly Ile Val His Trp Lys Pro Ser Glu Lys Leu Leu 195 200
205Glu Glu Glu Ala Thr Thr Tyr Thr Phe Ser Arg Ala Pro Ser Pro Thr
210 215 220Lys Pro Gln Ala Glu Thr Gly Ser Gly Cys225
23045172PRTMus musculus 45Met Arg Ile Phe Arg Pro Trp Arg Leu Arg
Cys Pro Ala Leu His Leu1 5 10 15Pro Ser Phe Pro Thr Phe Ser Ile Lys
Cys Ser Leu Pro Pro Leu Pro 20 25 30Thr Asp Glu Asp Met Cys Lys Ser
Val Thr Thr Gly Glu Trp Lys Lys 35 40 45Val Phe Tyr Glu Lys Met Glu
Glu Val Lys Pro Ala Asp Ser Trp Asp 50 55 60Phe Ile Ile Asp Pro Asn
Leu Lys His Asn Val Leu Ala Pro Gly Trp65 70 75 80Lys Gln Tyr Leu
Glu Leu His Ala Ser Gly Arg Phe His Cys Ser Trp 85 90 95Cys Trp His
Thr Trp Gln Ser Pro His Val Val Ile Leu Phe His Met 100 105 110Tyr
Leu Asp Lys Ala Gln Arg Ala Gly Ser Val Arg Met Arg Val Phe 115 120
125Lys Gln Leu Cys Tyr Glu Cys Gly Thr Ala Arg Leu Asp Glu Ser Ser
130 135 140Met Leu Glu Glu Asn Ile Glu Ser Leu Val Asp Asn Leu Ile
Thr Ser145 150 155 160Leu Arg Glu Gln Cys Tyr Gly Glu Arg Gly Gly
His 165 17046227PRTMus musculus 46Met Cys Lys Ser Val Thr Thr Gly
Glu Trp Lys Lys Val Phe Tyr Glu1 5 10 15Lys Met Glu Glu Val Lys Pro
Ala Asp Ser Trp Asp Phe Ile Ile Asp 20 25 30Pro Asn Leu Lys His Asn
Val Leu Ala Pro Gly Trp Lys Gln Tyr Leu 35 40 45Glu Leu His Ala Ser
Gly Arg Phe His Cys Ser Trp Cys Trp His Thr 50 55 60Trp Gln Ser Pro
His Val Val Ile Leu Phe His Met Tyr Leu Asp Lys65 70 75 80Ala Gln
Arg Ala Gly Ser Val Arg Met Arg Val Phe Lys Gln Leu Cys 85 90 95Tyr
Glu Cys Gly Thr Ala Arg Leu Asp Glu Ser Ser Met Leu Glu Glu 100 105
110Asn Ile Glu Ser Leu Val Asp Asn Leu Ile Thr Ser Leu Arg Glu Gln
115 120 125Cys Tyr Gly Glu Arg Gly Gly His Tyr Arg Ile His Val Ala
Ser Arg 130 135 140Gln Asp Asn Arg Arg His Arg Gly Glu Phe Cys Glu
Ala Cys Gln Glu145 150 155 160Gly Ile Val His Trp Lys Pro Ser Glu
Lys Leu Leu Glu Glu Glu Ala 165 170 175Thr Thr Tyr Thr Phe Ser Arg
Ala Pro Ser Pro Thr Lys Pro Gln Ala 180 185 190Glu Thr Gly Ser Gly
Cys Asn Phe Cys Ser Ile Pro Trp Cys Leu Phe 195 200 205Trp Ala Thr
Val Leu Met Leu Ile Ile Tyr Leu Gln Phe Ser Phe Arg 210 215 220Thr
Ser Val22547227PRTHomo sapiens 47Met Cys Lys Ser Val Thr Thr Asp
Glu Trp Lys Lys Val Phe Tyr Glu1 5 10 15Lys Met Glu Glu Ala Lys Pro
Ala Asp Ser Trp Asp Leu Ile Ile Asp 20 25 30Pro Asn Leu Lys His Asn
Val Leu Ser Pro Gly Trp Lys Gln Tyr Leu 35 40 45Glu Leu His Ala Ser
Gly Arg Phe His Cys Ser Trp Cys Trp His Thr 50 55 60Trp Gln Ser Pro
Tyr Val Val Ile Leu Phe His Met Phe Leu Asp Arg65 70 75 80Ala Gln
Arg Ala Gly Ser Val Arg Met Arg Val Phe Lys Gln Leu Cys 85 90 95Tyr
Glu Cys Gly Thr Ala Arg Leu Asp Glu Ser Ser Met Leu Glu Glu 100 105
110Asn Ile Glu Gly Leu Val Asp Asn Leu Ile Thr Ser Leu Arg Glu Gln
115 120 125Cys Tyr Gly Glu Arg Gly Gly Gln Tyr Arg Ile His Val Ala
Ser Arg 130 135 140Gln Asp Asn Arg Arg His Arg Gly Glu Phe Cys Glu
Ala Cys Gln Glu145 150 155 160Gly Ile Val His Trp Lys Pro Ser Glu
Lys Leu Leu Glu Glu Glu Ala 165 170 175Thr Thr Tyr Thr Phe Ser Arg
Ala Pro Ser Pro Thr Lys Ser Gln Asp 180 185 190Gln Thr Gly Ser Gly
Trp Asn Phe Cys Ser Ile Pro Trp Cys Leu Phe 195 200 205Trp Ala Thr
Val Leu Leu Leu Ile Ile Tyr Leu Gln Phe Ser Phe Arg 210 215 220Ser
Ser Val22548212PRTMus musculus 48Met Arg Ile Phe Arg Pro Trp Arg
Leu Arg Cys Pro Ala Leu His Leu1 5 10 15Pro Ser Phe Pro Thr Phe Ser
Ile Lys Cys Ser Leu Pro Pro Leu Pro 20 25 30Thr Asp Glu Asp Met Cys
Lys Ser Val Thr Thr Gly Glu Trp Lys Lys 35 40 45Val Phe Tyr Glu Lys
Met Glu Glu Val Lys Pro Ala Asp Ser Trp Asp 50 55 60Phe Ile Ile Asp
Pro Asn Leu Lys His Asn Val Leu Ala Pro Gly Trp65 70 75 80Lys Gln
Tyr Leu Glu Leu His Ala Ser Gly Arg Phe His Cys Ser Trp 85 90 95Cys
Trp His Thr Trp Gln Ser Pro His Val Val Ile Leu Phe His Met 100 105
110Tyr Leu Asp Lys Ala Gln Arg Ala Gly Ser Val Arg Met Arg Val Phe
115 120 125Lys Gln Leu Cys Tyr Glu Cys Gly Thr Ala Arg Leu Asp Glu
Ser Ser 130 135 140Met Leu Glu Glu Asn Ile Glu Ser Leu Val Asp Asn
Leu Ile Thr Ser145 150 155 160Leu Arg Glu Gln Cys Tyr Gly Glu Arg
Gly Gly His Tyr Arg Ile His 165 170 175Val Ala Ser Arg Gln Asp Asn
Arg Arg His Arg Gly Glu Phe Cys Glu 180 185 190Ala Cys Gln Glu Gly
Ile Val His Trp Lys Pro Ser Glu Lys Leu Leu 195 200 205Glu Glu Glu
Ala 21049193PRTMus musculus 49Met Arg Ile Phe Arg Pro Trp Arg Leu
Arg Cys Pro Ala Leu His Leu1 5 10 15Pro Ser Phe Pro Thr Phe Ser Ile
Lys Cys Ser Leu Pro Pro Leu Pro 20 25 30Thr Asp Glu Asp Met Cys Lys
Ser Val Thr Thr Gly Glu Trp Lys Lys 35 40 45Val Phe Tyr Glu Lys Met
Glu Glu Val Lys Pro Ala Asp Ser Trp Asp 50 55 60Phe Ile Ile Asp Pro
Asn Leu Lys His Asn Val Leu Ala Pro Gly Trp65 70 75 80Lys Gln Tyr
Leu Glu Leu His Ala Ser Gly Arg Phe His Cys Ser Trp 85 90 95Cys Trp
His Thr Trp Gln Ser Pro His Val Val Ile Leu Phe His Met 100 105
110Tyr Leu Asp Lys Ala Gln Arg Ala Gly Ser Val Arg Met Arg Val Phe
115 120 125Lys Gln Leu Cys Tyr Glu Cys Gly Thr Ala Arg Leu Asp Glu
Ser Ser 130 135 140Met Leu Glu Glu Asn Ile Glu Ser Leu Val Asp Asn
Leu Ile Thr Ser145 150 155 160Leu Arg Glu Gln Cys Tyr Gly Glu Arg
Gly Gly His Tyr Arg Ile His 165 170 175Val Ala Ser Arg Gln Asp Asn
Arg Arg His Arg Gly Glu Phe Cys Glu 180 185 190Ala50253PRTMus
musculus 50Met Arg Ile Phe Arg Pro Trp Arg Leu Arg Cys Pro Ala Leu
His Leu1 5 10 15Pro Ser Phe Pro Thr Phe Ser Ile Lys Cys Ser Leu Pro
Pro Leu Pro 20 25 30Thr Asp Glu Asp Met Cys Lys Ser Val Thr Thr Gly
Glu Trp Lys Lys 35 40 45Val Phe Tyr Glu Lys Met Glu Glu Val Lys Pro
Ala Asp Ser Trp Asp 50 55 60Phe Ile Ile Asp Pro Asn Leu Lys His Asn
Val Leu Ala Pro Gly Trp65 70 75 80Lys Gln Tyr Leu Glu Leu His Ala
Ser Gly Arg Phe His Cys Ser Trp 85 90 95Cys Trp His Thr Trp Gln Ser
Pro His Val Val Ile Leu Phe His Met 100 105 110Tyr Leu Asp Lys Ala
Gln Arg Ala Gly Ser Val Arg Met Arg Val Phe 115 120 125Lys Gln Leu
Cys Tyr Glu Cys Gly Thr Ala Arg Leu Asp Glu Ser Ser 130 135 140Met
Leu Glu Glu Asn Ile Glu Ser Leu Val Asp Asn Leu Ile Thr Ser145 150
155 160Leu Arg Glu Gln Cys Tyr Gly Glu Arg Gly Gly His Tyr Arg Ile
His 165 170 175Val Ala Ser Arg Gln Asp Asn Arg Arg His Arg Gly Glu
Phe Cys Glu 180 185 190Ala Cys Gln Glu Gly Ile Val His Trp Lys Pro
Ser Glu Lys Leu Leu 195 200 205Glu Glu Glu Ala Thr Thr Tyr Thr Phe
Ser Arg Ala Pro Ser Pro Thr 210 215 220Lys Pro Gln Ala Glu Thr Gly
Ser Gly Cys Asn Phe Cys Ser Ile Pro225 230 235 240Trp Cys Leu Phe
Trp Ala Thr Val Leu Met Leu Ile Ile 245
2505148DNAArtificialSynthetic 51tatagaattc gcggccgctc gcgatttttt
tttttttttt tttttttt 48
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References