U.S. patent application number 10/957135 was filed with the patent office on 2005-02-17 for pain signaling molecules.
Invention is credited to Anderson, David J., Dong, Xinzhong, Han, Sang-kyou, Simon, Melvin I., Zylka, Mark.
Application Number | 20050037468 10/957135 |
Document ID | / |
Family ID | 29999194 |
Filed Date | 2005-02-17 |
United States Patent
Application |
20050037468 |
Kind Code |
A1 |
Anderson, David J. ; et
al. |
February 17, 2005 |
Pain signaling molecules
Abstract
The invention relates generally to novel genes expressed in
normal but not Neurogenin-1-deficient animals. The invention
relates specifically to a novel family of G protein-coupled
receptors and a novel family of two-transmembrane segment proteins
that are expressed in dorsal root ganglia, and a method of
screening for genes specifically expressed in nociceptive sensory
neurons.
Inventors: |
Anderson, David J.;
(Altadena, CA) ; Dong, Xinzhong; (Pasadena,
CA) ; Zylka, Mark; (Pasadena, CA) ; Han,
Sang-kyou; (Arcadia, CA) ; Simon, Melvin I.;
(San Marino, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
29999194 |
Appl. No.: |
10/957135 |
Filed: |
September 30, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10957135 |
Sep 30, 2004 |
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10183116 |
Jun 26, 2002 |
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10183116 |
Jun 26, 2002 |
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09849869 |
May 4, 2001 |
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09849869 |
May 4, 2001 |
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09704707 |
Nov 3, 2000 |
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60222344 |
Aug 1, 2000 |
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60202027 |
May 4, 2000 |
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Current U.S.
Class: |
435/69.1 ;
435/252.33; 435/254.2; 435/320.1; 435/325; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 14/70571 20130101;
G01N 33/5041 20130101; G01N 2500/02 20130101; A61K 38/00 20130101;
G01N 33/5082 20130101; C07K 14/705 20130101; G01N 33/5058 20130101;
G01N 33/54366 20130101; G01N 2333/726 20130101 |
Class at
Publication: |
435/069.1 ;
435/320.1; 435/325; 530/350; 536/023.5; 435/252.33; 435/254.2 |
International
Class: |
C07K 014/705 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule encoding a G protein coupled
receptor and comprising a sequence having at least 70% sequence
identity to (1) the nucleic acid molecule of SEQ ID NO: 34, or (2)
the complement of the nucleic acid molecule of (1).
2. An isolated nucleic acid molecule that hybridizes under
stringent conditions to the isolated nucleic acid molecule of claim
1.
3. The isolated nucleic acid molecule of claim 1 operably linked to
an expression control element.
4. The isolated nucleic acid molecule of claim 3 operably linked to
a promoter element.
5. A vector comprising the isolated nucleic acid molecule of claim
4.
6. A host cell comprising the vector of claim 5.
7. The host cell of claim 6 wherein said host cell is a prokaryotic
cell.
8. The host cell of claim 7 wherein said host cell is an E.
coli.
9. The host cell of claim 6 wherein said host cell is a eukaryotic
cell.
10. The host cell of claim 9 wherein said host cell is a hamster
embryonic kidney (HEK) cell.
11. The host cell of claim 9 wherein said host cell is a yeast
cell.
12. A method for producing an MrgD polypeptide comprising culturing
the host cell of claim 6 under conditions in which the protein
encoded by said nucleic acid is expressed.
13. An isolated nucleic acid molecule according to claim 1 having
at least 80% sequence identity to (1) the nucleic acid molecule of
SEQ ID NO: 34, or (2) the complement of the nucleic acid molecule
of (1).
14. An isolated nucleic acid molecule according to claim 1 having
at least 90% sequence identity to (1) the nucleic acid molecule of
SEQ ID NO: 34, or (2) the complement of the nucleic acid molecule
of (1).
15. An isolated nucleic acid molecule that encodes a G protein
coupled receptor and comprises a sequence having at least 95%
sequence identity to (1) the nucleic acid molecule of SEQ ID NO:
34, or (2) the complement of the nucleic acid molecule of (1).
16. An isolated nucleic acid molecule comprising a sequence having
at least 99% sequence identity to (1) the nucleic acid molecule of
SEQ ID NO: 34, or (2) the complement of the nucleic acid molecule
of (1).
17. The isolated nucleic acid molecule of claim 16 operably linked
to an expression control element.
18. A vector comprising the isolated nucleic acid molecule of claim
16.
19. A host cell comprising the vector of claim 18.
20. The host cell of claim 18, wherein the host cell is selected
from the group consisting of eurkaryotic cells and prokaryotic
cells.
Description
[0001] This application claims priority under 35 U.S.C. .sctn.120
as a continuation of U.S. patent application Ser. No. 10/183,116,
filed Jun. 26, 2002, which in turn is a continuation-in-part of
U.S. patent application Ser. No. 09/849,869, filed May 4, 2001,
which is a continuation of U.S. patent application Ser. No.
09/704,707, filed Nov. 3, 2000 and under 35 U.S.C. .sctn.119(e) to
U.S. Provisional Applications 60/202,027, filed May 4, 2000,
60/222,344, filed Aug. 1, 2000, and 60/285,493, filed Apr. 19,
2001, which are herein incorporated by reference in their
entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates generally to novel genes expressed in
normal but not Neurogenin-1-deficient animals. The invention
relates specifically to a novel family of G protein-coupled
receptors and a novel family of two-transmembrane segment proteins
that are expressed in dorsal root ganglia, and a method of
screening for genes specifically expressed in nociceptive sensory
neurons.
[0004] 2. Description of the Related Art
[0005] The treatment of acute and chronic intractable pain is a
major target of drug development in the pharmaceutical industry.
Pain sensation is mediated by primary sensory neurons in the dorsal
root ganglia (DRG), which project peripherally to the skin and
centrally to the spinal cord. These neurons express signaling
molecules, such as receptors, ion channels and neuropeptides, which
are involved in pain sensation. One example is the so-called
Vanilloid Receptor-1 (VR-1), which is activated by capsaicin (chili
pepper) as well as by heat and acid. Such pain signaling molecules
may also influence pain sensation indirectly by acting as positive
or negative modulators of the sensory pathway. Searching for drugs
that interact with such signaling molecules, for example as
receptor agonists or antagonists, is an important approach to the
discovery of new therapeutics for the treatment of pain. New
candidate signaling molecules expressed by pain-sensing
("nociceptive") sensory neurons are therefore highly desirable
targets for new drug screening and drug discovery efforts. The
present inventors have previously identified a novel family of
basic helix-loop-helix (bHLH) transcription factors, called the
Neurogenins (Ngns), which are essential for the development of
sensory neurons in the DRG. Different Ngns are required for the
development of different subsets of sensory neurons. In particular,
Ngn1 is necessary for the development of most if not all
nociceptive sensory neurons. In Ngn1.sup.-/- mutant mice, although
DRG are still present, they are reduced in size and the majority of
nociceptive neurons, identified by expression of markers such as
trkA and VR-1, are missing (Ma et al. Genes&Develop, 13:
1717-1728, (1999)). These results suggested that the isolation of
genes expressed in wild-type (normal) but not Ngn1.sup.-/- DRG
might lead to the identification of novel drug target molecules
expressed in differentiating or mature nociceptive sensory
neurons.
[0006] While pain is usually a natural consequence of tissue
injury, as the healing process commences the pain and tenderness
associated with the injury resolve. However, some individuals
experience pain without an obvious injury or suffer protracted pain
after an initial insult. In addition, chronic or intractable pain
may occur in association with certain illnesses, such as, for
example, bone degenerative diseases, terminal cancer, AIDS, and
Reflex sympathetic dystrophy (RSD). Such patients may be unable to
receive relief with currently-available pain-relieving
(anti-nociceptive) drugs, such as opioid compounds, e.g. morphine,
due to problems such as dependence and tolerance. Therefore, there
is a great need for novel therapeutic agents for the treatment of
pain, in particular chronic pain.
SUMMARY OF THE INVENTION
[0007] The present inventors have carried out a screen for genes
expressed in wild-type but not Ngn1.sup.-/- DRG using positive
selection-based differential hybridization. This screen has
identified both known signaling molecules involved in nociceptive
neuron function, such as VR-1, and novel signaling molecules that
are highly specifically expressed in nociceptive sensory neurons.
The present invention therefore includes the discovery of new genes
that are expressed in normal mice but not in Ngn1 null mutant mice.
One family of novel genes isolated from the screen encodes a
receptor protein with 7 transmembrane segments, mrg, a
characteristic of G protein-coupled receptors. Subsequent staining
experiments (see FIG. 2, 2A-D) confirmed that mrg genes were
expressed specifically in subsets of nociceptive neurons in DRG.
Another novel gene family isolated in this screen, drg-12, encodes
a protein with two transmembrane segments.
[0008] In particular, the invention includes isolated nucleic acid
molecules selected from the group consisting of an isolated nucleic
acid molecule comprising a sequence having at least 70% sequence
identity to a nucleic acid molecule that encodes the MrgD
polypeptide with the amino acid sequence of SEQ ID NO: 49, isolated
nucleic acid molecules that hybridize to the complement of a
nucleic acid molecule comprising a sequence having at least 70%
sequence identity to a nucleic acid molecule that encodes the MrgD
polypeptide with the amino acid sequence of SEQ ID NO: 49, an
isolated nucleic acid molecule that that hybridizes under stringent
conditions to a nucleic acid molecule that encodes the MrgD
polypeptide of SEQ ID NO:49 and an isolated nucleic acid molecule
that hybridizes to the complement of a nucleic acid molecule that
encodes the MrgD polypeptide of SEQ ID NO: 49.
[0009] The present invention also includes the nucleic acid
molecules described above operably linked to one or more expression
control elements, including vectors comprising the isolated nucleic
acid molecules. The invention further includes host cells
transformed to contain the nucleic acid molecules of the invention
and methods for producing a protein comprising the step of
culturing a host cell transformed with a nucleic acid molecule of
the invention under conditions in which the protein is expressed.
The host cells may be prokaryotic cells, such as E. coli or
eukaryotic cells, such as hamster embyonic kidney (HEK) cells or
yeast cells.
[0010] The invention further provides an isolated Mrg polypeptide
selected from the group consisting of isolated polypeptides encoded
by the isolated nucleic acids described above and the human MrgD
polypeptide of SEQ ID NO: 35.
[0011] The MrgD polypeptide may be fused to a heterologous amino
acid sequence, such as an eptiope tag sequence or an immunoglobulin
constant domain sequence.
[0012] The invention further provides an isolated antibody that
specifically binds to a polypeptide of the invention, including
monoclonal and polyclonal antibodies, antibody fragments and
humanized antibodies.
[0013] In another aspect, the invention provides a composition of
matter comprising an MrgD polypeptide or an anti-MrgD antibody in
admixture with a pharmaceutically acceptable carrier. An article of
manufacture is also provided comprising the composition of matter,
a container, and instructions for using the composition of matter
to alter sensory perception in a mammal.
[0014] In a further aspect, the invention provides a method of
identifying a compound that can be used to alter pain perception in
a mammal. Test compounds are contacted with at least a portion of
an MrgD polypeptide of the invention. The MrgD polypeptide or the
test compound may be attached to a solid support, such as a
microtiter plate. In addition, either the test compound or the MrgD
polypeptide is preferably labelled.
[0015] Test compounds that are able to form complexes with the MrgD
polypeptide are identified. The effects of these compounds is
measured in an animal model of pain and compounds that alter pain
perception in the animal model are identified as useful in altering
pain perception in a mammal. The compound may enhance or decrease
the perception of pain.
[0016] In one embodiment the MrgD polypeptide is a native human
MrgD polypeptide, preferably the MrgD polypeptide of SEQ ID NO:
35.
[0017] In another embodiment the MrgD polypeptide may be apresent
in a cell membrane or a fraction of a cell membrane prepared from
cells expressing the MrgD polypeptide. In a further embodiment, the
MrgD polypeptide is present in an immunoadhesin.
[0018] The test compounds are preferably selected from the group
consisting of peptides, peptide mimetics, antibodies, small organic
molecules and small inorganic molecules. In a preferred embodiment
the test compounds are peptides. The peptides may be anchored to a
solid support by specific binding to an immobilized antibody. In
addition, the test compounds may be contained in a cellular
extract, particularly a cellular extract prepared from cells known
to express an MrgD polypeptide, such as dorsal root ganglion
cells.
[0019] In another aspect,the invention provides a method of
indentifying a compound that binds an MrgD polypeptide by
contacting an MrgD polypeptide or fragment with a test compound and
a ligand, such as an RFamide peptide, under condtions where binding
can occur. Preferably the MrgD polypeptide is contacted with the
RFamide peptide prior to being contacted with the test compound.
The ability of the test compound to interfere with biding of the
RFamide peptide tothe MrgD polypeptide is determined.
[0020] In one embodiment the MrgD polypeptide is a native human
MrgD polypeptide, preferably the MrgD polypeptide of SEQ ID NO:
35.
[0021] The invention also provides a method of identifying an MrgD
agonist that can be used to alter sensory perception in a mammal.
For example, the agonist may be used to enhance or decreast the
preception of pain. An MrgD polypeptide is expressed in a host cell
capable of producing a second messenger response. In one embodiment
the host cell is a eukaryotic cell, preferably a hamster embryonic
kidney (HEK) cell, more preferably an HEK cell that expresses Ga
15.
[0022] The host cell is contacted with one or more test compounds
and the second messenger response is measured. Compounds that
increase the measured second messenger response are identified as
agonists that can be used to alter sensory perception in a mammal.
In one embodiment measuring the second messenger response comprises
measuring a change in intercellular calcium concentration. This may
be done, for example, by using a FURA-2 indicator dye. In another
embodiment a second messenger response is measured by measuring the
flow of current across the cell membrane.
[0023] In another aspect, the invention provides a method for
identifying an MrgD polypeptide antagonist that is useful in
treating impaired sensory perception in a mammal. In particular the
method is useful for identifying antagonists that can alter the
perception of pain.
[0024] In one embodiment, an MrgD polypeptide, preferably the MrgD
polypeptide of SEQ ID NO: 35, is expressed in a host cell capable
of producing a second messenger response. The host cell is then
contacted with an RFamide peptide and one or more test compounds.
The second messenger response is measured, such as by the methods
described above, and compounds that alter the second messenger
response to the RFamide peptide are identified as agonists that are
useful in treating impaired sensory perception, such as pain.
[0025] In yet another aspect, the present invention provides a
method of identifying an anti-MrgD agonist antibody that can be
used to alter the perception of pain in a mammal. In one embodiment
the anti-MrgD agonist antibody the method is used to identify
anti-MrgD agonist antibodies that can be used to treat pain in a
mammal that is suffering from pain.
[0026] In a preferred embodiment, candidate antibodies are prepared
that specifically bind to an MrgD polypeptide, more preferably to
the MrgD polypeptide of SEQ ID NO: 35. An MrgD polypeptide,
preferably the MrgD polypeptide of SEQ ID NO: 35, is expressed in a
host cell known to be capable of producing a second messenger
response. The host cell is then contacted with a candidate antibody
and the second messenger response is measured. Antibodies that
increase the second messenger response are identfied as agonist
antibodies that can be used to treat pain in a mammal.
[0027] The invention also provides a method of treating pain in a
mammal, comprising administering to the mammal an MrgD agonist. In
one embodiment, the agonist is an agonist of the human MrgD
polypeptide of SEQ ID NO: 35.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 shows the alignment of a homologous region of the
amino acid sequences of SEQ ID NO: 2, 4, 6, 8, 10 and 12, and also
of two human members of the mrg family (SEQ ID NOS: 16 and 18).
[0029] FIG. 1A indicates that mrgs define a Novel G protein-couple
receptor Gene Family. Amino acid sequences of eight mouse
full-length mrg genes were aligned using ClustalW. Identical
residues in >50% of the predicted proteins are darkly shaded;
conservative substitutions are highlighted in light gray. The
approximate locations of predicted transmembrane domain 1-7 are
indicated on top of the sequences as TM1-TM7. The predicted
extracellular and cytoplasmic domains are indicated as E1-E7 and
C1-C7 respectively.
[0030] The microscopy images of in situ hybridization in FIG. 2
show the localization of antisense staining against a nucleotide of
SEQ ID NO: 2 ("mrg3") and of SEQ ID NO: 4 ("mrg4") in transverse
sections of dorsal root ganglia (DRG) from newborn wild type (WT)
and Neurogenin1 null mutant (Ngn1.sup.-/-) mice. White dashed lines
outline the DRG and black dashed lines outline the spinal cord.
Note that in the Ngn1.sup.-/- mutant, the size of the DRG is
severely reduced due to the loss of nociceptive sensory neurons,
identified using three other independent markers (trkA; VR-1 and
SNS-TTXi (Ma et al., (1999)). mrg3 is expressed in a subset of DRG
in WT mice (A) but is absent in the Ngn1.sup.-/- DRG (B). mrg4 is
expressed in a smaller subset of DRG than that of mrg3 (C). It is
also absent in the Ngn1.sup.-/- DRG (D). The loss of mrg-expressing
neurons in the Ngn1.sup.-/- DRG indicates that these neurons are
likely to be nociceptive.
[0031] FIG. 2A shows expression of mrgs in subsets of dorsal root
ganglia (DRG) neurons. Frozen transverse sections of DRG from
wild-type (a-i) and ngn1.sup.-/- (j) mutant new born mice were
annealed with antisense digoxigenin RNA probes, and hybridization
was visualized with an alkailine phosphatase-conjugated antibody.
Positive signals are shown as dark purple stainings. TrkA is
expressed in a large portion of wild-type DRG neurons (a) but
absent in ngn1.sup.-/- (data not shown). Each of the eight mrg
genes (b-i) is expressed in a small subset of neurons in wild-type
DRG in completely absent in Ngn1.sup.-/- DRG (j and data not
shown). Black dash line outlines the ngn1.sup.-/- mutant DRG.
[0032] FIG. 2B shows that mrgs are expressed by TrKa.sup.+
nociceptive neurons. Double labeling technique was used to
colocalize TrkA (b,e) and mrgs (a,d) in DRG neurons. During the
double labeling experiments frozen sections of wild-type DRG were
undergone in situ hybridizations with either mrg3 (a-c) or mrg5
(d-f) fluorescein-labeled antisense RNA probes followed by
anti-TrkA antibody immunostaining. The same two frames (a and b, d
and e) were digitally superimposed to reveal the extent of
colocalization (c, f). The white arrowheads indicate examples of
double positive cells.
[0033] FIG. 2C shows that mrgs and VR1 define two different
populations of nociceptive neurons in DRG. The combination of in
situ hybridizations with either mrg3 or mrg5 fluorescein-labeled
antisense RNA probes and anti-VR1 antibody immunostaining
demonstrated that neither mrg3 (a-c) nor mrg5 (d-f) were expressed
by VR1-positive neurons. In the merged images (c,f), there are no
colocalizations of VR1 with either mrg3 or mrg5. The white
arrowheads are pointed to mrgs-expressing but VR1-negative
nociceptive neurons.
[0034] FIG. 2D shows that mrgs are expressed by IB4.sup.+
nociceptive neurons. Double labeling technique was used to
colocalize IB4 (b,e) and mrgs (a,d) in DRG neurons. The expressions
of mrg3 and mrg5 were visualized by in situ hybridization as
described before. The same DRG sections were subsequently undergone
through FITC-conjugated lectin IB4 binding. In the merged images
(c,f), there are extensive overlappings between mrgs and IB4
stainings (yellow neurons indicated by arrowheads).
[0035] FIG. 3 compares the hydrophobicity plots predicting the
transmembrane regions of the amino acid sequence of (A) mrg3 (SEQ
ID NO: 2); (B) human1 gene (SEQ ID NO: 15); and (C) human2 gene
(SEQ ID NO: 17). More positive values indicate hydrophobicity.
[0036] FIG. 4 compares the hydrophobicity plots predicting the
transmembrane regions of the amino acid sequence of (A) mouse drg12
(SEQ ID NO: 14); (B) human drg12 (SEQ ID NO: 19)
[0037] FIG. 5 compares the hydrophobicity plots predicting the
transmembrane regions of the amino acid sequence of mrg9 (SEQ ID
NO: 21); mrg10 (SEQ ID NO: 23); mrg11 (SEQ ID NO: 25) and mrg12
(SEQ ID NO: 27).
[0038] FIG. 6A is an alignment of the amino acid sequences of
MRGA1-A8, deduced from nucleotide sequences of cDNA and BAC clones
from strain C57BL/6J mice. The predicted locations of the
transmembrane (TM1-TM7), extracellular (E1-E4), and cytoplasmic
(C1-C4) domains are indicated above the aligned sequences.
[0039] FIG. 6B depicts a phylogenetic analysis of MRG family
members identified from database searches. The protein sequences of
all MRGs were aligned using CLUSTALW (Thompson et al. Nucleic Acids
Res 22: 4673-80 (1994)). The dendrogram was generated with the
PHYLUP software package using the Neighbor-Joining method and 1,000
bootstrap trials. The horizontal length of the branches is
proportional to the number of amino acid changes. Vertical
distances are arbitrary. Mouse (m)Mrg genes with retrotransposon
sequences .about.650 nt 3' of their stop codon are highlighted
(L1). All genes that are predicted to encode pseudogenes are
indicated with the psi (.PSI.) symbol.
[0040] FIG. 6C shows the chromosomal organization of one mouse Mrg
cluster deduced from analysis of overlapping BAC clones. The
cluster contains four intact ORFs and three pseudogenes.
[0041] FIG. 7A shows the distribution of nociceptive sensory
neurons in a postnatal day 0 (P0) DRG as revealed by expression of
the NGF receptor trkA. This population is selectively eliminated in
Ngn1.sup.-/- mutants (Ma et al. Genes & Dev. 13: 1717-1728
(1999)).
[0042] FIG. 7B shows in situ hybridization with cRNA probes
detecting MrgA1. MrgA1 is expressed in a pattern similar to that of
trkA.sup.+ neurons on an adjacent section shown in FIG. 7A.
[0043] FIG. 7C-I shows in situ hybridization with cRNA probes
detecting MrgA2-MrgA8.
[0044] FIG. 7J shows that MrgA1 expression is eliminated in
Ngn1.sup.-/- mice, as is expression of other MrgA genes (not
shown). Remaining DRG neurons are present in the area delimited by
the dotted line, and can be visualized by expression of generic
neuronal markers.
[0045] FIG. 8 shows that expression of MrgAs is restricted to
non-peptidergic nociceptors that project to inner lamina II. Shown
are confocal microscopic images of in situ hybridizations using the
Mrg probes indicated, combined with fluorescent antibody detection
of trkA (A-D), substance P (I-L), CGRP (M-P), VR1 (Q-T) or staining
with fluorescent isolectin IB4 (IB4; E-H). MrgA.sup.+ or MrgD.sup.+
cells co-express trkA and IB4 (A-H, arrowheads), but most do not
express subP, CGRP or VR1 (I-T, arrowheads; arrows in I, M indicate
a minor subset of MrgA1.sup.+ neurons that co-express SubP and
CGRP).
[0046] FIG. 9 is a schematic illustration of the restriction of
MrgA (and MrgD) expression to non-peptidergic, IB4.sup.+, VR1.sup.-
sensory neurons that project to lamina IIi (Snider and McMahon
Neuron 20: 629-32 (1998)). Post-synaptic neurons in lamina IIi
express PKC.gamma..
[0047] FIG. 10 shows that individual sensory neurons co-express
multiple MrgAs. (A-C) double label in situ hybridization with MrgA1
(A) and A3 (B). (D-F) double labeling with MrgA1 (D) and MrgA4 (E).
In both cases, cells expressing MrgA3 or A4 are a subset of those
expressing MrgA1 (C, F, arrowheads). Arrows in (F) indicate
intranuclear dots of MrgA4 expression which may represent sites of
transcription. (G-I) Double label in situ with MrgA1 and MrgD. Some
overlap overlap between the two populations is seen (I, arrowhead),
while most cells express one receptor but not the other (I,
arrows). Approximately 15% of cells expressing either MrgA1 or MrgD
co-express both genes. Vertical bars to the right of panels (C, F,
I) represent a z-series viewed along the y-axis, horizontal bars
below the panels a z-series viewed along the x-axis. (J, K)
comparison of in situ hybridization signals obtained using a single
MrgA probe (J) and a mixture of 7 MrgA probes (K). Approximately 1%
of neurons were labeled by the MrgA4 probe, while .about.4.5% were
labeled by the mixed probe. The sum of the percentage of neurons
labeled by the individual MrgA2-8 probes is .about.6.6%, suggesting
that there is partial overlap within this population. (L) Venn
diagram illustrating combinations of gene expression revealed by in
situ analysis. The drawing is a conservative estimate of the number
of subsets, since we do not yet know, for example, whether MrgAs2-8
partially overlap with MrgD. The sizes of the circles are not
proportional.
[0048] FIG. 11 shows elevated intracellular free Ca.sup.++ elicited
by FLRF in HEK cells expressing MRGA1. (A, B) and (E, F) illustrate
Fura-2 fluorescence at 340 nm (A, E) and 380 nm (B, F) in
HEK-G.alpha..sub.15 cells expressing an MRGA1-GFP fusion protein
(A-D) or GFP alone (E-H). The images were taken 2 minutes after the
addition of 1 .mu.M of FLRFamide. The peri-nuclear, punctate
distribution of MRGA1-GFP revealed by intrinsic GFP fluorescence
(D, arrowheads) is characteristic of the ER-Golgi network,
indicating membrane integration and intracellular transport of the
receptors. In contrast, the control GFP protein is cytoplasmic (H).
The intracellular Ca.sup.2+ ([Ca.sup.2+].sub.i) release was
determined from the FURA-2 340 nM/380 nM emission ratio (C, G).
Note that MRGA1-expressing cells (but not surrounding untransfected
cells) show an elevated ratio of Fura-2 fluorescence at 340/380 nm
(C, arrowheads), indicating an increase in [Ca.sup.2+].sub.i. In
contrast, no such elevation is observed in control GFP-expressing
cells (G). The elevated 340/380 fluorescence seen in
MRGA1-expressing cells was dependent on the addition of ligand (not
shown).
[0049] FIG. 12A shows activation of MRGA receptors expressed in
heterologous cells by neuropeptide ligands. HEK-G.alpha..sub.15
cells (Offermanns and Simon. J Biol Chem 270: 15175-80 (1995))
expressing MRGA1 were tested with the indicated ligands at a
concentration of 1 .mu.M. The data indicate the mean percentages of
GFP-positive (i.e., transfected) cells showing calcium responses.
None of the agonists indicated showed any responses through
endogenous receptors in untransfected cells. Note that the RFamide
neuropeptides FMRF, FLRF and NPFF, as well as NPY, ACTH, CGRP-I and
-II and somatostatin (SST) produced the strongest responses.
[0050] FIG. 12B shows the ligand selectivity of MRGA1 expressed in
HEK cells lacking G.alpha..sub.15. The cells were exposed to
ligands at a concentration of 1 .mu.M as in (A).
[0051] FIG. 12C shows the ligand selectivity of MRGA4. The data
presented in FIGS. 12B and 12C indicate that the responses to the
most effective ligands do not depend on the presence of
G.alpha..sub.15. Note that MRGA1-expressing cells respond to FLRF
and NPFF but not to NPAF, while conversely MRGA4-expressing cells
respond to NPAF but not NPFF or FLRF
[0052] FIG. 12D shows dose-response curves for MRGA1 expressed in
HEK-G.alpha..sub.15 cells to selected RFamide neuropeptides. Each
data point represents the mean.+-.S.E.M. of at least 3 independent
determinations; at least 20 GFP.sup.+ cells were analyzed for each
determination. Responses at each ligand concentration were
normalized to the maximal response subsequently shown by the same
cells to a 5 .mu.M concentration of FLRF. MRGA1 (D) shows highest
sensitivity to FLRF (squares, EC.sub.50{tilde over ()}20 nM) and
lower sensitivity to NPFF (circles, EC.sub.50{tilde over ()}200
nM).
[0053] FIG. 12E shows dose-response curves for MRGA4 expressed in
HEK-G.alpha..sub.15 cells to selected RFamide neuropeptides. Each
data point represents the mean.+-.S.E.M. of at least 3 independent
determinations; at least 20 GFP.sup.+ cells were analyzed for each
determination. Responses at each ligand concentration were
normalized to the maximal response subsequently shown by the same
cells to a 5 .mu.M concentration of NPAF. MRGA4 is preferentially
activated by NPAF (triangles, EC.sub.50{tilde over ()}60 nM).
[0054] FIG. 12F shows dose-response curves for MAS1 expressed in
HEK-G.alpha..sub.15 cells to selected RFamide neuropeptides. Each
data point represents the mean.+-.S.E.M. of at least 3 independent
determinations; at least 20 GFP.sup.+ cells were analyzed for each
determination. Responses at each ligand concentration were
normalized to the maximal response subsequently shown by the same
cells to a 5 .mu.M concentration of NPFF. MAS1, like MRGA1, is
activated by NPFF with similar efficacy (EC.sub.50{tilde over
()}400 nM), but is not as well activated by FLRF (squares).
[0055] FIG. 13 depicts the expression pattern of mMrgB1 in a
sagital section of a newborn mouse. The staining pattern indicates
that the mMrgB1 gene is expressed in the scattered cells in the
epidermal layer of the skin, in the spleen and in the submandibular
gland.
[0056] FIG. 14 is a higher magnification of the mMrgB1 expression
in the spleen and skin depicted in FIG. 13.
[0057] FIG. 15 shows the expression of mMrgD in adult dorsal root
ganglia.
DETAILED DESCRIPTION OF THE INVENTION
I. General Description
[0058] As described above, the present invention is based on the
discovery of new genes that are expressed in the DRG of normal mice
but not in Ngn1 null mutant mice. One of the novel gene families
isolated from the screen encodes a receptor protein with 7
transmembrane segments, a characteristic of G protein-coupled
receptors. This novel 7 transmembrane receptor is most closely
related to the oncogene mas, and therefore was provisionally named
mas-related gene-3 (mrg3). mrg3 is now known as MrgA1, and the
terms are used interchangeably herein. Almost 50 members of the
Mas-related gene (Mrg) family have been identified, many of which
are specifically expressed in non-peptidergic nociceptors. Large
families of G protein-coupled receptors are also expressed in other
classes of sensory neurons, such as olfactory and gustatory
neurons.
[0059] The murine Mrg family of GPCRs contains three major
subfamilies (MrgA, B and C), each consisting of more than 10 highly
duplicated genes, as well as several single-copy genes such as
Mas1, Rta, MrgD and MrgE (FIG. 6B). The MrgA subfamily consists of
at least twenty members in mice: MrgA1 (SEQ ID NO: 2); MrgA2 (SEQ
ID NO: 4); MrgA3 (SEQ ID NO: 6); MrgA4. (SEQ ID NO: 11); MrgA5 (SEQ
ID NO: 21); MrgA6 (SEQ ID NO: 23); MrgA7 (SEQ ID NO: 25); MrgA8
(SEQ ID NO: 27); MrgA9 (SEQ ID NO: 53); MrgA10 (SEQ ID NO: 55);
MrgA11 (SEQ ID NO: 57); MrgA12 (SEQ ID NO: 59); MrgA13 (SEQ ID NO:
61); MrgA14 (SEQ ID NO: 63); MrgA15 (SEQ ID NO: 65); MrgA16 (SEQ ID
NO: 67); MrgA17 (SEQ ID NO: 69); MrgA18 (SEQ ID NO: 71); MrgA19
(SEQ ID NO: 73); MrgA20 (SEQ ID NO: 75). Four human sequences that
are most closes related to the MrgA subfamily have also been
identified:
1 MrgX1; (SEQ ID NO: 16) MrgX2; (SEQ ID NO: 18) MrgX3; (SEQ ID NO:
31) and MrgX4. (SEQ ID NO: 33)
[0060] The MrgB subfamily consists of at least twelve members in
mice:
2 MrgB1; (SEQ ID NO: 39) MrgB2; (SEQ ID NO: 41) MrgB3; (SEQ ID NO:
43) MrgB4; (SEQ ID NO: 45) MrgB5; (SEQ ID NO: 47) MrgB6; (SEQ ID
NO: 77) MrgB7; (SEQ ID NO: 79) MrgB8; (SEQ ID NO: 81) MrgB9; (SEQ
ID NO: 83) MrgB10; (SEQ ID NO: 85) MrgB11; (SEQ ID NO: 87) and
MrgB12. (SEQ ID NO: 89)
[0061] Ten members of the MrgC subfamily have been identified in
mice:
3 MrgC1; (SEQ ID NO: 91) MrgC2; (SEQ ID NO: 93) MrgC3; (SEQ ID NO:
95) MrgC4; (SEQ ID NO: 97) MrgC5; (SEQ ID NO: 99) MrgC6; (SEQ ID
NO: 101) MrgC7; (SEQ ID NO: 103) MrgC8; (SEQ ID NO: 105) MrgC9;
(SEQ ID NO: 107) and MrgC10. (SEQ ID NO: 109)
[0062] A single member of the MrgD subfamily has been identified in
mice, mMrgD (SEQ ID NO: 49) and its ortholog identified in humans,
hMrgD (SEQ ID NO: 35). Similarly, a single member of the MrgE
subfamily has been identified in mice, mMrgE (SEQ ID NO: 51) and
humans, hMrgE (SEQ ID NO: 37).
[0063] As is the case in other GPCR subfamilies, a number of the
Mrgs appear to be pseudogenes, including all members of the MrgC
subfamily. The presence of L1 retrotransposon elements near several
Mrg genes raises the possibility that pseudogene expansion may have
been driven by L1-mediated transduction (Goodier et al. Hum Mol
Genet 9: 653-7 (2000)).
[0064] In contrast to the murine MrgA and B subfamilies, which
together contain almost 40 intact coding sequences, only four
intact human MrgX sequences were identified. The remaining 10 human
Mrg sequences appear to be pseudogenes. Inclusion of other related
receptors such as hMrgD and hMas1 brings the total number of intact
human coding sequences in this family to nine (FIG. 6B).
[0065] Prior to the present invention, the primary nociceptive
sensory neurons were thought not to specifically discriminate among
different chemical stimuli, but rather to detect noxious stimuli of
various modalities by virtue of broadly tuned receptors such as VR1
(Tominaga et al. Neuron 21: 531-43 (1998)). The expression of Mrgs
reveals an unexpected degree of molecular diversification among
nociceptive sensory neurons. Approximately 13-14% of sensory
neurons express MrgA1, while 17-18% express MrgD and the overlap
between these two populations is only 15%. The MrgA1.sup.+
population seems to include most or all neurons expressing MrgA2-8.
However, these latter MrgA genes are not all expressed in the same
neurons. Thus the 8 MrgA genes and MrgD define at least 6 different
neuronal subpopulations, and the remaining 16 MrgA genes add even
greater diversity.
[0066] It is striking that both MrgA and D are expressed in
IB4.sup.+, VR1.sup.- sensory neurons. IB4.sup.+ neurons are known
to project to lamina IIi (Snider and McMahon Neuron 20: 629-32
(1998)), which has been implicated in chronic pain, such as that
accompanying nerve injury (Malmberg et al. Science 278: 279-83
(1997)). VR1 is activated both by thermal stimuli and chemical
stimuli such as capsaicin (Caterina et al. Nature 389: 816-824
(1997); Tominaga et al. Neuron 21: 531-43 (1998)), but VR1.sup.+
neurons are dispensable for the detection of noxious mechanical
stimuli (Caterina et al. Science 288: 306-13 (2000)). This
indicates that one of the functions of MrgA.sup.+ neurons is the
detection of noxious mechanical stimuli accompanying neuropathic or
inflammatory pain.
[0067] The existence of a family of putative G protein-coupled
receptors specifically expressed in nociceptive sensory neurons
suggests that these molecules are primary mediators or modulators
of pain sensation. It is therefore of great interest to identify
ligands, both endogenous and synthetic, that modulate the activity
of these receptors, for the management of chronic intractable pain.
Indeed, ligand screens in heterologous cell expression systems
indicate that these receptors can interact with RF-amide
neuropeptides of which the prototypic member is the molluscan
cardioexcitatory peptide FMRF-amide (Price and Greenberg Science
197: 670-671 (1977)). Mammalian RF-amide peptides include NPFF and
NPAF, which are derived from a common pro-peptide precursor
expressed in neurons of laminae I and II of the dorsal spinal cord
(Vilim et al. Mol Pharmacol 55: 804-11 (1999)). The expression of
this neuropeptide FF precursor in the synaptic termination zone of
Mrg-expressing sensory neurons, the ability of NPAF and NPFF to
activate these receptors in functional assays, and the presence of
binding sites for such peptides on primary sensory afferents in the
dorsal horn (Gouarderes et al. Synapse 35: 45-52 (2000)), together
indicate that these neuropeptides are ligands for Mrg receptors in
vivo. As intrathecal injection of NPFF/NPAF peptides produces
long-lasting antinociceptive effects in several chronic pain models
(reviewed in Panula et al. Brain Res 848: 191-6 (1999)), including
neuropathic pain (Xu et al. Peptides 20: 1071-7 (1999)), these data
further indicate that Mrgs are directly involved in the modulation
of pain.
[0068] One possibility for the extent of diversity among Mrgs
expressed by murine nociceptors is that different Mrgs are
expressed by sensory neurons that innervate different peripheral
targets, such as gut, skin, hair follicles, blood vessels, bones
and muscle. These targets may secrete different ligands for
different Mrgs. Another possibility is that neurons expressing
different Mrgs respond to a common modulator of peripheral
nociceptor sensitivity, but with different affinities. Such a
mechanism could, for example, provide a gradual restoration of
normal sensitivities among the population of nociceptors during
wound healing, as the concentration of such modulators gradually
returned to baseline. Such a graded response might be coupled to,
or even determine the activation thresholds of different subsets of
nociceptors. Another novel gene family isolated in this screen,
drg-12 encodes a protein with two putative transmembrane segments.
Drg12 was identified from both mice (SEQ ID NO: 14) and in humans
(SEQ ID NO: 29). In situ hybridization indicates that, like the mrg
genes, this gene is also specifically expressed in a subset of DRG
sensory neurons. As it is a membrane protein it may also be
involved in signaling by these neurons. Although there are no
obvious homologies between this protein and other known proteins,
it is noteworthy that two purinergic receptors specifically
expressed in nociceptive sensory neurons (P.sub.2X.sub.2 and
P.sub.2X.sub.3) have a similar bipartite transmembrane topology.
Therefore it is likely that the family drg-12 also encodes a
receptor or ion channel involved in nociceptive sensory
transduction or its modulation.
[0069] The proteins of the invention can serve as therapeutics and
as a target for agents that modulate their expression or activity,
for example in the treatment of chronic intractable pain and
neuropathic pain. For example, agents may be identified which
modulate biological processes associated with nociception such as
the reception, transduction and transmission of pain signals.
II. Specific Embodiments
[0070] A. Definitions
[0071] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. See,
e.g. Singleton et al., Dictionary of Microbiology and Molecular
Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994);
Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold
Springs Harbor Press (Cold Springs Harbor, N.Y. 1989). For purposes
of the present invention, the following terms are defined
below.
[0072] As used herein, the "protein" or "polypeptide" refers, in
part, to a protein that has the amino acid sequence depicted in SEQ
ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 19, 21, 23, 25, 27, 29, 31,
33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,
67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,
101, 103, 105, 107 and 109. The terms also refer to naturally
occurring allelic variants and proteins that have a slightly
different amino acid sequence than those specifically recited
above. Allelic variants, though possessing a slightly different
amino acid sequence than those recited above, will still have the
same or similar biological functions associated with the
protein.
[0073] Identity or homology with respect to amino acid sequences is
defined herein as the percentage of amino acid residues in the
candidate sequence that are identical with the known peptides,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent homology, and not considering any
conservative substitutions as part of the sequence identity (see
section B for the relevant parameters). Fusion proteins, or
N-terminal, C-terminal or internal extensions, deletions, or
insertions into the peptide sequence shall not be construed as
affecting homology.
[0074] Proteins can be aligned using CLUSTALW (Thompson et al.
Nucleic Acids Res 22:4673-80 (1994)) and homology or identity at
the nucleotide or amino acid sequence level may be determined by
BLAST (Basic Local Alignment Search Tool) analysis using the
algorithm employed by the programs blastp, blastn, blastx, tblastn
and tblastx (Karlin , et al. Proc. Natl. Acad. Sci. USA 87:
2264-2268 (1990) and Altschul, S. F. J. Mol. Evol. 36: 290-300
(1993), fully incorporated by reference) which are tailored for
sequence similarity searching. The approach used by the BLAST
program is to first consider similar segments between a query
sequence and a database sequence, then to evaluate the statistical
significance of all matches that are identified and finally to
summarize only those matches which satisfy a preselected threshold
of significance. For a discussion of basic issues in similarity
searching of sequence databases, see Altschul et al. (Nature
Genetics 6: 119-129 (1994)) which is fully incorporated by
reference. The search parameters for histogram, descriptions,
alignments, expect (i.e., the statistical significance threshold
for reporting matches against database sequences), cutoff, matrix
and filter are at the default settings. The default scoring matrix
used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix
(Henikoff, et al. Proc. Natl. Acad. Sci. USA 89: 10915-10919
(1992), fully incorporated by reference). For blastn, the scoring
matrix is set by the ratios of M (i.e., the reward score for a pair
of matching residues) to N (i.e., the penalty score for mismatching
residues), wherein the default values for M and N are 5 and -4,
respectively. Four blastn parameters were adjusted as follows: Q=10
(gap creation penalty); R=10 (gap extension penalty); wink=1
(generates word hits at every winkth position along the query); and
gapw=16 (sets the window width within which gapped alignments are
generated). The equivalent Blastp parameter settings were Q=9; R=2;
wink=1; and gapw=32. A Bestfit comparison between sequences,
available in the GCG package version 10.0, uses DNA parameters
GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty) and
the equivalent settings in protein comparisons are GAP=8 and
LEN=2.
[0075] "Variants" are biologically active polypeptides having an
amino acid sequence which differs from the sequence of a native
sequence polypeptide of the present invention, such as that shown
in FIG. 1 for mrg3 (SEQ ID NO: 2), by virtue of an insertion,
deletion, modification and/or substitution of one or more amino
acid residues within the native sequence. Variants include peptide
fragments of at least 5 amino acids, preferably at least 10 amino
acids, more preferably at least 15 amino acids, even more
preferably at least 20 amino acids that retain a biological
activity of the corresponding native sequence polypeptide. Variants
also include polypeptides wherein one or more amino acid residues
are added at the N- or C-terminus of, or within, a native sequence.
Further, variants also include polypeptides where a number of amino
acid residues are deleted and optionally substituted by one or more
different amino acid residues.
[0076] As used herein, a "conservative variant" refers to
alterations in the amino acid sequence that do not adversely affect
the biological functions of the protein. A substitution, insertion
or deletion is said to adversely affect the protein when the
altered sequence prevents or disrupts a biological function
associated with the protein. For example, the overall charge,
structure or hydrophobic/hydrophilic properties of the protein can
be altered without adversely affecting a biological activity.
Accordingly, the amino acid sequence can be altered, for example to
render the peptide more hydrophobic or hydrophilic, without
adversely affecting the biological activities of the protein.
[0077] As used herein, the "family of proteins" related to the
amino acid sequences of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,
53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,
87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 and 109 includes
proteins that have been isolated from the dorsal root ganglia of
organisms in addition to mice and humans. The methods used to
identify and isolate other members of the family of proteins
related to these proteins, such as the disclosed mouse and human
proteins, are described below.
[0078] Unless indicated otherwise, the term "Mrg" when used herein
refers to any one or more of the mammalian mas-related gene (Mrg)
receptors (i.e. MrgA1-8, MrgB, MrgC, MrgD, MrgE, MrgX1-4 and any
other members of the mas-related gene (Mrg) family now known or
identified in the future), including native sequence mammalian,
such as murine or human, Mrg receptors, Mrg receptor variants; Mrg
receptor extracellular domain; and chimeric Mrg receptors (each of
which is defined herein). The term specifically includes native
sequence murine Mrg receptors of the MrgA family, such as SEQ ID
NOs: 2, 4, 6 12, 21, 23, 25, 27, 53, 55, 57, 59, 61, 63, 65, 67,
69, 71, 73, and 75; native sequence murine Mrg receptors of the
MrgB family, such as SEQ ID NOs: 39, 41, 43, 45, 47, 77, 79, 81,
83, 85, 87, and 89; native sequence murine Mrg receptors of the
MrgC family, such as SEQ ID NOs: 91, 93, 95, 97, 99, 101, 103, 105,
107 and 109; native sequence murine Mrg receptors of the MrgD
family, such as SEQ ID NO: 49; native sequence murine Mrg receptors
of the MrgE family, such as SEQ ID NO: 51; their human homologues,
and the native sequence human Mrg receptors termed "MrgX" of SEQ ID
NOs: 16, 18, 31 and 33.
[0079] The terms "mas-related gene", "mrg" and "Mrg" are used
interchangeably herein. Further, the terms mrg3, MrgA1 and mMrgA1
are used interchangeably, as are the terms mrg4, MrgA2 and mMrgA2,
the terms mrg5, MrgA3 and mMrgA3, the terms mrg8, MrgA4 and mMrgA4,
the terms mrg9, MrgA5 and mMrgA5, the terms mrg10, MrgA6 and
mMrgA6, the terms mrg11, MrgA7 and mMrgA7, the terms mrgl2, MrgA8
and mMrgA8, the terms human1, MrgX1 and hMrgX1, the terms human2,
MrgX2 and hMrgX2, the terms human 4, MrgX3 and hMrgX3, and the
terms human5, MrgX4 and hMrgX4. These terms all refer to native
sequence Mrg proteins as described herein as well as functional
derivatives, including amino acid sequence variants thereof.
[0080] A "native" or "native sequence" Mrg or drg-12 receptor has
the amino acid sequence of a naturally occurring Mrg or drg-12
receptor in any mammalian species (including humans), irrespective
of its mode of preparation. Accordingly, a native or native
sequence Mrg or drg-12 receptor may be isolated from nature,
produced by techniques of recombinant DNA technology, chemically
synthesized, or produced by any combinations of these or similar
methods. Native Mrg and drg-12 receptors specifically include
polypeptides having the amino acid sequence of naturally occurring
allelic variants, isoforms or spliced variants of these receptors,
known in the art or hereinafter discovered.
[0081] The "extracellular domain" (ECD) is a form of the Mrg or
drg-12 receptor which is essentially free of the transmembrane and
cytoplasmic domains, i.e., has less than 1% of such domains,
preferably 0.5 to 0% of such domains, and more preferably 0.1 to 0%
of such domains. Ordinarily, the ECD will have an amino acid
sequence having at least about 60% amino acid sequence identity
with the amino acid sequence of one or more of the ECDs of a native
Mrg or drg-12 protein, for example as indicated in FIG. 1A for mrg3
(E1, E2 etc . . . ), preferably at least about 65%, more preferably
at least about 75%, even more preferably at least about 80%, even
more preferably at least about 90%, with increasing preference of
95%, to at least 99% amino acid sequence identity, and finally to
100% identity, and thus includes polypeptide variants as defined
below.
[0082] The first predicted extracellular domain (ECD1) comprises
approximately amino acids 1 to 21 for MrgA1, 1 to 21 for MrgA2, 1
to 21 for MrgA3, 1 to 21 for MrgA4, 1 to 3 for MrgA5, 1 to 17 for
MrgA6, 1 to 21 for MrgA7 and 1 to 21 for MrgA8. The second
predicted extracellular domain (ECD2) comprises approximately amino
acids 70 to 87 for MrgA1, 70 to 88 for MrgA2, 70 to 88 for MrgA3,
70 to 88 for MrgA4, 52 to 70 for MrgA5, 66 to 84 for MrgA6, 70 to
88 for MrgA7 and 70 to 88 for MrgA8. The third predicted
extracellular domain (ECD3) comprises approximately amino acids 149
to 160 for MrgA1, 150 to 161 for MrgA2, 150 to 161 for MrgA3, 150
to 161 for MrgA4, 132 to 144 for MrgA5, 146 to 157 for MrgA6, 150
to 161 for MrgA7 and 150 to 161 for MrgA8. The fourth predicted
extracellular domain (ECD4) comprises approximately amino acids 222
to 2244 for MrgA1, 223 to 245 for MrgA2, 223 to 242 for MrgA3, 223
to 245 for MrgA4, 205 to 225 for MrgA5, 219 to 241 for MrgA6, 223
to 245 for MrgA7 and 223 to 245 for MrgA8.
[0083] The term "drg-12" when used herein refers to any one or more
of the mammalian drg-12 receptors now known or identified in the
future, including native sequence mammalian, such as murine or
human, drg-12 receptors, drg-12 receptor variants; drg-12 receptor
extracellular domain; and chimeric drg-12 receptors (each of which
is defined herein). The term specifically includes native sequence
murine drg-12 receptor, such as SEQ ID NO: 14, and any human
homologues, such as human drg-12 (SEQ ID NO: 29).
[0084] As used herein, "nucleic acid" is defined as RNA or DNA that
encodes a protein or peptide as defined above, is complementary to
a nucleic acid sequence encoding such peptides, hybridizes to such
a nucleic acid and remains stably bound to it under appropriate
stringency conditions, exhibits at least about 50%, 60%, 70%, 75%,
85%, 90% or 95% nucleotide sequence identity across the open
reading frame, or encodes a polypeptide sharing at least about 50%,
60%, 70% or 75% sequence identity, preferably at least about 80%,
and more preferably at least about 85%, and even more preferably at
least about 90 or 95% or more identity with the peptide sequences.
Specifically contemplated are genomic DNA, cDNA, mRNA and antisense
molecules, as well as nucleic acids based on alternative backbones
or including alternative bases whether derived from natural sources
or synthesized. Such hybridizing or complementary nucleic acids,
however, are defined further as being novel and unobvious over any
prior art nucleic acid including that which encodes, hybridizes
under appropriate stringency conditions, or is complementary to
nucleic acid encoding a protein according to the present
invention.
[0085] As used herein, the terms nucleic acid, polynucleotide and
nucleotide are interchangeable and refer to any nucleic acid,
whether composed of phosphodiester linkages or modified linkages
such as phosphotriester, phosphoramidate, siloxane, carbonate,
carboxymethylester, acetamidate, carbamate, thioether, bridged
phosphoramidate, bridged methylene phosphonate, bridged
phosphoramidate, bridged phosphoramidate, bridged methylene
phosphonate, phosphorothioate, methylphosphonate,
phosphorodithioate, bridged phosphorothioate or sultone linkages,
and combinations of such linkages.
[0086] The terms nucleic acid, polynucleotide and nucleotide also
specifically include nucleic acids composed of bases other than the
five biologically occurring bases (adenine, guanine, thymine,
cytosine and uracil). For example, a polynucleotide of the
invention might contain at least one modified base moiety which is
selected from the group including but not limited to
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-carboxymethylaminomethyl-2-thiouridin- e,
5-carboxymethylaminomethyl-uracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiour- acil,
beta-D-mannosylqueosine, 5N-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[0087] Furthermore, a polynucleotide used in the invention may
comprise at least one modified sugar moiety selected from the group
including but not limited to arabinose, 2-fluoroarabinose,
xylulose, and hexose.
[0088] "Stringent conditions" are those that (1) employ low ionic
strength and high temperature for washing, for example, 0.015 M
NaCl/0.0015 M sodium citrate/0.1% SDS at 50.degree. C., or (2)
employ during hybridization a denaturing agent such as formamide,
for example, 50% (vol/vol) formamide with 0.1% bovine serum
albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium
phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate
at 42.degree. C. Another example is use of 50% formamide,
5.times.SSC (0.75M NaCl, 0.075 M sodium citrate), 50 mM sodium
phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5.times. Denhardt's
solution, sonicated salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and
10% dextran sulfate at 42.degree. C., with washes at 42.degree. C.
in 0.2.times.SSC and 0.1% SDS. A skilled artisan can readily
determine and vary the stringency conditions appropriately to
obtain a clear and detectable hybridization signal.
[0089] As used herein, a nucleic acid molecule is said to be
"isolated" when the nucleic acid molecule is substantially
separated from contaminant nucleic acid molecules encoding other
polypeptides.
[0090] As used herein, a fragment of an encoding nucleic acid
molecule refers to a small portion of the entire protein coding
sequence. The size of the fragment will be determined by the
intended use. For example, if the fragment is chosen so as to
encode an active portion of the protein, the fragment will need to
be large enough to encode the functional region(s) of the protein.
For instance, fragments which encode peptides corresponding to
predicted antigenic regions may be prepared (see FIGS. 3 and 4). If
the fragment is to be used as a nucleic acid probe or PCR primer,
then the fragment length is chosen so as to obtain a relatively
small number of false positives during probing/priming (see the
discussion in Section H).
[0091] Highly related gene homologs are polynucleotides encoding
proteins that have at least about 60% amino acid sequence identity
with the amino acid sequence of a naturally occurring native
sequence polynucleotide of the invention, such as MrgA1 (SEQ ID NO:
2), preferably at least about 65%, 70%, 75%, 80%, with increasing
preference of at least about 85% to at least about 99% amino acid
sequence identity, in 1% increments.
[0092] The term "mammal" is defined as an individual belonging to
the class Mammalia and includes, without limitation, humans,
domestic and farm animals, and zoo, sports, or pet animals, such as
sheep, dogs, horses, cats or cows. Preferably, the mammal herein is
human.
[0093] "Functional derivatives" include amino acid sequence
variants, and covalent derivatives of the native polypeptides as
long as they retain a qualitative biological activity of the
corresponding native polypeptide.
[0094] By "Mrg ligand" is meant a molecule which specifically binds
to and preferably activates an Mrg receptor. Examples of Mrg
ligands include, but are not limited to RF-amide neuropeptides,
such as FMRF, FLRF, NPAF, NPFF, and RFRP-1 for MrgA receptors, such
as MrgA1. The ability of a molecule to bind to Mrg can be
determined, for example, by the ability of the putative ligand to
bind to membrane fractions prepared from cells expressing Mrg.
[0095] Similarly, a drg-12 ligand is a molecule which specifically
binds to and preferably activates a drg-12 receptor.
[0096] A "chimeric" molecule is a polypeptide comprising a
full-length polypeptide of the present invention, a variant, or one
or more domains of a polypeptide of the present invention fused or
bonded to a heterologous polypeptide. The chimeric molecule will
generally share at least one biological property in common with a
naturally occurring native sequence polypeptide. An example of a
chimeric molecule is one that is epitope tagged for purification
purposes. Another chimeric molecule is an immunoadhesin.
[0097] The term "epitope-tagged" when used herein refers to a
chimeric polypeptide comprising Mrg or drg-12 fused to a "tag
polypeptide". The tag polypeptide has enough residues to provide an
epitope against which an antibody can be made, yet is short enough
such that it does not interfere with the biological activity of the
Mrg or drg-12. The tag polypeptide preferably is fairly unique so
that the antibody against it does not substantially cross-react
with other epitopes. Suitable tag polypeptides generally have at
least six amino acid residues and usually between about 8-50 amino
acid residues (preferably between about 9-30 residues). Preferred
are poly-histidine sequences, which bind nickel, allowing isolation
of the tagged protein by Ni-NTA chromatography as described (See,
e.g., Lindsay et al. Neuron 17:571-574 (1996)).
[0098] "Agonists" are molecules or compounds that stimulate one or
more of the biological properties of a polypeptide of the present
invention. These may include, but are not limited to, small organic
and inorganic molecules, peptides, peptide mimetics and agonist
antibodies.
[0099] The term "antagonist" is used in the broadest sense and
refers to any molecule or compound that blocks, inhibits or
neutralizes, either partially or fully, a biological activity
mediated by a receptor of the present invention by preventing the
binding of an agonist. Antagonists may include, but are not limited
to, small organic and inorganic molecules, peptides, peptide
mimetics and neutralizing antibodies.
[0100] The proteins of the present invention are preferably in
isolated form. As used herein, a protein is said to be isolated
when physical, mechanical or chemical methods are employed to
remove the protein from cellular constituents that are normally
associated with the protein. A skilled artisan can readily employ
standard purification methods to obtain an isolated protein. In
some instances, isolated proteins of the invention will have been
separated or purified from many cellular constituents, but will
still be associated with cellular membrane fragrnents or membrane
constituents.
[0101] Thus, "isolated Mrg" and "isolated drg-12" means Mrg or
drg-12 polypeptide, respectively, that has been purified from a
protein source or has been prepared by recombinant or synthetic
methods and purified. Purified Mrg or drg-12 is substantially free
of other polypeptides or peptides. "Substantially free" here means
less than about 5%, preferably less than about 2%, more preferably
less than about 1%, even more preferably less than about 0.5%, most
preferably less than about 0.1% contamination with other source
proteins.
[0102] "Essentially pure" protein means a composition comprising at
least about 90% by weight of the protein, based on total weight of
the composition, preferably at least about 95% by weight, more
preferably at least about 90% by weight, even more preferably at
least about 95% by weight. "Essentially homogeneous" protein means
a composition comprising at least about 99% by weight of protein,
based on total weight of the composition.
[0103] "Biological property" is a biological or immunological
activity, where biological activity refer to a biological function
(either inhibitory or stimulatory) caused by a native sequence or
variant polypeptide molecule herein, other than the ability to
induce the production of an antibody against an epitope within such
polypeptide, where the latter property is referred to as
immunological activity. Biological properties specifically include
the ability to bind a naturally occurring ligand of the receptor
molecules herein, preferably specific binding, and even more
preferably specific binding with high affinity.
[0104] "Antibodies" (Abs) and "immunoglobulins" (Igs) are
glycoproteins having the same structural characteristics. While
antibodies exhibit binding specificity to a specific antigen,
immunoglobulins include both antibodies and other antibody-like
molecules that lack antigen specificity. Polypeptides of the latter
kind are, for example, produced at low levels by the lymph system
and at increased levels by myelomas.
[0105] "Native antibodies" and "native immunoglobulins" are usually
heterotetrameric glycoproteins, composed of two identical light (L)
chains and two identical heavy (H) chains. Each light chain is
linked to a heavy chain by one covalent disulfide bond. while The
number of disulfide linkages varies among the heavy chains of
different immunoglobulin isotypes. Each heavy and light chain also
has regularly spaced intra-chain disulfide bridges. Each heavy
chain has at one end a variable domain (V.sub.H) followed by a
number of constant domains. Each light chain has a variable domain
at one end (V.sub.L) and a constant domain at its other end. The
constant domain of the light chain is aligned with the first
constant domain of the heavy chain, and the light-chain variable
domain is aligned with the variable domain of the heavy chain.
Particular amino acid residues are believed to form an interface
between the light- and heavy-chain variable domains.
[0106] The term "antibody" herein is used in the broadest sense and
specifically covers human, non-human (e.g. murine) and humanized
monoclonal antibodies (including full length monoclonal
antibodies), polyclonal antibodies, multi-specific antibodies
(e.g., bispecific antibodies), and antibody fragments so long as
they exhibit the desired biological activity.
[0107] "Antibody fragments" comprise a portion of a full-length
antibody, generally the antigen binding or variable domain thereof.
Examples of antibody fragments include Fab, Fab', F(ab').sub.2, and
Fv fragments; diabodies; linear antibodies; single-chain antibody
molecules; and multi-specific antibodies formed from antibody
fragments.
[0108] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of antibodies wherein the
individual antibodies comprising the population are identical
except for possible naturally occurring mutations that may be
present in minor amounts. Monoclonal antibodies are highly specific
and are directed against a single antigenic site. In addition,
monoclonal antibodies may be made by any method known in the art.
For example, the monoclonal antibodies to be used in accordance
with the present invention may be made by the hybridoma method
first described by Kohler et al., Nature 256:495 (1975), or may be
made by recombinant DNA methods (see, e.g., U.S. Pat. No.
4,816,567). The "monoclonal antibodies" may also be isolated from
phage antibody libraries using the techniques described in Clackson
et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol.
222:581-597 (1991), for example.
[0109] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass.
Fragments of chimeric antibodies are also included provided they
exhibit the desired biological activity (U.S. Pat. No. 4,816,567;
and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855
(1984)).
[0110] "Humanized" forms of non-human (e.g., murine) antibodies are
antibodies that contain minimal sequence derived from non-human
immunoglobulin. Humanized antibodies are generally human
immunoglobulins in which hypervariable region residues are replaced
by hypervariable region residues from a non-human species such as
mouse, rat, rabbit or non-human primate having the desired
specificity, affinity, and capacity. Framework region (FR) residues
of the human immunoglobulin may be replaced by corresponding
non-human residues. In addition, humanized antibodies may comprise
residues that are not found in either the recipient antibody or in
the donor antibody. In general, the humanized antibody will
comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the
hypervariable regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FRs are those of
a human immunoglobulin sequence. The humanized antibody optionally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin. For further
details, see Jones et al., Nature 321:522-525 (1986); Reichmann et
al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.
2:593-596 (1992).
[0111] The term "epitope" is used to refer to binding sites for
(monoclonal or polyclonal) antibodies on protein antigens.
[0112] By "agonist antibody" is meant an antibody which is a ligand
for a receptor of the invention and thus, able to activate and/or
stimulate one or more of the effector functions of native sequence
Mrg or drg-12.
[0113] By "neutralizing antibody" is meant an antibody molecule as
herein defined which is able to block or significantly reduce an
effector function of a polypeptide of the invention. For example, a
neutralizing antibody may inhibit or reduce Mrg or drg-12
activation by a known ligand.
[0114] The term "Mrg immunoadhesin" refers to a chimeric molecule
that comprises at least a portion of an Mrg or drg-12 molecule
(native or variant) and an immunoglobulin sequence. The
immunoglobulin sequence preferably, but not necessarily, is an
immunoglobulin constant domain. Immunoadhesins can possess many of
the properties of human antibodies. Since immunoadhesins can be
constructed from a human protein sequence with a desired
specificity linked to an appropriate human immunoglobulin hinge and
constant domain (Fc) sequence, the binding specificity of interest
can be achieved using entirely human components. Such
immunoadhesins are minimally immunogenic to the patient, and are
safe for chronic or repeated use. If the two arms of the
immunoadhesin structure have different specificities, the
immunoadhesin is called a "bispecific immunoadhesin" by analogy to
bispecific antibodies.
[0115] As used herein, "treatment" is a clinical intervention made
in response to a disease, disorder or physiological condition
manifested by a patient. The aim of treatment includes the
alleviation or prevention of symptoms, slowing or stopping the
progression or worsening of a disease, disorder, or condition and
the remission of the disease, disorder or condition. "Treatment"
refers to both therapeutic treatment and prophylactic or
preventative measures. Those in need of treatment include those
already affected by a disease or disorder or undesired
physiological condition as well as those in which the disease or
disorder or undesired physiological condition is to be prevented.
Specifically, treatment may alleviate pain, including pain
resulting from an existing condition or disorder, or to prevent
pain in situations where pain is likely to be experienced.
[0116] In the methods of the present invention, the term "control"
and grammatical variants thereof, are used to refer to the
prevention, partial or complete inhibition, reduction, delay or
slowing down of an unwanted event, such as the presence or onset of
pain.
[0117] The term "effective amount" refers to an amount sufficient
to effect beneficial or desirable clinical results. An effective
amount of an agonist or antagonist is an amount that is effective
to treat a disease, disorder or unwanted physiological
condition.
[0118] "Pain" is a sensory experience perceived by nerve tissue
distinct from sensations of touch, pressure, heat and cold. The
range of pain sensations, as well as the variation of perception of
pain by individuals, renders a precise definition of pain near
impossible. In the context of the present invention, "pain" is used
in the broadest possible sense and includes nociceptive pain, such
as pain related to tissue damage and inflammation, pain related to
noxious stimuli, acute pain, chronic pain, and neuropathic
pain.
[0119] "Acute pain" is often short-lived with a specific cause and
purpose; generally produces no persistent psychological reactions.
Acute pain can occur during soft tissue injury, and with infection
and inflammation. It can be modulated and removed by treating its
cause and through combined strategies using analgesics to treat the
pain and antibiotics to treat the infection.
[0120] "Chronic pain" is distinctly different from and more complex
than acute pain. Chronic pain has no time limit, often has no
apparent cause and serves no apparent biological purpose. Chronic
pain can trigger multiple psychological problems that confound both
patient and health care provider, leading to feelings of
helplessness and hopelessness. The most common causes of chronic
pain include low-back pain, headache, recurrent facial pain, pain
associated with cancer and arthritis pain.
[0121] The pain is termed "neuropathic" when it is taken to
represent neurologic dysfunction. "Neuropathic pain" has a complex
and variable etiology. It is typically characterized by
hyperalgesia (lowered pain threshold and enhanced pain perception)
and by allodynia (pain from innocuous mechanical or thermal
stimuli). Neuropathic pain is usually chronic and tends not to
respond to the same drugs as "normal pain" (nociceptive pain),
therefore, its treatment is much more difficult. Neuropathic pain
may develop whenever nerves are damaged, by trauma, by disease such
as diabetes, herpes zoster, or late-stage cancer, or by chemical
injury (e.g., as an untoward consequence of agents including the
false-nucleotide anti-HIV drugs). It may also develop after
amputation (including mastectomy). Examples of neuropathic pain
include monoradiculopathies, trigeminal neuralgia, postherpetic
neuralgia, complex regional pain syndromes and the various
peripheral neuropathies. This is in contrast with "normal pain" or
"nociceptive pain," which includes normal post-operative pain, pain
associated with trauma, and chronic pain of arthritis.
[0122] "Peripheral neuropathy" is a neurodegenerative disorder that
affects the peripheral nerves, most often manifested as one or a
combination of motor, sensory, sensorimotor, or autonomic
dysfunction. Peripheral neuropathies may, for example, be
characterized by the degeneration of peripheral sensory neurons,
which may result from a disease or disorder such as diabetes
(diabetic neuropathy), alcoholism and acquired immunodeficiency
syndrome (AIDS), from therapy such as cytostatic drug therapy in
cancer, or from genetic predisposition. Genetically acquired
peripheral neuropathies include, for example, Krabbe's disease,
Metachromatic leukodystrophy, and Charcot-Marie-Tooth (CMT)
Disease. Peripheral neuropathies are often accompanied by pain.
[0123] "Pharmaceutically acceptable" carriers, excipients, or
stabilizers are ones which are nontoxic to the cell or mammal being
exposed thereto at the dosages and concentrations employed. Often
the physiologically acceptable carrier is an aqueous pH buffered
solution such as phosphate buffer or citrate buffer. The
physiologically acceptable carrier may also comprise one or more of
the following: antioxidants including ascorbic acid, low molecular
weight (less than about 10 residues) polypeptides, proteins, such
as serum albumin, gelatin, immunoglobulins; hydrophilic polymers
such as polyvinylpyrrolidone, amino acids, carbohydrates including
glucose, mannose, or dextrins, chelating agents such as EDTA, sugar
alcohols such as mannitol or sorbitol, salt-forming counterions
such as sodium, and nonionic surfactants such as Tween.TM.,
polyethylene glycol (PEG), and Pluronics.TM..
[0124] "Peptide mimetics" are molecules which serve as substitutes
for peptides in interactions with the receptors of the present
invention (Morgan et al., Ann. Reports Med. Chem. 24:243-252
(1989)). Peptide mimetics, as used herein, include synthetic
structures that retain the structural and functional features of a
peptide. Peptide mimetics may or may not contain amino acids and/or
peptide bonds. The term, "peptide mimetics" also includes peptoids
and oligopeptoids, which are peptides or oligomers of N-substituted
amino acids (Simon et al., Proc. Natl. Acad. Sci. USA 89:9367-9371
(1972)). Further included as peptide mimetics are peptide
libraries, which are collections of peptides designed to be of a
given amino acid length and representing all conceivable sequences
of amino acids corresponding thereto.
[0125] A. Proteins Expressed in Primary Sensory Neurons of Dorsal
Root Ganglia
[0126] The present invention provides isolated mrg and drg-12
proteins, allelic variants of the proteins, and conservative amino
acid substitutions of the proteins. Polypeptide sequences of
several Mrg proteins of the present invention are provided in SEQ
ID NOs: 2, 4 ,6, 8, 10, 12, 16, 18, 21, 23, 25, 27, 31, 33, 35, 37,
39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,
73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,
105, 107 and 109. Polypeptide sequences of several drg-12 proteins
of the present invention are provided in SEQ ID NOs: 14, 19 and
29.
[0127] The proteins of the present invention further include
insertion, deletion or conservative amino acid substitution
variants of the sequences set forth in SEQ ID NOs: 2, 4, 6, 8, 10,
12, 14, 16, 18, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,
45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,
79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 and
109.
[0128] Ordinarily, the variants, allelic variants, the conservative
substitution variants, and the members of the protein family,
including corresponding homologues in other species, will have an
amino acid sequence having at least about 50%, or about 60% to 75%
amino acid sequence identity with the sequences set forth in SEQ ID
NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 19, 21, 23, 25, 27, 29, 31,
33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,
67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,
101, 103, 105, 107 or 109, more preferably at least about 80%, even
more preferably at least about 90%, and most preferably at least
about 95% sequence identity with said sequences.
[0129] The proteins of the present invention include molecules
having the amino acid sequence disclosed in SEQ ID NOs: 2, 4, 6, 8,
10, 12, 14, 16, 18, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,
43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,
77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107
and 109; fragments thereof having a consecutive sequence of at
least about 3, 4, 5, 6, 10, 15, 20, 25, 30, 35 or more amino acid
residues of the protein; amino acid sequence variants wherein one
or more amino acid residues has been inserted N- or C-terminal to,
or within, the disclosed coding sequence; and amino acid sequence
variants of the disclosed sequence, or their fragments as defined
above, that have been substituted by another residue. Such
fragments, also referred to as peptides or polypeptides, may
contain antigenic regions, functional regions of the protein
identified as regions of the amino acid sequence which correspond
to known protein domains, as well as regions of pronounced
hydrophilicity. The regions are all easily identifiable by using
commonly available protein sequence analysis software such as
MACVECTOR.TM. (Oxford Molecular).
[0130] Contemplated variants further include those containing
predetermined mutations by, e.g., homologous recombination,
site-directed or PCR mutagenesis, and the corresponding proteins of
other animal species, including but not limited to rabbit, rat,
porcine, bovine, ovine, equine, human and non-human primate
species, and the alleles or other naturally occurring variants of
the family of proteins; and derivatives wherein the protein has
been covalently modified by substitution, chemical, enzymatic, or
other appropriate means with a moiety other than a naturally
occurring amino acid (for example a detectable moiety such as an
enzyme or radioisotope).
[0131] Protein domains such as a ligand binding domain, an
extracellular domain, a transmembrane domain (e.g. comprising seven
membrane spanning segments and cytosolic loops or two membrane
spanning domains and cytosolic loops), the transmembrane domain and
a cytoplasmic domain and an active site may all be found in the
proteins or polypeptides of the invention. Such domains are useful
for making chimeric proteins and for in vitro assays of the
invention.
[0132] Variations in native sequence proteins of the present
invention or in various domains identified therein, can be made,
for example, using any techniques known in the art. Variation can
be achieved, for example, by substitution of at least one amino
acid with any other amino acid in one or more of the domains of the
protein. A change in the amino acid sequence of a protein of the
invention as compared with a native sequence protein may be
produced by a substitution, deletion or insertion of one or more
codons encoding the protein. A comparison of the sequence of the
Mrg or drg-12 polypeptide to be changed with that of homologous
known protein molecules may provide guidance as to which amino acid
residues may be inserted, substituted or deleted without affecting
a desired biological activity. In particular, it may be beneficial
to minimize the number of amino acid sequence changes made in
regions of high homology. Amino acid substitutions can be the
result of replacing one amino acid with another amino acid having
similar structural and/or chemical properties, such as the
replacement of a leucine with a serine, i.e., conservative amino
acid replacements. Insertions or deletions may optionally be in the
range of about 1 to 5 amino acids. The variation allowed may be
determined by systematically making insertions, deletions or
substitutions of amino acids in the sequence and testing the
resulting variants for activity exhibited by the full-length or
mature native sequence.
[0133] Polypeptide fragments are also useful in the methods of the
present invention. Such fragments may be truncated at the
N-terminus or C-terminus, or may lack internal residues, for
example, when compared with a full-length native protein. Certain
fragments lack amino acid residues that are not essential for a
desired biological activity of the Mrg or drg-12 polypeptide.
[0134] Mrg or drg-12 fragments may be prepared by any of a number
of conventional techniques. Desired peptide fragments may be
chemically synthesized or generated by enzymatic digestion, such as
by treating the protein with an enzyme known to cleave proteins at
sites defined by particular amino acid residues. Alternatively, the
DNA encoding the protein may be digested with suitable restriction
enzymes and the desired fragment isolated. Yet another suitable
technique involves isolating and amplifying a DNA fragment encoding
a desired polypeptide fragment, by polymerase chain reaction (PCR).
Oligonucleotides that define the desired termini of the DNA
fragment are employed at the 5' and 3' primers in the PCR.
Preferably, Mrg or drg-12 polypeptide fragments share at least one
biological and/or immunological activity with a native Mrg or
drg-12 polypeptide, respectively.
[0135] In making amino acid sequence variants that retain the
required biological properties of the corresponding native
sequences, the hydropathic index of amino acids may be considered.
For example, it is known that certain amino acids may be
substituted for other amino acids having a similar hydropathic
index or score without significant change in biological activity.
Thus, isoleucine, which has a hydropathic index of +4.5, can
generally be substituted for valine (+4.2) or leucine (+3.8),
without significant impact on the biological activity of the
polypeptide in which the substitution is made. Similarly, usually
lysine (-3.9) can be substituted for arginine (-4.5), without the
expectation of any significant change in the biological properties
of the underlying polypeptide. Other considerations for choosing
amino acid substitutions include the similarity of the side-chain
substituents, for example, size, electrophilic character, charge in
various amino acids. In general, alanine, glycine and serine;
arginine and lysine; glutamate and aspartate; serine and threonine;
and valine, leucine and isoleucine are interchangeable, without the
expectation of any significant change in biological properties.
Such substitutions are generally referred to as conservative amino
acid substitutions, and are the preferred type of substitutions
within the polypeptides of the present invention.
[0136] Non-conservative substitutions will entail exchanging a
member of one class of amino acids for another class. Such
substituted residues also may be introduced into the conservative
substitution sites or, more preferably, into the remaining
(non-conserved) sites.
[0137] The variations can be made using methods known in the art
such as site-directed mutagenesis, alanine scanning mutagenesis,
and PCR mutagenesis. Site-directed mutagenesis (Carter et al.,
Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res.,
10:6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34:315
(1985)), restriction selection mutagenesis (Wells et al., Philos.
Trans. R. Soc. London SerA, 317:415 (1986)) or other known
techniques can be performed on cloned DNA to produce the Mrg or
drg-12 variant DNA.
[0138] Scanning amino acid analysis can be employed to identify one
or more amino acids that can be replaced without a significant
impact on biological activity. Among the preferred scanning amino
acids are relatively small, neutral amino acids. Such amino acids
include alanine, glycine, serine, and cysteine. Alanine is
preferred because, in addition to being the most common amino acid,
it eliminates the side-chain beyond the beta-carbon and is
therefore less likely to alter the main-chain conformation of the
variant (Cunningham and Wells, Science, 244: 1081-1085 (1989)).
Further, alanine is frequently found in both buried and exposed
positions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.);
Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does
not yield adequate amounts of variation, an isoteric amino acid can
be used.
[0139] As described below, members of the family of proteins can be
used: 1) to identify agents which modulate at least one activity of
the protein; 2) to identify binding partners for the protein, 3) as
an antigen to raise polyclonal or monoclonal antibodies, 4) as a
therapeutic target, 5) as diagnostic markers to specific
populations of pain sensing neurons and 6) as targets for structure
based ligand identification.
[0140] B. Nucleic Acid Molecules
[0141] The present invention further provides nucleic acid
molecules that encode the mrg or drg-12 proteins having SEQ ID NO:
2, 4, 6, 8, 10, 12, 14, 16, 18, 19, 21, 23, 25, 27, 29, 31, 33, 35,
37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69,
71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,
103, 105, 107 or 109 and the related polypeptides herein described,
preferably in isolated form. cDNAs encoding eight full-length
variants of Mrg receptors (mMrgA1-8) are provided in FIG. 6A (SEQ
ID NO: 1, 3, 5, 11, 20, 22, 24, 26).
[0142] Preferred molecules are those that hybridize under the above
defined stringent conditions to the complement of SEQ ID NO: 1, 3,
5, 7, 9, 11, 13, 15, 17, 20, 22, 24, 26 or 28, 30, 32, 34, 36, 38,
40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,
7274, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,
104, 106 or 108 and which encode a functional peptide. Preferred
hybridizing molecules are those that hybridize under the above
conditions to the complement strand of the open reading frame or
coding sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 20,
22, 24, 26 or 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,
54, 56, 58, 60, 62, 64, 66, 68, 70, 7274, 76, 78, 80, 82, 84, 86,
88, 90, 92, 94, 96, 98, 100, 102, 104, 106 or 108.
[0143] It is not intended that the methods of the present invention
be limited by the source of the polynucleotide. The polynucleotide
can be from a human or non-human mammal, derived from any
recombinant source, synthesized in vitro or by chemical synthesis.
The nucleotide may be DNA or RNA and may exist in a
double-stranded, single-stranded or partially double-stranded
form.
[0144] Nucleic acids useful in the present invention include, by
way of example and not limitation, oligonucleotides such as
antisense DNAs and/or RNAs; ribozymes; DNA for gene therapy; DNA
and/or RNA chimeras; various structural forms of DNA including
single-stranded DNA, double-stranded DNA, supercoiled DNA and/or
triple-helix DNA; Z-DNA; and the like. The nucleic acids may be
prepared by any conventional means typically used to prepare
nucleic acids in large quantity. For example, DNAs and RNAs may be
chemically synthesized using commercially available reagents and
synthesizers by methods that are well-known in the art (see, e.g.,
Gait, 1985, Oligonucleotide Synthesis: A Practical Approach, IRL
Press, Oxford, England).
[0145] Any mRNA transcript encoded by Mrg or drg-12 nucleic acid
sequences may be used in the methods of the present invention,
including in particular, mRNA transcripts resulting from
alternative splicing or processing of mRNA precursors.
[0146] Nucleic acids having modified nucleoside linkages may also
be used in the methods of the present invention. Modified nucleic
acids may, for example, have greater resistance to degradation.
Such nucleic acids may be synthesized using reagents and methods
that are well known in the art. For example, methods for
synthesizing nucleic acids containing phosphonate phosphorothioate,
phosphorodithioate, phosphoramidate methoxyethyl phosphoramidate,
formacetal, thioformacetal, diisopropylsilyl, acetamidate,
carbamate, dimethylene-sulfide (--CH.sub.2--S--CH.sub.2),
dimethylene-sulfoxide (--CH.sub.2--SO--CH.sub.- 2),
dimethylene-sulfone (--CH.sub.2--SO.sub.2--CH.sub.2), 2'-O-alkyl,
and 2'-deoxy-2'-fluoro phosphorothioate internucleoside linkages
are well known in the art.
[0147] In some embodiments of the present invention, the nucleotide
used is an a-anomeric nucleotide. An a-anomeric nucleotide forms
specific double-stranded hybrids with complementary RNA in which,
contrary to the usual .beta.-units, the strands run parallel to
each other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641).
The nucleotide may be a 2'-0-methylribonucleotide (Inoue et al.,
1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA
analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).
[0148] Means for purifying the nucleic acids of the present
invention are well known in the art and the skilled artisan will be
able to choose the most appropriate method of purification for the
particular circumstances. Such a choice may be made, in part, based
on the size of the DNA, the amount to be purified and the desired
purity. For example, the nucleic acids can be purified by reverse
phase or ion exchange HPLC, size exclusion chromatography or gel
electrophoresis.
[0149] Isolated or purified polynucleotides having at least 10
nucleotides (i.e., a hybridizable portion) of an Mrg or drg-12
coding sequence or its complement may also be used in the methods
of the present invention. In other embodiments, the polynucleotides
contain at least 25 (continuous) nucleotides, 50 nucleotides, 100
nucleotides, 150 nucleotides, or 200 nucleotides of an Mrg coding
sequence, or a full-length Mrg coding sequence. Nucleic acids can
be single or double stranded. Additionally, the invention relates
to polynucleotides that selectively hybridize to a complement of
the foregoing coding sequences. In preferred embodiments, the
polynucleotides contain at least 10, 25, 50, 100, 150 or 200
nucleotides or the entire length of an Mrg coding sequence.
[0150] Nucleotide sequences that encode a mutant of an Mrg protein,
peptide fragments of Mrg, truncated forms of Mrg, and Mrg fusion
proteins may also be useful in the methods of the present
invention. Nucleotides encoding fusion proteins may include, but
are not limited to, full length Mrg sequences, truncated forms of
Mrg, or nucleotides encoding peptide fragments of Mrg fused to an
unrelated protein or peptide, such as for example, a domain fused
to an Ig Fc domain or fused to an enzyme such as a fluorescent
protein or a luminescent protein which can be used as a marker.
[0151] Furthermore, polynucleotide variants that have been
generated, at least in part, by some form of directed evolution,
such as gene shuffling or recursive sequence recombination may be
used in the methods of the present invention. For example, using
such techniques novel sequences can be generated encoding proteins
similar to Mrg or drg-12 but having altered functional or
structural characteristics.
[0152] Highly related gene homologs of the Mrg encoding
polynucleotide sequences described above may also be useful in the
present invention. Highly related homologs can encode proteins
sharing functional activities with Mrg proteins.
[0153] The present invention further provides fragments of the
encoding nucleic acid molecule. Fragments of the encoding nucleic
acid molecules of the present invention (i.e., synthetic
oligonucleotides) that are used as probes or specific primers for
the polymerase chain reaction (PCR), or to synthesize gene
sequences encoding proteins of the invention, can easily be
synthesized by chemical techniques, for example, the
phosphotriester method of Matteucci, et al., (J. Am. Chem. Soc.
103:3185-3191, 1981) or using automated synthesis methods. In
addition, larger DNA segments can readily be prepared by well known
methods, such as synthesis of a group of oligonucleotides that
define various modular segments of the gene, followed by ligation
of oligonucleotides to build the complete modified gene.
[0154] The encoding nucleic acid molecules of the present invention
may further be modified so as to contain a detectable label for
diagnostic and probe purposes. A variety of such labels are known
in the art and can readily be employed with the encoding molecules
herein described. Suitable labels include, but are not limited to,
biotin, radiolabeled nucleotides and the like. A skilled artisan
can readily employ any such label to obtain labeled variants of the
nucleic acid molecules of the invention.
[0155] Any nucleotide sequence which encodes the amino acid
sequence of a protein of the invention can be used to generate
recombinant molecules which direct the expression of the protein,
as described in more detail below. In addition, the methods of the
present invention may also utilize a fusion polynucleotide
comprising an Mrg or drg-12 coding sequence and a second coding
sequence for a heterologous protein.
[0156] C. Isolation of other Related Nucleic Acid Molecules
[0157] As described above, the identification and characterization
of a nucleic acid molecule encoding an mrg or drg-12 protein allows
a skilled artisan to isolate nucleic acid molecules that encode
other members of the same protein family in addition to the
sequences herein described
[0158] Essentially, a skilled artisan can readily use the amino
acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 19, 21,
23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,
57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,
91, 93, 95, 97, 99, 101, 103, 105, 107 or 109 to generate antibody
probes to screen expression libraries prepared from appropriate
cells. Typically, polyclonal antiserum from mammals such as rabbits
immunized with the purified protein (as described below) or
monoclonal antibodies can be used to probe a mammalian cDNA or
genomic expression library, such as a lambda gtll library, to
obtain the appropriate coding sequence for other members of the
protein family. The cloned cDNA sequence can be expressed as a
fusion protein, expressed directly using its own control sequences,
or expressed by constructions using control sequences appropriate
to the particular host used for expression of the protein.
[0159] Alternatively, a portion of the coding sequence herein
described can be synthesized and used as a probe to retrieve DNA
encoding a member of the Mrg protein family from cells derived from
any mammalian organism, particularly cells believed to express Mrg
proteins. Oligomers containing approximately 18-20 nucleotides
(encoding about a 6-7 amino acid stretch) are prepared and used to
screen genomic DNA or cDNA libraries to obtain hybridization under
stringent conditions or conditions of sufficient stringency to
eliminate an undue level of false positives. Oligonucleotides
corresponding to either the 5' or 3' terminus of the coding
sequence may be used to obtain longer nucleotide sequences.
[0160] It may be necessary to screen multiple cDNA libraries to
obtain a full-length cDNA. In addition, it may be necessary to use
a technique such as the RACE (Rapid Amplification of cDNA Ends)
technique to obtain the complete 5' terminal coding region. RACE is
a PCR-based strategy for amplifying the 5' end of incomplete cDNAs.
To obtain the 5' end of the cDNA, PCR is carried out on
5'-RACE-Ready cDNA using an anchor primer and a 3' primer. A second
PCR is then carried out using the anchored primer and a nested 3'
primer. Once a full length cDNA sequence is obtained, it may be
translated into amino acid sequence and examined for identifiable
regions such as a continuous open reading frame flanked by
translation initiation and termination sites, a potential signal
sequence and finally overall structural similarity to the protein
sequences disclosed herein.
[0161] Related nucleic acid molecules may also be retrieved by
using pairs of oligonucleotide primers in a polymerase chain
reaction (PCR) to selectively clone an encoding nucleic acid
molecule. The oligonucleotide primers may be degenerate
oligonucleotide primer pools designed on the basis of the protein
coding sequences disclosed herein. The template for the reaction
may be cDNA obtained by reverse transcription (RT) of mRNA prepared
from, for example, human or non-human cell lines or tissues known
or suspected to express an Mrg or drg-12 gene allele, such as DRG
tissue. A PCR denature/anneal/extend cycle for using such PCR
primers is well known in the art and can readily be adapted for use
in isolating other encoding nucleic acid molecules.
[0162] The PCR product may be subcloned and sequenced to ensure
that the amplified sequences represent the sequences of an Mrg or
drg-12 coding sequence. The PCR fragment may then be used to
isolate a full-length cDNA clone by a variety of methods. For
example, the amplified fragment may be labeled and used to screen a
cDNA library. Alternatively, the labeled fragment may be used to
isolate genomic clones via the screening of a genomic library.
[0163] PCR technology may also be utilized to isolate full-length
cDNA sequences. RNA may be isolated, from an appropriate cellular
or tissue source, such as dorsal root ganglion (DRG) and an RT
reaction may be carried out using an oligonucleotide primer
specific for the most 5' end of the amplified fragment to prime
first strand synthesis. The resulting RNA/DNA hybrid may then be
"tailed" with guanines in a terminal transferase reaction, the
hybrid may be digested with RNAase H, and second strand synthesis
may then be primed with a poly-C primer. This allows isolation of
cDNA sequences upstream of the amplified fragment.
[0164] Nucleic acid molecules encoding other members of the mrg and
drg-12 families may also be identified in existing genomic or other
sequence information using any available computational method,
including but not limited to: PSI-BLAST (Altschul, et al. (1997)
Nucleic Acids Res. 25:3389-3402); PHI-BLAST (Zhang, et al. (1998),
Nucleic Acids Res. 26:3986-3990), 3D-PSSM (Kelly et al. J. Mol.
Biol. 299(2): 499-520 (2000)); and other computational analysis
methods (Shi et al. Biochem. Biophys. Res. Commun. 262(1):132-8
(1999) and Matsunami et. al. Nature 404(6778):601-4 (2000).
[0165] A cDNA clone of a mutant or allelic variant of an Mrg or
drg-12 gene may also be isolated. A possible source of a mutant or
variant protein is tissue known to express Mrg or drg-12, such as
DRG tissue, obtained from an individual putatively carrying a
mutant or variant form of Mrg or drg-12. Such an individual may be
identified, for example, by a demonstration of increased or
decreased responsiveness to painful stimuli. In one embodiment, a
mutant or variant Mrg or drg-12 gene may be identified by PCR. The
first cDNA strand may be synthesized by hybridizing an oligo-dT
oligonucleotide to mRNA isolated from the tissue putatively
carrying a variant and extending the new strand with reverse
transcriptase. The second strand of the cDNA is then synthesized
using an oligonucleotide that hybridizes specifically to the 5' end
of the normal gene. Using these two primers, the product is then
amplified via PCR, cloned into a suitable vector, and subjected to
DNA sequence analysis through methods well known to those of skill
in the art. By comparing the. DNA sequence of the mutant Mrg allele
to that of the normal Mrg allele, the mutation(s) responsible for
any loss or alteration of function of the mutant Mrg gene product
can be ascertained.
[0166] Alternatively, a genomic library can be constructed using
DNA obtained from an individual suspected of or known to carry a
mutant Mrg allele, or a cDNA library can be constructed using RNA
from a tissue known, or suspected, to express a mutant Mrg allele.
An unimpaired Mrg gene or any suitable fragment thereof may then be
labeled and used as a probe to identify the corresponding mutant
Mrg allele in such libraries. Clones containing the mutant Mrg gene
sequences may then be purified and subjected to sequence analysis
according to methods well known to those of skill in the art.
[0167] Additionally, an expression library can be constructed
utilizing cDNA synthesized from, for example, RNA isolated from a
tissue known, or suspected, to express a mutant Mrg allele in an
individual suspected of carrying such a mutant allele. In this
manner, gene products made by the putatively mutant tissue may be
expressed and screened using standard antibody screening techniques
in conjunction with antibodies raised against the normal Mrg gene
product, as described, below.
[0168] D. Recombinant DNA Molecules Containing a Nucleic Acid
Molecule
[0169] The present invention further provides recombinant DNA
molecules (rDNAs) that contain a coding sequence. As used herein, a
rDNA molecule is a DNA molecule that has been subjected to
molecular manipulation in situ. Methods for generating rDNA
molecules are well known in the art, for example, see Sambrook et
al., Molecular Cloning: A Laboratory Manual, 2nd edition, 1989;
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. In
the preferred rDNA molecules, a coding DNA sequence is operably
linked to expression control sequences and/or vector sequences.
[0170] Thus the present invention also contemplates DNA vectors
that contain any of the Mrg or drg-12 coding sequences and/or their
complements, optionally associated with a regulatory element that
directs the expression of the coding sequences. The choice of
vector and/or expression control sequences to which one of the
protein family encoding sequences of the present invention is
operably linked depends directly, as is well known in the art, on
the functional properties desired, e.g., protein expression, and
the host cell to be transformed. A vector contemplated by the
present invention is at least capable of directing the replication
or insertion into the host chromosome, and preferably also
expression, of the structural gene included in the rDNA
molecule.
[0171] Both cloning and expression vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. In cloning vectors this sequence is one that
enables the vector to replicate independently of the host
chromosomal DNA, and includes origins of replication or
autonomously replicating sequences. Such sequences are well known
for a variety of bacteria, yeast, and viruses. The origin of
replication from the plasmid pBR322 is suitable for most
Gram-negative bacteria, the 2.mu. plasmid origin is suitable for
yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or
BPV) are useful for cloning vectors in mammalian cells.
[0172] In addition to being capable of replication in at least one
class of organism most expression vectors can be transfected into
another organism for expression. For example, a vector is
replicated in E. coli and then the same vector is transfected into
yeast or mammalian cells for expression.
[0173] DNA may also be amplified by insertion into the host genome.
For example, transfection of Bacillus with a vector comprising a
DNA sequence complementary to a Bacillus genomic sequence results
in homologous recombination with the genome and insertion of the
DNA from the vector. One disadvantage to this type of system is
that the recovery of genomic DNA encoding the protein of interest
is more complex than that of an exogenously replicated vector
because restriction enzyme digestion is required to excise the
DNA.
[0174] Expression control elements that are used for regulating the
expression of an operably linked protein encoding sequence are
known in the art and include, but are not limited to, inducible
promoters, constitutive promoters, secretion signals, and other
regulatory elements. Preferably, the inducible promoter is readily
controlled, such as being responsive to a nutrient in the host
cell's medium.
[0175] In one embodiment, the vector containing a coding nucleic
acid molecule will include a prokaryotic replicon, i.e., a DNA
sequence having the ability to direct autonomous replication and
maintenance of the recombinant DNA molecule extrachromosomally in a
prokaryotic host cell, such as a bacterial host cell, transformed
therewith. Such replicons are well known in the art. In addition,
vectors that include a prokaryotic replicon may also include a gene
whose expression confers a detectable marker such as a drug
resistance. Typical bacterial drug resistance genes are those that
confer resistance to ampicillin or tetracycline.
[0176] Vectors that include a prokaryotic replicon can further
include a prokaryotic or bacteriophage promoter capable of
directing the expression (transcription and translation) of the
coding gene sequences in a bacterial host cell, such as E. coli. A
promoter is an expression control element formed by a DNA sequence
that permits binding of RNA polymerase and transcription to occur.
Promoter sequences that are compatible with bacterial hosts are
typically provided in plasmid vectors containing convenient
restriction sites for insertion of a DNA segment of the present
invention. Typical of such vector plasmids are pUC8, pUC9, pBR322
and pBR329 available from BioRad Laboratories, (Richmond, Calif.),
pPL and pKK223 available from Pharmacia (Piscataway, N.J.).
[0177] Expression vectors compatible with eukaryotic cells,
preferably those compatible with vertebrate cells, can also be used
to form rDNA molecules that contain a coding sequence. Eukaryotic
cell expression vectors are well known in the art and are available
from several commercial sources. Typically, such vectors are
provided containing convenient restriction sites for insertion of
the desired DNA segment. Typical of such vectors are pSVL and
pKSV-10 (Pharmacia), pBPV-1/pML2d (International Biotechnologies,
Inc.), pTDT1 (ATCC, #31255), eukaryotic viral vectors such as
adenoviral or retroviral vectors, and the like eukaryotic
expression vectors.
[0178] Eukaryotic cell expression vectors used to construct the
rDNA molecules of the present invention may further include a
selectable marker that is effective in an eukaryotic cell,
preferably a drug resistance selection marker. This gene encodes a
factor necessary for the survival or growth of transformed host
cells grown in a selective culture medium. Host cells not
transformed with the vector containing the selection gene will not
survive in the culture medium. Typical selection genes encode
proteins that confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tetracycline,
complement auxotrophic deficiencies, or supply critical nutrients
withheld from the media. A preferred drug resistance marker is the
gene whose expression results in neomycin resistance, i.e., the
neomycin phosphotransferase (neo) gene. (Southern et al., J. Mol.
Anal. Genet. 1:327-341, 1982.) The selectable marker can optionally
be present on a separate plasmid and introduced by
co-transfection.
[0179] In one example of a selection system, mammalian cell
transformants are placed under selection pressure such that only
the transformants are able to survive by virtue of having taken up
the vector(s). Selection pressure is imposed by progressively
increasing the concentration of selection agent in the culture
medium, thereby stimulating amplification of both the selection
gene and the DNA that encodes the desired protein. Amplification is
the process by which genes in greater demand for the production of
a protein critical for growth are reiterated in tandem within the
chromosomes of successive generations of recombinant cells.
Increased quantities of the desired protein, such as Mrg, are
synthesized from the amplified DNA. Examples of amplifiable genes
include DHFR, thymidine kinase, metallothionein-I and -II,
adenosine deaminase, and ornithine decarboxylase.
[0180] Thus in one embodiment Chinese hamster ovary (CHO) cells
deficient in DHFR activity are prepared and propagated as described
by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). The
CHO cells are then transformed with the DHFR selection gene and
transformants are are identified by culturing in a culture medium
that contains methotrexate (Mtx), a competitive antagonist of DHFR.
The transformed cells are then exposed to increased levels of
methotrexate. This leads to the synthesis of multiple copies of the
DHFR gene, and, concomitantly, multiple copies of other DNA
comprising the expression vectors, such as the DNA encoding the
protein of interest, for example DNA encoding Mrg.
[0181] Alternatively, host cells can be transformed or
co-transformed with DNA sequences encoding a protein of interest
such as Mrg, wild-type DHFR protein, and another selectable marker
such as aminoglycoside 3'-phosphotransferase (APH). The
transformants can then be selected by growth in medium containing a
selection agent for the selectable marker such as an
aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418.
[0182] As mentioned above, expression and cloning vectors usually
contain a promoter that is recognized by the host organism and is
operably linked to the nucleic acid encoding the protein of
interest. Promoters are untranslated sequences located upstream
(5') to the start codon of a structural gene (generally within
about 100 to 1000 bp) and control the transcription and translation
of the particular nucleic acid sequence, such as an Mrg nucleic
acid sequence, to which they are operably linked. Promoters may be
inducible or constitutive. Inducible promoters initiate increased
levels of transcription from DNA under their control in response to
some change in culture conditions, such as a change in temperature.
Many different promoters are well known in the art, as are methods
for operably linking the promoter to the DNA encoding the protein
of interest. Both the native Mrg or drg-12 promoter sequence and
many heterologous promoters may be used to direct amplification
and/or expression of the Mrg or drg-12 DNA. However, heterologous
promoters are preferred, as they generally permit greater
transcription and higher yields of the desired protein as compared
to the native promoter.
[0183] Promoters suitable for use with prokaryotic hosts include,
for example, the .beta.-lactamase and lactose promoter systems
(Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature,
281:544 (1979)). However, other bacterial promoters are well known
in the art and are suitable. Promoters for use in bacterial systems
also will contain a Shine-Delgarno (S.D.) sequence operably linked
to the DNA encoding the protein of interest.
[0184] Promoter sequences that can be used in eukaryotic cells are
also well known. Virtually all eukaryotic genes have an AT-rich
region located approximately 25 to 30 bases upstream from the
transcription initiation site. Another sequence found 70 to 80
bases upstream from the start of transcription of many genes is a
CXCAAT region where X may be any nucleotide. At the 3' end of most
eukaryotic genes is an AATAAA sequence that may be the signal for
addition of the poly-A tail to the 3' end of the coding sequence.
All of these sequences may be inserted into eukaryotic expression
vectors.
[0185] Examples of suitable promoting sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase (Hitzeman
et al., J. Biol. Chem., 255:2073 (1980)) or other glycolytic
enzymes (Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland,
Biochemistry, 17:4900 (1978)).
[0186] Inducible promoters for use with yeast are also well known
and include the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in EP
73,657. Yeast enhancers also are advantageously used with yeast
promoters.
[0187] Mrg or drg-12 transcription from vectors in mammalian host
cells may also be controlled by promoters obtained from the genomes
of viruses such as polyoma virus, fowlpox virus, adenovirus, bovine
papilloma virus, avian sarcoma virus, cytomegalovirus, a
retrovirus, hepatitis-B virus and most preferably Simian Virus 40
(SV40), from heterologous mammalian promoters, e.g., the actin
promoter or an immunoglobulin promoter, from heat-shock promoters,
and from the promoter normally associated with the native sequence,
provided such promoters are compatible with the host cell
systems.
[0188] Transcription may be increased by inserting an enhancer
sequence into the vector. Enhancers are cis-acting elements of DNA,
usually about 10 to 300 bp in length, that act on a promoter to
increase its transcription. Many enhancer sequences are now known
from mammalian genes (globin, elastase, albumin, a-fetoprotein, and
insulin). Preferably an enhancer from a eukaryotic cell virus will
be used. Examples include the SV40 enhancer on the late side of the
replication origin (bp 100-270), the cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication
origin, and adenovirus enhancers. The enhancer may be spliced into
the vector at a position 5' or 3' to the protein-encoding sequence,
but is preferably located at a site 5' from the promoter.
[0189] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA.
These sequences are often found in the 5' and, occasionally 3',
untranslated regions of eukaryotic or viral DNAs or cDNAs and are
well known in the art.
[0190] Plasmid vectors containing one or more of the components
described above are readily constructed using standard techniques
well known in the art.
[0191] For analysis to confirm correct sequences in plasmids
constructed, the plasmid may be replicated in E. coli, purified,
and analyzed by restriction endonuclease digestion, and/or
sequenced by conventional methods.
[0192] Particularly useful in the preparation of proteins of the
present invention are expression vectors that provide for transient
expression in mammalian cells of DNA encoding Mrg or drg-12.
Transient expression involves the use of an expression vector that
is able to replicate efficiently in a host cell, such that the host
cell accumulates many copies of the expression vector and, in turn,
synthesizes high levels of a the polypeptide encoded by the
expression vector. Sambrook et al., supra, pp. 16.17-16.22.
Transient expression systems allow for the convenient positive
identification of polypeptides encoded by cloned DNAs, as well as
for the screening of such polypeptides for desired biological or
physiological properties. Thus, transient expression systems are
particularly useful in the invention for purposes of identifying
biologically active analogs and variants of the polypeptides of the
invention and for identifying agonists and antagonists thereof.
[0193] Other methods, vectors, and host cells suitable for
adaptation to the synthesis of Mrg or drg-12 in recombinant
vertebrate cell culture are well known in the art and are readily
adapted to the specific circumstances.
[0194] E. Host Cells Containing an Exogenously Supplied Coding
Nucleic Acid Molecule
[0195] The present invention further provides host cells
transformed with a nucleic acid molecule that encodes a protein of
the present invention. The host cell can be either prokaryotic or
eukaryotic but is preferably eukaryotic.
[0196] Eukaryotic cells useful for expression of a protein of the
invention are not limited, so long as the cell line is compatible
with cell culture methods and compatible with the propagation of
the expression vector and expression of the gene product. Such host
cells are capable of complex processing and glycosylation
activities. In principle, any higher eukaryotic cell culture is
workable, whether from vertebrate or invertebrate culture.
Preferred eukaryotic host cells include, but are not limited to,
yeast, insect and mammalian cells, preferably vertebrate cells such
as those from a mouse, rat, monkey or human cell line. Preferred
eukaryotic host cells include Chinese hamster ovary (CHO) cells
available from the ATCC as CCL61, NIH Swiss mouse embryo cells
(NIH/3T3) available from the ATCC as CRL 1658, baby hamster kidney
cells (BHK), HEK293 cells and the like eukaryotic tissue culture
cell lines.
[0197] Propagation of vertebrate cells in culture is a routine
procedure. See, e.g., Tissue Culture, Academic Press, Kruse and
Patterson, editors (1973). Additional examples of useful mammalian
host cell lines that can be readily cultured are monkey kidney CV1
line transformed by SV40 (COS-7, ATCC CRL 1651); mouse sertoli
cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); monkey
kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells
(VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA,
ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat
liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC
CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary
tumor (MMT 060562, ATCC CCL51).
[0198] Xenopus oocytes may also be directly injected with RNA
capable of expressing either the mrg or drg-12 proteins by standard
procedures (see Tominaga et al. Jpn J. Pharmacol. 83(1):20-4
(2000); Tominaga et al. Neuron 21(3):531-43 (1998) and Bisogno et
al. Biochem, Biophys. Res. Commun. 262(1):275-84 (1999)).
[0199] Examples of invertebrate cells that can be used as hosts
include plant and insect cells. Numerous baculoviral strains and
variants and corresponding permissive insect host cells are known
in the art and may be utilized in the methods of the present
invention. In addition, plant cell cultures are known and may be
transfected, for example, by incubation with Agrobacterium
tumefaciens, which has been manipulated to contain Mrg or drg-12
encoding DNA.
[0200] Any prokaryotic host can be used to express a rDNA molecule
encoding a protein or a protein fragment of the invention. Suitable
prokaryotes include eubacteria, such as Gram-negative or
Gram-positive organisms, for example, Enterobacteriaceae such as
Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella,
Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g.,
Serratia marcescans, and Shigella, as well as Bacilli such as B.
subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed
in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.
aeruginosa, and Streptomyces. The preferred prokaryotic host is E.
coli. In addition, it is preferably that the host cell secrete
minimal amounts of proteolytic enzymes.
[0201] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for Mrg- or drg-12-encoding vectors. For example, Saccharomyces
cerevisiae may be used. In addition a number of other genera,
species, and strains are commonly available and useful herein, such
as Schizosaccharomyces pombe (Beach et al. Nature 290:140 (1981);
EP 139,383); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et
al., supra) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574;
Louvencourt et al., J. Bacteriol., 737 (1983)), K. fragilis (ATCC
12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178),
K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den
Berg et al., supra), K. thermotolerans, and K. marxianus; yarrowia
(EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al. J.
Basic Microbiol., 28:265-278 (1988)); Candida; Trichoderma reesia
(EP 244,234); Neurospora crassa (Case et al. Proc. Natl. Acad. Sci.
USA, 76:5259-5263 (1979)); Schwanniomyces such as Schwanniomyces
occidentalis (EP 394,538); and filamentous fungi such as, e.g.,
Neurospora, Penicillium, Tolypocladium (WO 91/00357), and
Aspergillus hosts such as A. nidulans (Ballance et al. Biochem.
Biophys. Res. Commun., 112:284-289 (1983); Tilburn et al., Gene,
26:205-221 (1983); Yelton et al. Proc. Natl. Acad. Sci. USA,
81:1470-1474 (1984)) and A. niger (Kelly et al. EMBO J., 4:475-479
(1985)).
[0202] Transformation of appropriate cell hosts with a rDNA
molecule of the present invention is accomplished by well known
methods that typically depend on the type of vector used and host
system employed. With regard to transformation of prokaryotic host
cells, electroporation and salt treatment methods are typically
employed, see, for example, Cohen et al. Proc. Natl. Acad. Sci. USA
69:2110, (1972); and Maniatis et al., Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y. (1982). With regard to transformation of vertebrate
cells with vectors containing rDNAs, electroporation, cationic
lipid or salt treatment methods are typically employed, see, for
example, Graham et al. Virol. 52:456, (1973); Wigler et al. Proc.
Natl. Acad. Sci. USA 76:1373-76, (1979). The calcium phosphate
precipitation method is preferred. However, other methods of for
introducing DNA into cells may also be used, including nuclear
microinjection and bacterial protoplast fusion.
[0203] For transient expression of recombinant channels,
transformed host cells for the measurement of Na.sup.+ current or
intracellular Na.sup.+ levels are typically prepared by
co-transfecting constructs into cells such as HEK293 cells with a
fluorescent reporter plasmid (such as pGreen Lantern-1, Life
Technologies) using the calcium-phosphate precipitation technique
(Ukomadu et al. Neuron 8, 663-676 (1992)). After forty-eight hours,
cells with green fluorescence are selected for recording (Dib-Hajj
et al. FEBS Lett. 416, 11-14 (1997)). Similarly, for transient
expression of Mrg receptors and measurement of intracellular
Ca.sup.2+ changes in response to receptor activation as described
in Example 4, HEK cells can be co-transfected with Mrg expression
constructs and a fluorescent reporter plasmid. HEK293 cells are
typically grown in high glucose DMEM (Life Technologies)
supplemented with 10% fetal calf serum (Life Technologies).
[0204] Prokaryotic cells used to produce polypeptides of this
invention are cultured in suitable media as described generally in
Sambrook et al., supra.
[0205] The mammalian host cells used to produce the polypeptides of
this invention may be cultured in a variety of media, including but
not limited to commercially available media such as Ham's F10
(Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640
(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma). In
addition, any of the media described in Ham et al. Meth. Enz.,
58:44 (1979), Barnes et al. Anal. Biochem. 102:255 (1980), U.S.
Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as
culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleosides (such as adenosine and
thymidine), antibiotics, trace elements, and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations as determined by the
skilled practitioner. The culture conditions are those previously
used with the host cell selected for expression, and will be
apparent to the skilled artisan.
[0206] The host cells referred to in this disclosure encompass
cells in culture as well as cells that are within a host
animal.
[0207] Successfully transformed cells, i.e., cells that contain a
rDNA molecule of the present invention, can be identified by well
known techniques including the selection for a selectable marker.
For example, cells resulting from the introduction of an rDNA of
the present invention can be cloned to produce single colonies.
Cells from those colonies can be harvested, lysed and their DNA
content examined for the presence of the rDNA using a method such
as that described by Southern, J. Mol. Biol. 98:503, (1975), or
Berent et al., Biotech. 3:208, (1985) or the proteins produced from
the cell assayed via an immunological method as described
below.
[0208] Gene amplification and/or expression may be measured by any
technique known in the art, including Southern blotting, Northern
blotting to quantitate the transcription of mRNA (Thomas, Proc.
Natl. Acad. Sci. USA, 77:5201-5205 (1980)), dot blotting (DNA
analysis), or in situ hybridization, using an appropriately labeled
probe, based on the sequences provided herein. Various labels may
be employed, most commonly radioisotopes, particularly .sup.32P.
Immunological methods for measuring gene expression include
immunohistochemical staining of tissue sections or cells in
culture, as well as assaying protein levels in culture medium or
body fluids. With immunohistochemical staining techniques, a cell
sample is prepared by dehydration and fixation, followed by
reaction with labeled antibodies specific for the gene product,
where the labels are usually visually detectable, such as enzymatic
labels, fluorescent labels, luminescent labels, and the like.
[0209] Antibodies useful for immunohistochemical staining and/or
assay of sample fluids may be either monoclonal or polyclonal, and
may be prepared as described herein.
[0210] F. Production of Recombinant Proteins Using an rDNA
Molecule
[0211] The present invention further provides methods for producing
a protein of the invention using nucleic acid molecules herein
described. In general terms, the production of a recombinant form
of a protein typically involves the following steps:
[0212] A nucleic acid molecule is first obtained that encodes a mrg
or drg-12 protein of the invention, for example, nucleotides
115-1026 of SEQ ID NO: 1, nucleotides 115-1029 of SEQ ID NO: 1,
nucleotides 137-1051 of SEQ ID NO: 3, nucleotides 137-1054 of SEQ
ID NO: 3, nucleotides 165-1070 of SEQ ID NO: 5, nucleotides
165-1073 of SEQ ID NO: 5, nucleotides 1-450 of SEQ ID NO: 7,
nucleotides 1-459 of SEQ ID NO: 9, nucleotides 1820-2734 of SEQ ID
NO: 11, nucleotides 170-574 of SEQ ID NO: 13, nucleotides 170-577
of SEQ ID NO: 13, nucleotides 328-1293 of SEQ ID NO: 15,
nucleotides 328-1296 of SEQ ID NO:15, nucleotides 171-1160 of SEQ
ID NO: 17, nucleotides 171-1163 of SEQ ID NO:17, nucleotides 83-943
of SEQ ID NO: 20, nucleotides 83-946 of SEQ ID NO:20; nucleotides
16-918 of SEQ ID NO: 22, nucleotides 16-921 of SEQ ID NO: 22;
nucleotides 106-1020 of SEQ ID NO: 24, nucleotides 106-1023 of SEQ
ID NO: 24; nucleotides 45-959 of SEQ ID NO: 26, nucleotides 45-962
of SEQ ID NO: 26, nucleotides 1-405 of SEQ ID NO: 28 and
nucleotides 1-408 of SEQ ID NO: 28. If the encoding sequence is
uninterrupted by introns, as are these sequences, it is directly
suitable for expression in any host.
[0213] The nucleic acid molecule is then preferably placed in
operable linkage with suitable control sequences, as described
above, to form an expression unit containing the protein open
reading frame. The expression unit is used to transform a suitable
host and the transformed host is cultured under conditions that
allow the production of the recombinant protein. Optionally the
recombinant protein is isolated from the medium or from the cells;
recovery and purification of the protein may not be necessary in
some instances where some impurities may be tolerated or when the
recombinant cells are used, for instance, in high throughput
assays.
[0214] Each of the foregoing steps can be done in a variety of
ways. For example, the desired coding sequences may be obtained
from genomic fragments and used directly in appropriate hosts. The
construction of expression vectors that are operable in a variety
of hosts is accomplished using appropriate replicons and control
sequences, as set forth above. The control sequences, expression
vectors, and transformation methods are dependent on the type of
host cell used to express the gene and were discussed in detail
earlier. Suitable restriction sites can, if not normally available,
be added to the ends of the coding sequence so as to provide an
excisable gene to insert into these vectors. A skilled artisan can
readily adapt any host/expression system known in the art for use
with the nucleic acid molecules of the invention to produce
recombinant protein.
[0215] In one embodiment, Mrg or drg-12 may be produced by
homologous recombination. Briefly, primary human cells containing
an Mrg- or drg-12-encoding gene are transformed with a vector
comprising an amplifiable gene (such as dihydrofolate reductase
(DHFR)) and at least one flanking region of a length of at least
about 150 bp that is homologous with a DNA sequence at the locus of
the coding region of the Mrg or drg-12 gene. The amplifiable gene
must be located such that it does not interfere with expression of
the Mrg or drg-12 gene. Upon transformation the construct becomes
homologously integrated into the genome of the primary cells to
define an amplifiable region.
[0216] Transformed cells are then selected for by means of the
amplifiable gene or another marker present in the construct. The
presence of the marker gene establishes the presence and
integration of the construct into the host genome. PCR, followed by
sequencing or restriction fragment analysis may be used to confirm
that homologous recombination occurred.
[0217] The entire amplifiable region is then isolated from the
identified primary cells and transformed into host cells. Clones
are then selected that contain the amplifiable region, which is
then amplified by treatment with an amplifying agent. Finally, the
host cells are grown so as to express the gene and produce the
desired protein.
[0218] The proteins of this invention may be produced recombinantly
not only directly, but also as a fusion polypeptide with a
heterologous polypeptide. In one embodiment the heterologous
polypeptide may be a signal sequence. In general, the signal
sequence may be a component of the vector, or it may be a part of
the Mrg or drg-12 DNA that is inserted into the vector. The
heterologous signal sequence selected preferably is one that is
recognized and processed (i.e., cleaved by a signal peptidase) by
the host cell. For expression in prokaryotic host cells the signal
sequence may be a prokaryotic signal sequence selected, for
example, from the group consisting of the alkaline phosphatase,
penicillinase, lpp, and heat-stable enterotoxin II leaders. For
yeast secretion the native signal sequence may be substituted by,
e.g., the yeast invertase leader, a factor leader (including
Saccharomyces and Kluyveromyces a-factor leaders, or acid
phosphatase leader and the C. albicans glucoamylase leader). In
mammalian cell expression any native signal sequence is
satisfactory. Alternatively it may be substituted with a signal
sequence from related proteins, as well as viral secretory leaders,
for example, the herpes simplex gD signal. The DNA for such
precursor regions is ligated in reading frame to DNA encoding the
mature protein or a soluble variant thereof.
[0219] The heterologous polypeptide may also be a marker
polypeptide that can be used, for example, to identify the location
of expression of the fusion protein. The marker polypeptide may be
any known in the art, such as a fluorescent protein. A prefered
marker protein is green fluorescent protein (GFP).
[0220] G. Modifications of Mrg Polypeptides
[0221] Covalent modifications of Mrg and drg-12 and their
respective variants are included within the scope of this
invention. In one embodiment, specific amino acid residues of a
polypeptide of the invention are reacted with an organic
derivatizing agent. Derivatization with bifunctional agents is
useful, for instance, for crosslinking Mrg or Mrg fragments or
derivatives to a water-insoluble support matrix or surface for use
in methods for purifying anti-Mrg antibodies and identifying
binding partners and ligands. In addition, Mrg or Mrg fragments may
be crosslinked to each other to modulate binding specificity and
effector function. Many crosslinking agents are known in the art
and include, but are not limited to, 1,1-bis(diazoacetyl)-2-pheny-
lethane, glutaraldehyde, N-hydroxysuccinimide esters, bifunctional
maleimides such as bis-N-maleimido-1,8-octane and agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate.
[0222] Other contemplated modifications include deamidation of
glutaminyl and asparaginyl residues to the corresponding glutamyl
and aspartyl residues, respectively, hydroxylation of proline and
lysine, phosphorylation of hydroxyl groups of seryl or threonyl
residues, methylation of the .alpha.-amino groups of lysine,
arginine, and histidine side chains (T. E. Creighton, Proteins:
Structure and Molecular Properties, W.H. Freeman & Co., San
Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine,
and amidation of any C-terminal carboxyl group.
[0223] Modification of the glycosylation patterns of the
polypeptides of the invention are also contemplated. Methods for
altering the glycosylation pattern of polypeptides are well known
in the art. For example, one or more of the carbohydrate moities
found in native sequence Mrg or drg-12 may be removed chemically,
enzymatically or by modifying the glycosylation site.
Alternatively, additional gycosylation can be added, such as by
manipulating the composition of the carbohydrate moities directly
or by adding glycosylation sites not present in the native sequence
Mrg or drg-12 by altering the amino acid sequence.
[0224] Another type of covalent modification of the polypeptides of
the invention comprises linking the polypeptide or a fragment or
derivative thereof to one of a variety of nonproteinaceous
polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or
polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.
4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337.
[0225] The polypeptides of the present invention may also be
modified in a way to form a chimeric molecule comprising Mrg or
drg-12 fused to another, heterologous polypeptide or amino acid
sequence.
[0226] In one embodiment, such a chimeric molecule comprises a
fusion of the Mrg or drg-12 with a tag polypeptide that provides an
epitope to which an anti-tag antibody can selectively bind. The
epitope tag is generally placed at the amino- or carboxyl-terminus
of the polypeptide. The epitope tag allows for identification of
the chimeric protein as well as purification of the chimeric
protein by affinity purification using an anti-tag antibody or
another type of affinity matrix that binds to the epitope tag. A
number of tag polypeptides and their respective antibodies are well
known in the art. Well known tags include poly-histidine (poly-his)
or poly-histidine-glycine (poly-his-gly) tags; the flue HA tag
polypeptide (Field et al., Mol. Cell. Biol., 8:2159-2165 (1988));
the c-myc tag (Evan et al., Molecular and Cellular Biology,
5:3610-3616 (1985)); the Herpes Simplex virus glycoprotein D (gD)
tag (Paborsky et al., Protein Engineering, 3(6):547-553 (1990)) and
the Flag-peptide (Hopp et al., BioTechnology, 6:1204-1210
(1988)).
[0227] In another embodiment, the chimeric molecule comprises a
fusion of Mrg or drg-12 with an immunoglobulin or a particular
region of an immunoglobulin. To produce an immunoadhesin, the
polypeptide of the invention or a fragment or specific domain(s)
thereof could be fused to the Fc region of an IgG molecule.
Typically the fusion is to an immunoglobulin heavy chain constant
region sequence. Mrg- or drg-12-immunoglobulin chimeras for use in
the present invention are normally prepared from nucleic acid
encoding one or more extracellular domains, or fragments thereof,
of an Mrg or drg-12 receptor fused C-terminally to nucleic acid
encoding the N-terminus of an immunoglobulin constant domain
sequence. N-terminal fusions are also possible.
[0228] While not required in the immunoadhesins of the present
invention, an immunoglobulin light chain might be present either
covalently linked to an Mrg- or drg-12-immunoglobulin heavy chain
fusion polypeptide, or directly fused to Mrg or drg-12. In order to
obtain covalent association, DNA encoding an immunoglobulin light
chain may be coexpressed with the DNA encoding the Mrg- or
drg-12-immunoglobulin heavy chain fusion protein. Upon secretion,
the hybrid heavy chain and the light chain will be covalently
associated to provide an immunoglobulin-like structure comprising
two disulfide-linked immunoglobulin heavy chain-light chain
pairs.
[0229] Bispecific immunoadhesins may also be made. Such
immunoadhesins may combine an Mrg or drg-12 domain and a domain,
such as the extracellular domain, from another receptor.
Alternatively, the immunoadhesins herein might comprise portions of
two different Mrg receptors, each fused to an immunoglobulin heavy
chain constant domain sequence.
[0230] In yet another embodiment, the chimeric molecule of the
present invention comprises a fusion of Mrg or drg-12 or a fragment
or domain(s) thereof, with a heterologous receptor or fragment or
domain(s) thereof. The heterologous receptor may be a related Mrg
or drg-12 family member, or may be completely unrelated. The
heterologous protein fused to the Mrg or drg-12 protein may be
chosen to obtain a fusion protein with a desired ligand specificity
or a desired affinity for a particular ligand or to obtain a fusion
protein with a desired effector function.
[0231] H. Methods of Using Mrgs or Drgs as Molecular or Diagnostic
Probes
[0232] The sequences and antibodies, proteins and peptides of the
present invention may be used as molecular probes for the detection
of cells or tissues related to or involved with sensory perception,
especially perception of pain. Although many methods may be used to
detect the nucleic acids or proteins of the invention in situ,
preferred probes include antisense molecules and anti-mrg or
anti-drg-12 antibodies.
[0233] Probes for the detection of the nucleic acids or proteins of
the invention may find use in the identification of the involvement
of Mrg or drg-12 proteins in particular disease states, such as
glaucoma or chronic pain, or in enhanced or inhibited sensory
perception. In particular, probes of the present invention may be
useful in determining if Mrg or drg-12 expression is increased or
decreased in patients demonstrating changes in sensory perception,
such as in patients with allodynia, hyperalgesia or chronic pain,
or patients with a disease or disorder, such as glaucoma. A
determination of decreased expression or overexpression of a
polypeptide of the invention may be useful in identifying a
therapeutic approach to treating the disorder, such as by
administering Mrg or drg-12 agonists or antagonists.
[0234] Determination of changes in Mrg or drg-12 expression levels
in animal models of disease states, particularly pain, may also be
useful in identifying the types of disorders that might be
effectively treated by compounds that modify expression or
activity.
[0235] Further, the probes of the invention, including antisense
molecules and antibodies, may be used to detect the expression of
mutant or variant forms of Mrg or drg-12 variants. The ability to
detect such variants may be useful in identifying the role that the
variants play in particular disease states and in the symptoms
experienced by particular patients. Identification of the
involvement of a variant of Mrg or drg-12 in a disease or disorder
may suggest a therapeutic approach for treatment of the disease or
disorder, such as gene therapy or the administration of agonists or
antagonists known to bind the receptor variant.
[0236] In addition, probes of the invention may be used to
determine the exact expression patterns of the various Mrg and
drg-12 family members, including the relationship of one to
another. For example, the microscopy images of in situ
hybridization in FIG. 2 show the localization of antisense staining
against a nucleotide of SEQ ID NO:2 ("mrg3") and of SEQ ID NO:4
("mrg4") in transverse sections of dorsal root ganglia (DRG) from
newborn wild type (WT) and Neurogenin1 null mutant (Ngn1.sup.-/-)
mice. White dashed lines outline the DRG and black dashed lines
outline the spinal cord. Note that in the Ngn1.sup.-/- mutant, the
size of the DRG is severely reduced due to the loss of nociceptive
sensory neurons, identified using three other independent markers
(trkA; VR-1 and SNS-TTXi (Ma et al., (1999)). mrg3 is expressed in
a subset of DRG in WT mice (A) but is absent in the Ngn1.sup.-/-
DRG (B). mrg4 is expressed in a smaller subset of DRG than that of
mrg3 (C). It is also absent in the Ngn1.sup.-/- DRG (D). The loss
of mrg-expressing neurons in the Ngn1.sup.-/- DRG indicates that
these neurons are likely to be nociceptive.
[0237] Expression of mrgs in subsets of dorsal root ganglia (DRG)
neurons are shown in FIG. 2A. Frozen transverse sections of DRG
from wild-type (a-i) and ngn1.sup.-/- (j) mutant new born mice were
annealed with antisense digoxigenin RNA probes, and hybridization
was visualized with an alkaline phosphatase-conjugated antibody.
Positive signals are shown as dark purple stainings. TrkA is
expressed in a large portion of wild-type DRG neurons (a) but
absent in ngn1.sup.-/- (data not shown). Each of the eight mrg
genes (b-i) is expressed in a small subset of neurons in wild-type
DRG in completely absent in ngn1.sup.-/- DRG (j and data not
shown). Black dash line outlines the ngn1.sup.-/- mutant DRG.
[0238] In FIG. 2B, mrgs are expressed by TrKa.sup.+ nociceptive
neurons. Double labeling technique was used to colocalize TrkA
(green; [b,e]) and mrgs (red; [a,d]) in DRG neurons. During the
double labeling experiments frozen sections of wild-type DRG were
undergone in situ hybridizations with either mrg3 (a-c) or mrg5
(d-f) fluorescein-labeled antisense RNA probes followed by
anti-TrkA antibody immunostaining. The same two frames (a and b, d
and e) were digitally superimposed to reveal the extent of
colocalization (c, f). The colocalizations of TrkA with either mrg3
or mrg5 appear yellow in merged images (c, f, respectively). The
white arrowheads indicate examples of double positive cells.
[0239] In FIG. 2C, mrgs and VR1 define two different populations of
nociceptive neurons in DRG. The combination of in situ
hybridizations (red) with either mrg3 or mrg5 fluorescein-labeled
antisense RNA probes and anti-VR1 antibody immunostaining (green)
demonstrated that neither mrg3 (a-c) nor mrg5 (d-f) were expressed
by VR1-positive neurons. In the merged images (c,f), there are no
colocalizations of VR1 with either mrg3 or mrg5. The white
arrowheads are pointed to mrgs-expressing but VR1-negative
nociceptive neurons.
[0240] In FIG. 2D mrgs are shown to be expressed by IB4.sup.+
nociceptive neurons. Double labeling technique was used to
colocalize IB4 (green; [b,e]) and mrgs (red; [a,d]) in DRG neurons.
The expressions of mrg3 and mrg5 were visualized by in situ
hybridization as described before. The same DRG sections were
subsequently undergone through FITC-conjugated lectin IB4 binding.
In the merged images (c,f), there are extensive overlappings
between mrgs and IB4 stainings (yellow neurons indicated by
arrowheads).
[0241] Information about the expression patterns of the receptors
of the invention in normal tissue and tissue taken from animal
models of disease or patients suffering from a disease or disorder
will be useful in further defining the biological function of the
receptors and in tailoring treatment regimens to the specific
receptor or combination of receptors involved in a particular
disease or disorder.
[0242] I. Methods to Identify Binding Partners
[0243] As discussed in more detail below, several peptides have
been putatively identified as endogenous ligands for Mrg receptors.
In particular the RF-amide peptides, including NPAF and NPFF, have
been shown to efficiently stimulate several of the Mrg receptors.
In order to identify additional new ligands for the Mrg receptors
and ligands for drg-12, it is first necessary to indentify
compounds that bind to these receptors. Thus, another embodiment of
the present invention provides methods of isolating and identifying
binding partners or ligands of proteins of the invention.
Macromolecules that interact with Mrg are referred to, for purposes
of this discussion, as "binding partners." While the discussion
below is specficially directed to identifying binding partners for
Mrg receptors, it is contemplated that the assays of the invention
may be used to identify binding partners for drg-12 as well.
[0244] Receptor binding can be tested using Mrg receptors isolated
from their native source or synthesized directly. However, Mrg
receptors obtained by the recombinant methods described above are
preferred.
[0245] The compounds which may be screened in accordance with the
invention include, but are not limited to polypeptides, peptides,
including but not limited to members of random peptide libraries;
(see, e.g., Lam, K. S. et al., 1991, Nature 354:82-84; Houghten, R.
et al., 1991, Nature 354:84-86) and combinatorial chemistry-derived
molecular libraries made of D- and/or L-configuration amino acids,
phosphopeptides (including, but not limited to members of random or
partially degenerate, directed phosphopeptide libraries; see, e.g.,
Songyang, Z. et al., 1993, Cell 72:767-778), peptide mimetics,
antibodies (including, but not limited to, polyclonal, monoclonal,
humanized, anti-idiotypic, chimeric or single chain antibodies,
FAb, F(abN).sub.2 and FAb expression library fragments, and
epitope-binding fragments thereof), and small organic or inorganic
molecules.
[0246] The ability of candidate or test compounds to bind Mrg
receptors can be measured directly or indirectly, such as in
competitive binding assays. In competitive binding experiments, the
concentration of the test compound necessary to displace 50% of
another compound bound to the receptor (IC.sub.50) is used as a
measure of binding affinity. In these experiments the other
compound is a ligand known to bind to the Mrg receptor with high
affinity, such as an RF-amide peptide.
[0247] A variety of assay formats may be employed, including
biochemical screening assays, immunoassays, cell-based assays and
protein-protein binding assays, all of which are well characterized
in the art. In one embodiment the assay involves anchoring the test
compound onto a solid phase, adding the non-immobilized component
comprising the Mrg receptor, and detecting Mrg/test compound
complexes anchored on the solid phase at the end of the reaction.
In an alternative embodiment, the Mrg may be anchored onto a solid
surface, and the test compound, which is not anchored. In both
situations either the test compound or the Mrg receptor is labeled,
either directly or indirectly, to allow for identification of
complexes. For example, an Mrg-Ig immunoadhesin may be anchored to
a solid support and contacted with one or more test compounds.
[0248] Microtiter plates are preferably utilized as the solid phase
and the anchored component may be immobilized by non-covalent or
covalent attachments. Non-covalent attachment may be accomplished
by simply coating the solid surface with a solution of the protein
and drying. Alternatively, an immobilized antibody, preferably a
monoclonal antibody, specific for the protein to be immobilized may
be used to anchor the protein to the solid surface.
[0249] Alternatively, a reaction can be conducted in a liquid
phase, the reaction products separated from unreacted components,
and complexes detected; e.g., using an immobilized antibody
specific for either Mrg polypeptide, peptide or fusion protein or
the test compound to anchor any complexes formed in solution, and a
labeled antibody specific for the other component of the possible
complex to detect anchored complexes.
[0250] In one embodiment of these methods, a protein of the
invention or a fragment of a protein of the invention, for
instance, an extracellular domain fragment, is mixed with one or
more potential binding partners, or an extract or fraction of a
cell, under conditions that allow the association of potential
binding partners with the protein of the invention. After mixing,
peptides, polypeptides, proteins or other molecules that have
become associated with a protein of the invention are separated
from the mixture. The binding partner that bound to the protein of
the invention can then be removed, identified and further analyzed.
To identify and isolate a binding partner, the entire Mrg protein,
for instance a protein comprising the entire amino acid sequence of
SEQ ID NOs: 2, 4, 6, 8, 10, 12, 16, 18, 21, 23, 25, 27, 31, 33, 35,
37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69,
71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,
103, 105, 107 or 109 can be used. Alternatively, a fragment of the
Mrg polypeptide can be used.
[0251] As used herein, a cellular extract refers to a preparation
or fraction which is made from a lysed or disrupted cell. The
preferred source of cellular extracts will be cells derived from
DRG. Alternatively, cellular extracts may be prepared from cells
derived from any tissue, including normal human kidney tissue, or
available cell lines, particularly kidney derived cell lines.
[0252] A variety of methods can be used to obtain an extract of a
cell. Cells can be disrupted using either physical or chemical
disruption methods. Examples of physical disruption methods
include, but are not limited to, sonication and mechanical
shearing. Examples of chemical lysis methods include, but are not
limited to, detergent lysis and enzyme lysis. A skilled artisan can
readily adapt methods for preparing cellular extracts in order to
obtain extracts for use in the present methods.
[0253] Once an extract of a cell is prepared, the extract is mixed
with the protein of the invention under conditions in which
association of the protein with the binding partner can occur.
Alternatively, one or more known compounds or molecules can be
mixed with the protein of the invention. A variety of conditions
can be used, the most preferred being conditions that closely
resemble conditions found in the cytoplasm of a human cell.
Features such as osmolarity, pH, temperature, and the concentration
of cellular extract used, can be varied to optimize the association
of the protein with the binding partner.
[0254] After mixing under appropriate conditions, the bound complex
is separated from the mixture. A variety of techniques can be
utilized to separate the mixture. For example, antibodies specific
to a protein of the invention can be used to immunoprecipitate the
binding partner complex. Alternatively, standard chemical
separation techniques such as chromatography and density/sediment
centrifugation can be used.
[0255] After removal of non-associated cellular constituents found
in the extract, and/or unbound compounds or molecules, the binding
partner can be dissociated from the complex using conventional
methods. For example, dissociation can be accomplished by altering
the salt concentration or pH of the mixture.
[0256] To aid in separating associated binding partner pairs from
the mixed extract, the protein of the invention can be immobilized
on a solid support. For example, the protein can be attached to a
nitrocellulose matrix or acrylic beads. Attachment of the protein
to a solid support aids in separating peptide/binding partner pairs
from other constituents found in the extract. The identified
binding partners can be either a single protein or a complex made
up of two or more proteins or any other macromolecule.
[0257] Alternatively, binding partners may be identified using a
Far-Western assay according to the procedures of Takayama et al.
Methods Mol. Biol. 69:171-84 (1997) or Sauder et al. J Gen. Virol.
77(5): 991-6 or identified through the use of epitope tagged
proteins or GST fusion proteins.
[0258] Binding partners may also be identified in whole cell
binding assays that are well known in the art. In one embodiment,
an Mrg receptor is expressed in cells in which it is not normally
expressed, such as COS cells. The cells expressing Mrg are then
contacted with a potential binding partner that has previously been
labeled, preferably with radioactivity or a fluorescent marker. The
cells are then washed to remove unbound material and the binding of
the potential binding partner to the cells is assessed, for example
by collecting the cells on a filter and counting radioactivity. The
amount of binding of the potential binding partner to untransfected
cells or mock transfected cells is subtracted as background.
[0259] This type of assay may be carried out in several alternative
ways. For example, in one embodiment it is done using cell membrane
fractions from cells transfected with an Mrg or known to express an
Mrg, rather than whole cells. In another embodiment purified Mrg is
refolded in lipids to produce membranes that are used in the
assay.
[0260] Alternatively, the nucleic acid molecules of the invention
can be used in cell based systems to detect protein-protein
interactions (see WO99/55356). These systems have been used to
identify other protein partner pairs and can readily be adapted to
employ the nucleic acid molecules herein described.
[0261] Any method suitable for detecting protein-protein
interactions may be employed for identifying proteins, including
but not limited to soluble, transmembrane or intracellular
proteins, that interact with Mrg receptors. Among the traditional
methods which may be employed are co-immunoprecipitation,
crosslinking and co-purification through gradients or
chromatographic columns to identify proteins that interact with
Mrg. For such assays, the Mrg component can be a full-length
protein, a soluble derivative thereof, a peptide corresponding to a
domain of interest, or a fusion protein containing some region of
Mrg.
[0262] Methods may be employed which result in the simultaneous
identification of genes that encode proteins capable of interacting
with Mrg. These methods include, for example, probing expression
libraries, using labeled Mrg or a variant thereof.
[0263] One method of detecting protein interactions in vivo that
may be used to identify Mrg binding partners is the yeast
two-hybrid system. This system is well known in the art and is
commercially available from Clontech (Palo Alto, Calif.).
[0264] Briefly, two hybrid proteins are employed, one comprising
the DNA-binding domain of a transcription activator protein fused
to the Mrg receptor, or a polypeptide, peptide, or fusion protein
therefrom, and the other comprising the transcription activator
protein's activation domain fused to an unknown target protein.
These proteins are expressed in a strain of the yeast Saccharomyces
cerevisiae that contains a reporter gene (e.g., HBS or lacZ) whose
regulatory region contains the transcription activator's binding
site. While either hybrid protein alone cannot activate
transcription of the reporter gene, interaction of the two hybrid
proteins reconstitutes the functional activator protein and results
in expression of the reporter gene, which is detected by an assay
for the reporter gene product.
[0265] The target protein is preferably obtained from tissue or
cells known to express the Mrg receptor, such as DRG cells. For
example, a cDNA library prepared from DRG cells may be used.
[0266] Binding partners may also be identified by their ability to
interfere with or disrupt the interaction of known ligands. Even if
they do not activate Mrg receptors, binding partners that interfere
with interactions with known ligands may be useful in regulating or
augmenting Mrg activity in the body and/or controlling disorders
associated with Mrg activity (or a deficiency thereof).
[0267] Compounds that interfere with the interaction between Mrg
and a known ligand may be identified by preparing a reaction
mixture containing Mrg, or some variant or fragment thereof, and a
known binding partner, such as an RF-amide peptide, under
conditions and for a time sufficient to allow the two to interact
and bind, thus forming a complex. In order to test a compound for
inhibitory activity, the reaction mixture is prepared in the
presence and absence of the test compound. The test compound may be
initially included in the reaction mixture, or may be added at a
time subsequent to the addition of the Mrg and its binding partner.
Control reaction mixtures are incubated without the test compound.
The formation of any complexes between the Mrg and the binding
partner is then detected. The formation of a complex in the control
reaction, but not in the reaction mixture containing the test
compound indicates that the compound interferes with the
interaction of the Mrg and the known binding partner. Additionally,
complex formation within reaction mixtures containing the test
compound and normal Mrg protein may also be compared to complex
formation within reaction mixtures containing the test compound and
a mutant Mrg. This comparison may be important in those cases
wherein it is desirable to identify compounds that specifically
disrupt interactions of mutant, or mutated, Mrg but not the normal
proteins.
[0268] The order of addition of reactants can be varied to obtain
different information about the compounds being tested. For
example, test compounds that interfere with the interaction by
competition can be identified by conducting the binding reaction in
the presence of the test substance. In this case the test compound
is added to the reaction mixture prior to, or simultaneously with,
Mrg and the known binding partner. Alternatively, test compounds
that have the ability to disrupt preformed complexes can be
identified by adding the test compound to the reaction mixture
after complexes have been formed.
[0269] In an alternate embodiment of the invention, a preformed
complex of Mrg and an interactive binding partner is prepared in
which either the Mrg or its binding partners is labeled, but the
signal generated by the label is quenched due to formation of the
complex (see, e.g., U.S. Pat. No. 4,109,496 to Rubenstein which
utilizes this approach for immunoassays). The addition of a test
compound that competes with and displaces one of the species from
the preformed complex results in the generation of a signal above
background. In this way, test substances which disrupt the
interaction can be identified. Whole cells expressing Mrg, membrane
fractions prepared from cells expressing Mrg or membranes
containing refolded Mrg may be used in the assays described above.
However, these same asays can be employed using peptide fragments
that correspond to the binding domains of Mrg and/or the
interactive or binding partner (in cases where the binding partner
is a protein), in place of one or both of the full length proteins.
Any number of methods routinely practiced in the art can be used to
identify and isolate the binding sites. These methods include, but
are not limited to, mutagenesis of the gene encoding an Mrg protein
and screening for disruption of binding of a known ligand.
[0270] The compounds identified can be useful, for example, in
modulating the activity of wild type and/or mutant Mrg; can be
useful in elaborating the biological function of Mrg receptors; can
be utilized in screens for identifying compounds that disrupt
normal Mrg receptor interactions or may themselves disrupt or
activate such interactions; and can be useful therapeutically.
[0271] J. Methods to Identify Agents that Modulate the Expression
of a Nucleic Acid.
[0272] Another embodiment of the present invention provides methods
for identifying agents that modulate the expression of a nucleic
acid encoding a mrg or drg-12 protein of the invention or another
protein involved in an mrg or drg-12 mediated pathway. These agents
may be, but are not limited to, peptides, peptide mimetics, and
small organic molecules that are able to gain entry into an
appropriate cell (e.g., in the DRG) and affect the expression of a
gene. Agents that modulate the expression of Mrg or drg-12 or a
protein in an mrg mediated pathway may be useful therapeutically,
for example to increase or decrease sensory perception, such as the
perception of pain, to treat glaucoma, or to increase or decrease
wound healing.
[0273] Such assays may utilize any available means of monitoring
for changes in the expression level of the nucleic acids of the
invention. As used herein, an agent is said to modulate the
expression of a nucleic acid of the invention, for instance a
nucleic acid encoding the protein having the sequence of SEQ ID
NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 19, 21, 23, 25, 27, 29, 31,
33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,
67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,
101, 103, 105, 107 or 109 if it is capable of up- or
down-regulating expression of the gene or mRNA levels nucleic acid
in a cell.
[0274] In one assay format, cell lines that contain reporter gene
fusions between the open reading frames and/or the 5' or 3'
regulatory sequences of a gene of the invention and any assayable
fusion partner may be prepared. Numerous assayable fusion partners
are known and readily available including the firefly luciferase
gene and the gene encoding chloramphenicol acetyltransferase (Alam
et al. Anal. Biochem. 188:245-254 (1990)). Cell lines containing
the reporter gene fusions are then exposed to the agent to be
tested under appropriate conditions and time. Differential
expression of the reporter gene between samples exposed to the
agent and control samples identifies agents which modulate the
expression of a nucleic acid encoding a mrg or drg-12 protein.
[0275] Additional assay formats may be used to monitor the ability
of the agent to modulate the expression of a nucleic acid encoding
a mrg or drg-12 protein of the invention. For instance, mRNA
expression may be monitored directly by hybridization to the
nucleic acids of the invention. Cell lines are exposed to the agent
to be tested under appropriate conditions and time and total RNA or
mRNA is isolated by standard procedures such those disclosed in
Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Ed.
Cold Spring Harbor Laboratory Press, 1989).
[0276] Probes to detect differences in RNA expression levels
between cells exposed to the agent and control cells may be
prepared from the nucleic acids of the invention. It is preferable,
but not necessary, to design probes which hybridize only with
target nucleic acids under conditions of high stringency. Only
highly complementary nucleic acid hybrids form under conditions of
high stringency. Accordingly, the stringency of the assay
conditions determines the amount of complementarity which should
exist between two nucleic acid strands in order to form a hybrid.
Stringency should be chosen to maximize the difference in stability
between the probe:target hybrid and potential probe:non-target
hybrids.
[0277] Probes may be designed from the nucleic acids of the
invention through methods known in the art. For instance, the G+C
content of the probe and the probe length can affect probe binding
to its target sequence. Methods to optimize probe specificity are
commonly available in Sambrook et al. (Molecular Cloning: A
Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, NY,
1989) or Ausubel et al. (Current Protocols in Molecular Biology,
Greene Publishing Co., NY, 1995).
[0278] Hybridization conditions are modified using known methods,
such as those described by Sambrook et al. and Ausubel et al., as
required for each probe. Hybridization of total cellular RNA or RNA
enriched for polyA RNA can be accomplished in any available format.
For instance, total cellular RNA or RNA enriched for polyA RNA can
be affixed to a solid support and the solid support exposed to at
least one probe comprising at least one, or part of one of the
sequences of the invention under conditions in which the probe will
specifically hybridize. Alternatively, nucleic acid fragments
comprising at least one, or part of one of the sequences of the
invention can be affixed to a solid support, such as a silicon chip
or porous glass wafer. The wafer can then be exposed to total
cellular RNA or polyA RNA from a sample under conditions in which
the affixed sequences will specifically hybridize. Such wafers and
hybridization methods are widely available, for example, those
disclosed by Beattie (WO 95/11755). By examining for the ability of
a given probe to specifically hybridize to an RNA sample from an
untreated cell population and from a cell population exposed to the
agent, agents which up or down regulate the expression of a nucleic
acid encoding a mrg or drg-12 are identified.
[0279] Hybridization for qualitative and quantitative analysis of
mRNAs may also be carried out by using a RNase Protection Assay
(i.e., RPA, see Ma et al. Methods 10: 273-238 (1996)). Briefly, an
expression vehicle comprising cDNA encoding the gene product and a
phage specific DNA dependent RNA polymerase promoter (e.g., T7, T3
or SP6 RNA polymerase) is linearized at the 3' end of the cDNA
molecule, downstream from the phage promoter, wherein such a
linearized molecule is subsequently used as a template for
synthesis of a labeled antisense transcript of the cDNA by in vitro
transcription. The labeled transcript is then hybridized to a
mixture of isolated RNA (i.e., total or fractionated mRNA) by
incubation at 45.degree. C. overnight in a buffer comprising 80%
formamide, 40 mM Pipes, pH 6.4, 0.4 M NaCl and 1 mM EDTA. The
resulting hybrids are then digested in a buffer comprising 40
.mu.g/ml ribonuclease A and 2 .mu.g/ml ribonuclease. After
deactivation and extraction of extraneous proteins, the samples are
loaded onto urea/polyacrylamide gels for analysis.
[0280] In another assay format, products, cells or cell lines are
first be identified which express mrg or drg-12 gene products
physiologically. Cells and/or cell lines so identified would be
expected to comprise the necessary cellular machinery such that the
fidelity of modulation of the transcriptional apparatus is
maintained with regard to exogenous contact of agent with
appropriate surface transduction mechanisms and/or the cytosolic
cascades. Such cells or cell lines -are then transduced or
transfected with an expression vehicle (e.g., a plasmid or viral
vector) construct comprising an operable non-translated 5' or
3'-promoter containing end of the structural gene encoding the
instant gene products fused to one or more antigenic fragments,
which are peculiar to the instant gene products, wherein said
fragments are under the transcriptional control of said promoter
and are expressed as polypeptides whose molecular weight can be
distinguished from the naturally occurring polypeptides or may
further comprise an immunologically distinct tag. Such a process is
well known in the art.
[0281] Cells or cell lines transduced or transfected as outlined
above are then contacted with agents under appropriate conditions;
for example, the agent comprises a pharmaceutically acceptable
excipient and is contacted with cells comprised in an aqueous
physiological buffer such as phosphate buffered saline (PBS) at
physiological pH, Eagles balanced salt solution (BSS) at
physiological pH, PBS or BSS comprising serum or conditioned media
comprising PBS or BSS and/or serum incubated at 37.degree. C. Said
conditions may be modulated as deemed necessary by one of skill in
the art. Subsequent to contacting the cells with the agent, said
cells will be disrupted and the polypeptides of the lysate are
fractionated such that a polypeptide fraction is pooled and
contacted with an antibody to be further processed by immunological
assay (e.g., ELISA, immunoprecipitation or Western blot). The pool
of proteins isolated from the "agent-contacted" sample will be
compared with a control sample where only the excipient is
contacted with the cells and an increase or decrease in the
immunologically generated signal from the "agent-contacted" sample
compared to the control will be used to distinguish the
effectiveness of the agent.
[0282] The probes described above for identifying differential
expression of Mrg mRNA in response to applied agents can also be
used to identify differential expression of Mrg mRNA in populations
of mammals, for example populations with differing levels of
sensory perception. Methods for identifying differential expression
of genes are well known in the art. In one embodiment, mRNA is
prepared from tissue or cells taken from patients exhibiting
altered sensory perception, such as patients experiencing
neuropathic pain, or suffering from a disease or disorder in which
the Mrg receptor may play a role, such as glaucoma, and Mrg
expression levels are quantified using the probes described above.
The Mrg expression levels may then be compared to those in other
populations to determine the role that Mrg expression is playing in
the alteration of sensory perception and to determine whether
treatment aimed at increasing or decreasing Mrg expression levels
would be appropriate.
[0283] K. Methods to Identify Agents that Modulate Protein Levels
or at Least One Activity of the Proteins of DRG Primary Sensory
Neurons.
[0284] Another embodiment of the present invention provides methods
for identifying agents or conditions that modulate protein levels
and/or at least one activity of a mrg or drg-12 protein of the
invention, including agonists and antagonists. Such methods or
assays may utilize any means of monitoring or detecting the desired
activity.
[0285] In one format, the relative amounts of a protein of the
invention between a cell population that has been exposed to the
agent to be tested compared to an unexposed control cell population
may be assayed. In this format, probes such as specific antibodies
are used to monitor the differential expression of the protein in
the different cell populations. Cell lines or populations are
exposed to the agent to be tested under appropriate conditions and
time. Cellular lysates may be prepared from the exposed cell line
or population and a control, unexposed cell line or population. The
cellular lysates are then analyzed with the probe.
[0286] In another embodiment, animals known to express Mrg or
drg-12 receptors are subjected to a particular environmental
stimulus and any change produced in Mrg or drg-12 protein
expression by exposure to the stimulus is measured. Transgenic
animals, such as transgenic mice, produced to express a particular
Mrg in a particular location may be used. The environmental
stimulus is not limited and may be, for example, exposure to
stressful conditions, or exposure to noxious or painful stimuli.
Differences in Mrg receptor expression levels in response to
environmental stimuli may provide insight into the biological role
of Mrgs and possible treatments for diseases or disorders related
to the stimuli used.
[0287] Antibody probes are prepared by immunizing suitable
mammalian hosts in appropriate immunization protocols using the
peptides, polypeptides or proteins of the invention if they are of
sufficient length, or, if desired, or if required to enhance
immunogenicity, conjugated to suitable carriers. Methods for
preparing immunogenic conjugates with carriers such as BSA, KLH, or
other carrier proteins are well known in the art. In some
circumstances, direct conjugation using, for example, carbodiimide
reagents may be effective; in other instances linking reagents such
as those supplied by Pierce Chemical Co. (Rockford, Ill.), may be
desirable to provide accessibility to the hapten. The hapten
peptides can be extended at either the amino or carboxy terminus
with a cysteine residue or interspersed with cysteine residues, for
example, to facilitate linking to a carrier. Administration of the
immunogens is conducted generally by injection over a suitable time
period and with use of suitable adjuvants, as is generally
understood in the art. During the immunization schedule, titers of
antibodies are taken to determine adequacy of antibody
formation.
[0288] While the polyclonal antisera produced in this way may be
satisfactory for some applications, for pharmaceutical
compositions, use of monoclonal preparations is preferred.
Immortalized cell lines which secrete the desired monoclonal
antibodies may be prepared using the standard method of Kohler and
Milstein Nature 256:495-497 (1975)) or modifications which effect
immortalization of lymphocytes or spleen cells, as is generally
known. The immortalized cell lines secreting the desired antibodies
are screened by immunoassay in which the antigen is the peptide
hapten, polypeptide or protein. When the appropriate immortalized
cell culture secreting the desired antibody is identified, the
cells can be cultured either in vitro or by production in ascites
fluid.
[0289] The desired monoclonal antibodies are then recovered from
the culture supernatant or from the ascites supernatant. Fragments
of the monoclonals or the polyclonal antisera which contain the
immunologically significant portion can be used as antagonists, as
well as the intact antibodies. Use of immunologically reactive
fragments, such as the Fab, Fab', of F(ab').sub.2 fragments is
often preferable, especially in a therapeutic context, as these
fragments are generally less immunogenic than the whole
immunoglobulin.
[0290] The antibodies or fragments may also be produced, using
current technology, by recombinant means. Antibody regions that
bind specifically to the desired regions of the protein can also be
produced in the context of chimeras with multiple species origin,
such as humanized antibodies as discussed in more detail below.
[0291] 1. Identification of Agonists and Antagonists
[0292] The present invention provides for assays to identify
compounds that serve as agonists or antagonists of one or more of
the biological properties of Mrg and/or drg-12. Mrg agonists and
antagonists may be useful in the prevention and treatment of
problems associated with sensory perception, particularly
nociception. Mrg agonists and antagonists may alter sensory
perception, particularly the perception of pain. For example,
compounds identified as Mrg receptor agonists may be used to
stimulate Mrg receptor activation and thus may be effective in
treating mammals suffering from pain by reducing the perception of
pain. Compounds that are identified as Mrg receptor antagonists may
be used, for example, to decrease the effector functions of Mrg
receptors. This may be useful in cases where the Mrg receptors
contain a mutation that produces increased responsiveness, or in
cases of Mrg receptor overexpression. For instance, Mrg receptor
antagonists may be useful in increasing the sensitivity of mammals
to pain where appropriate, such as in diseases involving decreased
sensory responsiveness, like some forms of diabetes.
[0293] Assays for identifying agonists or antagonsts may be done in
vitro or in vivo, by monitoring the response of a cell following
binding of the ligand to the receptor. An agonist will produce a
cellular response, while an antagonist will have no effect on
cellular response but will be capable of preventing cellular
response to a known agonist.
[0294] a. Small Molecules
[0295] Small molecules may have the ability to act as Mrg agonists
or antagonists and thus may be screened for an effect on a
biological activity of Mrg. Small molecules preferably have a
molecular weight of less than 10 kD, more preferably less than 5 kD
and even more preferably less than 2 kD. Such small molecules may
include naturally occurring small molecules, synthetic organic or
inorganic compounds, peptides and peptide mimetics. However, small
molecules in the present invention are not limited to these forms.
Extensive libraries of small molecules are commercially available
and a wide variety of assays are well known in the art to screen
these molecules for the desired activity.
[0296] Candidate Mrg agonist and antagonist small molecules are
preferably first identified in an assay that allows for the rapid
identification of potential agonists and antagonists. An example of
such an assay is a binding assay wherein the ability of the
candidate molecule to bind to the Mrg receptor is measured, such as
those described above. In another example, the ability of candidate
molecules to interfere with the binding of a known ligand, for
example FMRFamide to MrgA1, is measured. Candidate molecules that
are identified by their ability to bind to Mrg proteins or
interfere with the binding of known ligands are then tested for
their ability to stimulate one or more biological activities.
[0297] The activity of the proteins of the invention may be
monitored in cells expressing the mrg and/or drg-12 proteins of the
invention by assaying for physiological changes in the cells upon
exposure to the agent or agents to be tested. Such physiological
changes include but are not limited to the flow of current across
the membrane of the cell.
[0298] In one embodiment the protein is expressed in a cell that is
capable of producing a second messenger response and that does not
normally express Mrg or drg-12. The cell is then contacted with the
compound of interest and changes in the second messenger response
are measured. Methods to monitor or assay these changes are readily
available. For instance, the mrg genes of the invention may be
expressed in cells expressing Ga15, a G protein a subunit that
links receptor activation to increases in intracellular calcium
[Ca.sup.2+] which can be monitored at the single cell level using
the FURA-2 calcium indicator dye as disclosed in Chandrashekar et
al. Cell 100:703-711, (2000). This assay is described in more
detail in Example 5.
[0299] Similar assays may also be used to identify inhibitors or
antagonists of Mrg or drg-12 activation. For example, cells
expressing Mrg or drg-12 and capable of producing a quantifiable
response to receptor activation are contacted with a known Mrg or
drg-12 activator and the compound to be tested. In one embodiment,
HEK cells expressing Ga15 and MrgA1 are contacted with FMRFamide
and the compound to be tested. The cellular response is measured,
in this case increase in [Ca.sup.2+]. A decreased response compared
to the known activator by itself indicates that the compound acts
as an inhibitor of activation.
[0300] While such assays may be formatted in any manner,
particularly preferred formats are those that allow high-throughput
screening (HTP). In HTP assays of the invention, it is possible to
screen thousands of different modulators or ligands in a single
day. For instance, each well of a microtiter plate can be used to
run a separate assay, for instance an assay based on the ability of
the test compounds to modulate receptor activation derived
increases in intracellular calcium as described above.
[0301] Agents that are assayed in the above method can be randomly
selected or rationally selected or designed. As used herein, an
agent is said to be randomly selected when the agent is chosen
randomly without considering the specific sequences involved in the
association of the a protein of the invention alone or with its
associated substrates, binding partners, etc. An example of
randomly selected agents is the use a chemical library or a peptide
combinatorial library, or a growth broth of an organism.
[0302] As used herein, an agent is said to be rationally selected
or designed when the agent is chosen on a nonrandom basis which
takes into account the sequence of the target site and/or its
conformation in connection with the agent's action. Sites of
interest might be peptides within the membrane spanning regions,
cytoplasmic and extracellular peptide loops between these
transmembrane regions, or selected sequences within the N-terminal
extracellular domain or C-terminal intracellular domain. Agents can
be rationally selected or rationally designed by utilizing the
peptide sequences that make up these sites.
[0303] The agents of the present invention can be, as examples,
peptides, small molecules, vitamin derivatives, as well as
carbohydrates. Dominant negative proteins, DNAs encoding these
proteins, antibodies to these proteins, peptide fragments of these
proteins or mimics of these proteins may be introduced into cells
to affect function. "Mimic" used herein refers to the modification
of a region or several regions of a peptide molecule to provide a
structure chemically different from the parent peptide but
topographically and functionally similar to the parent peptide (see
Grant G A. in: Meyers (ed.) Molecular Biology and Biotechnology
(New York, VCH Publishers, 1995), pp. 659-664). A skilled artisan
can readily recognize that there is no limit as to the structural
nature of the agents of the present invention.
[0304] The peptide agents of the invention can be prepared using
standard solid phase (or solution phase) peptide synthesis methods,
as is known in the art. In addition, the DNA encoding these
peptides may be synthesized using commercially available
oligonucleotide synthesis instrumentation and produced
recombinantly using standard recombinant production systems. The
production using solid phase peptide synthesis is necessitated if
non-gene-encoded amino acids are to be included.
[0305] b. Antibodies
[0306] Another class of agents of the present invention are
antibodies immunoreactive with critical positions of proteins of
the invention. These antibodies may be human or non-human,
polyclonal or monoclonal and may serve as agonist antibodies or
neutralizing antibodies. They include amino acid sequence variants,
glycosylation variants and fragments of antibodies. Antibody agents
are obtained by immunization of suitable mammalian subjects with
peptides, containing as antigenic regions, those portions of the
protein intended to be targeted by the antibodies. General
techniques for the production of such antibodies and the selection
of agonist or neutralizing antibodies are well known in the
art.
[0307] The antibodies of the present invention can be polyclonal
antibodies, monoclonal antibodies, chimeric antibodies, humanized
antibodies, human antibodies, heteroconjugate antibodies, or
antibody fragments. In addition, the antibodies can be made by any
method known in the art, including recombinant methods.
[0308] Mrg agonist and neutralizing antibodies may be preliminarily
identified based on their ability to bind the Mrg receptor. For
example, Western blot techniques well known in the art may be used
to screen a variety of antibodies for their ability to bind Mrg.
Mrg agonist and neutralizing antibodies are then identified from
the group of candidate antibodies based on their biological
activity. In one embodiment, Mrg agonist antibodies are identified
by their ability to induce activation of a second messenger system
in cells expressing the Mrg protein and comprising a second
messenger system. For example, HEK cells overexpressing Ga15 and
transfected with mrg may be contacted with a potential Mrg agonist
antibody. An increase in intracellular calcium, measured as
described in Example 5, would indicate that the antibody is an
agonist antibody.
[0309] Identification of a neutralizing antibody involves
contacting a cell expressing Mrg with a known Mrg ligand, such as
an RF-amide peptide, and the candidate antibody and observing the
effect of the antibody on Mrg activation. In one embodiment, Mrg
receptors expressed in HEK cells overexpressing Ga 15 are contacted
with an Mrg ligand such as FMRFamide and the candidate neutralizing
antibody. A decrease in responsiveness to the ligand, measured as
described in Example 5, would indicate that the antibody is a
neutralizing antibody.
[0310] c. Other Antagonists
[0311] The Mrg or drg-12 antagonists are not limited to Mrg or
drg-12 ligands. Other antagonists include variants of a native Mrg
or drg-12 receptor that retains the ability to bind an endogenous
ligand but is not able to mediate a biological response. Soluble
receptors and immunoadhesins that bind Mrg or drg-12 ligands may
also be antagonists, as may antibodies that specifically bind a
ligand near its binding site and prevent its interaction with the
native receptor. These antagonists may be identified in the assays
described above.
[0312] d. Computer Modeling
[0313] Computer modeling and searching technologies permit
identification of compounds, or the improvement of already
identified compounds, that can modulate Mrg receptor expression or
activity. Once an agonist or antagonist is identified, the active
sites or regions, such as ligand binding sites, are determined. The
active site can be identified using methods known in the art
including, for example, by determing the effect of various amino
acid substitutions or deletions on ligand binding or from study of
complexes of the relevant compound or composition with its natural
ligand, such as with X-ray crystallography.
[0314] Next, the three dimensional geometric structure of the
active site is determined such as by X-ray crystallography, NMR,
chemical crosslinking or other methods known in the art. Computer
modeling can be utilized to make predictions about the structure
where the experimental results are not clear. Examples of molecular
modeling systems are the CHARMm and QUANTA programs (Polygen
Corporation, Waltham, Mass.). Once a predicted structure is
determined, candidate modulating compounds can be identified by
searching databases containing compounds along with information on
their molecular structure in an effort to find compuonds that have
structures capable of interacting with the active site. The
compounds found from this search are potential modulators of the
activity of the proteins of the present invention and can be tested
in the assays described above.
[0315] The agonistic or antagonistic activity of test compounds
identified in cell based assays as described above can be further
elucidated in assays using animals, for example transgenic animals
that overexpress Mrg receptors as described in more detail below.
In one embodiment, the effect of administration of potential Mrg
antagonists or agonists on the responsiveness of such transgenic
animals to sensory stimuli, such as noxious or painful stimuli, is
measured. The therapeutic utility of such compounds may be
confirmed by testing in these types of experiments or in animal
models of particular disorders, for example animal models of
neuropathic pain.
[0316] L. Uses for Agents that Modulate at Least One Activity of
the Proteins.
[0317] As provided in the Examples, the mrg or drg-12 proteins and
nucleic acids of the invention, are expressed in the primary
nociceptive sensory neurons of DRG. In addition the Mrg receptors
are expressed in specialized skin cells that play a role in wound
repair. Further, proteins homologous to Mrg receptors are expressed
in the trabecular meshwork of the eye and a role for them has been
suggested in the regulation of pressure in the eye (Gonzalez et al.
Invest. Ophth. Vis. Sci. 41: 3678-3693 (2000)). Thus, the present
invention further provides compositions containing one or more
agents that modulate expression or at least one activity of a
protein of the invention. For example, the invention provides
ligands that directly activate Mrg receptors.
[0318] Agents that modulate, up-or-down-regulate the expression of
the protein or agents such as agonists or antagonists of at least
one activity of the protein may be used to modulate biological and
pathologic processes associated with the protein's function and
activity. Several agents that activate the Mrg receptors are
identified in the examples, including the RF-amide peptides. Thus
the present invention provides methods to treat impaired sensory
perception, such as pain, including neuropathic pain, as well as to
promote wound healing, to restore normal sensitivity following
injury and to treat ocular conditions, particularly those
associated with pressure, such as glaucoma.
[0319] As described in the Figures and Examples, expression of a
protein of the invention may be associated with biological
processes of nociception, which may also be considered pathological
processes. As used herein, an agent is said to modulate a
biological or pathological process when the agent alters the
degree, severity or nature of the process. For instance, the
neuronal transmission of pain signals may be prevented or modulated
by the administration of agents which up-regulate down-regulate or
modulate in some way the expression or at least one activity of a
protein of the invention.
[0320] The pain that may be treated by the proteins of the present
invention and agonists and antagonists thereof, is not limited in
any way and includes pain associated with a disease or disorder,
pain associated with tissue damage, pain associated with
inflammation, pain associated with noxious stimuli of any kind, and
neuropathic pain, including pain associated with peripheral
neuropathies, as well as pain without an identifiable source. The
pain may be subjective and does not have to be associated with an
objectively quantifiable behavior or response.
[0321] In addition to treating pain, the compounds and methods of
the present invention may be useful for increasing or decreasing
sensory responses. It may be useful to increase responsiveness to
stimuli, including noxious stimuli and painful stimuli, in some
disease states that are characterized by a decreased responsiveness
to stimuli, for example in diabetes.
[0322] Certain conditions, such as chronic disease states
associated with pain and peripheral neuropathies and particlularly
conditions resulting from a defective Mrg gene, can benefit from an
increase in the responsiveness to Mrg receptor ligands. Thus these
condition may be treated by increasing the number of functional Mrg
receptors in cells of patients suffering from such conditions. This
could be increasing the expression of Mrg receptor in cells through
gene therapy using Mrg-encoding nucleic acid. This includes both
gene therapy where a lasting effect is achieved by a single
treatment, and gene therapy where the increased expression is
transient. Selective expression of Mrg in appropriate cells may be
achieved by using Mrg genes controlled by tissue specific or
inducible promoters or by producing localized infection with
replication defective viruses carrying a recombinant Mrg gene, or
by any other method known in the art.
[0323] In a further embodiment, patients that suffer from an excess
of Mrg, hypersensitivity to Mrg ligands or excessive activation of
Mrg may be treated by administering an effective amount of
anti-sense RNA or anti-sense oligodeoxyribonucleotides
corresponding to the Mrg gene coding region, thereby decreasing
expression of Mrg.
[0324] As used herein, a subject to be treated can be any mammal,
so long as the mammal is in need of modulation of a pathological or
biological process mediated by a protein of the invention. For
example, the subject may be experiencing pain or may be
anticipating a painful event, such as surgery. The invention is
particularly useful in the treatment of human subjects.
[0325] In the therapeutic methods of the present invention the
patient is administered an effective amount of a composition of the
present invention, such as an Mrg protein, peptide fragment, Mrg
variant, Mrg agonist, Mrg antagonist, or anti-Mrg antibody of the
invention.
[0326] The agents of the present invention can be provided alone,
or in combination with other agents that modulate a particular
biological or pathological process. For example, an agent of the
present invention can be administered in combination with other
known drugs or may be combined with analgesic drugs or
non-analgesic drugs used during the treatment of pain that occurs
in the presence or absence of one or more other pathological
processes. As used herein, two or more agents are said to be
administered in combination when the two agents are administered
simultaneously or are administered independently in a fashion such
that the agents will act at the same time.
[0327] The agents of the present invention are administered to a
mammal, preferably to a human patient, in accord with known
methods. Thus the agents of the present invention can be
administered via parenteral, subcutaneous, intravenous,
intramuscular, intraperitoneal, intracerebrospinal,
intra-articular, intrasynovial, intrathecal, transdermal, topical,
inhalation or buccal routes. They may be administered continuously
by infusion or by bolus injection. Generally, where the disorder
permits the agents should be delivered in a site-specific manner.
Alternatively, or concurrently, administration may be by the oral
route. The dosage administered will be dependent upon the age,
health, and weight of the recipient, kind of concurrent treatment,
if any, frequency of treatment, and the nature of the effect
desired.
[0328] The toxicity and therapeutic efficacy of agents of the
present invention can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals. While agents
that exhibit toxic side effects can be used, care should be taken
to design a delivery system that targets such compounds to the
desired site of action in order to reduce side effects.
[0329] While individual needs vary, determination of optimal ranges
of effective amounts of each component is within the skill of the
art. For the prevention or treatment of disease, the appropriate
dosage of agent will depend on the type of disease to be treated,
the severity and course of the disease, whether the agent is
administered for preventive or therapeutic purposes, previous
therapy, the patient's clinical history and response to the agent,
and the discretion of the attending physician. Therapeutic agents
are suitably administered to the patient at one time or over a
series of treatments. Typical dosages comprise 0.1 to 100 .mu.g/kg
body wt. The preferred dosages comprise 0.1 to 10 .mu.g/kg body wt.
The most preferred dosages comprise 0.1 to 1 .mu.g/kg body wt. For
repeated administrations over several days or longer, depending on
the condition, the treatment is sustained until a desired
suppression of disease symptoms occurs. The progress of this
therapy is easily monitored by conventional techniques and
assays.
[0330] In addition to the pharmacologically active agent, the
compositions of the present invention may contain suitable
pharmaceutically acceptable carriers comprising excipients and
auxiliaries that facilitate processing of the active compounds into
preparations which can be used pharmaceutically for delivery to the
site of action. Suitable formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form, for example, water-soluble salts. In addition, suspensions of
the active compounds as appropriate oily injection suspensions may
be administered. Suitable lipophilic solvents or vehicles include
fatty oils, for example, sesame oil, or synthetic fatty acid
esters, for example, ethyl oleate or triglycerides. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension include, for example, sodium
carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the
suspension may also contain stabilizers. Liposomes can also be used
to encapsulate the agent for delivery into the cell. The agent can
also be prepared as a sustained-release formulation, including
semipermeable matrices of solid hydrophobic polymers containing the
protein. The sustained release preparation may take the form of a
gel, film or capsule.
[0331] The pharmaceutical formulation for systemic administration
according to the invention may be formulated for enteral,
parenteral or topical administration. Indeed, all three types of
formulations may be used simultaneously to achieve systemic
administration of the active ingredient.
[0332] Suitable formulations for oral administration include hard
or soft gelatin capsules, pills, tablets, including coated tablets,
elixirs, suspensions, syrups or inhalations and controlled release
forms thereof.
[0333] In practicing the methods of this invention, the compounds
of this invention may be used alone or in combination with other
therapeutic or diagnostic agents. In certain preferred embodiments,
the compounds of this invention may be co-administered along with
other compounds typically prescribed for these conditions according
to generally accepted medical practice. The compounds of this
invention can be utilized in vivo, ordinarily in mammals, such as
humans, sheep, horses, cattle, pigs, dogs, cats, rats and mice, or
in vitro. When used in vivo, the compounds must be sterile. This is
readily accomplished by filtration through sterile filtration
membranes.
[0334] a. Articles of Manufacture
[0335] In another embodiment of the invention, an article of
manufacture containing materials useful for the treatment of the
disorders described above is provided. The article of manufacture
comprises a container and a label or package insert(s) on or
associated with the container. Suitable containers include, for
example, bottles, vials, syringes, etc. The containers may be
formed from a variety of materials such as glass or plastic. The
container holds a composition which is effective for treating the
condition and may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). At least one
active agent in the composition is an Mrg agonist. The label or
package insert indicates that the composition is used for treating
the condition of choice, such as to treat impaired sensory
perception, for example to reduce neuropathic pain. In one
embodiment, the label or package inserts indicates that the
composition comprising the Mrg agonist can be used to treat pain,
glaucoma or to accelerate wound healing.
[0336] M. Transgenic Animals
[0337] Transgenic animals containing mutant, knock-out or modified
genes corresponding to the mrg and/or drg-12 sequences are also
included in the invention. Transgenic animals are genetically
modified animals into which recombinant, exogenous or cloned
genetic material has been experimentally transferred. Such genetic
material is often referred to as a "transgene". The nucleic acid
sequence of the transgene, in this case a form of SEQ ID NOS: 1, 3,
5, 7, 9, 11, 13, 15, 17, 20, 22, 24, 26 or 28, 30, 32, 34, 36, 38,
40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,
7274, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,
104, 106 or 108 may be integrated either at a locus of a genome
where that particular nucleic acid sequence is not otherwise
normally found or at the normal locus for the transgene. In
addition the transgene may encode a non-funtional variant. The
transgene may consist of nucleic acid sequences derived from the
genome of the same species or of a different species than the
species of the target animal.
[0338] The term "germ cell line transgenic animal" refers to a
transgenic animal in which the genetic alteration or genetic
information was introduced into a germ line cell, thereby
conferring the ability of the transgenic animal to transfer the
genetic information to offspring. If such offspring in fact possess
some or all of that alteration or genetic information, then they
too are transgenic animals.
[0339] The alteration or genetic information may be foreign to the
species of animal to which the recipient belongs, foreign only to
the particular individual recipient, or may be genetic information
already possessed by the recipient. In the last case, the altered
or introduced gene may be expressed differently than the native
gene.
[0340] Transgenic animals can be produced by a variety of different
methods including transfection, electroporation, microinjection,
gene targeting in embryonic stem cells and recombinant viral and
retroviral infection (see, e.g., U.S. Pat. No. 4,736,866; U.S. Pat.
No. 5,602,307; Mullins et al. Hypertension 22(4):630-633 (1993);
Brenin et al. Surg. Oncol. 6(2)99-110 (1997); Tuan (ed.),
Recombinant Gene Expression Protocols, Methods in Molecular Biology
No. 62, Humana Press (1997)).
[0341] A number of recombinant or transgenic mice have been
produced, including those which express an activated oncogene
sequence (U.S. Pat. No. 4,736,866); express simian SV40 T-antigen
(U.S. Pat. No. 5,728,915); lack the expression of interferon
regulatory factor 1 (IRF-1) (U.S. Pat. No. 5,731,490); exhibit
dopaminergic dysfunction (U.S. Pat. No. 5,723,719); express at
least one human gene which participates in blood pressure control
(U.S. Pat. No. 5,731,489); display greater similarity to the
conditions existing in naturally occurring Alzheimer's disease
(U.S. Pat. No. 5,720,936); have a reduced capacity to mediate
cellular adhesion (U.S. Pat. No. 5,602,307); possess a bovine
growth hormone gene (Clutter et al. Genetics 143(4):1753-1760
(1996)); or, are capable of generating a fully human antibody
response (McCarthy The Lancet 349(9049):405 (1997)).
[0342] While mice and rats remain the animals of choice for most
transgenic experimentation, in some instances it is preferable or
even necessary to use alternative animal species. Transgenic
procedures have been successfully utilized in a variety of
non-murine animals, including sheep, goats, pigs, dogs, cats,
monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs (see,
e.g., Kim et al. Mol. Reprod. Dev. 46(4): 515-526 (1997); Houdebine
Reprod. Nutr. Dev. 35(6):609-617 (1995); Petters Reprod. Fertil.
Dev. 6(5):643-645 (1994); Schnieke et al. Science
278(5346):2130-2133 (1997); and Amoah J. Animal Science
75(2):578-585 (1997)).
[0343] The method of introduction of nucleic acid fragments into
recombination competent mammalian cells can be by any method that
favors co-transformation of multiple nucleic acid molecules.
Detailed procedures for producing transgenic animals are readily
available to one skilled in the art, including the disclosures in
U.S. Pat. No. 5,489,743 and U.S. Pat. No. 5,602,307.
[0344] It is contemplated that mice lacking a particular Mrg or
drg-12 gene, or in which expression of a particular Mrg or drg-12
has been increased or decreased will be used in an assay for
determining how Mrgs influence behavior, including sensory
responses, particularly responses to painful stimuli. In
particular, transgenic mice will be used to determine if Mrg
mediates the response to a particular type of noxious stimuli, such
as mechanical, thermal or chemical. Thus in one embodiment
transgenic mice lacking native Mrg receptors, or in which Mrg
receptor expression levels have been modified, will be tested to
determine their sensitivity to pressure, temperature, and other
noxious stimuli. Assays for determining sensitivity to stimuli are
well known in the art. These include, but are not limited to,
assays that measure responsiveness to mechanical pain (von Frey
hairs or tail pinch), thermal pain (latency to lick or jump in the
hot plate assay), chemical pain (latency to lick when a noxious
substance such as capsaicin or formalin is injected in the paw),
visceral pain (abdominal stretching in response to intraperitoneal
injection of acetic acid) and neuropathic pain. For example, mice
in which one or more Mrgs have been deleted will be tested for
their responsiveness to a variety of painful stimuli of varying
intensity. By determining the sensory responses that are mediated
by the Mrg receptors, therapeutic agents known to stimulate or
inhibit Mrg receptors can be chosen for the treatment of disease
states known to involve these types of responses. In addition,
therapeutics specifically aimed at treating disorders involving
these responses can be developed by targeting the Mrg
receptors.
[0345] In one embodiment, transgenic mice expressing one or more
human Mrg proteins are produced. The expression pattern of the
human Mrg protein may then be determined and the effect of the
expression of the human Mrg protein on various sensory modalities
may be investigated. Further, the efficacy of potential therapeutic
agents may be investigated in these mice.
[0346] In addition, the effects of changes in the expression levels
of specific Mrg proteins can be investigated in animal models of
disease states. By identifying the effect of increasing or
decreasing Mrg receptor levels and activation, therapeutic regimens
useful in treating the diseases can be developed. In one
embodiment, mice in which Mrg receptor expression levels have been
increased or decreased are tested in models of neuropathic
pain.
[0347] Further, mice in which Mrg expression levels have been
manipulated may be tested for their ability to respond to compounds
known to modulate responsiveness to pain, such as analgesics. In
this way the role of Mrg in the sensation of pain may be further
elucidated. For example, a lack of response to a known analgesic in
the transgenic mice lacking Mrg would indicate that the Mrg
receptors play a role in mediating the action of the analgesic.
[0348] Another preferred transgenic mouse is one in which the Mrg
gene is modified to express a marker or tracer such as green
fluorescent protein (GFP). By examining the expression pattern of
the marker or tracer, the exact location and projection of Mrg
containing neurons and other cells can be mapped. This information
will be compared to the location and projection of neurons and
other cells whose involvment in specific disease states has
previously been identified. In this way additional therapeutic uses
for the compounds of the present invention may be realized.
[0349] N. Diagnostic Methods
[0350] As described in the Examples, the genes and proteins of the
invention may be used to diagnose or monitor the presence or
absence of sensory neurons and of biological or pathological
activity in sensory neurons. For instance, expression of the genes
or proteins of the invention may be used to differentiate between
normal and abnormal sensory neuronal activities associated with
acute pain, chronic intractable pain, or allodynia. Expression
levels can also be used to differentiate between various stages or
the severity of neuronal abnormalities. One means of diagnosing
pathological states of sensory neurons involved in pain
transmission using the nucleic acid molecules or proteins of the
invention involves obtaining tissue from living subjects. These
subjects may be non-human animal models of pain.
[0351] The use of molecular biological tools has become routine in
forensic technology. For example, nucleic acid probes may be used
to determine the expression of a nucleic acid molecule comprising
all or at least part of the sequences of the invention in
forensic/pathology specimens. Further, nucleic acid assays may be
carried out by any means of conducting a transcriptional profiling
analysis. In addition to nucleic acid analysis, forensic methods of
the invention may target the proteins of the invention to determine
up or down regulation of the genes (Shiverick et al., Biochim
Biophys Acta 393(1): 124-33 (1975)).
[0352] Methods of the invention may involve treatment of tissues
with collagenases or other proteases to make the tissue amenable to
cell lysis (Semenov et al., Biull Eksp Biol Med 104(7): 113-6
(1987)). Further, it is possible to obtain biopsy samples from
different regions of the kidney or other tissues for analysis.
[0353] Assays to detect nucleic acid or protein molecules of the
invention may be in any available format. Typical assays for
nucleic acid molecules include hybridization or PCR based formats.
Typical assays for the detection of proteins, polypeptides or
peptides of the invention include the use of antibody probes in any
available format such as in situ binding assays, etc. See Harlow et
al., Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, 1988 and Section G. In preferred embodiments, assays
are carried-out with appropriate controls.
[0354] The above methods may also be used in other diagnostic
protocols, including protocols and methods to detect disease states
in other tissues or organs.
[0355] O. Methods of Identifying other Genes Expressed in Primary
Nociceptive Sensory Neurons.
[0356] As described in the Examples, the mrg and drg-12 genes of
the invention have been identified RNA using a
suppression-PCR-based method (Clontech) to enrich for genes
expressed in the DRG of wild type but not Ngn1 mutant mice. This
general method may be used to identify and isolate other DRG
specific genes by producing transgenic mice that do not express
other genes required for the development or presence of the
nociceptive subset of DRG neurons. For instance, Trk.sup.-/- mice
may be used in the methods of the invention to isolate other genes
associated with nociceptive DRG neurons (see Lindsay Philos. Trans
R. Soc. Lond. B. Biol. Sci. 351(1338): 365-73 (1996) and Walsh et
al. J. Neurosci. 19(10): 4155-68).
[0357] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilized the
compounds of the present invention and practice the claimed
methods. The following working examples therefore, specifically
point out preferred embodiments of the present invention, and are
not to be construed as limiting in any way the remainder of the
disclosure.
EXAMPLES
Example 1
Positive Selection-Based Differential Hybridization between Wild
Type and Ngn1.sup.-/- DRG to Identify Candidate Genes Involved in
Nociception
[0358] Previous studies have shown that Neurogenin1 (Ngn1), a bHLH
transcription factor (Ma et al. Cell 87: 43-52 (1996)), is required
for cell fate determination of nociceptive sensory neurons in
dorsal root ganglia (DRG) (Ma et al. Genes & Dev. 13: 1717-1728
(1999)). In Ngn1.sup.-/- mutant mouse embryos most if not all
trkA.sup.+ neurons, which include the nociceptive subclass, fail to
be generated. This mutant phenotype was exploited to isolate genes
specifically expressed in such neurons, by subtracting cDNAs from
neonatal wild-type and Ngn1.sup.-/- DRG. Genes expressed in the
former but not the latter cDNA population are specific to
trkA.sup.+ nociceptive neurons.
[0359] Total RNA was isolated from the dorsal root ganglia (DRG) of
newborn wild type or Ngn1.sup.-/- mice (see Ma et al. Genes
Develop. 13:1717-1728 (1999), Fode et al. Neuron 20:483-494 (1998)
and Ma et al. Neuron 20:469-482 (1998)). A suppression-PCR-based
method (Clontech) was then used to enrich for genes expressed in
wild type but not Ngn1 mutant DRG. Briefly, cDNA was synthesized
from the RNA using Superscript reverse transcriptase (Gibco) with
oligo dT primers, and was amplified with the Smart PCR
Amplification Kit (Clontech). The amplified wild-type and
Ngn1.sup.-/- DRG cDNAs were used as tester and driver,
respectively, in the PCR-Select subtractive hybridization protocol
(Clontech). Differential screening by dot blot analysis identified
several clones, which were enriched in cDNA from wild-type DRG
compared to that from Ngn1.sup.-/- DRG. These clones were analyzed
further by nucleotide sequencing and in situ hybridization.
[0360] Approximately 1,600 positives were identified in the primary
screen, and of these 142 were sequenced. Fifty of these represented
known genes, and 92 represented new genes (see Table 2). Among the
known genes were several signaling molecules specifically expressed
in nociceptive sensory neurons. These included VR-1, calcitonin
gene-related peptide (CGRP), the tetrodotoxin-insensitive sodium
channel (SNS-TTXi) and diacylglycerol kinase. Among the new genes
were several encoding proteins with structural features
characteristic of ion channels or receptors, which were revealed by
in situ hybridization to be specifically expressed in a subset of
DRG sensory neurons. These molecules are described in more detail
in Examples 2 and 3.
4TABLE 2 Summary of results of the differential hybridization
screening for genes involved in pain sensation. # of times isolated
from the screen Name A. Known genes: 13 NaN 9 Diacylglycerol kinase
7 Synaptophysin Iia 5 Vanilloid receptor1 3 GluR5-2c 2 CGRP 2 CLIM1
1 SNS-TTXi 1 Alpha N-catenin I 1 Brain Na channel III 1 NICA6 1
Secretogranin B. Novel genes: 2 Mrg3 (a novel G-protein-coupled
receptor) 2 DRG12 Note: Previous studies have shown that the genes
with bolded letters are expressed specifically in nociceptors.
Example 2
A Novel Family of Putative G Protein-Coupled Receptors Specifically
Expressed in Nociceptive Sensory Neurons
[0361] Among the novel genes isolated from the screen were two
independent clones encoding a receptor protein with 7 transmembrane
segments (SEQ ID NO: 1), a characteristic of G protein-coupled
receptors. The novel 7 transmembrane receptor isolated is most
closely related to the oncogene mas, and therefore has been named
mas-related gene-3 (mrg3). mrg3 is also known as mas-related gene
A1, or MrgA1. A complete coding sequence for mrg3 has been deduced
from the genomic DNA sequence (FIG. 1A and SEQ ID NO: 2). MrgA1
shows significant homology (35% identity) to MAS1 (Young et al.
Cell 45: 711-9 (1986)). It also shares significant homology (30-35%
identity) with two other mammalian GPCRs, called Mas-related gene 1
(MRG1) (Monnot et al. Mol Endocrinol 5: 1477-87 (1991)) and rat
thoracic aorta (RTA) (Ross et al. Proc Natl Acad Sci USA 87: 3052-6
(1990)).
[0362] Such G protein-coupled receptors are expressed in other
classes of sensory neurons, such as olfactory and gustatory
neurons, but molecules in this class had not previously been
described in DRG sensory neurons, with the exception of the
Protease-Activated Receptors (PARs).
[0363] Further screening of mouse DRG cDNA library and mouse
genomic library by using mrg3 DNA as a probe has identified nine
additional closely related genes named mrg4 (MrgA2), mrg5 (MrgA3),
mrg6, mrg7, mrg8 (MrgA4), mrg9 (MrgA5), mrg10 (MrgA6), mrg11
(MrgA7), and mrg12 (MrgA8). Among them, mrg4, 5 and mrg 8-12
contain full-length open reading frames (see FIG. 1). Two human
homologues were found by searching databases using the blast
program. The protein alignment of the eight mrg genes, mrg3-8 and
human1-2, suggested that they define a novel G protein-coupled
receptor gene family (FIG. 1A).
[0364] In particular MrgA1-4 were isolated from a P0 mouse DRG cDNA
library and clones containing the entire ORFs of MRGsA5-8 were
isolated from a mouse genomic BAC library arrayed on filters
(Incyte Genomics). FIG. 6A shows an alignment of the polypeptide
sequence of MrgA1-8 and indicates the transmembrane domains as well
as the cytoplasmic and extracellular loops. In addition, other
mouse MrgAs, as well as other human Mrg sequences, were identified
by searching the Celera mouse and human (Venter et al. Science 291:
1304-51 (2001)) genomic databases, using the TBLASTN program with
MrgA1 as the query. Table 3 shows that the MrgA genes are highly
homologous to each other. This high degree of homology combined
with the presence of certain characteristic conserved residues
indicates that they define a novel subfamily of the MAS family of
GPCRs.
[0365] To identify additional members of the mouse Mrg family,
TBLASTN searches were run against the Celera mouse fragment
database (indexed Jan. 7, 2001; 18,251,375 fragments) using MRGA1
and MRGA4 protein sequences as queries. These searches identified
299 unique mouse genomic DNA fragments. The sequences of these
fragments were downloaded and assembled into contigs with GELMERGE
(GCG Wisconsin Package) under stringent conditions (90% identity,
20 nt minimum overlap). GELMERGE was run again (80% identity, 20 nt
minimum overlap) to reduce the dataset further. The consensus
nucleotide sequence from each contig was then queried against the
Celera mouse fragment database with BLASTN to identify additional
sequences for assembly (final n=536 fragments). The consensus
sequences from the final assembly were placed into a FASTA
formatted database. This database was then searched with TFASTY
using MRGA1 as query to identify the potential coding regions from
each consensus sequence, regardless of whether the error-prone
genomic sequence introduced stop codons or frameshifts into the
proteins (Pearson, W. R. (1999). Flexible similarity searching with
the FASTA3 program package. In Bioinformatics Methods and
Protocols, S. Misener and S. A. Krawetz, eds. (Totowa, N.J.: Humana
Press), pp. 185-219). The protein sequences from these searches
were then combined into a single FASTA formatted file for
phylogenetic analysis.
[0366] Using this analysis, 16 additional members of the murine
MrgA subfamily were identified (FIG. 6B). In addition to this
subfamily, two closely related Mrg subfamilies called MrgB and
MrgC, were also discovered (FIG. 6B). To confirm the existence of
an ORF in the mouse MrgB genes, high-fidelity PCR was used to
amplify mMrgB1-5, mMrgD, and mMrgE from C57B1/6 mouse genomic DNA.
Several independent clones were sequenced and confirmed the ORF
predictions. The presence of numerous stop codons and frame shifts
in the assembled Celera sequence indicated that the mMrgC genes are
pseudogenes.
[0367] The MrgB subfamily contains 14 genes, whereas MrgC has 12
members. The percent sequence identity within each of these
subfamilies is greater than 50% (Table 3). Strikingly, all 12 MrgC
members appear to be pseudogenes (FIG. 1B, "?"), as they contain
multiple premature stop codons, frameshift mutations or both.
Together, therefore, the MrgA and MrgB subfamilies comprise 36
intact ORFs.
5TABLE 3 Similarity and identity between murine MRG subfamilies
mMRG mMRG mMRG mMRG mMRG mMRG mMRG mMRG mMRG A1 A2 A3 B1 B2 B3 C1
C2 C3 mMRGA1 -- 77.9 73.1 48.1 46.3 43.6 44.9 46.7 47.8 mMRGA2 87.5
-- 71.8 42.4 45.4 42.7 41.5 44.5 43.5 mMRGA3 85.1 83.1 -- 47.9 46.8
44.2 46.0 49.8 46.6 mMRGB1 72.1 66.8 70.2 -- 57.6 50.0 42.9 47.1
45.3 mMRGB2 68.7 67.7 69.4 72.7 -- 53.5 41.8 44.4 43.1 mMRGB3 65.2
65.7 64.6 69.5 73.5 -- 37.0 38.8 36.4 mMRGC1 69.5 65.2 70.9 64.4
67.0 63.3 -- 76.0 79.1 mMRGC2 69.8 72.5 74.2 69.4 70.8 65.7 81.4 --
78.8 mMRGC3 70.9 67.2 71.0 66.2 69.5 64.6 86.1 86.3 --
[0368] Percent identity (top-right, bold) and percent similarity
(bottom-left) between the protein sequences are indicated. "hMRG"
indicates a human MRG amino acid sequence; "mMRG" indicates a
murine MRG sequence. "hMRGX" is used to indicate a human homolog of
mMRGA and mMRGB sequences (FIG. 1B). Values were derived from
global alignments using the GAP program in the GCG package.
[0369] Searches of the Celera (Venter et al. Science 291: 1304-51
(2001)) and public (Consortium. Nature 409: 860-921 (2001)) genomic
sequence databases, using both BLAST (Altschul et al. Journal of
Molecular Biology 215: 403-410 (1990)) and Hidden Markov Models
(HMMs (Eddy. Bioinformatics 14, 755-63 (1998)), revealed 4 closely
related (.about.50% identity) full-length human genes, and at least
10 human pseudogenes. Briefly, TBLASTN searches were run against
the Celera human genome database (Venter et al. Science 291:
1304-51 (2001)) using the mMrgA1 protein sequence as the query. The
genomic sequences that were identified in this search were
downloaded, placed into a FASTA formatted database and searched
with TFASTY to identify a non-redundant set of proteins. With the
exception of hMrgX3, hMrgE, and hMrg.PSI.8, all human Mrgs were
independently identified from a similar analysis of the public
human genome sequence (Consortium. Nature 409: 860-921 (2001)).
Human MrgX1-4 sequences were independently verified from
PCR-amplified products derived from human BAC clones containing the
genes.
[0370] Although the human genes appear to be more similar to the
murine MrgA subfamily than the MrgB subfamily in the phylogenetic
tree (FIG. 6B, hMrgX1-4), in the absence of clear orthologous pairs
we currently refer to them as hMrgX genes. In addition to the MrgA,
B and C subfamilies, a number of additional Mas1-related orphan
GPCRs were identified by this search, including those we refer to
as Mrgs D-F (FIG. 6B). Several of these sequences, such as MrgD,
have clear human orthologs (FIG. 6B, hMrgD). At the protien level
hMrgD and mMrgD are 58% identical and 73% similar, while at the
nucleotide level they are 73% identical. All together, we
identified almost 45 murine and 9 human intact coding sequences
belonging to this family.
6TABLE 4 Similarity and identity between human and murine MRGs hMRG
hMRG mMRG mMRG mMRG mMRG mMRG X2 E A1 B4 B1 D E hMRGX2 -- 40.2 55.6
50.1 53.4 40.5 38.8 hMRGE 62.8 -- 36.6 32.8 32.8 33.9 76.5 mMRGA1
74.8 57.7 -- 48.1 48.1 37.1 39.7 mMRGB4 71.0 58.0 70.4 -- 54.5 34.8
36.6 mMRGB1 73.5 60.5 72.1 74.1 -- 36.5 33.8 mMRGD 61.1 57.6 59.5
64.2 61.3 -- 35.1 mMRGE 59.0 84.0 62.5 63.7 59.1 59.3 --
[0371] Percent identity (top-right, bold) and percent similarity
(bottom-left) between the protein sequences are indicated. "hMRG"
indicates a human MRG amino acid sequence; "mMRG" indicates a
murine MRG sequence. "hMRGX" is used to indicate a human homolog of
mMRGA and mMRGB sequences (FIG. 1B). Values were derived from
global alignments using the GAP program in the GCG package.
[0372] MRG receptors have short (3-21 amino acid) N-termini with no
apparent signal peptide, which are predicted to be located
extracellularly. The transmembrane domains and intracellular
domains are highly conserved suggesting that the receptors have a
shared function. The most divergent regions of MRGA-family
receptors appear localized to the extracellular loops (FIG. 6A),
suggesting that these receptors recognize different ligands, or the
same ligand but with different affinities. Interestingly, we
identified 12 single nucleotide polymorphisms in the MrgA1 coding
sequence between murine strains C57BL/6J and 129SvJ. These 12
changes resulted in 6 amino acid substitutions, all of which were
either conservative, or which substituted residues expressed at the
same position by other family members.
[0373] A large mouse genomic contig was built by analyzing
overlapping BAC clones containing MrgA sequences (FIG. 6C). There
are 7 MrgA genes, including 3 pseudogenes, residing in this contig.
Such clustering is a common feature of GPCR-encoding gene families
(Xie et al. Mamm Genome 11: 1070-8 (2000)). Strikingly, all of the
human Mrg genes (with the exception of Mas1 and Mrg1) are located
on chromosome 11, which also contains 50% of all human olfactory
receptors genes. All of the MrgA genes in the murine BAC contig
(FIG. 6C) encode intact ORFs with N-terminal methionines, like many
other GPCR-encoding genes. Using the Celera mouse genome database,
sequences flanking each MrgA coding region were obtained and
analyzed. This analysis revealed that at least six MrgA genes have
L1 retrotransposon sequences located .about.650 bp downstream of
their coding sequences (FIG. 6B, indicated by "L1").
[0374] All of the eight full-length mas-related genes, mrg3-5 and
mrg8-12, are enriched in nociceptive sensory neurons as indicated
by their expression in a subset of DRG sensory neurons which are
eliminated in ngn1.sup.-/- mutant DRG (FIG. 2 and 2A).
Example 3
A Novel Two-Transmembrane Segment Protein Specifically Expressed in
Nociceptive Sensory Neurons
[0375] Another novel gene isolated in this screen, drg12 (SEQ ID
NO: 13), encodes a protein with two putative transmembrane segments
(SEQ ID NO: 14). In situ hybridization indicates that, like the mrg
genes, this gene is also specifically expressed in a subset of DRG
sensory neurons. Although there are no obvious homologies between
this protein and other sequences in the database, it is noteworthy
that two purinergic receptors specifically expressed in nociceptive
sensory neurons (P.sub.2X.sub.2 and P.sub.2X.sub.3) have a similar
bipartite transmembrane topology. Therefore it is likely that drg12
also encodes a receptor or ion channel involved in nociceptive
sensory transduction or its modulation. The hydrophobicity of a
homologous region of a drg12 human sequence (SEQ ID NO: 19) is
compared with the hydrophobicity of mouse drg12 in FIG. 4.
Example 4
Mrg and Drg-12 Genes are Specifically Expressed in Nociceptive
Sensory Neurons
[0376] The prediction of function for mrg-family and drg-12 genes
is based on their structure and expression pattern, taken together
with the identification of ligands as described below. To determine
whether Mrg proteins are expressed in DRG neurons, in situ
hybridization using dioxygenin-labeled riboprobes was performed.
Briefly, tissue was obtained from P0 mouse pups and fixed in 4%
paraformaldehyde overnight at 4.degree. C., cryoprotected in 30%
sucrose overnight and embedded in OCT. Tissue sections were cut
transversely on a cryostat at 18 .mu.m. Non-isotopic in situ
hybridization on frozen sections was performed as previously
described using cRNA probes (Ma et al. Cell 87: 43-52 (1996); Perez
et al. Development 126: 1715-1728 (1999)). Eight MrgAs, 5 MrgBs and
MrgD were used as probes. At least 10 DRGs were analyzed to count
the number of neurons positive for each probe.
[0377] Mrg and drg12 genes, including all eight MrgAs (MrgA1-8),
are expressed in subsets of small-diameter sensory neurons in the
dorsal root ganglia (DRG) of the mouse (FIG. 7B-I). Importantly,
the expression of all eight MrgAs was virtually absent in the DRGs
of Ngn1.sup.-/- animals (FIG. 7J), consistent with the design of
the substractive hybridization screen. Among the eight MrgA clones
examined, MrgA1 has the widest expression within sensory neurons in
DRGs (13.5%). Other MrgAs are only expressed in several cells per
DRG section (ranging from 0.2-1.5% of DRG neurons). This
differential abundance may explain why only MrgA1 was isolated in
the original screen. No obvious differences in the expression
patterns of MrgA1-8 were noticed in DRGs from different axial
levels. This expression is highly specific, in that expression of
these genes has thus far not been detected in any other tissue of
the body or in any other region of the nervous system thus far
examined.
[0378] Like the MrgA genes, MrgD was also specifically expressed in
a subset of DRG sensory neurons (see below, FIG. 15). In contrast,
MrgB1-5 were not detectably expressed in DRGs. However, mMrgB1
expression has been observed in scattered cells in the epidermal
layer of skin in newborn mice, as well as in the spleen and the
submandibular gland (FIGS. 13 and 14). These cells appear to be
immune cells that play a role in wound repair. mMrgB2 also shows
this expression pattern. In contrast, mMrgB3, mMrgB4 and mMrgB5 do
not appear to be expressed in any of these tissues.
[0379] Using Northern blot analysis, human MrgD was found to be
expressed in human dorsal root gangli neurons. A Northern blot
containing 20 .mu.g total RNA from human DRG neurons was hybridized
with a human MrgD probe and a transcript of 4.4 kb was detected.
Further analysis indicated that human MrgD is not expresed in human
brain, heart, skeletal muscle, thymus, colon, spleen, kidney,
liver, small intestine, placenta, lung or peripheral blood
leukocytes. Thus, like mMrgD, human MrgD shows highly restricted
expression in pain sensing neurons. In addition, the data indicate
that the mouse and human MrgD are functional orthologs.
[0380] These results indicate that Mrg and drg12 genes are
expressed in primary sensory neurons. However, DRG contain
different classes of neurons subserving different types of
sensation: e.g., heat, pain, touch and body position. Independent
identification is provided by the fact that the neurons that
express the mrg-family and drg12 genes are largely or completely
eliminated in Ngn1.sup.-/- DRG (FIG. 2), because the Ngn1 mutation
is independently known to largely or completely eliminate the
nociceptive (noxious stimuli-sensing) subset of DRG neurons,
identified by expression of the independent markers trkA, VR-1 and
SNS-TTXi (Ma et. al. Genes & Dev. 13: 1717-1728 (1999)). The
loss of mrg- and drg12-expressing neurons in Ngn1.sup.-/- mutant
DRG therefore indicates that these genes are very likely expressed
in nociceptive sensory neurons. Although small numbers of sensory
neurons of other classes (trkB.sup.+ and trkC.sup.+) are eliminated
in the Ngn1.sup.-/- mutant as well, mrg and drg12 genes are
unlikely to be expressed in these classes of sensory neurons,
because if they were then the majority of mrg- and drg12-expressing
sensory neurons would be predicted to be spared in the Ngn1.sup.-/-
mutant, and that is not the case.
[0381] The lack of expression of MrgAs in DRGs from Ngn1.sup.-/-
mice is consistent with the idea that they are expressed in
cutaneous sensory neurons. Furthermore, the distribution of
MrgA1.sup.+ cells was similar to that of neurons expressing trkA, a
marker of nociceptive sensory neurons (McMahon et al. Neuron 12:
1161-71 (1994); Snider and Silos-Santiago Philos Trans R Soc Lond B
Biol Sci 351: 395-403 (1996)) (FIG. 7A, B). To directly determine
whether MrgA genes are expressed in trkA.sup.+ cells, in situ
hybridization was performed for MrgA1, A3 and A4 in conjunction
with immunolabeling using anti-trkA antibodies, on neonatal DRG.
Fluorescein-UTP-labeled cRNA probes were detected with alkaline
phospatase- (AP-) conjugated anti-fluorescein antibody (1:2000,
Roche) and developed with Fast Red (Roche) to generate a red
fluorescent signal. After the fluorescent in situ hybridization was
performed, sections were incubated in primary antibodies against
TrkA (1:5000, gift from Dr. Louis Reichardt), VR1 (1:5000, gift
from Dr. D. Julius), CGRP (1:500, Chemicon), or SubstanceP (1:1000,
Diasorin). All antibodies were diluted in 1.times.PBS containing 1%
normal goat serum and 0.1% TritonX-100. Primary antibody
incubations were carried out overnight at 4.degree. C. Secondary
antibodies used were goat-anti-rabbit-IgG conjugated to Alexa 488
(1:250, Molecular Probes). For double-labeling with Griffonia
simplicifolia IB4 lectin, sections were incubated with 12.5
.mu.g/ml FITC-conjugated IB4 lectin (Sigma) following in situ
hybridization.
[0382] Double labeling experiment using mrgs antisense RNA probes
with anti-trkA antibodies confirmed that mrgs, specifically MrgAs,
are co-expressed by trkA+ nociceptive neurons in DRG (see FIG. 7B
and FIG. 8A-C). Similar results were obtained for MrgD (FIG. 8D).
Taken together, these data indicate that MrgAs and MrgD are
specifically expressed by nociceptive sensory neurons in DRG.
[0383] Further experiments were carried out to determine whether
Mrgs are expressed in particular subsets of nociceptors. Additional
double labeling experiments using mrgs antisense RNA probes with
anit-VR1 and isolectin B4 (IB4)-labeling, as described above, have
shown that mrgs are preferentially expressed by IB4+ nociceptive
neurons but not VR1-expressing nociceptive neurons (FIGS. 2C and
2D). In particular, combined fluorescent labeling for IB4 together
with in situ hybridization with MrgA1, A3, A4 and MrgD probes
clearly showed that these receptors are expressed by IB4.sup.+
neurons (FIG. 8E-H), and may be restricted to this subset. This
result indicates that these Mrgs are expressed by non-peptidergic
nociceptive neurons that project to lamina IIi (Snider and McMahon
Neuron 20: 629-32 (1998)). Consistent with this assignment, the
majority (90%) of MrgA1+, and all MrgA3.sup.+, A4.sup.+ and
MrgD.sup.+ cells, lack substance P expression (FIG. 8I-L).
Similarly, the majority (70%) of MrgA1.sup.+, and all MrgA3.sup.+,
A4.sup.+ and MrgD.sup.+ cells, do not express CGRP (FIG. 8M-P),
another neuropeptide expressed by C-fiber nociceptors. Previous
studies had shown that IB4.sup.+ nociceptive neurons were involved
in neuropathic pain resulting from nerve injury (Malmberg, A. B. et
al. Science 278: 279-83 (1997)). Neuropathic pain including
postherpetic neuralgia, reflex sympathetic dystrophy, and phantom
limb pain is the most difficult pain to be managed. Mrgs may play
essential roles in mediating neuropathic pain and may provide
alternative solutions to manage neuropathic pain.
[0384] Recent studies have provided evidence for the existence of
two neurochemically and functionally distinct subpopulations of
IB4.sup.+ nociceptors: those that express the vanilloid receptor
VR1 (Caterina et al. Science 288: 306-13 (1997)), and those that do
not (Michael and Priestley J Neurosci 19: 1844-54 (1999); Stucky
and Lewin J Neurosci 19: 6497-505 (1999)). Strikingly, in situ
hybridization with MrgA or D probes combined with anti-VR1 antibody
immunostaining indicated that the MrgA1, A3, A4 and D-expressing
cell population was mutually exclusive with VR1.sup.+ cells (FIG.
8Q-T). In summary, these expression data demonstrate that MrgA and
D genes are expressed in the subclass of nonpeptidergic cutaneous
sensory neurons that are IB4.sup.+ and VR1.sup.- (FIG. 9).
[0385] MrgA1 is Co-Expressed with other MrgA Genes
[0386] MrgA1 is more broadly expressed than are the other MrgA
genes (FIG. 2), suggesting MrgA1 and MrgA2-8 are expressed by
different or overlapping subsets of nociceptors. Double-label in
situ hybridization studies using probes labeled with digoxigenin
and fluorescein indicated that most or all neurons expressing MrgA3
or MrgA4 co-express MrgA1 (FIG. 10A-F). Interestingly, the
fluorescent in situ hybridization signals for MrgA3 and A4 using
tyramide amplification often appeared as dots within nuclei that
were circumscribed by the cytoplasmic expression of MrgA1 mRNA,
detected by Fast Red (FIG. 10F). Such dots were not observed using
the less-sensitive Fast Red detection method, and were only
observed in the nuclei of MrgA1.sup.+ cells. Similar intranuclear
dots have previously been observed in studies of pheromone receptor
gene expression, and have been suggested to represent sites of
transcription (Pantages and Dulac Neuron 28: 835-845 (2000)). The
results for MrgA1, 3 and 4 indicate that those neurons that express
the rarer MrgA genes (MrgA2-8) are a subset of those that express
MrgA1.
[0387] To address the question of whether MrgsA2-A8 are expressed
in the same or in different neurons, the number of neurons labeled
by single probes was compared to that labeled by a mixture of all 7
probes (Buck and Axel Cell 65: 175-187 (1991)). Approximately
3-fold more neurons (4.5% vs. 1%) were labeled by the mixed probe
than by an individual probe to MrgA4 (FIG. 10J, K), indicating that
these genes are not all co-expressed in the same population of
neurons. However, the percentage of neurons labeled by the mixed
probe (4.5%) was less than the sum of the percentage of neurons
labeled by each of the 7 individual probes (6.6%), indicating that
there is some overlap in the expression of MrgA2-A8. In additon,
higher signal intensity was observed in individual neurons using
the mixed probe, than using a single probe.
[0388] Double-labeling experiments with MrgA1 and MrgD probes were
also performed. These proteins share only 60% sequence similarity,
as shown in FIG. 6B and Table 3. The results of these experiments
indicated only partial overlap between neurons expressing these two
receptors (FIG. 10G-I). Approximately 15% (118/786) of neurons
expressing either MrgA1 or MrgD co-expressed both genes.
Thirty-four percent (118/344) of MrgA1.sup.+ cells co-expressed
MrgD, while 26.7% (118/442) of MrgD.sup.+ cells co-expressed
MrgA1.
[0389] Taken together, these data indicate the existence of at
least three distinct subpopulations of IB4.sup.+, VR1.sup.- sensory
neurons: MrgA1.sup.+MrgD.sup.+; MrgA1.sup.+MrgD.sup.- and
MrgA1.sup.- MrgD.sup.+. The MrgA1.sup.+ subset is further
subdivided into different subsets expressing one or more of the
MrgsA2-A8.
[0390] Mrg-Family Genes Encode Putative G-Protein Coupled Receptors
(GPCRs).
[0391] Hydrophobicity plots of the encoded amino acid sequences of
the mrg-family genes predicts membrane proteins with 7
transmembrane segments. Such a structure is characteristic of
receptors that signal through "G-proteins." G proteins are a family
of cytoplasmic molecules that activate or inhibit enzymes involved
in the generation or degradation of "second messenger" molecules,
such as cyclic nucleotides (cAMP, cGMP), IP.sub.3 and intracellular
free calcium (Ca.sup.++). Such second messenger molecules then
activate or inhibit other molecules involved in intercellular
signaling, such as ion channels and other receptors.
[0392] G protein-coupled receptors (GPCRs) constitute one of the
largest super-families of membrane receptors, and contain many
subfamilies of receptors specific for different ligands. These
ligands include neurotransmitters and neuropeptides manufactured by
the body (e.g., noradrenaline, adrenaline, dopamine; and substance
P, somatostatin, respectively), as well as sensory molecules
present in the external world (odorants, tastants).
[0393] Although the mrg-family genes are highly homologous, the
most divergent regions were the extracellular domains (see FIG.
6A). The variability of the extracellular domains of mrg family
suggests that they may recognize different ligands.
[0394] The fact that the mrg-family genes encode GPCRs, and are
specifically expressed in nociceptive sensory neurons, suggest that
these receptors are involved, directly or indirectly, in the
sensation or modulation of pain, heat or other noxious stimuli.
Therefore the mrg-encoded receptors are useful as targets for
identifying drugs that effect the sensation or modulation of pain,
heat or other noxious stimuli. The nature of the most useful type
of drug (agonistic or antagonistic) will reflect the nature of the
normal influence of these receptors on the sensation of such
noxious stimuli. For example, if mrg-encoded receptors normally act
negatively, to inhibit or suppress pain, then agonistic drugs would
provide useful therapeutics; conversely, if the receptors normally
act positively, to promote or enhance pain, then antagonistic drugs
would provide useful therapeutics. There might even be certain
clinical settings in which it would be useful to enhance
sensitivity to noxious stimuli, for example in peripheral sensory
neuropathies associated with diabetes.
[0395] The nature of the influence of mrg-encoded GPCRs on pain
sensation may be revealed by the phenotypic consequences of
targeted mutation of these genes in mice. For example, if such mice
displayed enhanced sensitivity to noxious stimuli, then it could be
concluded that the receptors normally function to inhibit or
suppress pain responses, and vice-versa. Alternatively,
high-throughput screens may be used to identify small molecules
that bind tightly to the mrg-encoded receptors. Such molecules
would be expected to fall into two categories: agonists and
antagonists. Agonists would be identified by their ability to
activate intracellular second messenger pathways in a
receptor-dependent manner, while antagonists would inhibit them.
Testing of such drugs in animal models of pain sensitivity will
then reveal further information concerning the function of the
GPCRs: for example, if the molecules behave as receptor antagonists
in vitro, and they suppress sensitivity or responsiveness to
noxious stimuli in vivo, then it may be concluded that the receptor
normally functions to promote or enhance pain sensation.
Conversely, if receptor agonists suppress, while antagonists
enhance, pain sensation in vivo, then it may be concluded that the
receptor normally functions to suppress or inhibit pain
sensation.
[0396] Drg12 Encodes a Putative Transmembrane Signaling
Molecule
[0397] Hydrophobicity plots of the encoded amino acid sequence of
the drg12 gene predicts a membrane protein with 2 transmembrane
segments. The membrane localization of this protein has been
verified by immuno-staining of cultured cells transfected with an
epitope-tagged version of the polypeptide. Although the DRG12 amino
acid sequence has no homology to known families of proteins, its
bipartite transmembrane structure strongly suggests that it is
involved in some aspect of intercellular signaling, for example as
a receptor, ion channel or modulator of another receptor or ion
channel. This prediction is supported by the precedent that two
known receptors with a similar bipartite transmembrane topology,
the purinergic P.sub.2X.sub.2 and P.sub.2X.sub.3 receptors, are
like DRG12, specifically expressed in nociceptive sensory
neurons.
[0398] Based on this structural data, and its specific expression
in nociceptive sensory neurons, it is probable that DRG12 is
involved, directly or indirectly, in the sensation or modulation of
noxious stimuli. Accordingly, the drg12-encoded protein is a useful
target for the development of novel therapeutics for the treatment
of pain.
Example 5
Mrg Proteins are Receptors for Neuropeptides
[0399] As discussed above, the structure of the proteins encoded by
Mrg genes indicates that they function as receptors. To identify
ligands for the Mrg receptors, selected MrgA genes were tested in a
calcium release assay. MrgA genes, including MrgA1 and MrgA4, were
cloned into a eukaryotic expression vector and transfected into
human embryonic kidney (HEK) 293 cells. HEK-293 cells were obtained
from the ATCC and cultured in DMEM supplemented with 10% fetal
bovine serum. An HEK293-G.alpha..sub.15 cell line stably expressing
G.alpha..sub.15 was provided by Aurora Biosciences Corporation and
grown on Matrigel.TM. (growth factor reduced Matrigel, Becton
Dickinson, diluted 1:200 with serum-free DMEM)-coated flasks and
maintained at 37.degree. C. in DMEM (GibcoBRL) supplemented with
10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 0.1 mM
non-essential amino acids, 1 mM sodium pyruvate, 25 mM HEPES and 3
.mu.g/ml blastcidin-S. For transfection, cells were seeded on
Matrigel-coated 35 mm glass-bottom dishes (Bioptech Inc., Butler,
Pa.). After 16-24 hr, cells were transfected using FuGENE 6
(Roche). Transfection efficiencies were estimated by visualization
of GFP fused to the C-terminus of MrgA1 and A4, and were typically
>60%. Fusing GFP to the C-termini of the MrgA coding sequences
additionally allowed for visual confirmation of the intracellular
distribution of the receptors and their membrane integration in the
transfected cells (FIG. 11D).
[0400] To increase the sensitivity of the calcium release assay, in
some experiments the MRGA-GFP fusion proteins were expressed in HEK
293 cells modified to express G.alpha..sub.15, which couples GPCRs
to a signal transduction pathway leading to the release of
intracellular free Ca.sup.2+ (Offermann and Simon J Biol Chem 270:
15175-80 (1995)). This calcium release can be monitored
ratiometrically using Fura-2 as a fluorescent indicator dye (Tsien
et al. Cell Calcium 6: 145-57 (1985)) (FIG. 11A-C). This
heterologous expression system has been previously used to identify
ligands for taste receptors (Chandrashekar et al. Cell 100: 703-11
(2000)).
[0401] Because MRGAs exhibit the highest sequence similarity to
peptide hormone receptors, approximately 45 candidate peptides were
screened for their ability to activate MRGA1, using the
intracellular Ca.sup.2+-release assay. Briefly, transfected cells
were washed once in Hank's balanced salt solution with 11 mM
D-glucose and 10 mM HEPES, pH 7.4 (assay buffer) and loaded with 2
.mu.M Fura-2 AM (Molecular Probes) at room temperature for 90 min,
with rotation. Loaded cells were washed twice with assay buffer and
placed on a micro-perfusion chamber (Bioptech). The chamber was
mounted on top of a Olympus IMT2 inverted microscope, and imaged
with an Olympus DPlanApo 40.times. oil immersion objective lens.
Samples were illuminated by a 75 W xenon bulb, and a
computer-controlled filter changer (Lambda-10; Shutter Instruments)
was used to switch the excitation wavelength. A cooled CCD camera
(Photometric) was used in detecting fluorescence. GFP-positive
cells within a field were identified using an excitation wavelength
of 400 nm, a dichroic 505 nm long-pass filter and an emitter
bandpass of 535 nm (Chroma Technology). In the same field, calcium
measurements were performed at an excitation wavelength of 340 nm
and 380 nm, and an emission wavelength of 510 mn. Agonists were
diluted in assay buffer and solution changes accomplished by
micro-perfusion pump (Bioptech). Fura-2 fluorescence signals (340
nm, 380 nm and the 340/380 ratio) originating from GFP-positive
cells were continuously monitored at 0.4- or 1-second intervals and
collected using Axon Imaging Workbench 4.0 software (Axon).
Instrument calibration was carried out with standard calcium
solutions (Molecular probes) in glass bottom dishes (MatTek
Corp.).
[0402] At a concentration of 1 .mu.M, numerous neuropeptides
produced some level of activation of MrgA1-expressing cells (FIG.
12A). These included ACTH, CGRP-I and -II, NPY and somatostatin
(SST). Nevertheless, many other peptide hormones did not activate
MRGA1, including angiotensins I-III and neurokinins A and B,
alpha-MSH and gamma2-MSH (FIG. 12A and data not shown). MrgA1 was
only very weakly activated by ecosanoid ligands such as
Prostaglandin-E1 and Arachidonic Acid (data not shown).
[0403] The most efficient responses in MrgA1-expressing HEK cells
were elicited by RFamide peptides, including FLRF and the molluscan
cardioactive neuropeptide FMRFamide (Price and Greenberg Science
197: 670-671 (1977)) (Phe-Met-Arg-Phe-amide) (FIG. 11C, 12A). Two
mammalian RFamide peptides, NPAF and NPFF, which are cleaved from a
common pro-peptide precursor (Vilim et al. Mol Pharmacol 55: 804-11
(1999)) were then tested. The response of MrgA1-expressing cells to
NPFF at 1 .mu.M was similar to that seen with FMRFamide, while that
to NPAF was significantly lower (FIG. 12A). MrgA1 was also weakly
activated by two other RFamide ligands, .gamma..sub.l-MSH and
schistoFLRF (data not shown).
[0404] In order to examine further the specificity of activation of
MrgA1 and A4, the top candidate ligands emerging from the intial
screen were tested on these same receptors expressed in HEK cells
lacking Ga.alpha..sup.15. MrgA1 and A4 expressed in this system
retained responses to RFamide peptides (FIG. 12B, C), demonstrating
that the intracellular Ca.sup.2+ release responses seen in the
initial screen are not dependent on the presence of exogenous
G.alpha..sub.15. This indicates that MrgAs act in HEK cells via Gq
or Gi. The response of MrgA1-expressing HEK cells to NPFF was lower
than that to FLRF (FIG. 12B), and there was no response to NPAF.
Conversely, MrgA4-expressing cells responded to NPAF, but not to
NPFF or FLRF (FIG. 12C). In both cases, the response to NPY seen in
G.alpha..sub.15-expressing cells (FIG. 11A) was lost completely,
while those to CGRP-II and ACTH were considerably diminished.
[0405] In order to determine the lowest concentrations of RFamide
ligands capable of activating MrgA1 and A4, dose-response
experiments were carried out in HEK cells expressing
G.alpha..sub.15, which afforded greater sensitivity (FIG. 12D, E).
These experiments indicated that MrgA1 could be activated by FLRF
at nanomolar concentrations (FIG. 12D; EC.sub.50{tilde over ()}20
nM), and by NPFF at about an order of magnitude higher
concentration (FIG. 12D; EC.sub.50{tilde over ()}20 nM), whereas
NPAF was much less effective. In contrast, MrgA4 was well activated
by NPAF (FIG. 12E; EC.sub.50{tilde over ()}60 nM), and much more
weakly activated by FLRF and NPFF. Neither receptor showed strong
activation in response to RFRP-1, -2 or -3, a series of RFamide
ligands produced from a different precursor (Hinuma et al. Nat Cell
Biol 2: 703-8 (2000)). These data confirm that MrgA1 and MrgA4
display different selectivities towards different RFamide ligands
in this system. By contrast, these receptors responded similarly to
ACTH (EC.sub.50.about.60- and 200 nM for MrgA1 and A4,
respectively; data not shown).
[0406] Finally, given the sequence similarity between MRGA
receptors and MAS1, the responsiveness of cells expressing
exogenous Mas1 to NPFF, NPAF and FLRF was tested. MAS1 showed a
profile distinct from both MrgA1 and MrgA4 (FIG. 12F): like MrgA1,
it was activated by NPFF at a similar concentration of the peptide
(EC.sub.50{tilde over ()}400 nM), but unlike MrgA1 it was poorly
activated by FLRF. In contrast to MrgA4, MAS1 did not respond well
to NPAF. No response was detected in MAS1-expressing cells upon
exposure to Angiotensins I and II, ligands which have been
previously reported to activate this receptor (Jackson, T. R., et
al. Nature 335: 437-40 (1988)). Nor did MAS1 respond to ACTH. Thus,
MAS1, MrgA1 and MrgA4 expressed in this heterologous system are all
activated by RFamide family ligands, but with differing
ligand-sensitivities and -selectivities (Table 4).
7TABLE 4 Selectivity of activation of Mas-related GPCRs by RF-amide
ligands in HEK cells A. Ligand receptor FLRF NPFF NPAF MRGA1 +++ ++
+/- MRGA4 +/- +/- +++ MAS1 +/- ++ +/-
[0407] Relative efficacy of activation of the indicated receptors
by the indicated ligands is shown. For quantification, see FIG. 6.
"+++" indicates 10 nM<EC.sub.50<100 nM; "++" indicates 100
nM<EC.sub.50<500 nM; "+/-" indicates weak response seen at 1
.mu.M. For details see FIG. 6.
[0408] A novel family consisting of close to 50 MAS1 related
g-protein coupled receptors has been identified. The specific
expression of several classes of these receptors in a subset of
nociceptive sensory neurons indicates that these receptors play a
role in the sensation or modulation of pain. Consistently, these
receptors have been shown to be activated by RFamide neuropeptides,
which are known to mediate analgesia. As a result, these receptors
provide a novel target for anti-nociceptive drugs.
[0409] Although the present invention has been described in detail
with reference to examples above, it is understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
following claims. All cited patents, patent applications and
publications referred to in this application are herein
incorporated by reference in their entirety.
Sequence CWU 1
1
109 1 1088 DNA Mus Musculus CDS (115)...(1026) 1 acagaagcca
gagagctaca tccagcaaga ggaatggggg aaagcagcac ctgtgcaggg 60
tttctagccc taaacacatc ggcctcgcca acagcaccca caacaactaa tcca atg 117
Met 1 gac aat acc atc cct gga ggt atc aac atc acg att ctg atc cca
aac 165 Asp Asn Thr Ile Pro Gly Gly Ile Asn Ile Thr Ile Leu Ile Pro
Asn 5 10 15 ttg atg atc atc atc ttc gga ctg gtc ggg ctg aca gga aat
ggc att 213 Leu Met Ile Ile Ile Phe Gly Leu Val Gly Leu Thr Gly Asn
Gly Ile 20 25 30 gtg ttc tgg ctc ctg ggc ttc tgt ttg cac agg aac
gcc ttc tca gtc 261 Val Phe Trp Leu Leu Gly Phe Cys Leu His Arg Asn
Ala Phe Ser Val 35 40 45 tac atc cta aac tta gct cta gct gac ttc
ttc ttc ctc cta ggt cac 309 Tyr Ile Leu Asn Leu Ala Leu Ala Asp Phe
Phe Phe Leu Leu Gly His 50 55 60 65 atc ata gat tcc ata ctg ctt ctt
ctc aat gtt ttc tac cca att acc 357 Ile Ile Asp Ser Ile Leu Leu Leu
Leu Asn Val Phe Tyr Pro Ile Thr 70 75 80 ttt ctc ttg tgc ttt tac
acg atc atg atg gtt ctc tat atc gca ggc 405 Phe Leu Leu Cys Phe Tyr
Thr Ile Met Met Val Leu Tyr Ile Ala Gly 85 90 95 ctg agc atg ctc
agt gcc atc agc act gag cgc tgc ctg tct gta ctg 453 Leu Ser Met Leu
Ser Ala Ile Ser Thr Glu Arg Cys Leu Ser Val Leu 100 105 110 tgc ccc
atc tgg tat cac tgt cac cgc cca gaa cac aca tca act gtc 501 Cys Pro
Ile Trp Tyr His Cys His Arg Pro Glu His Thr Ser Thr Val 115 120 125
atg tgt gct gtc atc tgg gtc ctg tcc ctg ttg atc tgc att ctg aat 549
Met Cys Ala Val Ile Trp Val Leu Ser Leu Leu Ile Cys Ile Leu Asn 130
135 140 145 agt tat ttc tgc ggt ttc tta aat acc caa tat aaa aat gaa
aat ggg 597 Ser Tyr Phe Cys Gly Phe Leu Asn Thr Gln Tyr Lys Asn Glu
Asn Gly 150 155 160 tgt ctg gca ttg aac ttc ttt act gct gca tac ctg
atg ttt ttg ttt 645 Cys Leu Ala Leu Asn Phe Phe Thr Ala Ala Tyr Leu
Met Phe Leu Phe 165 170 175 gtg gtc ctc tgt ctg tcc agc ctg gct ctg
gtg gcc agg ttg ttc tgt 693 Val Val Leu Cys Leu Ser Ser Leu Ala Leu
Val Ala Arg Leu Phe Cys 180 185 190 ggt act ggg cag ata aag ctt acc
aga ttg tat gta acc att att ctg 741 Gly Thr Gly Gln Ile Lys Leu Thr
Arg Leu Tyr Val Thr Ile Ile Leu 195 200 205 agc att ttg gtt ttt ctc
ctt tgc gga ttg ccc ttt ggc atc cac tgg 789 Ser Ile Leu Val Phe Leu
Leu Cys Gly Leu Pro Phe Gly Ile His Trp 210 215 220 225 ttt ctg tta
ttc aag att aag gat gat ttt cat gta ttt gat ctt gga 837 Phe Leu Leu
Phe Lys Ile Lys Asp Asp Phe His Val Phe Asp Leu Gly 230 235 240 ttt
tat ctg gca tca gtt gtc ctg act gct att aat agc tgt gcc aac 885 Phe
Tyr Leu Ala Ser Val Val Leu Thr Ala Ile Asn Ser Cys Ala Asn 245 250
255 ccc atc att tac ttc ttc gtg gga tcc ttc agg cat cgg ttg aag cac
933 Pro Ile Ile Tyr Phe Phe Val Gly Ser Phe Arg His Arg Leu Lys His
260 265 270 cag acc ctc aaa atg gtt ctc cag aat gca ctg caa gac act
cct gag 981 Gln Thr Leu Lys Met Val Leu Gln Asn Ala Leu Gln Asp Thr
Pro Glu 275 280 285 aca gcc aaa atc atg gtg gag atg tca aga agc aaa
tca gag cca 1026 Thr Ala Lys Ile Met Val Glu Met Ser Arg Ser Lys
Ser Glu Pro 290 295 300 tgatgaagag cctttgcctg gcccttagaa gtggctttgg
ggtgagcatt gccctgctgc 1086 ac 1088 2 304 PRT Mus Musculus 2 Met Asp
Asn Thr Ile Pro Gly Gly Ile Asn Ile Thr Ile Leu Ile Pro 1 5 10 15
Asn Leu Met Ile Ile Ile Phe Gly Leu Val Gly Leu Thr Gly Asn Gly 20
25 30 Ile Val Phe Trp Leu Leu Gly Phe Cys Leu His Arg Asn Ala Phe
Ser 35 40 45 Val Tyr Ile Leu Asn Leu Ala Leu Ala Asp Phe Phe Phe
Leu Leu Gly 50 55 60 His Ile Ile Asp Ser Ile Leu Leu Leu Leu Asn
Val Phe Tyr Pro Ile 65 70 75 80 Thr Phe Leu Leu Cys Phe Tyr Thr Ile
Met Met Val Leu Tyr Ile Ala 85 90 95 Gly Leu Ser Met Leu Ser Ala
Ile Ser Thr Glu Arg Cys Leu Ser Val 100 105 110 Leu Cys Pro Ile Trp
Tyr His Cys His Arg Pro Glu His Thr Ser Thr 115 120 125 Val Met Cys
Ala Val Ile Trp Val Leu Ser Leu Leu Ile Cys Ile Leu 130 135 140 Asn
Ser Tyr Phe Cys Gly Phe Leu Asn Thr Gln Tyr Lys Asn Glu Asn 145 150
155 160 Gly Cys Leu Ala Leu Asn Phe Phe Thr Ala Ala Tyr Leu Met Phe
Leu 165 170 175 Phe Val Val Leu Cys Leu Ser Ser Leu Ala Leu Val Ala
Arg Leu Phe 180 185 190 Cys Gly Thr Gly Gln Ile Lys Leu Thr Arg Leu
Tyr Val Thr Ile Ile 195 200 205 Leu Ser Ile Leu Val Phe Leu Leu Cys
Gly Leu Pro Phe Gly Ile His 210 215 220 Trp Phe Leu Leu Phe Lys Ile
Lys Asp Asp Phe His Val Phe Asp Leu 225 230 235 240 Gly Phe Tyr Leu
Ala Ser Val Val Leu Thr Ala Ile Asn Ser Cys Ala 245 250 255 Asn Pro
Ile Ile Tyr Phe Phe Val Gly Ser Phe Arg His Arg Leu Lys 260 265 270
His Gln Thr Leu Lys Met Val Leu Gln Asn Ala Leu Gln Asp Thr Pro 275
280 285 Glu Thr Ala Lys Ile Met Val Glu Met Ser Arg Ser Lys Ser Glu
Pro 290 295 300 3 1234 DNA Mus musculus CDS (137)...(1051) 3
tctgtagtga ctgtatcttt ccttctacac aagccagtga gctacatcca acaagaggat
60 tggggaaagc aatggtgaag catttcttgc ctttaagacc tcagcctcac
caacagcacc 120 agtgacaaca aatcca atg gac gaa acc ctc cct gga agt
atc aac att agg 172 Met Asp Glu Thr Leu Pro Gly Ser Ile Asn Ile Arg
1 5 10 att ctg atc cca aaa ttg atg atc atc atc ttc gga ctg gtc gga
ctg 220 Ile Leu Ile Pro Lys Leu Met Ile Ile Ile Phe Gly Leu Val Gly
Leu 15 20 25 atg gga aac gcc att gtg ttc tgg ctc ctg ggc ttc cac
ttg cgc aag 268 Met Gly Asn Ala Ile Val Phe Trp Leu Leu Gly Phe His
Leu Arg Lys 30 35 40 aat gac ttc tca ctc tac atc cta aac ttg gcc
cgg gct gac ttc ctt 316 Asn Asp Phe Ser Leu Tyr Ile Leu Asn Leu Ala
Arg Ala Asp Phe Leu 45 50 55 60 ttc ctc ctc agt agt atc ata gct tcc
acc ctg ttt ctt ctc aaa gtt 364 Phe Leu Leu Ser Ser Ile Ile Ala Ser
Thr Leu Phe Leu Leu Lys Val 65 70 75 tcc tac ctc agc atc atc ttt
cac ttg tgc ttt aac acc att atg atg 412 Ser Tyr Leu Ser Ile Ile Phe
His Leu Cys Phe Asn Thr Ile Met Met 80 85 90 gtt gtc tac atc aca
ggg ata agc atg ctc agt gcc atc agc act gag 460 Val Val Tyr Ile Thr
Gly Ile Ser Met Leu Ser Ala Ile Ser Thr Glu 95 100 105 tgc tgc ctg
tct gtc ctg tgc ccc acc tgg tat cgc tgc cac cgt cca 508 Cys Cys Leu
Ser Val Leu Cys Pro Thr Trp Tyr Arg Cys His Arg Pro 110 115 120 gta
cat aca tca act gtc atg tgt gct gtg atc tgg gtc cta tcc ctg 556 Val
His Thr Ser Thr Val Met Cys Ala Val Ile Trp Val Leu Ser Leu 125 130
135 140 ttg atc tgc att ctg aat agc tat ttc tgt gct gtc tta cat acc
aga 604 Leu Ile Cys Ile Leu Asn Ser Tyr Phe Cys Ala Val Leu His Thr
Arg 145 150 155 tat gat aat gac aat gag tgt ctg gca act aac atc ttt
acc gcc tcg 652 Tyr Asp Asn Asp Asn Glu Cys Leu Ala Thr Asn Ile Phe
Thr Ala Ser 160 165 170 tac atg ata ttt ttg ctt gtg gtc ctc tgt ctg
tcc agc ctg gct ctg 700 Tyr Met Ile Phe Leu Leu Val Val Leu Cys Leu
Ser Ser Leu Ala Leu 175 180 185 ctg gcc agg ttg ttc tgt ggc gct ggg
cag atg aag ctt acc aga ttt 748 Leu Ala Arg Leu Phe Cys Gly Ala Gly
Gln Met Lys Leu Thr Arg Phe 190 195 200 cat gtg acc atc ttg ctg acc
ctt ttg gtt ttt ctc ctc tgc ggg ttg 796 His Val Thr Ile Leu Leu Thr
Leu Leu Val Phe Leu Leu Cys Gly Leu 205 210 215 220 ccc ttt gtc atc
tac tgc atc ctg tta ttc aag att aag gat gat ttc 844 Pro Phe Val Ile
Tyr Cys Ile Leu Leu Phe Lys Ile Lys Asp Asp Phe 225 230 235 cat gta
tta gat gtt aat ttt tat cta gca tta gaa gtc ctg act gct 892 His Val
Leu Asp Val Asn Phe Tyr Leu Ala Leu Glu Val Leu Thr Ala 240 245 250
att aac agc tgt gcc aac ccc atc atc tac ttc ttc gtg ggc tct ttc 940
Ile Asn Ser Cys Ala Asn Pro Ile Ile Tyr Phe Phe Val Gly Ser Phe 255
260 265 aga cat cag ttg aag cac cag acc ctc aaa atg gtt ctc cag agt
gca 988 Arg His Gln Leu Lys His Gln Thr Leu Lys Met Val Leu Gln Ser
Ala 270 275 280 ctg cag gac act cct gag aca gct gaa aac atg gta gag
atg tca agt 1036 Leu Gln Asp Thr Pro Glu Thr Ala Glu Asn Met Val
Glu Met Ser Ser 285 290 295 300 aac aaa gca gag cct tgatgaagag
cctctacctg gacctcagag gtggctttgg 1091 Asn Lys Ala Glu Pro 305
agtgagcact gccctgctgc acttgaccac tgtccactct tctctcagct tactgatttg
1151 acatgcctca gtggtccacc aacaacttca acatctctcc actaacttag
tttttctacc 1211 cctcctgaat aaaagcatta atc 1234 4 305 PRT Mus
musculus 4 Met Asp Glu Thr Leu Pro Gly Ser Ile Asn Ile Arg Ile Leu
Ile Pro 1 5 10 15 Lys Leu Met Ile Ile Ile Phe Gly Leu Val Gly Leu
Met Gly Asn Ala 20 25 30 Ile Val Phe Trp Leu Leu Gly Phe His Leu
Arg Lys Asn Asp Phe Ser 35 40 45 Leu Tyr Ile Leu Asn Leu Ala Arg
Ala Asp Phe Leu Phe Leu Leu Ser 50 55 60 Ser Ile Ile Ala Ser Thr
Leu Phe Leu Leu Lys Val Ser Tyr Leu Ser 65 70 75 80 Ile Ile Phe His
Leu Cys Phe Asn Thr Ile Met Met Val Val Tyr Ile 85 90 95 Thr Gly
Ile Ser Met Leu Ser Ala Ile Ser Thr Glu Cys Cys Leu Ser 100 105 110
Val Leu Cys Pro Thr Trp Tyr Arg Cys His Arg Pro Val His Thr Ser 115
120 125 Thr Val Met Cys Ala Val Ile Trp Val Leu Ser Leu Leu Ile Cys
Ile 130 135 140 Leu Asn Ser Tyr Phe Cys Ala Val Leu His Thr Arg Tyr
Asp Asn Asp 145 150 155 160 Asn Glu Cys Leu Ala Thr Asn Ile Phe Thr
Ala Ser Tyr Met Ile Phe 165 170 175 Leu Leu Val Val Leu Cys Leu Ser
Ser Leu Ala Leu Leu Ala Arg Leu 180 185 190 Phe Cys Gly Ala Gly Gln
Met Lys Leu Thr Arg Phe His Val Thr Ile 195 200 205 Leu Leu Thr Leu
Leu Val Phe Leu Leu Cys Gly Leu Pro Phe Val Ile 210 215 220 Tyr Cys
Ile Leu Leu Phe Lys Ile Lys Asp Asp Phe His Val Leu Asp 225 230 235
240 Val Asn Phe Tyr Leu Ala Leu Glu Val Leu Thr Ala Ile Asn Ser Cys
245 250 255 Ala Asn Pro Ile Ile Tyr Phe Phe Val Gly Ser Phe Arg His
Gln Leu 260 265 270 Lys His Gln Thr Leu Lys Met Val Leu Gln Ser Ala
Leu Gln Asp Thr 275 280 285 Pro Glu Thr Ala Glu Asn Met Val Glu Met
Ser Ser Asn Lys Ala Glu 290 295 300 Pro 305 5 1312 DNA Mus musculus
CDS (165)...(1070) 5 cgcggccgcg tcgacaagaa atattctgta gtgactgtat
ccttccttct acacaagcca 60 gcaagctaca tccagcaaga ggaatgggag
aaagcaacac cagtgcaggg tttctggccc 120 gaaacacctc agcctcgaca
atgacaccca caacaacaaa ttca atg aac gaa acc 176 Met Asn Glu Thr 1
atc cct gga agt att gac atc gag acc ctg atc cca gac ttg atg atc 224
Ile Pro Gly Ser Ile Asp Ile Glu Thr Leu Ile Pro Asp Leu Met Ile 5
10 15 20 atc atc ttc gga ctg gtc ggg ctg aca gga aat gcg att gtg
ttc tgg 272 Ile Ile Phe Gly Leu Val Gly Leu Thr Gly Asn Ala Ile Val
Phe Trp 25 30 35 ctc ctt ggc ttc cgc atg cac agg act gcc ttc tta
gtc tac atc cta 320 Leu Leu Gly Phe Arg Met His Arg Thr Ala Phe Leu
Val Tyr Ile Leu 40 45 50 aac ttg gcc ctg gct gac ttc ctc ttc ctt
ctc tgt cac atc ata aat 368 Asn Leu Ala Leu Ala Asp Phe Leu Phe Leu
Leu Cys His Ile Ile Asn 55 60 65 tcc aca gtg gat ctt ctc aag ttt
acc cta ccc aaa gga att ttt gcc 416 Ser Thr Val Asp Leu Leu Lys Phe
Thr Leu Pro Lys Gly Ile Phe Ala 70 75 80 ttt tgt ttt cac act atc
aaa agg gtt ctc tat atc aca ggc ctg agc 464 Phe Cys Phe His Thr Ile
Lys Arg Val Leu Tyr Ile Thr Gly Leu Ser 85 90 95 100 atg ctc agt
gcc atc agc act gag cgc tgc ctg tct gtc ctg tgc ccc 512 Met Leu Ser
Ala Ile Ser Thr Glu Arg Cys Leu Ser Val Leu Cys Pro 105 110 115 atc
tgg tat cac tgc cgc cgc cca gaa cac aca tca act gtc atg tgt 560 Ile
Trp Tyr His Cys Arg Arg Pro Glu His Thr Ser Thr Val Met Cys 120 125
130 gct gtg atc tgg gtc ctg tcc ctg ttg atc tgc att ctg gat ggt tat
608 Ala Val Ile Trp Val Leu Ser Leu Leu Ile Cys Ile Leu Asp Gly Tyr
135 140 145 ttc tgc ggt tac tta gat aac cat tat ttc aat tac tct gtg
tgt cag 656 Phe Cys Gly Tyr Leu Asp Asn His Tyr Phe Asn Tyr Ser Val
Cys Gln 150 155 160 gca tgg gac atc ttt atc gga gca tac ctg atg ttt
ttg ttt gta gtc 704 Ala Trp Asp Ile Phe Ile Gly Ala Tyr Leu Met Phe
Leu Phe Val Val 165 170 175 180 ctc tgt ctg tcc acc ctg gct cta ctg
gcc agg ttg ttc tgt ggt gct 752 Leu Cys Leu Ser Thr Leu Ala Leu Leu
Ala Arg Leu Phe Cys Gly Ala 185 190 195 agg aat atg aaa ttt acc aga
tta ttc gtg acc atc atg ctg acc gtt 800 Arg Asn Met Lys Phe Thr Arg
Leu Phe Val Thr Ile Met Leu Thr Val 200 205 210 ttg gtt ttt ctt ctc
tgt ggg ttg ccc tgg ggc atc acc tgg ttc ctg 848 Leu Val Phe Leu Leu
Cys Gly Leu Pro Trp Gly Ile Thr Trp Phe Leu 215 220 225 tta ttc tgg
att gca cct ggt gtg ttt gta cta gat tat agc cct ctt 896 Leu Phe Trp
Ile Ala Pro Gly Val Phe Val Leu Asp Tyr Ser Pro Leu 230 235 240 ctg
gtc cta act gct att aac agc tgt gcc aac ccc att att tac ttc 944 Leu
Val Leu Thr Ala Ile Asn Ser Cys Ala Asn Pro Ile Ile Tyr Phe 245 250
255 260 ttc gtg ggc tcc ttc agg caa cgg ttg aat aaa cag acc ctc aaa
atg 992 Phe Val Gly Ser Phe Arg Gln Arg Leu Asn Lys Gln Thr Leu Lys
Met 265 270 275 gtt ctc cag aaa gcc ctg cag gac act cct gag aca cct
gaa aac atg 1040 Val Leu Gln Lys Ala Leu Gln Asp Thr Pro Glu Thr
Pro Glu Asn Met 280 285 290 gtg gag atg tca aga aac aaa gca gag ccg
tgatgaagag cctctgccta 1090 Val Glu Met Ser Arg Asn Lys Ala Glu Pro
295 300 gacttcagag gtggatttgg agtgagcact gccctgctgc acttgaccac
tgtccactct 1150 cctctcagct tactgacttg acatgcctca ctggtccacc
aacaccttcc aaagctctcc 1210 actgacttag tatttatacc tctcccaaac
aatagcatta ttcaaaaact ataatttctg 1270 catccttctt tacattaata
aaattcccat actaagttca aa 1312 6 302 PRT Mus musculus 6 Met Asn Glu
Thr Ile Pro Gly Ser Ile Asp Ile Glu Thr Leu Ile Pro 1 5 10 15 Asp
Leu Met Ile Ile Ile Phe Gly Leu Val Gly Leu Thr Gly Asn Ala 20 25
30 Ile Val Phe Trp Leu Leu Gly Phe Arg Met His Arg Thr Ala Phe Leu
35 40 45 Val Tyr Ile Leu Asn Leu Ala Leu Ala Asp Phe Leu Phe Leu
Leu Cys 50 55 60 His Ile Ile Asn Ser Thr Val Asp Leu Leu Lys Phe
Thr Leu Pro Lys 65 70 75 80 Gly Ile Phe Ala Phe Cys Phe His Thr Ile
Lys Arg Val Leu Tyr Ile 85 90 95 Thr Gly Leu Ser Met Leu Ser Ala
Ile Ser Thr Glu Arg Cys Leu Ser 100 105 110 Val Leu Cys Pro Ile Trp
Tyr His Cys Arg Arg Pro Glu His Thr Ser 115 120 125 Thr Val Met Cys
Ala Val Ile Trp Val Leu Ser Leu Leu Ile Cys Ile 130 135 140 Leu
Asp
Gly Tyr Phe Cys Gly Tyr Leu Asp Asn His Tyr Phe Asn Tyr 145 150 155
160 Ser Val Cys Gln Ala Trp Asp Ile Phe Ile Gly Ala Tyr Leu Met Phe
165 170 175 Leu Phe Val Val Leu Cys Leu Ser Thr Leu Ala Leu Leu Ala
Arg Leu 180 185 190 Phe Cys Gly Ala Arg Asn Met Lys Phe Thr Arg Leu
Phe Val Thr Ile 195 200 205 Met Leu Thr Val Leu Val Phe Leu Leu Cys
Gly Leu Pro Trp Gly Ile 210 215 220 Thr Trp Phe Leu Leu Phe Trp Ile
Ala Pro Gly Val Phe Val Leu Asp 225 230 235 240 Tyr Ser Pro Leu Leu
Val Leu Thr Ala Ile Asn Ser Cys Ala Asn Pro 245 250 255 Ile Ile Tyr
Phe Phe Val Gly Ser Phe Arg Gln Arg Leu Asn Lys Gln 260 265 270 Thr
Leu Lys Met Val Leu Gln Lys Ala Leu Gln Asp Thr Pro Glu Thr 275 280
285 Pro Glu Asn Met Val Glu Met Ser Arg Asn Lys Ala Glu Pro 290 295
300 7 450 DNA Mus musculus CDS (1)...(450) 7 ctg tgc cgg atc tgg
tat cac tgc cgc cgc cca gaa cac aca tca act 48 Leu Cys Arg Ile Trp
Tyr His Cys Arg Arg Pro Glu His Thr Ser Thr 1 5 10 15 gtc atg tgt
gct gtc atc tgg gtc ctg tcc ctg ttg atc tgc att ctg 96 Val Met Cys
Ala Val Ile Trp Val Leu Ser Leu Leu Ile Cys Ile Leu 20 25 30 aat
agt tat ttc tgc ggt ttc tta aat acc caa tat aaa aat gaa aat 144 Asn
Ser Tyr Phe Cys Gly Phe Leu Asn Thr Gln Tyr Lys Asn Glu Asn 35 40
45 ggg tgt ctg gca ttg agc ttc ttt act gct gca tac ctg atg ttt ttg
192 Gly Cys Leu Ala Leu Ser Phe Phe Thr Ala Ala Tyr Leu Met Phe Leu
50 55 60 ttt gtg gtc ctc tgt ctg tcc agc ctg gct ctg gtg gcc agg
ttg ttc 240 Phe Val Val Leu Cys Leu Ser Ser Leu Ala Leu Val Ala Arg
Leu Phe 65 70 75 80 tgt ggt gct agg aat atg aaa ttt acc aga tta ttc
gtg acc atc atg 288 Cys Gly Ala Arg Asn Met Lys Phe Thr Arg Leu Phe
Val Thr Ile Met 85 90 95 ctg acc gtt ttg gtt ttt ctt ctc tgt ggg
ttg ccc tgg ggc atc acc 336 Leu Thr Val Leu Val Phe Leu Leu Cys Gly
Leu Pro Trp Gly Ile Thr 100 105 110 tgg ttc ctg tta ttc tgg att gca
cct ggt gtg ttt gta cta gat tat 384 Trp Phe Leu Leu Phe Trp Ile Ala
Pro Gly Val Phe Val Leu Asp Tyr 115 120 125 agc cct ctt ctg gtc cta
act gct att aac agc tgt gcc aac ccc att 432 Ser Pro Leu Leu Val Leu
Thr Ala Ile Asn Ser Cys Ala Asn Pro Ile 130 135 140 att tac ttc ttc
gtc ggc 450 Ile Tyr Phe Phe Val Gly 145 150 8 150 PRT Mus musculus
8 Leu Cys Arg Ile Trp Tyr His Cys Arg Arg Pro Glu His Thr Ser Thr 1
5 10 15 Val Met Cys Ala Val Ile Trp Val Leu Ser Leu Leu Ile Cys Ile
Leu 20 25 30 Asn Ser Tyr Phe Cys Gly Phe Leu Asn Thr Gln Tyr Lys
Asn Glu Asn 35 40 45 Gly Cys Leu Ala Leu Ser Phe Phe Thr Ala Ala
Tyr Leu Met Phe Leu 50 55 60 Phe Val Val Leu Cys Leu Ser Ser Leu
Ala Leu Val Ala Arg Leu Phe 65 70 75 80 Cys Gly Ala Arg Asn Met Lys
Phe Thr Arg Leu Phe Val Thr Ile Met 85 90 95 Leu Thr Val Leu Val
Phe Leu Leu Cys Gly Leu Pro Trp Gly Ile Thr 100 105 110 Trp Phe Leu
Leu Phe Trp Ile Ala Pro Gly Val Phe Val Leu Asp Tyr 115 120 125 Ser
Pro Leu Leu Val Leu Thr Ala Ile Asn Ser Cys Ala Asn Pro Ile 130 135
140 Ile Tyr Phe Phe Val Gly 145 150 9 459 DNA Mus musculus CDS
(1)...(459) 9 ctg tgc ccg acg tgg tat cgc tgc cac cgt cca gta cat
aca tca act 48 Leu Cys Pro Thr Trp Tyr Arg Cys His Arg Pro Val His
Thr Ser Thr 1 5 10 15 gtc atg tgt gct gtg atc tgg gtc cta tcc ctg
ttg atc tgc att ctg 96 Val Met Cys Ala Val Ile Trp Val Leu Ser Leu
Leu Ile Cys Ile Leu 20 25 30 aat agc tat ttc tgt gct gtc tta cat
acc aga tat gat aat gac aat 144 Asn Ser Tyr Phe Cys Ala Val Leu His
Thr Arg Tyr Asp Asn Asp Asn 35 40 45 gag tgt ctg gca act aac atc
ttt acc gcc tcg tac atg ata ttt ttg 192 Glu Cys Leu Ala Thr Asn Ile
Phe Thr Ala Ser Tyr Met Ile Phe Leu 50 55 60 ctt gtg gtc ctc tgt
ctg tcc agc ctg gct ctg ctg gcc agg ttg ttc 240 Leu Val Val Leu Cys
Leu Ser Ser Leu Ala Leu Leu Ala Arg Leu Phe 65 70 75 80 tgt ggc gct
ggg cag atg aag ctt acc aga ttt cat gtg acc atc ttg 288 Cys Gly Ala
Gly Gln Met Lys Leu Thr Arg Phe His Val Thr Ile Leu 85 90 95 ctg
acc ctt ttg gtt ttt ctc ctc tgc ggg ttg ccc ttt gtc atc tac 336 Leu
Thr Leu Leu Val Phe Leu Leu Cys Gly Leu Pro Phe Val Ile Tyr 100 105
110 tgc atc ctg tta ttc aag att aag gat gat ttc cat gta tta gat gtt
384 Cys Ile Leu Leu Phe Lys Ile Lys Asp Asp Phe His Val Leu Asp Val
115 120 125 aat ctt tat cta gca tta gaa gtc ctg act gct att aac agc
tgt gcc 432 Asn Leu Tyr Leu Ala Leu Glu Val Leu Thr Ala Ile Asn Ser
Cys Ala 130 135 140 aac ccc atc atc tac ttc ttc gtc gga 459 Asn Pro
Ile Ile Tyr Phe Phe Val Gly 145 150 10 153 PRT Mus musculus 10 Leu
Cys Pro Thr Trp Tyr Arg Cys His Arg Pro Val His Thr Ser Thr 1 5 10
15 Val Met Cys Ala Val Ile Trp Val Leu Ser Leu Leu Ile Cys Ile Leu
20 25 30 Asn Ser Tyr Phe Cys Ala Val Leu His Thr Arg Tyr Asp Asn
Asp Asn 35 40 45 Glu Cys Leu Ala Thr Asn Ile Phe Thr Ala Ser Tyr
Met Ile Phe Leu 50 55 60 Leu Val Val Leu Cys Leu Ser Ser Leu Ala
Leu Leu Ala Arg Leu Phe 65 70 75 80 Cys Gly Ala Gly Gln Met Lys Leu
Thr Arg Phe His Val Thr Ile Leu 85 90 95 Leu Thr Leu Leu Val Phe
Leu Leu Cys Gly Leu Pro Phe Val Ile Tyr 100 105 110 Cys Ile Leu Leu
Phe Lys Ile Lys Asp Asp Phe His Val Leu Asp Val 115 120 125 Asn Leu
Tyr Leu Ala Leu Glu Val Leu Thr Ala Ile Asn Ser Cys Ala 130 135 140
Asn Pro Ile Ile Tyr Phe Phe Val Gly 145 150 11 2853 DNA Mus
musculus CDS (1820)...(2734) 11 caaggattct acaaacccaa gtatgcaagt
caacaatcta aatataattt gttccttttg 60 aagttagtgg ttcaatataa
cagacaaata catcatgccc tgaaattagc tttgaacaat 120 gctaagccca
taatgggaag taaaagattt gcttggttcc cactttcttc cttttctatt 180
ccgtttggac catagtggct agtgtctctt acaagatcac aagaaggagg ctctgcattt
240 atttctgagt gcctgtctgc atcctccttt ggcctggagg tcctctatga
aatcctgaag 300 taagaaagaa atgttccaga ctctgatttt tcttcctaga
ccaatgctat tcccttccat 360 gttgccaaca acttctcatc actctttctg
tactttcttt tagctgggtg gtttcttaat 420 ctacagtatt gactgtcatg
tcaaagttgg gtattttttg gctttagata tttcttctct 480 ggcttttctc
ccatccacac ataatcaaaa cactgaggtg atgacactaa gggactgctc 540
aaaggaaaag ggtgggttcc tgggctttgg ggttattaat aatttgcctg tcctctgcca
600 gcctctatca actcccctaa aacacaaaaa taattgttcc tagcaggcaa
gcacgacctg 660 acaattaatt aatgatcata aaaagtgcat tataaacatc
tgaaaacctc ataataaaac 720 tcaacacctt atacagtgag tatgttgtgg
ggtctgcata aatccaacaa aactccaatg 780 gagtggtact cagctattaa
aaatgaggaa ttcacgaaat tcttagccaa atgattagaa 840 gtagaaaata
tgatcctgag tgagaaaaga acaggcttgg tatgtactca ctgataagtg 900
gatactagcc caaaagctgc aaataatcag gataaaattc acagaccaca tgaacctcaa
960 taagaaggaa gaccaaagta tgggcgtttc ggtccttctt agaaggagaa
caaaatactc 1020 ccaagagcaa atatggagat aaagtgtaga acaggcacta
aaggaaaagt cacccagaga 1080 atgttccacc tggggattca tcccatatac
agttaccaaa cccagacact cttatggatg 1140 ccaaggagtg aatgctgaca
tagctgtttc ctaagaggcc atgccagaca cttacaaata 1200 cagaggccca
agttagcaac caaccattag actgagcaca gggttcctaa tagaggagtc 1260
agagaaagga ctgagggagt tgaaggggtt tgcatcccca taagaaaaac aacaacatga
1320 accaacaaga cactctcccc accaaccccc tgaactccta gggactaagc
catcaacaaa 1380 agagtacaca tggctccaga tgcatatgtt gcagaggatg
gccatatcat gcattgatgg 1440 aagaggtcct tgaacctatg aaggttctat
tgatgcccca gtgtaaggga atcgagggca 1500 gagaggtgga agtgggtgtg
tgggttgagc aacaccctca cagaagcagg gggagggagg 1560 atgagatggg
ggtttccagg aaggggggaa gcaggaaagg ggataacatt ttaaatttaa 1620
atatagaaaa tatccaatac aaaacatttt gaacaaacaa caaaaaactc acaaaaacaa
1680 caacaacaaa aaaaagaaat taaaagttgt gttcatagtg aaggcctcat
ttcttctttg 1740 tgttcccagc aacaccagtg cagggtttct ggccctaaac
acctcagcct cggcaatggc 1800 acccacaaca acaaatcca atg aac gaa acc atc
cct gga agt att gac atc 1852 Met Asn Glu Thr Ile Pro Gly Ser Ile
Asp Ile 1 5 10 gag acc ctg atc cca aac ttg atg atc atc atc ttc gga
ctg gtc ggg 1900 Glu Thr Leu Ile Pro Asn Leu Met Ile Ile Ile Phe
Gly Leu Val Gly 15 20 25 ctg aca gga aat gtc att ttg ttt tgg ctc
ctg ggc ttc cac ttg cac 1948 Leu Thr Gly Asn Val Ile Leu Phe Trp
Leu Leu Gly Phe His Leu His 30 35 40 agg aat gcc ttc tta gtc tac
atc cta aac ttg gcc ctg gct gac ttc 1996 Arg Asn Ala Phe Leu Val
Tyr Ile Leu Asn Leu Ala Leu Ala Asp Phe 45 50 55 ctc ttc ctt ctc
tgt cac atc ata aat tcc aca atg ctt ctt ctc aag 2044 Leu Phe Leu
Leu Cys His Ile Ile Asn Ser Thr Met Leu Leu Leu Lys 60 65 70 75 gtt
cac cta ccc aac aat att ttg aac cat tgc ttt gac atc atc atg 2092
Val His Leu Pro Asn Asn Ile Leu Asn His Cys Phe Asp Ile Ile Met 80
85 90 aca gtt ctc tac atc aca ggc ctg agc atg ctc agt gcc atc agc
act 2140 Thr Val Leu Tyr Ile Thr Gly Leu Ser Met Leu Ser Ala Ile
Ser Thr 95 100 105 gag cgc tgc ctg tct gtc ctg tgc ccc atc tgg tat
cgg tgc cgc cgc 2188 Glu Arg Cys Leu Ser Val Leu Cys Pro Ile Trp
Tyr Arg Cys Arg Arg 110 115 120 cca gaa cac aca tca act gtc ctg tgt
gct gtg atc tgg ttc ctg ccc 2236 Pro Glu His Thr Ser Thr Val Leu
Cys Ala Val Ile Trp Phe Leu Pro 125 130 135 ctg ttg atc tgc att ctg
aat gga tat ttc tgt cat ttc ttt ggt ccc 2284 Leu Leu Ile Cys Ile
Leu Asn Gly Tyr Phe Cys His Phe Phe Gly Pro 140 145 150 155 aaa tat
gta att gac tct gtg tgt ctg gca acg aac ttc ttt atc aga 2332 Lys
Tyr Val Ile Asp Ser Val Cys Leu Ala Thr Asn Phe Phe Ile Arg 160 165
170 aca tac ccg atg ttt ttg ttt ata gtc ctc tgt ctg tcc acc ctg gct
2380 Thr Tyr Pro Met Phe Leu Phe Ile Val Leu Cys Leu Ser Thr Leu
Ala 175 180 185 ctg ctg gcc agg ttg ttc tgt ggt ggt ggg aag acg aaa
ttt acc aga 2428 Leu Leu Ala Arg Leu Phe Cys Gly Gly Gly Lys Thr
Lys Phe Thr Arg 190 195 200 tta ttc gtg acc atc atg ctg acc gtt ttg
gtt ttt ctt ctc tgt ggg 2476 Leu Phe Val Thr Ile Met Leu Thr Val
Leu Val Phe Leu Leu Cys Gly 205 210 215 ttg ccc ctg ggc ttc ttc tgg
ttt ctg gtg ccg tgg att aac cgt gat 2524 Leu Pro Leu Gly Phe Phe
Trp Phe Leu Val Pro Trp Ile Asn Arg Asp 220 225 230 235 ttc agt gta
cta gat tat ata ctt ttt cag aca tca ctt gtc cta act 2572 Phe Ser
Val Leu Asp Tyr Ile Leu Phe Gln Thr Ser Leu Val Leu Thr 240 245 250
tct gtt aac agc tgt gcc aac ccc atc att tac ttc ttt gtg ggc tcc
2620 Ser Val Asn Ser Cys Ala Asn Pro Ile Ile Tyr Phe Phe Val Gly
Ser 255 260 265 ttc agg cat cgg ttg aag cac aag acc ctc aaa atg gtt
ctc cag agt 2668 Phe Arg His Arg Leu Lys His Lys Thr Leu Lys Met
Val Leu Gln Ser 270 275 280 gca ttg cag gac act cct gag aca cct gaa
aac atg gtg gag atg tca 2716 Ala Leu Gln Asp Thr Pro Glu Thr Pro
Glu Asn Met Val Glu Met Ser 285 290 295 aga agc aaa gca gag ccg
tgatgaagag cctctacctg gacctcagag 2764 Arg Ser Lys Ala Glu Pro 300
305 gtggctttgg attgagcact gccctgctgc acttgaccac tgtccactct
cctctcagct 2824 tactgacttt ggatgcctca gtggtccaa 2853 12 305 PRT Mus
musculus 12 Met Asn Glu Thr Ile Pro Gly Ser Ile Asp Ile Glu Thr Leu
Ile Pro 1 5 10 15 Asn Leu Met Ile Ile Ile Phe Gly Leu Val Gly Leu
Thr Gly Asn Val 20 25 30 Ile Leu Phe Trp Leu Leu Gly Phe His Leu
His Arg Asn Ala Phe Leu 35 40 45 Val Tyr Ile Leu Asn Leu Ala Leu
Ala Asp Phe Leu Phe Leu Leu Cys 50 55 60 His Ile Ile Asn Ser Thr
Met Leu Leu Leu Lys Val His Leu Pro Asn 65 70 75 80 Asn Ile Leu Asn
His Cys Phe Asp Ile Ile Met Thr Val Leu Tyr Ile 85 90 95 Thr Gly
Leu Ser Met Leu Ser Ala Ile Ser Thr Glu Arg Cys Leu Ser 100 105 110
Val Leu Cys Pro Ile Trp Tyr Arg Cys Arg Arg Pro Glu His Thr Ser 115
120 125 Thr Val Leu Cys Ala Val Ile Trp Phe Leu Pro Leu Leu Ile Cys
Ile 130 135 140 Leu Asn Gly Tyr Phe Cys His Phe Phe Gly Pro Lys Tyr
Val Ile Asp 145 150 155 160 Ser Val Cys Leu Ala Thr Asn Phe Phe Ile
Arg Thr Tyr Pro Met Phe 165 170 175 Leu Phe Ile Val Leu Cys Leu Ser
Thr Leu Ala Leu Leu Ala Arg Leu 180 185 190 Phe Cys Gly Gly Gly Lys
Thr Lys Phe Thr Arg Leu Phe Val Thr Ile 195 200 205 Met Leu Thr Val
Leu Val Phe Leu Leu Cys Gly Leu Pro Leu Gly Phe 210 215 220 Phe Trp
Phe Leu Val Pro Trp Ile Asn Arg Asp Phe Ser Val Leu Asp 225 230 235
240 Tyr Ile Leu Phe Gln Thr Ser Leu Val Leu Thr Ser Val Asn Ser Cys
245 250 255 Ala Asn Pro Ile Ile Tyr Phe Phe Val Gly Ser Phe Arg His
Arg Leu 260 265 270 Lys His Lys Thr Leu Lys Met Val Leu Gln Ser Ala
Leu Gln Asp Thr 275 280 285 Pro Glu Thr Pro Glu Asn Met Val Glu Met
Ser Arg Ser Lys Ala Glu 290 295 300 Pro 305 13 3391 DNA Mus
musculus CDS (170)...(574) 13 ccgaaaacca acaaaataga accgcgggtg
cctttctcca gctgggatga aggacttgag 60 cagaaactca ttgccagctt
cctccctacg cgagagccga ctgagtccca ggtccccagt 120 cttcccccgg
gacgttgtgc acggtgccca ttcttgagca gccacaaca atg gag gtg 178 Met Glu
Val 1 ctc ccc aag gcc ctg gag gta gac gag agg tct cca gag tcc aag
gac 226 Leu Pro Lys Ala Leu Glu Val Asp Glu Arg Ser Pro Glu Ser Lys
Asp 5 10 15 ctg ctg ccc agc cag aca gcc agc tcc ctg tgc atc agt tcc
aga agt 274 Leu Leu Pro Ser Gln Thr Ala Ser Ser Leu Cys Ile Ser Ser
Arg Ser 20 25 30 35 gag tct gtc tgg acc acc aca ccc aaa agc aac tgg
gaa atc tac cac 322 Glu Ser Val Trp Thr Thr Thr Pro Lys Ser Asn Trp
Glu Ile Tyr His 40 45 50 aag ccc atc atc atc atg tca gtg gga gct
gcc att ctg ctc ttt ggc 370 Lys Pro Ile Ile Ile Met Ser Val Gly Ala
Ala Ile Leu Leu Phe Gly 55 60 65 gtg gcc atc acc tgt gtg gcc tac
atc ttg gaa gag aag cat aaa gtt 418 Val Ala Ile Thr Cys Val Ala Tyr
Ile Leu Glu Glu Lys His Lys Val 70 75 80 gtg caa gtg ctc agg atg
ata ggg cct gcc ttc ctg tcc ctg gga ctc 466 Val Gln Val Leu Arg Met
Ile Gly Pro Ala Phe Leu Ser Leu Gly Leu 85 90 95 atg atg ctg gtg
tgt ggg ctg gtg tgg gtc ccc ata atc aaa aag aag 514 Met Met Leu Val
Cys Gly Leu Val Trp Val Pro Ile Ile Lys Lys Lys 100 105 110 115 cag
aag caa agg cag aag tcc aac ttc ttc caa agc ctc aag ttc ttc 562 Gln
Lys Gln Arg Gln Lys Ser Asn Phe Phe Gln Ser Leu Lys Phe Phe 120 125
130 ctc ctg aac cgc tgatgactgg ttgtccagaa gatctgctaa ccaataagca 614
Leu Leu Asn Arg 135 gcctcctacc ttctcttcgg gtaccacaaa gttgatccag
gcaaaccctc ctcttggccc 674 tgtggacagg atagagctca gggcttcacc
ctcatacaac ctagcagcat tgctgactga 734 gtctcacctg gtttccatag
ctgtggatgc tgtgcccttg gatactttca ttaccctcat 794 ccctggcacc
tgcattcagc catcagccat cccattctct ctgccaaggg caatgtgtgc 854
atgctaggaa attctttggg ggttgactac attcccaagg agaacttgta tgttacggtt
914 gtgtgcctga tcttagattc ccatctacat ccttctggaa ccaaaagtga
ccaagcagat 974 aaggctgact tcagtcccat tgggtttgac agccttggct
ccctccttgg atgggacatt 1034
gactaacatt acaagagaaa ggatatgtct catgtatcac acattccaaa atctggacag
1094 tgatggggct gggggtgagg gaaacactgt ctagagtaaa ccattcctct
gggagtaatc 1154 tggaacttat acagtgaagg aagttagctc ctaaatatat
gatattggca caagaggcaa 1214 tatgcaggct aagaggtatc aacacttccc
cttgatcctc caatgcgctt cttgcagaat 1274 gcctttatat tagcaattag
ccaagaacaa atgctctttg ttctaacttc cttccccacc 1334 acatctctgc
gtctacacag ctccagaaca gaaggacggg aggccacaga tgtgacctgt 1394
aagatcatct ccttctcctg tcaatcaaga cctaacctga aattgaatgc catgtccgac
1454 tcacgctgca tggggtttta gagataggtt cactggaaaa aaggaaatct
cagcctccct 1514 cctccctgtt cctccctacc aaacaagcaa gtatttattg
agtttccttc tctaggccta 1574 cgttgggaac agccagaccc agtctctgat
gtcatcttat ttccaaaagt gaaagaggga 1634 aaaacatggc caagccaact
ggcaatactc catactgagt tcttagggtg gccatgggaa 1694 cacatggatc
taacaaatgt acaggaagat agatttctgg agaccatgtt caccccttct 1754
gaatatgaag gggaaggaag tgtttggaat gagcaagatg tgcaaggtag tcagcaactg
1814 ccttgcatgt ggagaagcta aggggaaaga gacagggtgg ggttaggatt
ccgcatagct 1874 cccggatgct attccatcct ctcttgccta cttcccccct
gcttccccag gtaccttaca 1934 tccagctact ccttggtaca ctgcaggctt
ctggggtcaa tagggactgg gaggggcatc 1994 tccagagggc ctaacaagta
gatataaccc aagaggtaag taccctcaaa acttcattat 2054 agtcaccaag
acacctttag gcaaaagacc gggcacctat aagaaatttc caaagctgtt 2114
ccaggcaagg ccaggccaga gagcagagga aggtacctag tagcaaagtg aatgacaaga
2174 gctgcattgg ttcaggttga ctcttcatcc ttaacctttg ggcatttggg
aacactatgg 2234 caaacaacct ccaacaggtc tccagatatc tcaaccattc
acagtacttc tataggcagt 2294 tagaatccac cacctttgtt cctgttgcat
tgtgggacat tcctcggagg aagtatttgt 2354 tttgtggaat caacacacac
acacacacgc acagagagag agagagagag agagagagag 2414 agagagagag
agagagagag agagaaagaa agagaaagaa agaaagaaag agaaagagac 2474
tgactcccta actaaaaagt cagagtttgg gaagcctgtg gcctttcaaa gctcacttaa
2534 gaatatcatg ttcctcatta agactcacat catcgagccc aggccctgca
gtccacccat 2594 tccctgaata caggcagctc aggaccaacc ctggggttgt
tgaaatactg cctagtgctt 2654 ccacgaatgt ctaatgcctc catgacaggg
ctttcagacc actcctttct cctgacatgg 2714 aaggacagcc ctggggtgga
gcctctcaat cttctgtgcc ttcatgaaag ggaacacaca 2774 gatgagctca
cagccagctc acttggaatc cgcaccccat gcacctcatt gtcctgagag 2834
ctcattgtct gggcacagct gtgggaagac ctttgcagat ctcactttca agtatgtctc
2894 aacagaaggg agtttgggga taatcacgat gccaggaaat cttcaagttc
tagacatctt 2954 tcatagccac atcagtacct gttccccaac ccctgcccct
caaggtaagt acttagcaaa 3014 caaaatcaaa gagcctttga gaaaatatcc
caaatactgg ttaactcccc cggccttgca 3074 ccaaactccc cacaaaagtg
atagtcagga agtgagcaga gtcacaccca acatcttgga 3134 aaattttgcc
aaagaccatt gcctcatgaa aactggggtg gggataacct gtgagtgcag 3194
ccgggttgga tgccgtgtct ctgcaacaaa gcattctggg tagtgatttc agtcatctca
3254 gaagacaaga gcaacatcca cagcaccatc ccaccggact gtattacggg
cttctgtcgc 3314 tcttctgttt tggagaattt aatctaaccc aacgcctaat
ggaatcaatg tcgtattgaa 3374 ctgtattctg tttaaaa 3391 14 135 PRT Mus
musculus 14 Met Glu Val Leu Pro Lys Ala Leu Glu Val Asp Glu Arg Ser
Pro Glu 1 5 10 15 Ser Lys Asp Leu Leu Pro Ser Gln Thr Ala Ser Ser
Leu Cys Ile Ser 20 25 30 Ser Arg Ser Glu Ser Val Trp Thr Thr Thr
Pro Lys Ser Asn Trp Glu 35 40 45 Ile Tyr His Lys Pro Ile Ile Ile
Met Ser Val Gly Ala Ala Ile Leu 50 55 60 Leu Phe Gly Val Ala Ile
Thr Cys Val Ala Tyr Ile Leu Glu Glu Lys 65 70 75 80 His Lys Val Val
Gln Val Leu Arg Met Ile Gly Pro Ala Phe Leu Ser 85 90 95 Leu Gly
Leu Met Met Leu Val Cys Gly Leu Val Trp Val Pro Ile Ile 100 105 110
Lys Lys Lys Gln Lys Gln Arg Gln Lys Ser Asn Phe Phe Gln Ser Leu 115
120 125 Lys Phe Phe Leu Leu Asn Arg 130 135 15 2040 DNA Homo
sapiens CDS (328)...(1293) 15 gcccaggata gagtaatcat cgggtccaca
gccctggcta gatgagtggg ggtgttttga 60 tcctaatgtt attcccatgt
tagcacagaa cttgtgtggc agtagagaga ggtcaggctt 120 cagagtcagc
aagaactgga tttcaaactg gatttgagga cccccacctt ttgataggtg 180
acttattctc tgtgagtctc tgatctgccc tctttaaatg aggaagtaaa tcccacatgg
240 cagggtggtg gggagaatca gagatcatac agctggtgat cacaactggt
ttctgtttcc 300 agggtcacca gactagggtt tctgagc atg gat cca acc atc
tca acc ttg gac 354 Met Asp Pro Thr Ile Ser Thr Leu Asp 1 5 aca gaa
ctg aca cca atc aac gga act gag gag act ctt tgc tac aag 402 Thr Glu
Leu Thr Pro Ile Asn Gly Thr Glu Glu Thr Leu Cys Tyr Lys 10 15 20 25
cag acc ttg agc ctc acg gtg ctg acg tgc atc gtt tcc ctt gtc ggg 450
Gln Thr Leu Ser Leu Thr Val Leu Thr Cys Ile Val Ser Leu Val Gly 30
35 40 ctg aca gga aac gca gtt gtg ctc tgg ctc ctg ggc tgc cgc atg
cgc 498 Leu Thr Gly Asn Ala Val Val Leu Trp Leu Leu Gly Cys Arg Met
Arg 45 50 55 agg aac gcc ttc tcc atc tac atc ctc aac ttg gcc gca
gca gac ttc 546 Arg Asn Ala Phe Ser Ile Tyr Ile Leu Asn Leu Ala Ala
Ala Asp Phe 60 65 70 ctc ttc ctc agc ggc cgc ctt ata tat tcc ctg
tta agc ttc atc agt 594 Leu Phe Leu Ser Gly Arg Leu Ile Tyr Ser Leu
Leu Ser Phe Ile Ser 75 80 85 atc ccc cat acc atc tct aaa atc ctc
tat cct gtg atg atg ttt tcc 642 Ile Pro His Thr Ile Ser Lys Ile Leu
Tyr Pro Val Met Met Phe Ser 90 95 100 105 tac ttt gca ggc ctg agc
ttt ctg agt gcc gtg agc acc gag cgc tgc 690 Tyr Phe Ala Gly Leu Ser
Phe Leu Ser Ala Val Ser Thr Glu Arg Cys 110 115 120 ctg tcc gtc ctg
tgg ccc atc tgg tac cgc tgc cac cgc ccc aca cac 738 Leu Ser Val Leu
Trp Pro Ile Trp Tyr Arg Cys His Arg Pro Thr His 125 130 135 ctg tca
gcg gtg gtg tgt gtc ctg ctc tgg gcc ctg tcc ctg ctg cgg 786 Leu Ser
Ala Val Val Cys Val Leu Leu Trp Ala Leu Ser Leu Leu Arg 140 145 150
agc atc ctg gag tgg atg tta tgt ggc ttc ctg ttc agt ggt gct gat 834
Ser Ile Leu Glu Trp Met Leu Cys Gly Phe Leu Phe Ser Gly Ala Asp 155
160 165 tct gct tgg tgt caa aca tca gat ttc atc aca gtc gcg tgg ctg
att 882 Ser Ala Trp Cys Gln Thr Ser Asp Phe Ile Thr Val Ala Trp Leu
Ile 170 175 180 185 ttt tta tgt gtg gtt ctc tgt ggg tcc agc ctg gtc
ctg ctg atc agg 930 Phe Leu Cys Val Val Leu Cys Gly Ser Ser Leu Val
Leu Leu Ile Arg 190 195 200 att ctc tgt gga tcc cgg aag ata ccg ctg
acc agg ctg tac gtg acc 978 Ile Leu Cys Gly Ser Arg Lys Ile Pro Leu
Thr Arg Leu Tyr Val Thr 205 210 215 atc ctg ctc aca gta ctg gtc ttc
ctc ctc tgt ggc ctg ccc ttt ggc 1026 Ile Leu Leu Thr Val Leu Val
Phe Leu Leu Cys Gly Leu Pro Phe Gly 220 225 230 att cag ttt ttc cta
ttt tta tgg atc cac gtg gac agg gaa gtc tta 1074 Ile Gln Phe Phe
Leu Phe Leu Trp Ile His Val Asp Arg Glu Val Leu 235 240 245 ttt tgt
cat gtt cat cta gtt tct att ttc ctg tcc gct ctt aac agc 1122 Phe
Cys His Val His Leu Val Ser Ile Phe Leu Ser Ala Leu Asn Ser 250 255
260 265 agt gcc aac ccc atc att tac ttc ttc gtg ggc tcc ttt agg cag
cgt 1170 Ser Ala Asn Pro Ile Ile Tyr Phe Phe Val Gly Ser Phe Arg
Gln Arg 270 275 280 caa aat agg cag aac ctg aag ctg gtt ctc cag agg
gct ctg cag gac 1218 Gln Asn Arg Gln Asn Leu Lys Leu Val Leu Gln
Arg Ala Leu Gln Asp 285 290 295 gcg tct gag gtg gat gaa ggt gga ggg
cag ctt cct gag gaa atc ctg 1266 Ala Ser Glu Val Asp Glu Gly Gly
Gly Gln Leu Pro Glu Glu Ile Leu 300 305 310 gag ctg tcg gga agc aga
ttg gag cag tgaggaagag cctctgccct 1313 Glu Leu Ser Gly Ser Arg Leu
Glu Gln 315 320 gtcagacagg actttgagag caacactgcc ctgccaccct
tgacaattat atgcgttttt 1373 cttagccttc tgcctcagaa atgtctcagt
ggttcctcaa ggtcttcaaa tagatgttta 1433 tctaacctga cagttgcggt
tttcacccat ggaaagcatt agtctgacag tacaatgttt 1493 agattctcct
tgatattacc aacacatttt ccctgttatc tcacactgaa tctttcctac 1553
agaacacttt ttctgcaatt ttctttgtaa taaaaggagt tcctgtacaa aaccctaaaa
1613 cactctttat acttctttcc tacctgatag catcaaaaag gaagattcct
tattaatctc 1673 tcagactatg ttcccctgaa aatcatgttc ccttctatga
ctggaggcat tactgcagtt 1733 agaagctcga ttcttaataa gtgagttctg
ctatctctac attccattga attctcagat 1793 acagagcaaa ataatgtcct
tagagacaga ctctctcttc ataaaaacac tctcacctat 1853 tggttttata
aaaagtcttc ccctgtcatt tgttcacagc atggtgatat gttggccttg 1913
gtttctagta aagacaactg tggccccttc cccttgagaa cttttaagtg cttatttagc
1973 tcttcctgga ctaatggacc agtgaggagc ccataaatgt gccccagttc
tattttggcc 2033 attggaa 2040 16 322 PRT Homo sapiens 16 Met Asp Pro
Thr Ile Ser Thr Leu Asp Thr Glu Leu Thr Pro Ile Asn 1 5 10 15 Gly
Thr Glu Glu Thr Leu Cys Tyr Lys Gln Thr Leu Ser Leu Thr Val 20 25
30 Leu Thr Cys Ile Val Ser Leu Val Gly Leu Thr Gly Asn Ala Val Val
35 40 45 Leu Trp Leu Leu Gly Cys Arg Met Arg Arg Asn Ala Phe Ser
Ile Tyr 50 55 60 Ile Leu Asn Leu Ala Ala Ala Asp Phe Leu Phe Leu
Ser Gly Arg Leu 65 70 75 80 Ile Tyr Ser Leu Leu Ser Phe Ile Ser Ile
Pro His Thr Ile Ser Lys 85 90 95 Ile Leu Tyr Pro Val Met Met Phe
Ser Tyr Phe Ala Gly Leu Ser Phe 100 105 110 Leu Ser Ala Val Ser Thr
Glu Arg Cys Leu Ser Val Leu Trp Pro Ile 115 120 125 Trp Tyr Arg Cys
His Arg Pro Thr His Leu Ser Ala Val Val Cys Val 130 135 140 Leu Leu
Trp Ala Leu Ser Leu Leu Arg Ser Ile Leu Glu Trp Met Leu 145 150 155
160 Cys Gly Phe Leu Phe Ser Gly Ala Asp Ser Ala Trp Cys Gln Thr Ser
165 170 175 Asp Phe Ile Thr Val Ala Trp Leu Ile Phe Leu Cys Val Val
Leu Cys 180 185 190 Gly Ser Ser Leu Val Leu Leu Ile Arg Ile Leu Cys
Gly Ser Arg Lys 195 200 205 Ile Pro Leu Thr Arg Leu Tyr Val Thr Ile
Leu Leu Thr Val Leu Val 210 215 220 Phe Leu Leu Cys Gly Leu Pro Phe
Gly Ile Gln Phe Phe Leu Phe Leu 225 230 235 240 Trp Ile His Val Asp
Arg Glu Val Leu Phe Cys His Val His Leu Val 245 250 255 Ser Ile Phe
Leu Ser Ala Leu Asn Ser Ser Ala Asn Pro Ile Ile Tyr 260 265 270 Phe
Phe Val Gly Ser Phe Arg Gln Arg Gln Asn Arg Gln Asn Leu Lys 275 280
285 Leu Val Leu Gln Arg Ala Leu Gln Asp Ala Ser Glu Val Asp Glu Gly
290 295 300 Gly Gly Gln Leu Pro Glu Glu Ile Leu Glu Leu Ser Gly Ser
Arg Leu 305 310 315 320 Glu Gln 17 1300 DNA Homo sapiens CDS
(171)...(1160) 17 tccctggccc ttaataaatg acttaatctc ttcaagcctc
tgatttcctc tcctgtaaaa 60 caggggcggt aattaccaca taacaggctg
gtcatgaaaa tcagtgaaca tgcagcaggt 120 gctcaagtct tgtttttgtt
tccaggggca ccagtggagg ttttctgagc atg gat 176 Met Asp 1 cca acc acc
ccg gcc tgg gga aca gaa agt aca aca gtg aat gga aat 224 Pro Thr Thr
Pro Ala Trp Gly Thr Glu Ser Thr Thr Val Asn Gly Asn 5 10 15 gac caa
gcc ctt ctt ctg ctt tgt ggc aag gag acc ctg atc ccg gtc 272 Asp Gln
Ala Leu Leu Leu Leu Cys Gly Lys Glu Thr Leu Ile Pro Val 20 25 30
ttc ctg atc ctt ttc att gcc ctg gtc ggg ctg gta gga aac ggg ttt 320
Phe Leu Ile Leu Phe Ile Ala Leu Val Gly Leu Val Gly Asn Gly Phe 35
40 45 50 gtg ctc tgg ctc ctg ggc ttc cgc atg cgc agg aac gcc ttc
tct gtc 368 Val Leu Trp Leu Leu Gly Phe Arg Met Arg Arg Asn Ala Phe
Ser Val 55 60 65 tac gtc ctc agc ctg gcc ggg gcc gac ttc ctc ttc
ctc tgc ttc cag 416 Tyr Val Leu Ser Leu Ala Gly Ala Asp Phe Leu Phe
Leu Cys Phe Gln 70 75 80 att ata aat tgc ctg gtg tac ctc agt aac
ttc ttc tgt tcc atc tcc 464 Ile Ile Asn Cys Leu Val Tyr Leu Ser Asn
Phe Phe Cys Ser Ile Ser 85 90 95 atc aat ttc cct agc ttc ttc acc
act gtg atg acc tgt gcc tac ctt 512 Ile Asn Phe Pro Ser Phe Phe Thr
Thr Val Met Thr Cys Ala Tyr Leu 100 105 110 gca ggc ctg agc atg ctg
agc acc gtc agc acc gag cgc tgc ctg tcc 560 Ala Gly Leu Ser Met Leu
Ser Thr Val Ser Thr Glu Arg Cys Leu Ser 115 120 125 130 gtc ctg tgg
ccc atc tgg tat cgc tgc cgc cgc ccc aga cac ctg tca 608 Val Leu Trp
Pro Ile Trp Tyr Arg Cys Arg Arg Pro Arg His Leu Ser 135 140 145 gcg
gtc gtg tgt gtc ctg ctc tgg gcc ctg tcc cta ctg ctg agc atc 656 Ala
Val Val Cys Val Leu Leu Trp Ala Leu Ser Leu Leu Leu Ser Ile 150 155
160 ttg gaa ggg aag ttc tgt ggc ttc tta ttt agt gat ggt gac tct ggt
704 Leu Glu Gly Lys Phe Cys Gly Phe Leu Phe Ser Asp Gly Asp Ser Gly
165 170 175 tgg tgt cag aca ttt gat ttc atc act gca gcg tgg ctg att
ttt tta 752 Trp Cys Gln Thr Phe Asp Phe Ile Thr Ala Ala Trp Leu Ile
Phe Leu 180 185 190 ttc atg gtt ctc tgt ggg tcc agt ctg gcc ctg ctg
gtc agg atc ctc 800 Phe Met Val Leu Cys Gly Ser Ser Leu Ala Leu Leu
Val Arg Ile Leu 195 200 205 210 tgt ggc tcc agg ggt ctg cca ctg acc
agg ctg tac ctg acc atc ctg 848 Cys Gly Ser Arg Gly Leu Pro Leu Thr
Arg Leu Tyr Leu Thr Ile Leu 215 220 225 ctc aca gtg ctg gtg ttc ctc
ctc tgc ggc ctg ccc ttt ggc att cag 896 Leu Thr Val Leu Val Phe Leu
Leu Cys Gly Leu Pro Phe Gly Ile Gln 230 235 240 tgg ttc cta ata tta
tgg atc tgg aag gat tct gat gtc tta ttt tgt 944 Trp Phe Leu Ile Leu
Trp Ile Trp Lys Asp Ser Asp Val Leu Phe Cys 245 250 255 cat att cat
cca gtt tca gtt gtc ctg tca tct ctt aac agc agt gcc 992 His Ile His
Pro Val Ser Val Val Leu Ser Ser Leu Asn Ser Ser Ala 260 265 270 aac
ccc atc att tac ttc ttc gtg ggc tct ttt agg aag cag tgg cgg 1040
Asn Pro Ile Ile Tyr Phe Phe Val Gly Ser Phe Arg Lys Gln Trp Arg 275
280 285 290 ctg cag cag ccg atc ctc aag ctg gct ctc cag agg gct ctg
cag gac 1088 Leu Gln Gln Pro Ile Leu Lys Leu Ala Leu Gln Arg Ala
Leu Gln Asp 295 300 305 att gct gag gtg gat cac agt gaa gga tgc ttc
cgt cag ggc acc ccg 1136 Ile Ala Glu Val Asp His Ser Glu Gly Cys
Phe Arg Gln Gly Thr Pro 310 315 320 gag atg tcg aga agc agt ctg gtg
tagagatgga cagcctctac ttccatcaga 1190 Glu Met Ser Arg Ser Ser Leu
Val 325 330 tatatgtggc tttgagaggc aactttgccc ctgtctgtct gatttgctga
actttctcag 1250 tcctgatttt aaaacagtta agagagtcct tgtgaggatt
aagtgagaca 1300 18 330 PRT Homo sapiens 18 Met Asp Pro Thr Thr Pro
Ala Trp Gly Thr Glu Ser Thr Thr Val Asn 1 5 10 15 Gly Asn Asp Gln
Ala Leu Leu Leu Leu Cys Gly Lys Glu Thr Leu Ile 20 25 30 Pro Val
Phe Leu Ile Leu Phe Ile Ala Leu Val Gly Leu Val Gly Asn 35 40 45
Gly Phe Val Leu Trp Leu Leu Gly Phe Arg Met Arg Arg Asn Ala Phe 50
55 60 Ser Val Tyr Val Leu Ser Leu Ala Gly Ala Asp Phe Leu Phe Leu
Cys 65 70 75 80 Phe Gln Ile Ile Asn Cys Leu Val Tyr Leu Ser Asn Phe
Phe Cys Ser 85 90 95 Ile Ser Ile Asn Phe Pro Ser Phe Phe Thr Thr
Val Met Thr Cys Ala 100 105 110 Tyr Leu Ala Gly Leu Ser Met Leu Ser
Thr Val Ser Thr Glu Arg Cys 115 120 125 Leu Ser Val Leu Trp Pro Ile
Trp Tyr Arg Cys Arg Arg Pro Arg His 130 135 140 Leu Ser Ala Val Val
Cys Val Leu Leu Trp Ala Leu Ser Leu Leu Leu 145 150 155 160 Ser Ile
Leu Glu Gly Lys Phe Cys Gly Phe Leu Phe Ser Asp Gly Asp 165 170 175
Ser Gly Trp Cys Gln Thr Phe Asp Phe Ile Thr Ala Ala Trp Leu Ile 180
185 190 Phe Leu Phe Met Val Leu Cys Gly Ser Ser Leu Ala Leu Leu Val
Arg 195 200 205 Ile Leu Cys Gly Ser Arg Gly Leu Pro Leu Thr Arg Leu
Tyr Leu Thr 210 215 220 Ile Leu Leu Thr Val Leu Val Phe Leu Leu Cys
Gly Leu Pro Phe Gly 225 230 235 240 Ile Gln Trp Phe Leu Ile Leu Trp
Ile Trp Lys Asp Ser Asp Val Leu 245 250 255 Phe Cys His Ile His Pro
Val Ser Val Val Leu Ser Ser Leu Asn Ser 260 265 270
Ser Ala Asn Pro Ile Ile Tyr Phe Phe Val Gly Ser Phe Arg Lys Gln 275
280 285 Trp Arg Leu Gln Gln Pro Ile Leu Lys Leu Ala Leu Gln Arg Ala
Leu 290 295 300 Gln Asp Ile Ala Glu Val Asp His Ser Glu Gly Cys Phe
Arg Gln Gly 305 310 315 320 Thr Pro Glu Met Ser Arg Ser Ser Leu Val
325 330 19 135 PRT Homo sapiens 19 Met Glu Thr Leu Pro Lys Val Leu
Glu Val Asp Glu Lys Ser Pro Glu 1 5 10 15 Ala Lys Asp Leu Leu Pro
Ser Gln Thr Ala Ser Ser Leu Cys Ile Ser 20 25 30 Ser Arg Ser Glu
Ser Val Trp Thr Thr Thr Pro Arg Ser Asn Trp Glu 35 40 45 Ile Tyr
Arg Lys Pro Ile Val Ile Met Ser Val Gly Gly Ala Ile Leu 50 55 60
Leu Phe Gly Val Val Ile Thr Cys Leu Ala Tyr Thr Leu Lys Leu Ser 65
70 75 80 Asp Lys Ser Leu Ser Ile Leu Lys Met Val Gly Pro Gly Phe
Leu Ser 85 90 95 Leu Gly Leu Met Met Leu Val Cys Gly Leu Val Trp
Val Pro Ile Ile 100 105 110 Lys Lys Lys Gln Lys His Arg Gln Lys Ser
Asn Phe Leu Arg Ser Leu 115 120 125 Lys Ser Phe Phe Leu Thr Arg 130
135 20 970 DNA Mus musculus CDS (83)...(943) 20 gtgtcaccaa
cagcacccac aacaaatcca atggacaaac ctctttggaa gtatggacat 60
ctggattctg acccgaaact ag atg atc atc ata ttc aga ctg gtt ggg atg
112 Met Ile Ile Ile Phe Arg Leu Val Gly Met 1 5 10 aca gga aat gcc
att gtg ttc tgg ctc ctg ggc ttc agc ttg cac agg 160 Thr Gly Asn Ala
Ile Val Phe Trp Leu Leu Gly Phe Ser Leu His Arg 15 20 25 aat gcc
ttc tca gtc tac att tta aac ttg gcc ctt gct gac ttc gtc 208 Asn Ala
Phe Ser Val Tyr Ile Leu Asn Leu Ala Leu Ala Asp Phe Val 30 35 40
ttc ctc ctc tgt cac atc ata gat tcc atg ctg ctt ctt ctc act gtt 256
Phe Leu Leu Cys His Ile Ile Asp Ser Met Leu Leu Leu Leu Thr Val 45
50 55 ttc tac ccc aac aat atc ttt tct ggg tac ttt tac acc atc atg
acg 304 Phe Tyr Pro Asn Asn Ile Phe Ser Gly Tyr Phe Tyr Thr Ile Met
Thr 60 65 70 gtt ccc tac atc gca ggc ctg agc atg ctc agt gcc atc
agc act gag 352 Val Pro Tyr Ile Ala Gly Leu Ser Met Leu Ser Ala Ile
Ser Thr Glu 75 80 85 90 ctc tgc ctg tct gtc ctg tgc ccc atc tgg tat
cgc tgc cac cac cca 400 Leu Cys Leu Ser Val Leu Cys Pro Ile Trp Tyr
Arg Cys His His Pro 95 100 105 gaa cac aca tca act gtc atg tgt gct
gcg ata tgg gtc ctg ccc ctg 448 Glu His Thr Ser Thr Val Met Cys Ala
Ala Ile Trp Val Leu Pro Leu 110 115 120 ttg gtc tgc att ctg aat agg
tat ttc tgc agt ttc tta gat atc aat 496 Leu Val Cys Ile Leu Asn Arg
Tyr Phe Cys Ser Phe Leu Asp Ile Asn 125 130 135 tat aac aat gac aaa
cag tgt ctg gca tca aac ttc ttt act aga gca 544 Tyr Asn Asn Asp Lys
Gln Cys Leu Ala Ser Asn Phe Phe Thr Arg Ala 140 145 150 tac ctg atg
ttt ttg ttt gtg gtc ctt tgt ctg tcc agc atg gct ctg 592 Tyr Leu Met
Phe Leu Phe Val Val Leu Cys Leu Ser Ser Met Ala Leu 155 160 165 170
ctg gcc agg ttg ttc tgt ggc act ggg cag atg aag ctt acc aga ttg 640
Leu Ala Arg Leu Phe Cys Gly Thr Gly Gln Met Lys Leu Thr Arg Leu 175
180 185 tac gtg acc atc atg ctg act gtt ttg ggt ttt ctc ctc tgt ggg
ttg 688 Tyr Val Thr Ile Met Leu Thr Val Leu Gly Phe Leu Leu Cys Gly
Leu 190 195 200 ccc ttt gtc atc tac tac ttc ctg tta ttc aat att aag
gat ggt ttt 736 Pro Phe Val Ile Tyr Tyr Phe Leu Leu Phe Asn Ile Lys
Asp Gly Phe 205 210 215 tgt tta ttt gat ttt aga ttt tat atg tca aca
cat gtc ctg act gct 784 Cys Leu Phe Asp Phe Arg Phe Tyr Met Ser Thr
His Val Leu Thr Ala 220 225 230 att aac aac tgt gcc aac ccc ata att
tac ttt ttc gag ggc tcc ttc 832 Ile Asn Asn Cys Ala Asn Pro Ile Ile
Tyr Phe Phe Glu Gly Ser Phe 235 240 245 250 agg cat cag ttg aag cac
cag acc ctc aaa atg gtt ctc cag agt gta 880 Arg His Gln Leu Lys His
Gln Thr Leu Lys Met Val Leu Gln Ser Val 255 260 265 ctg cag gac act
cct gag ata gct gaa aat atg gtg gag atg tca aga 928 Leu Gln Asp Thr
Pro Glu Ile Ala Glu Asn Met Val Glu Met Ser Arg 270 275 280 aac ata
cca aag cca tgatgaaaag cctttgcctg gacctca 970 Asn Ile Pro Lys Pro
285 21 287 PRT Mus musculus 21 Met Ile Ile Ile Phe Arg Leu Val Gly
Met Thr Gly Asn Ala Ile Val 1 5 10 15 Phe Trp Leu Leu Gly Phe Ser
Leu His Arg Asn Ala Phe Ser Val Tyr 20 25 30 Ile Leu Asn Leu Ala
Leu Ala Asp Phe Val Phe Leu Leu Cys His Ile 35 40 45 Ile Asp Ser
Met Leu Leu Leu Leu Thr Val Phe Tyr Pro Asn Asn Ile 50 55 60 Phe
Ser Gly Tyr Phe Tyr Thr Ile Met Thr Val Pro Tyr Ile Ala Gly 65 70
75 80 Leu Ser Met Leu Ser Ala Ile Ser Thr Glu Leu Cys Leu Ser Val
Leu 85 90 95 Cys Pro Ile Trp Tyr Arg Cys His His Pro Glu His Thr
Ser Thr Val 100 105 110 Met Cys Ala Ala Ile Trp Val Leu Pro Leu Leu
Val Cys Ile Leu Asn 115 120 125 Arg Tyr Phe Cys Ser Phe Leu Asp Ile
Asn Tyr Asn Asn Asp Lys Gln 130 135 140 Cys Leu Ala Ser Asn Phe Phe
Thr Arg Ala Tyr Leu Met Phe Leu Phe 145 150 155 160 Val Val Leu Cys
Leu Ser Ser Met Ala Leu Leu Ala Arg Leu Phe Cys 165 170 175 Gly Thr
Gly Gln Met Lys Leu Thr Arg Leu Tyr Val Thr Ile Met Leu 180 185 190
Thr Val Leu Gly Phe Leu Leu Cys Gly Leu Pro Phe Val Ile Tyr Tyr 195
200 205 Phe Leu Leu Phe Asn Ile Lys Asp Gly Phe Cys Leu Phe Asp Phe
Arg 210 215 220 Phe Tyr Met Ser Thr His Val Leu Thr Ala Ile Asn Asn
Cys Ala Asn 225 230 235 240 Pro Ile Ile Tyr Phe Phe Glu Gly Ser Phe
Arg His Gln Leu Lys His 245 250 255 Gln Thr Leu Lys Met Val Leu Gln
Ser Val Leu Gln Asp Thr Pro Glu 260 265 270 Ile Ala Glu Asn Met Val
Glu Met Ser Arg Asn Ile Pro Lys Pro 275 280 285 22 1024 DNA Mus
musculus CDS (16)...(918) 22 ccagtgcacg aaacc atg cat aga agt atc
agc atc agg att ctg ata aca 51 Met His Arg Ser Ile Ser Ile Arg Ile
Leu Ile Thr 1 5 10 aac ttg atg atc gtc atc ctc gga cta gtc ggg ctg
aca gga aac gcc 99 Asn Leu Met Ile Val Ile Leu Gly Leu Val Gly Leu
Thr Gly Asn Ala 15 20 25 att gtg ttc tgg ctc ctg ctc ttc cgc ttg
cgc agg aac gcc ttc tca 147 Ile Val Phe Trp Leu Leu Leu Phe Arg Leu
Arg Arg Asn Ala Phe Ser 30 35 40 atc tac atc cta aac ttg gcc ctg
gct gac ttc ctc ttc ctc ctc tgc 195 Ile Tyr Ile Leu Asn Leu Ala Leu
Ala Asp Phe Leu Phe Leu Leu Cys 45 50 55 60 cac atc ata gct tcc aca
gag cat att ctc acg ttt tcc tcc ccc aac 243 His Ile Ile Ala Ser Thr
Glu His Ile Leu Thr Phe Ser Ser Pro Asn 65 70 75 agt atc ttt atc
aat tgc ctt tac acc ttc agg gtg ctt ctc tac atc 291 Ser Ile Phe Ile
Asn Cys Leu Tyr Thr Phe Arg Val Leu Leu Tyr Ile 80 85 90 gca ggc
ctg agc atg ctc agt gcc atc agc att gag cgc tgc ctg tct 339 Ala Gly
Leu Ser Met Leu Ser Ala Ile Ser Ile Glu Arg Cys Leu Ser 95 100 105
gtc atg tgc ccc atc tgg tat cgc tgc cac agc cca gaa cac aca tca 387
Val Met Cys Pro Ile Trp Tyr Arg Cys His Ser Pro Glu His Thr Ser 110
115 120 act gtc atg tgt gct atg atc tgg gtc ctg tct cta ttg ctc tgc
att 435 Thr Val Met Cys Ala Met Ile Trp Val Leu Ser Leu Leu Leu Cys
Ile 125 130 135 140 ctg tat agg tat ttc tgc ggc ttc ttg gat acc aaa
tat gaa gat gac 483 Leu Tyr Arg Tyr Phe Cys Gly Phe Leu Asp Thr Lys
Tyr Glu Asp Asp 145 150 155 tat ggg tgt cta gca atg aac ttc ctt act
acc gca tac ctg atg ttt 531 Tyr Gly Cys Leu Ala Met Asn Phe Leu Thr
Thr Ala Tyr Leu Met Phe 160 165 170 ttg ttt gta gtc ctc tgt gtg tcc
agc ctg gct ctg ctg gcc agg ttg 579 Leu Phe Val Val Leu Cys Val Ser
Ser Leu Ala Leu Leu Ala Arg Leu 175 180 185 ttc tgt ggc gct gga cgg
atg aag ctt acc aga tta tac gtg acc atc 627 Phe Cys Gly Ala Gly Arg
Met Lys Leu Thr Arg Leu Tyr Val Thr Ile 190 195 200 acg ctg acc ctt
ttg gtt ttt ctc ctc tgc ggg ttg ccc tgt ggc ttc 675 Thr Leu Thr Leu
Leu Val Phe Leu Leu Cys Gly Leu Pro Cys Gly Phe 205 210 215 220 tac
tgg ttc ctg tta tcc aaa att aag aat gtt ttt act gta ttt gaa 723 Tyr
Trp Phe Leu Leu Ser Lys Ile Lys Asn Val Phe Thr Val Phe Glu 225 230
235 ttt agt ctt tat ctg gca tca gtt gtc ctg act gct att aac agc tgt
771 Phe Ser Leu Tyr Leu Ala Ser Val Val Leu Thr Ala Ile Asn Ser Cys
240 245 250 gcc aac ccc atc att tac ttc ttt gtg ggc tca ttc agg cat
cgg ttg 819 Ala Asn Pro Ile Ile Tyr Phe Phe Val Gly Ser Phe Arg His
Arg Leu 255 260 265 aag cac cag acc ctc aaa atg gtt ctc cag agt gca
ctg cag gac act 867 Lys His Gln Thr Leu Lys Met Val Leu Gln Ser Ala
Leu Gln Asp Thr 270 275 280 cct gag aca cct gaa aac atg gtg gag atg
tca aga aac aaa gca gag 915 Pro Glu Thr Pro Glu Asn Met Val Glu Met
Ser Arg Asn Lys Ala Glu 285 290 295 300 ctg tgatgaagag cctctgcccg
gacctcagag gtggctttgg agtgagcact 968 Leu gccctgctgc acttggccac
tgtccactct cctctcagct tactcacttg gcatgc 1024 23 301 PRT Mus
musculus 23 Met His Arg Ser Ile Ser Ile Arg Ile Leu Ile Thr Asn Leu
Met Ile 1 5 10 15 Val Ile Leu Gly Leu Val Gly Leu Thr Gly Asn Ala
Ile Val Phe Trp 20 25 30 Leu Leu Leu Phe Arg Leu Arg Arg Asn Ala
Phe Ser Ile Tyr Ile Leu 35 40 45 Asn Leu Ala Leu Ala Asp Phe Leu
Phe Leu Leu Cys His Ile Ile Ala 50 55 60 Ser Thr Glu His Ile Leu
Thr Phe Ser Ser Pro Asn Ser Ile Phe Ile 65 70 75 80 Asn Cys Leu Tyr
Thr Phe Arg Val Leu Leu Tyr Ile Ala Gly Leu Ser 85 90 95 Met Leu
Ser Ala Ile Ser Ile Glu Arg Cys Leu Ser Val Met Cys Pro 100 105 110
Ile Trp Tyr Arg Cys His Ser Pro Glu His Thr Ser Thr Val Met Cys 115
120 125 Ala Met Ile Trp Val Leu Ser Leu Leu Leu Cys Ile Leu Tyr Arg
Tyr 130 135 140 Phe Cys Gly Phe Leu Asp Thr Lys Tyr Glu Asp Asp Tyr
Gly Cys Leu 145 150 155 160 Ala Met Asn Phe Leu Thr Thr Ala Tyr Leu
Met Phe Leu Phe Val Val 165 170 175 Leu Cys Val Ser Ser Leu Ala Leu
Leu Ala Arg Leu Phe Cys Gly Ala 180 185 190 Gly Arg Met Lys Leu Thr
Arg Leu Tyr Val Thr Ile Thr Leu Thr Leu 195 200 205 Leu Val Phe Leu
Leu Cys Gly Leu Pro Cys Gly Phe Tyr Trp Phe Leu 210 215 220 Leu Ser
Lys Ile Lys Asn Val Phe Thr Val Phe Glu Phe Ser Leu Tyr 225 230 235
240 Leu Ala Ser Val Val Leu Thr Ala Ile Asn Ser Cys Ala Asn Pro Ile
245 250 255 Ile Tyr Phe Phe Val Gly Ser Phe Arg His Arg Leu Lys His
Gln Thr 260 265 270 Leu Lys Met Val Leu Gln Ser Ala Leu Gln Asp Thr
Pro Glu Thr Pro 275 280 285 Glu Asn Met Val Glu Met Ser Arg Asn Lys
Ala Glu Leu 290 295 300 24 1045 DNA Mus musculus CDS (106)...(1020)
24 tttgtgttca tagtgaatga ctaatttctt ctttgtgttc ccagtgcaga
gtttctggcc 60 ctaaacacct cagcctcagc aatgtcaccc acgacaacaa gtcca atg
gac gaa acc 117 Met Asp Glu Thr 1 agc cct aga agt att gac atc gag
tca ctg atc cca aac ttg atg atc 165 Ser Pro Arg Ser Ile Asp Ile Glu
Ser Leu Ile Pro Asn Leu Met Ile 5 10 15 20 atc atc ttt gga ctg gtt
ggg ctg aca gga aat gcc att gtg ctc tgg 213 Ile Ile Phe Gly Leu Val
Gly Leu Thr Gly Asn Ala Ile Val Leu Trp 25 30 35 ctc ctg ggc ttc
tgc ttg cac agg aat gcc ttc tta gtc tac atc cta 261 Leu Leu Gly Phe
Cys Leu His Arg Asn Ala Phe Leu Val Tyr Ile Leu 40 45 50 aac ttg
gcc ctg gct gac ttc ctc ttc ctt ctc tgt cac ttc ata aat 309 Asn Leu
Ala Leu Ala Asp Phe Leu Phe Leu Leu Cys His Phe Ile Asn 55 60 65
tca gca atg ttt ctt ctc aag gtt cct ata ccc aac ggt atc ttt gtc 357
Ser Ala Met Phe Leu Leu Lys Val Pro Ile Pro Asn Gly Ile Phe Val 70
75 80 tat tgc ttt tac acc atc aaa atg gtt ctc tac atc aca ggc ctg
agc 405 Tyr Cys Phe Tyr Thr Ile Lys Met Val Leu Tyr Ile Thr Gly Leu
Ser 85 90 95 100 atg ctc agt gcc atc agc act gag cgc tgc ctt tct
gtc ctg tgc ccc 453 Met Leu Ser Ala Ile Ser Thr Glu Arg Cys Leu Ser
Val Leu Cys Pro 105 110 115 atc tgg tat cac tgc cgc cgc cca gaa cac
aca tca act gtc atg tgt 501 Ile Trp Tyr His Cys Arg Arg Pro Glu His
Thr Ser Thr Val Met Cys 120 125 130 gct gtg att tgg atc ttt tcc gtg
ttg atc tgc att ctg aaa gaa tat 549 Ala Val Ile Trp Ile Phe Ser Val
Leu Ile Cys Ile Leu Lys Glu Tyr 135 140 145 ttc tgt gat ttc ttt ggt
acc aaa ttg gga aat tac tat gtg tgt cag 597 Phe Cys Asp Phe Phe Gly
Thr Lys Leu Gly Asn Tyr Tyr Val Cys Gln 150 155 160 gca tcc aac ttc
ttt atg gga gca tac cta atg ttt ttg ttt gta gtc 645 Ala Ser Asn Phe
Phe Met Gly Ala Tyr Leu Met Phe Leu Phe Val Val 165 170 175 180 ctc
tgt ctg tcc acc ctg gct ctg ctg gcc agg ttg ttc tgt ggt gct 693 Leu
Cys Leu Ser Thr Leu Ala Leu Leu Ala Arg Leu Phe Cys Gly Ala 185 190
195 gag aag atg aaa ttt acc aga tta ttc gtg acc atc atg ctg acc att
741 Glu Lys Met Lys Phe Thr Arg Leu Phe Val Thr Ile Met Leu Thr Ile
200 205 210 ttg gtt ttt ctc ctc tgt ggg ttg cca tgg ggc ttc ttc tgg
ttc ctg 789 Leu Val Phe Leu Leu Cys Gly Leu Pro Trp Gly Phe Phe Trp
Phe Leu 215 220 225 tta atc tgg att aag ggt ggt ttt agt gta cta gat
tat aga ctt tat 837 Leu Ile Trp Ile Lys Gly Gly Phe Ser Val Leu Asp
Tyr Arg Leu Tyr 230 235 240 ttg gca tca att gtc cta act gtt gtt aac
agc tgt gcc aac ccc atc 885 Leu Ala Ser Ile Val Leu Thr Val Val Asn
Ser Cys Ala Asn Pro Ile 245 250 255 260 att tac ttc ttc gtg gga tca
ttc agg cat cgg ttg aag cac cag acc 933 Ile Tyr Phe Phe Val Gly Ser
Phe Arg His Arg Leu Lys His Gln Thr 265 270 275 ctc aaa atg gtt ctc
cag agt gca ctg cag gac act cct gag aca cat 981 Leu Lys Met Val Leu
Gln Ser Ala Leu Gln Asp Thr Pro Glu Thr His 280 285 290 gaa aac atg
gtg gag atg tca aga atc aaa gca gag cag tgatgaagag 1030 Glu Asn Met
Val Glu Met Ser Arg Ile Lys Ala Glu Gln 295 300 305 cctctgcctg
gacct 1045 25 305 PRT Mus musculus 25 Met Asp Glu Thr Ser Pro Arg
Ser Ile Asp Ile Glu Ser Leu Ile Pro 1 5 10 15 Asn Leu Met Ile Ile
Ile Phe Gly Leu Val Gly Leu Thr Gly Asn Ala 20 25 30 Ile Val Leu
Trp Leu Leu Gly Phe Cys Leu His Arg Asn Ala Phe Leu 35 40 45 Val
Tyr Ile Leu Asn Leu Ala Leu Ala Asp Phe Leu Phe Leu Leu Cys 50 55
60 His Phe Ile Asn Ser Ala Met Phe Leu Leu Lys Val Pro Ile Pro Asn
65 70 75 80 Gly Ile Phe Val Tyr Cys Phe Tyr Thr Ile Lys Met Val Leu
Tyr Ile 85 90 95 Thr Gly Leu Ser Met Leu Ser
Ala Ile Ser Thr Glu Arg Cys Leu Ser 100 105 110 Val Leu Cys Pro Ile
Trp Tyr His Cys Arg Arg Pro Glu His Thr Ser 115 120 125 Thr Val Met
Cys Ala Val Ile Trp Ile Phe Ser Val Leu Ile Cys Ile 130 135 140 Leu
Lys Glu Tyr Phe Cys Asp Phe Phe Gly Thr Lys Leu Gly Asn Tyr 145 150
155 160 Tyr Val Cys Gln Ala Ser Asn Phe Phe Met Gly Ala Tyr Leu Met
Phe 165 170 175 Leu Phe Val Val Leu Cys Leu Ser Thr Leu Ala Leu Leu
Ala Arg Leu 180 185 190 Phe Cys Gly Ala Glu Lys Met Lys Phe Thr Arg
Leu Phe Val Thr Ile 195 200 205 Met Leu Thr Ile Leu Val Phe Leu Leu
Cys Gly Leu Pro Trp Gly Phe 210 215 220 Phe Trp Phe Leu Leu Ile Trp
Ile Lys Gly Gly Phe Ser Val Leu Asp 225 230 235 240 Tyr Arg Leu Tyr
Leu Ala Ser Ile Val Leu Thr Val Val Asn Ser Cys 245 250 255 Ala Asn
Pro Ile Ile Tyr Phe Phe Val Gly Ser Phe Arg His Arg Leu 260 265 270
Lys His Gln Thr Leu Lys Met Val Leu Gln Ser Ala Leu Gln Asp Thr 275
280 285 Pro Glu Thr His Glu Asn Met Val Glu Met Ser Arg Ile Lys Ala
Glu 290 295 300 Gln 305 26 980 DNA Mus musculus CDS (45)...(959) 26
tagacacctc agcatatgca atggcaccca cgaccacaaa tcca atg gac aaa acc 56
Met Asp Lys Thr 1 atc ctt gga agt att gac atc gag acc ctg atc cga
cat ttg atg atc 104 Ile Leu Gly Ser Ile Asp Ile Glu Thr Leu Ile Arg
His Leu Met Ile 5 10 15 20 atc atc ttc gga ctg gtc ggg ctg aca gga
aat gcc att gtg ttc tgg 152 Ile Ile Phe Gly Leu Val Gly Leu Thr Gly
Asn Ala Ile Val Phe Trp 25 30 35 ctc ctg ggc ttc cac ttg cac agg
aat gcc ttc tta gtc tac atc ata 200 Leu Leu Gly Phe His Leu His Arg
Asn Ala Phe Leu Val Tyr Ile Ile 40 45 50 aac ttg gcc ctg gct gac
ttc ttc tat ctg ctc tgt cac atc ata aat 248 Asn Leu Ala Leu Ala Asp
Phe Phe Tyr Leu Leu Cys His Ile Ile Asn 55 60 65 tcc ata atg ttt
ctt ctc aag gtt ccc tca ccc aac att atc ttg gac 296 Ser Ile Met Phe
Leu Leu Lys Val Pro Ser Pro Asn Ile Ile Leu Asp 70 75 80 cat tgc
ttt tac acc atc atg ata gtt ctc tac atc aca ggc ctg agc 344 His Cys
Phe Tyr Thr Ile Met Ile Val Leu Tyr Ile Thr Gly Leu Ser 85 90 95
100 atg ctc agc gcc atc agc act gag cgc tgc ctg tct gtc ctg tgc ccc
392 Met Leu Ser Ala Ile Ser Thr Glu Arg Cys Leu Ser Val Leu Cys Pro
105 110 115 atc tgg tat cgc tgc cac cgt cca gaa cac aca tca act gtc
atg tgt 440 Ile Trp Tyr Arg Cys His Arg Pro Glu His Thr Ser Thr Val
Met Cys 120 125 130 gct gtg atc tgg gta atg tcc ctg ttg atc tct att
ctc aat gga tat 488 Ala Val Ile Trp Val Met Ser Leu Leu Ile Ser Ile
Leu Asn Gly Tyr 135 140 145 ttc tgt aat ttc tct agt ccc aaa tat gta
aat aac tct gtg tgt cag 536 Phe Cys Asn Phe Ser Ser Pro Lys Tyr Val
Asn Asn Ser Val Cys Gln 150 155 160 gca tca cac atc ttt atc aga aca
tac cca ata ttt ttg ttt gta ctc 584 Ala Ser His Ile Phe Ile Arg Thr
Tyr Pro Ile Phe Leu Phe Val Leu 165 170 175 180 ctc tgt ctg tcc acc
ctt gct ctg ctg gcc agg ttg ttc tct ggt gct 632 Leu Cys Leu Ser Thr
Leu Ala Leu Leu Ala Arg Leu Phe Ser Gly Ala 185 190 195 ggg aag agg
aaa ttt acc aga tta ttc gtg acc atc atg ctg gcc att 680 Gly Lys Arg
Lys Phe Thr Arg Leu Phe Val Thr Ile Met Leu Ala Ile 200 205 210 ttg
gtt ttt ctt ctc tgt ggg tta ccc ctg ggc ttc ttc tgg ttt ctg 728 Leu
Val Phe Leu Leu Cys Gly Leu Pro Leu Gly Phe Phe Trp Phe Leu 215 220
225 tca ccc tgg att gag gat cgt ttc att gta cta gat tat aga ctt ttt
776 Ser Pro Trp Ile Glu Asp Arg Phe Ile Val Leu Asp Tyr Arg Leu Phe
230 235 240 ttt gca tca gtt gtc cta act gtt gtt aac agc tgt gcc aac
ccc atc 824 Phe Ala Ser Val Val Leu Thr Val Val Asn Ser Cys Ala Asn
Pro Ile 245 250 255 260 att tac ttc ttt gtg ggc tcc ttc agg cat cgg
ttg aag caa cag acc 872 Ile Tyr Phe Phe Val Gly Ser Phe Arg His Arg
Leu Lys Gln Gln Thr 265 270 275 ctc aaa atg ttt ctc cag aga gca ctg
cag gac acc cct gag aca cct 920 Leu Lys Met Phe Leu Gln Arg Ala Leu
Gln Asp Thr Pro Glu Thr Pro 280 285 290 gaa aac atg gtg gag atg tca
aga agc aaa gca gag ccg tgatgaagag 969 Glu Asn Met Val Glu Met Ser
Arg Ser Lys Ala Glu Pro 295 300 305 cctcttccag g 980 27 305 PRT Mus
musculus 27 Met Asp Lys Thr Ile Leu Gly Ser Ile Asp Ile Glu Thr Leu
Ile Arg 1 5 10 15 His Leu Met Ile Ile Ile Phe Gly Leu Val Gly Leu
Thr Gly Asn Ala 20 25 30 Ile Val Phe Trp Leu Leu Gly Phe His Leu
His Arg Asn Ala Phe Leu 35 40 45 Val Tyr Ile Ile Asn Leu Ala Leu
Ala Asp Phe Phe Tyr Leu Leu Cys 50 55 60 His Ile Ile Asn Ser Ile
Met Phe Leu Leu Lys Val Pro Ser Pro Asn 65 70 75 80 Ile Ile Leu Asp
His Cys Phe Tyr Thr Ile Met Ile Val Leu Tyr Ile 85 90 95 Thr Gly
Leu Ser Met Leu Ser Ala Ile Ser Thr Glu Arg Cys Leu Ser 100 105 110
Val Leu Cys Pro Ile Trp Tyr Arg Cys His Arg Pro Glu His Thr Ser 115
120 125 Thr Val Met Cys Ala Val Ile Trp Val Met Ser Leu Leu Ile Ser
Ile 130 135 140 Leu Asn Gly Tyr Phe Cys Asn Phe Ser Ser Pro Lys Tyr
Val Asn Asn 145 150 155 160 Ser Val Cys Gln Ala Ser His Ile Phe Ile
Arg Thr Tyr Pro Ile Phe 165 170 175 Leu Phe Val Leu Leu Cys Leu Ser
Thr Leu Ala Leu Leu Ala Arg Leu 180 185 190 Phe Ser Gly Ala Gly Lys
Arg Lys Phe Thr Arg Leu Phe Val Thr Ile 195 200 205 Met Leu Ala Ile
Leu Val Phe Leu Leu Cys Gly Leu Pro Leu Gly Phe 210 215 220 Phe Trp
Phe Leu Ser Pro Trp Ile Glu Asp Arg Phe Ile Val Leu Asp 225 230 235
240 Tyr Arg Leu Phe Phe Ala Ser Val Val Leu Thr Val Val Asn Ser Cys
245 250 255 Ala Asn Pro Ile Ile Tyr Phe Phe Val Gly Ser Phe Arg His
Arg Leu 260 265 270 Lys Gln Gln Thr Leu Lys Met Phe Leu Gln Arg Ala
Leu Gln Asp Thr 275 280 285 Pro Glu Thr Pro Glu Asn Met Val Glu Met
Ser Arg Ser Lys Ala Glu 290 295 300 Pro 305 28 408 DNA Homo sapiens
CDS (1)...(405) 28 atg gag act ctc ccc aag gtt cta gag gtc gat gag
aag tct cca gaa 48 Met Glu Thr Leu Pro Lys Val Leu Glu Val Asp Glu
Lys Ser Pro Glu 1 5 10 15 gcc aag gac ctg ctg ccc agc cag acc gcc
agc tcc ctg tgc atc agc 96 Ala Lys Asp Leu Leu Pro Ser Gln Thr Ala
Ser Ser Leu Cys Ile Ser 20 25 30 tcc agg agc gag tct gtc tgg acc
acc acc ccc agg agt aac tgg gaa 144 Ser Arg Ser Glu Ser Val Trp Thr
Thr Thr Pro Arg Ser Asn Trp Glu 35 40 45 atc tac cgc aag ccc atc
gtt atc atg tca gtg ggc ggt gcc atc ctg 192 Ile Tyr Arg Lys Pro Ile
Val Ile Met Ser Val Gly Gly Ala Ile Leu 50 55 60 ctt ttc ggc gtg
gtc atc acc tgc ttg gcc tac acc ttg aag ctg agt 240 Leu Phe Gly Val
Val Ile Thr Cys Leu Ala Tyr Thr Leu Lys Leu Ser 65 70 75 80 gac aag
agt ctc tcc atc ctc aaa atg gta ggg cct ggc ttc ctg tcc 288 Asp Lys
Ser Leu Ser Ile Leu Lys Met Val Gly Pro Gly Phe Leu Ser 85 90 95
ctg gga ctc atg atg ctg gtg tgc ggg ctg gtg tgg gtg ccc atc atc 336
Leu Gly Leu Met Met Leu Val Cys Gly Leu Val Trp Val Pro Ile Ile 100
105 110 aaa aag aaa cag aag cac aga cag aag tcg aat ttc tta cgc agc
ctc 384 Lys Lys Lys Gln Lys His Arg Gln Lys Ser Asn Phe Leu Arg Ser
Leu 115 120 125 aag tcc ttc ttc ctg act cgc tga 408 Lys Ser Phe Phe
Leu Thr Arg 130 135 29 135 PRT Homo sapiens 29 Met Glu Thr Leu Pro
Lys Val Leu Glu Val Asp Glu Lys Ser Pro Glu 1 5 10 15 Ala Lys Asp
Leu Leu Pro Ser Gln Thr Ala Ser Ser Leu Cys Ile Ser 20 25 30 Ser
Arg Ser Glu Ser Val Trp Thr Thr Thr Pro Arg Ser Asn Trp Glu 35 40
45 Ile Tyr Arg Lys Pro Ile Val Ile Met Ser Val Gly Gly Ala Ile Leu
50 55 60 Leu Phe Gly Val Val Ile Thr Cys Leu Ala Tyr Thr Leu Lys
Leu Ser 65 70 75 80 Asp Lys Ser Leu Ser Ile Leu Lys Met Val Gly Pro
Gly Phe Leu Ser 85 90 95 Leu Gly Leu Met Met Leu Val Cys Gly Leu
Val Trp Val Pro Ile Ile 100 105 110 Lys Lys Lys Gln Lys His Arg Gln
Lys Ser Asn Phe Leu Arg Ser Leu 115 120 125 Lys Ser Phe Phe Leu Thr
Arg 130 135 30 1400 DNA Homo sapiens CDS (332)...(1297) 30
tcaggcccag gatagagtaa tcatcgggtc cacagcactg gctagatgag tgggggtgtt
60 ttgatcctaa tgttattccc atgttagcac agaacttgtg tggcagtaga
gagaggtcag 120 gcttcagagt cagcaagaac tggatttcaa actggatttg
aggaccccca ccttttgata 180 ggtgacttat tctctgtgag tctctgatct
gccctcttta aatgaggaag taaatcccac 240 atggcagggt ggtggggaga
atcagagatc atacagctgg tgatcacaac tggtttctgt 300 ttccagggtc
accagactgg ggtttctgag c atg gat tca acc atc cca gtc 352 Met Asp Ser
Thr Ile Pro Val 1 5 ttg ggt aca gaa ctg aca cca atc aac gga cgt gag
gag act cct tgc 400 Leu Gly Thr Glu Leu Thr Pro Ile Asn Gly Arg Glu
Glu Thr Pro Cys 10 15 20 tac aag cag acc ctg agc ttc acg ggg ctg
acg tgc atc gtt tcc ctt 448 Tyr Lys Gln Thr Leu Ser Phe Thr Gly Leu
Thr Cys Ile Val Ser Leu 25 30 35 gtc gcg ctg aca gga aac gcg gtt
gtg ctc tgg ctc ctg ggc tgc cgc 496 Val Ala Leu Thr Gly Asn Ala Val
Val Leu Trp Leu Leu Gly Cys Arg 40 45 50 55 atg cgc agg aac gct gtc
tcc atc tac atc ctc aac ctg gtc gcg gcc 544 Met Arg Arg Asn Ala Val
Ser Ile Tyr Ile Leu Asn Leu Val Ala Ala 60 65 70 gac ttc ctc ttc
ctt agc ggc cac att ata tgt tcg ccg tta cgc ctc 592 Asp Phe Leu Phe
Leu Ser Gly His Ile Ile Cys Ser Pro Leu Arg Leu 75 80 85 atc aat
atc cgc cat ccc atc tcc aaa atc ctc agt cct gtg atg acc 640 Ile Asn
Ile Arg His Pro Ile Ser Lys Ile Leu Ser Pro Val Met Thr 90 95 100
ttt ccc tac ttt ata ggc cta agc atg ctg agc gcc atc agc acc gag 688
Phe Pro Tyr Phe Ile Gly Leu Ser Met Leu Ser Ala Ile Ser Thr Glu 105
110 115 cgc tgc ctg tcc atc ctg tgg ccc atc tgg tac cac tgc cgc cgc
ccc 736 Arg Cys Leu Ser Ile Leu Trp Pro Ile Trp Tyr His Cys Arg Arg
Pro 120 125 130 135 aga tac ctg tca tcg gtc atg tgt gtc ctg ctc tgg
gcc ctg tcc ctg 784 Arg Tyr Leu Ser Ser Val Met Cys Val Leu Leu Trp
Ala Leu Ser Leu 140 145 150 ctg cgg agt atc ctg gag tgg atg ttc tgt
gac ttc ctg ttt agt ggt 832 Leu Arg Ser Ile Leu Glu Trp Met Phe Cys
Asp Phe Leu Phe Ser Gly 155 160 165 gct gat tct gtt tgg tgt gaa acg
tca gat ttc att aca atc gcg tgg 880 Ala Asp Ser Val Trp Cys Glu Thr
Ser Asp Phe Ile Thr Ile Ala Trp 170 175 180 ctg gtt ttt tta tgt gtg
gtt ctc tgt ggg tcc agc ctg gtc ctg ctg 928 Leu Val Phe Leu Cys Val
Val Leu Cys Gly Ser Ser Leu Val Leu Leu 185 190 195 gtc agg att ctc
tgt gga tcc cgg aag atg ccg ctg acc agg ctg tac 976 Val Arg Ile Leu
Cys Gly Ser Arg Lys Met Pro Leu Thr Arg Leu Tyr 200 205 210 215 gtg
acc atc ctc ctc aca gtg ctg gtc ttc ctc ctc tgt ggc ctg ccc 1024
Val Thr Ile Leu Leu Thr Val Leu Val Phe Leu Leu Cys Gly Leu Pro 220
225 230 ttt ggc att cag tgg gcc ctg ttt tcc agg atc cac ctg gat tgg
aaa 1072 Phe Gly Ile Gln Trp Ala Leu Phe Ser Arg Ile His Leu Asp
Trp Lys 235 240 245 gtc tta ttt tgt cat gtg cat cta gtt tcc att ttc
ctg tcc gct ctt 1120 Val Leu Phe Cys His Val His Leu Val Ser Ile
Phe Leu Ser Ala Leu 250 255 260 aac agc agt gcc aac ccc atc att tac
ttc ttc gtg ggc tcc ttt agg 1168 Asn Ser Ser Ala Asn Pro Ile Ile
Tyr Phe Phe Val Gly Ser Phe Arg 265 270 275 cag cgt caa aat agg cag
aac ctg aag ctg gtt ctc cag agg gct ctg 1216 Gln Arg Gln Asn Arg
Gln Asn Leu Lys Leu Val Leu Gln Arg Ala Leu 280 285 290 295 cag gac
acg cct gag gtg gat gaa ggt gga ggg tgg ctt cct cag gaa 1264 Gln
Asp Thr Pro Glu Val Asp Glu Gly Gly Gly Trp Leu Pro Gln Glu 300 305
310 acc ctg gag ctg tcg gga agc aga ttg gag cag tgaggaagaa
cctctgccct 1317 Thr Leu Glu Leu Ser Gly Ser Arg Leu Glu Gln 315 320
gtcagacagg actttgagag caatgctgcc ctgccaccct tgacaattat atgcattttt
1377 cttagccttc tgcctcagaa atg 1400 31 322 PRT Homo sapiens 31 Met
Asp Ser Thr Ile Pro Val Leu Gly Thr Glu Leu Thr Pro Ile Asn 1 5 10
15 Gly Arg Glu Glu Thr Pro Cys Tyr Lys Gln Thr Leu Ser Phe Thr Gly
20 25 30 Leu Thr Cys Ile Val Ser Leu Val Ala Leu Thr Gly Asn Ala
Val Val 35 40 45 Leu Trp Leu Leu Gly Cys Arg Met Arg Arg Asn Ala
Val Ser Ile Tyr 50 55 60 Ile Leu Asn Leu Val Ala Ala Asp Phe Leu
Phe Leu Ser Gly His Ile 65 70 75 80 Ile Cys Ser Pro Leu Arg Leu Ile
Asn Ile Arg His Pro Ile Ser Lys 85 90 95 Ile Leu Ser Pro Val Met
Thr Phe Pro Tyr Phe Ile Gly Leu Ser Met 100 105 110 Leu Ser Ala Ile
Ser Thr Glu Arg Cys Leu Ser Ile Leu Trp Pro Ile 115 120 125 Trp Tyr
His Cys Arg Arg Pro Arg Tyr Leu Ser Ser Val Met Cys Val 130 135 140
Leu Leu Trp Ala Leu Ser Leu Leu Arg Ser Ile Leu Glu Trp Met Phe 145
150 155 160 Cys Asp Phe Leu Phe Ser Gly Ala Asp Ser Val Trp Cys Glu
Thr Ser 165 170 175 Asp Phe Ile Thr Ile Ala Trp Leu Val Phe Leu Cys
Val Val Leu Cys 180 185 190 Gly Ser Ser Leu Val Leu Leu Val Arg Ile
Leu Cys Gly Ser Arg Lys 195 200 205 Met Pro Leu Thr Arg Leu Tyr Val
Thr Ile Leu Leu Thr Val Leu Val 210 215 220 Phe Leu Leu Cys Gly Leu
Pro Phe Gly Ile Gln Trp Ala Leu Phe Ser 225 230 235 240 Arg Ile His
Leu Asp Trp Lys Val Leu Phe Cys His Val His Leu Val 245 250 255 Ser
Ile Phe Leu Ser Ala Leu Asn Ser Ser Ala Asn Pro Ile Ile Tyr 260 265
270 Phe Phe Val Gly Ser Phe Arg Gln Arg Gln Asn Arg Gln Asn Leu Lys
275 280 285 Leu Val Leu Gln Arg Ala Leu Gln Asp Thr Pro Glu Val Asp
Glu Gly 290 295 300 Gly Gly Trp Leu Pro Gln Glu Thr Leu Glu Leu Ser
Gly Ser Arg Leu 305 310 315 320 Glu Gln 32 1604 DNA Homo sapiens
CDS (433)...(1398) 32 tgcatggtct tccttcctgt ccatggatga ccagtcctag
tcacgagtgt gtcacaacca 60 cctctttgtg tatctgaatt cctccacctg
aaagaaaatt tcagacccag gatagattaa 120 tcatcgggtc caaagccctg
gccggatgag tgggggtgtt ttgatcctaa tgttattccc 180 atgtcagcac
agaacttgtg tggcagtaga gagatgtcag gcttcagagt caacaagaac 240
tggatttcaa actggatttg aggaccccca cctttggtaa gtgacttatt atctgcgagc
300 ctctgtttct ctcttcttta aatgaggaca gtaaatccca tacggcaggg
tggtggggag 360 aatcagagat gatacagctg gtgatcacat ctggtttgtg
ttcccagggg caccagacta 420 gagtttctga gc atg gat cca acc gtc cca gtc
ttc ggt aca aaa ctg aca 471 Met Asp Pro Thr Val Pro Val Phe Gly Thr
Lys Leu Thr 1 5 10 cca atc aac gga cgt gag gag act cct tgc
tac aat cag acc ctg agc 519 Pro Ile Asn Gly Arg Glu Glu Thr Pro Cys
Tyr Asn Gln Thr Leu Ser 15 20 25 ttc acg gtg ctg acg tgc atc att
tcc ctt gtc gga ctg aca gga aac 567 Phe Thr Val Leu Thr Cys Ile Ile
Ser Leu Val Gly Leu Thr Gly Asn 30 35 40 45 gcg gta gtg ctc tgg ctc
ctg ggc tac cgc atg cgc agg aac gct gtc 615 Ala Val Val Leu Trp Leu
Leu Gly Tyr Arg Met Arg Arg Asn Ala Val 50 55 60 tcc atc tac atc
ctc aac ctg gcc gca gca gac ttc ctc ttc ctc agc 663 Ser Ile Tyr Ile
Leu Asn Leu Ala Ala Ala Asp Phe Leu Phe Leu Ser 65 70 75 ttc cag
att ata cgt tcg cca tta cgc ctc atc aat atc agc cat ctc 711 Phe Gln
Ile Ile Arg Ser Pro Leu Arg Leu Ile Asn Ile Ser His Leu 80 85 90
atc cgc aaa atc ctc gtt tct gtg atg acc ttt ccc tac ttt aca ggc 759
Ile Arg Lys Ile Leu Val Ser Val Met Thr Phe Pro Tyr Phe Thr Gly 95
100 105 ctg agt atg ctg agc gcc atc agc acc gag cgc tgc ctg tct gtt
ctg 807 Leu Ser Met Leu Ser Ala Ile Ser Thr Glu Arg Cys Leu Ser Val
Leu 110 115 120 125 tgg ccc atc tgg tac cgc tgc cgc cgc ccc aca cac
ctg tca gcg gtc 855 Trp Pro Ile Trp Tyr Arg Cys Arg Arg Pro Thr His
Leu Ser Ala Val 130 135 140 gtg tgt gtc ctg ctc tgg ggc ctg tcc ctg
ctg ttt agt atg ctg gag 903 Val Cys Val Leu Leu Trp Gly Leu Ser Leu
Leu Phe Ser Met Leu Glu 145 150 155 tgg agg ttc tgt gac ttc ctg ttt
agt ggt gct gat tct agt tgg tgt 951 Trp Arg Phe Cys Asp Phe Leu Phe
Ser Gly Ala Asp Ser Ser Trp Cys 160 165 170 gaa acg tca gat ttc atc
cca gtc gcg tgg ctg att ttt tta tgt gtg 999 Glu Thr Ser Asp Phe Ile
Pro Val Ala Trp Leu Ile Phe Leu Cys Val 175 180 185 gtt ctc tgt gtt
tcc agc ctg gtc ctg ctg gtc agg atc ctc tgt gga 1047 Val Leu Cys
Val Ser Ser Leu Val Leu Leu Val Arg Ile Leu Cys Gly 190 195 200 205
tcc cgg aag atg ccg ctg acc agg ctg tac gtg acc atc ctg ctc aca
1095 Ser Arg Lys Met Pro Leu Thr Arg Leu Tyr Val Thr Ile Leu Leu
Thr 210 215 220 gtg ctg gtc ttc ctc ctc tgc ggc ctg ccc ttc ggc att
ctg ggg gcc 1143 Val Leu Val Phe Leu Leu Cys Gly Leu Pro Phe Gly
Ile Leu Gly Ala 225 230 235 cta att tac agg atg cac ctg aat ttg gaa
gtc tta tat tgt cat gtt 1191 Leu Ile Tyr Arg Met His Leu Asn Leu
Glu Val Leu Tyr Cys His Val 240 245 250 tat ctg gtt tgc atg tcc ctg
tcc tct cta aac agt agt gcc aac ccc 1239 Tyr Leu Val Cys Met Ser
Leu Ser Ser Leu Asn Ser Ser Ala Asn Pro 255 260 265 atc att tac ttc
ttc gtg ggc tcc ttt agg cag cgt caa aat agg cag 1287 Ile Ile Tyr
Phe Phe Val Gly Ser Phe Arg Gln Arg Gln Asn Arg Gln 270 275 280 285
aac ctg aag ctg gtt ctc cag agg gct ctg cag gac aag cct gag gtg
1335 Asn Leu Lys Leu Val Leu Gln Arg Ala Leu Gln Asp Lys Pro Glu
Val 290 295 300 gat aaa ggt gaa ggg cag ctt cct gag gaa agc ctg gag
ctg tcg gga 1383 Asp Lys Gly Glu Gly Gln Leu Pro Glu Glu Ser Leu
Glu Leu Ser Gly 305 310 315 agc aga ttg ggg cca tgagggagag
cctctgccct gtcagtcaga cgggactttg 1438 Ser Arg Leu Gly Pro 320
agagcaacac tgtcctgcca cccttgacaa ttacatgcgt ttttcttagc gtttcgcctc
1498 agaaatgtct cagtggtaac tcaaggtctt caaataaatg tttatctaac
ctgacagttg 1558 cagttttcac ccatggaaag cattagtctg acagtacaat gtttgg
1604 33 322 PRT Homo sapiens 33 Met Asp Pro Thr Val Pro Val Phe Gly
Thr Lys Leu Thr Pro Ile Asn 1 5 10 15 Gly Arg Glu Glu Thr Pro Cys
Tyr Asn Gln Thr Leu Ser Phe Thr Val 20 25 30 Leu Thr Cys Ile Ile
Ser Leu Val Gly Leu Thr Gly Asn Ala Val Val 35 40 45 Leu Trp Leu
Leu Gly Tyr Arg Met Arg Arg Asn Ala Val Ser Ile Tyr 50 55 60 Ile
Leu Asn Leu Ala Ala Ala Asp Phe Leu Phe Leu Ser Phe Gln Ile 65 70
75 80 Ile Arg Ser Pro Leu Arg Leu Ile Asn Ile Ser His Leu Ile Arg
Lys 85 90 95 Ile Leu Val Ser Val Met Thr Phe Pro Tyr Phe Thr Gly
Leu Ser Met 100 105 110 Leu Ser Ala Ile Ser Thr Glu Arg Cys Leu Ser
Val Leu Trp Pro Ile 115 120 125 Trp Tyr Arg Cys Arg Arg Pro Thr His
Leu Ser Ala Val Val Cys Val 130 135 140 Leu Leu Trp Gly Leu Ser Leu
Leu Phe Ser Met Leu Glu Trp Arg Phe 145 150 155 160 Cys Asp Phe Leu
Phe Ser Gly Ala Asp Ser Ser Trp Cys Glu Thr Ser 165 170 175 Asp Phe
Ile Pro Val Ala Trp Leu Ile Phe Leu Cys Val Val Leu Cys 180 185 190
Val Ser Ser Leu Val Leu Leu Val Arg Ile Leu Cys Gly Ser Arg Lys 195
200 205 Met Pro Leu Thr Arg Leu Tyr Val Thr Ile Leu Leu Thr Val Leu
Val 210 215 220 Phe Leu Leu Cys Gly Leu Pro Phe Gly Ile Leu Gly Ala
Leu Ile Tyr 225 230 235 240 Arg Met His Leu Asn Leu Glu Val Leu Tyr
Cys His Val Tyr Leu Val 245 250 255 Cys Met Ser Leu Ser Ser Leu Asn
Ser Ser Ala Asn Pro Ile Ile Tyr 260 265 270 Phe Phe Val Gly Ser Phe
Arg Gln Arg Gln Asn Arg Gln Asn Leu Lys 275 280 285 Leu Val Leu Gln
Arg Ala Leu Gln Asp Lys Pro Glu Val Asp Lys Gly 290 295 300 Glu Gly
Gln Leu Pro Glu Glu Ser Leu Glu Leu Ser Gly Ser Arg Leu 305 310 315
320 Gly Pro 34 966 DNA Homo sapiens 34 atgaaccaga ctttgaatag
cagtgggacc gtggagtcag ccctaaacta ttccagaggg 60 agcacagtgc
acacggccta cctggtgctg agctccctgg ccatgttcac ctgcctgtgc 120
gggatggcag gcaacagcat ggtgatctgg ctgctgggct ttcgaatgca caggaacccc
180 ttctgcatct atatcctcaa cctggcggca gccgacctcc tcttcctctt
cagcatggct 240 tccacgctca gcctggaaac ccagcccctg gtcaatacca
ctgacaaggt ccacgagctg 300 atgaagagac tgatgtactt tgcctacaca
gtgggcctga gcctgctgac ggccatcagc 360 acccagcgct gtctctctgt
cctcttccct atctggttca agtgtcaccg gcccaggcac 420 ctgtcagcct
gggtgtgtgg cctgctgtgg acactctgtc tcctgatgaa cgggttgacc 480
tcttccttct gcagcaagtt cttgaaattc aatgaagatc ggtgcttcag ggtggacatg
540 gtccaggccg ccctcatcat gggggtctta accccagtga tgactctgtc
cagcctgacc 600 ctctttgtct gggtgcggag gagctcccag cagtggcggc
ggcagcccac acggctgttc 660 gtggtggtcc tggcctctgt cctggtgttc
ctcatctgtt ccctgcctct gagcatctac 720 tggtttgtgc tctactggtt
gagcctgccg cccgagatgc aggtcctgtg cttcagcttg 780 tcacgcctct
cctcgtccgt aagcagcagc gccaaccccg tcatctactt cctggtgggc 840
agccggagga gccacaggct gcccaccagg tccctgggga ctgtgctcca acaggcgctt
900 cgcgaggagc ccgagctgga aggtggggag acgcccaccg tgggcaccaa
tgagatgggg 960 gcttga 966 35 321 PRT Homo sapiens 35 Met Asn Gln
Thr Leu Asn Ser Ser Gly Thr Val Glu Ser Ala Leu Asn 1 5 10 15 Tyr
Ser Arg Gly Ser Thr Val His Thr Ala Tyr Leu Val Leu Ser Ser 20 25
30 Leu Ala Met Phe Thr Cys Leu Cys Gly Met Ala Gly Asn Ser Met Val
35 40 45 Ile Trp Leu Leu Gly Phe Arg Met His Arg Asn Pro Phe Cys
Ile Tyr 50 55 60 Ile Leu Asn Leu Ala Ala Ala Asp Leu Leu Phe Leu
Phe Ser Met Ala 65 70 75 80 Ser Thr Leu Ser Leu Glu Thr Gln Pro Leu
Val Asn Thr Thr Asp Lys 85 90 95 Val His Glu Leu Met Lys Arg Leu
Met Tyr Phe Ala Tyr Thr Val Gly 100 105 110 Leu Ser Leu Leu Thr Ala
Ile Ser Thr Gln Arg Cys Leu Ser Val Leu 115 120 125 Phe Pro Ile Trp
Phe Lys Cys His Arg Pro Arg His Leu Ser Ala Trp 130 135 140 Val Cys
Gly Leu Leu Trp Thr Leu Cys Leu Leu Met Asn Gly Leu Thr 145 150 155
160 Ser Ser Phe Cys Ser Lys Phe Leu Lys Phe Asn Glu Asp Arg Cys Phe
165 170 175 Arg Val Asp Met Val Gln Ala Ala Leu Ile Met Gly Val Leu
Thr Pro 180 185 190 Val Met Thr Leu Ser Ser Leu Thr Leu Phe Val Trp
Val Arg Arg Ser 195 200 205 Ser Gln Gln Trp Arg Arg Gln Pro Thr Arg
Leu Phe Val Val Val Leu 210 215 220 Ala Ser Val Leu Val Phe Leu Ile
Cys Ser Leu Pro Leu Ser Ile Tyr 225 230 235 240 Trp Phe Val Leu Tyr
Trp Leu Ser Leu Pro Pro Glu Met Gln Val Leu 245 250 255 Cys Phe Ser
Leu Ser Arg Leu Ser Ser Ser Val Ser Ser Ser Ala Asn 260 265 270 Pro
Val Ile Tyr Phe Leu Val Gly Ser Arg Arg Ser His Arg Leu Pro 275 280
285 Thr Arg Ser Leu Gly Thr Val Leu Gln Gln Ala Leu Arg Glu Glu Pro
290 295 300 Glu Leu Glu Gly Gly Glu Thr Pro Thr Val Gly Thr Asn Glu
Met Gly 305 310 315 320 Ala 36 767 DNA Homo sapiens CDS (2)...(716)
36 c cac atg gtg gcc atc gtc ccc gac ttg ctg caa ggc cgg ctg gac
ttc 49 His Met Val Ala Ile Val Pro Asp Leu Leu Gln Gly Arg Leu Asp
Phe 1 5 10 15 ccg ggc ttc gtg cag acc agc ctg gca acg ctg cgc ttc
ttc tgc tac 97 Pro Gly Phe Val Gln Thr Ser Leu Ala Thr Leu Arg Phe
Phe Cys Tyr 20 25 30 atc gtg ggc ctg agt ctc ctg gcg gcc gtc agc
gtg gag cag tgc ctg 145 Ile Val Gly Leu Ser Leu Leu Ala Ala Val Ser
Val Glu Gln Cys Leu 35 40 45 gcc gcc ctc ttc cca gcc tgg tac tcg
tgc cgc cgc cca cgc cac ctg 193 Ala Ala Leu Phe Pro Ala Trp Tyr Ser
Cys Arg Arg Pro Arg His Leu 50 55 60 acc acc tgt gtg tgc gcc ctc
acc tgg gcc ctc tgc ctg ctg ctg cac 241 Thr Thr Cys Val Cys Ala Leu
Thr Trp Ala Leu Cys Leu Leu Leu His 65 70 75 80 ctg ctg ctc agc agc
gcc tgc acc cag ttc ttc ggg gag ccc agc cgc 289 Leu Leu Leu Ser Ser
Ala Cys Thr Gln Phe Phe Gly Glu Pro Ser Arg 85 90 95 cac ttg tgc
cgg acg ctg tgg ctg gtg gca gcg gtg ctg ctg gct ctg 337 His Leu Cys
Arg Thr Leu Trp Leu Val Ala Ala Val Leu Leu Ala Leu 100 105 110 ctg
tgt tgc acc atg tgt ggg gcc agc ctt atg ctg ctg ctg cgg gtg 385 Leu
Cys Cys Thr Met Cys Gly Ala Ser Leu Met Leu Leu Leu Arg Val 115 120
125 gag cga ggc ccc cag cgg ccc cca ccc cgg ggc ttc cct ggg ctc atc
433 Glu Arg Gly Pro Gln Arg Pro Pro Pro Arg Gly Phe Pro Gly Leu Ile
130 135 140 ctc ctc acc gtc ctc ctc ttc ctc ttc tgc ggc ctg ccc ttc
ggc atc 481 Leu Leu Thr Val Leu Leu Phe Leu Phe Cys Gly Leu Pro Phe
Gly Ile 145 150 155 160 tac tgg ctg tcc cgg aac ctg ctc tgg tac atc
ccc cac tac ttc tac 529 Tyr Trp Leu Ser Arg Asn Leu Leu Trp Tyr Ile
Pro His Tyr Phe Tyr 165 170 175 cac ttc agc ttc ctc atg gcc gcc gtg
cac tgc gcg gcc aag ccc gtc 577 His Phe Ser Phe Leu Met Ala Ala Val
His Cys Ala Ala Lys Pro Val 180 185 190 gtc tac ttc tgc ctg ggc agt
gcc cag ggc cgc agg ctg ccc ctc cgg 625 Val Tyr Phe Cys Leu Gly Ser
Ala Gln Gly Arg Arg Leu Pro Leu Arg 195 200 205 ctg gtc ctc cag cga
gcg ctg gga gac gag gct gag ctg ggg gcc gtc 673 Leu Val Leu Gln Arg
Ala Leu Gly Asp Glu Ala Glu Leu Gly Ala Val 210 215 220 agg gag acc
tcc cgc cgg ggc ctg gtg gac ata gca gcc tga g 716 Arg Glu Thr Ser
Arg Arg Gly Leu Val Asp Ile Ala Ala * 225 230 235 cctggggcc
cccgacccca gctgcagccc ccgtgaggca agagggtgac t 767 37 237 PRT Homo
sapiens 37 His Met Val Ala Ile Val Pro Asp Leu Leu Gln Gly Arg Leu
Asp Phe 1 5 10 15 Pro Gly Phe Val Gln Thr Ser Leu Ala Thr Leu Arg
Phe Phe Cys Tyr 20 25 30 Ile Val Gly Leu Ser Leu Leu Ala Ala Val
Ser Val Glu Gln Cys Leu 35 40 45 Ala Ala Leu Phe Pro Ala Trp Tyr
Ser Cys Arg Arg Pro Arg His Leu 50 55 60 Thr Thr Cys Val Cys Ala
Leu Thr Trp Ala Leu Cys Leu Leu Leu His 65 70 75 80 Leu Leu Leu Ser
Ser Ala Cys Thr Gln Phe Phe Gly Glu Pro Ser Arg 85 90 95 His Leu
Cys Arg Thr Leu Trp Leu Val Ala Ala Val Leu Leu Ala Leu 100 105 110
Leu Cys Cys Thr Met Cys Gly Ala Ser Leu Met Leu Leu Leu Arg Val 115
120 125 Glu Arg Gly Pro Gln Arg Pro Pro Pro Arg Gly Phe Pro Gly Leu
Ile 130 135 140 Leu Leu Thr Val Leu Leu Phe Leu Phe Cys Gly Leu Pro
Phe Gly Ile 145 150 155 160 Tyr Trp Leu Ser Arg Asn Leu Leu Trp Tyr
Ile Pro His Tyr Phe Tyr 165 170 175 His Phe Ser Phe Leu Met Ala Ala
Val His Cys Ala Ala Lys Pro Val 180 185 190 Val Tyr Phe Cys Leu Gly
Ser Ala Gln Gly Arg Arg Leu Pro Leu Arg 195 200 205 Leu Val Leu Gln
Arg Ala Leu Gly Asp Glu Ala Glu Leu Gly Ala Val 210 215 220 Arg Glu
Thr Ser Arg Arg Gly Leu Val Asp Ile Ala Ala 225 230 235 38 1361 DNA
Mus musculus CDS (48)...(1064) 38 tctttttttt ttttcattgc agaactgaga
ttgcaccact cctgaaa atg gac tta 56 Met Asp Leu 1 gtc atc caa gac tgg
acc att aat att aca gca ctg aaa gaa agc aat 104 Val Ile Gln Asp Trp
Thr Ile Asn Ile Thr Ala Leu Lys Glu Ser Asn 5 10 15 gac aat gga ata
tca ttt tgt gaa gtt gtg tct cgt acc atg act ttt 152 Asp Asn Gly Ile
Ser Phe Cys Glu Val Val Ser Arg Thr Met Thr Phe 20 25 30 35 ctt tcc
ctc atc att gcc tta gtt ggg ctg gtt gga aat gcc aca gtg 200 Leu Ser
Leu Ile Ile Ala Leu Val Gly Leu Val Gly Asn Ala Thr Val 40 45 50
tta tgg ttt ctg ggc ttc cag atg agc agg aat gcc ttc tct gtc tac 248
Leu Trp Phe Leu Gly Phe Gln Met Ser Arg Asn Ala Phe Ser Val Tyr 55
60 65 atc ctc aac ctt gct ggt gct gac ttt gtc ttc atg tgc ttt caa
att 296 Ile Leu Asn Leu Ala Gly Ala Asp Phe Val Phe Met Cys Phe Gln
Ile 70 75 80 gta cat tgt ttt tat att atc tta gac atc tac ttc atc
ccc act aat 344 Val His Cys Phe Tyr Ile Ile Leu Asp Ile Tyr Phe Ile
Pro Thr Asn 85 90 95 ttt ttt tca tct tac act atg gtg tta aac att
gct tac ctt agt ggt 392 Phe Phe Ser Ser Tyr Thr Met Val Leu Asn Ile
Ala Tyr Leu Ser Gly 100 105 110 115 ctg agc atc ctc act gtc att agc
act gaa cgc ttc cta tct gtc atg 440 Leu Ser Ile Leu Thr Val Ile Ser
Thr Glu Arg Phe Leu Ser Val Met 120 125 130 tgg ccc atc tgg tac cgc
tgc caa cgc cca agg cac aca tca gct gtc 488 Trp Pro Ile Trp Tyr Arg
Cys Gln Arg Pro Arg His Thr Ser Ala Val 135 140 145 ata tgt act gtg
ctt tgg gtc ttg tcc ctg gtg ttg agc ctc ctg gaa 536 Ile Cys Thr Val
Leu Trp Val Leu Ser Leu Val Leu Ser Leu Leu Glu 150 155 160 gga aag
gaa tgt ggc ttc cta tat tac act agt ggc cct ggt ttg tgt 584 Gly Lys
Glu Cys Gly Phe Leu Tyr Tyr Thr Ser Gly Pro Gly Leu Cys 165 170 175
aag aca ttt gat tta atc act act gca tgg tta att gtt tta ttt gtg 632
Lys Thr Phe Asp Leu Ile Thr Thr Ala Trp Leu Ile Val Leu Phe Val 180
185 190 195 gtt ctc ttg gga tcc agt ctg gcc ttg gtg ctt acc atc ttc
tgt ggc 680 Val Leu Leu Gly Ser Ser Leu Ala Leu Val Leu Thr Ile Phe
Cys Gly 200 205 210 tta cac aag gtt cct gtg acc agg ttg tat gtg acc
att gtg ttt aca 728 Leu His Lys Val Pro Val Thr Arg Leu Tyr Val Thr
Ile Val Phe Thr 215 220 225 gtg ctt gtc ttc ctg atc ttt ggt ctg ccc
tat ggg atc tac tgg ttc 776 Val Leu Val Phe Leu Ile Phe Gly Leu Pro
Tyr Gly Ile Tyr Trp Phe 230 235 240 ctc tta gag tgg att agg gaa ttt
cat gat aat aaa cct tgt ggt ttt 824 Leu Leu Glu Trp Ile Arg Glu Phe
His Asp Asn Lys Pro Cys Gly Phe 245 250 255 cgt aac gtg aca ata ttt
ctg tcc tgt att aac agc tgt gcc aac ccc 872 Arg Asn Val Thr Ile Phe
Leu Ser Cys Ile Asn Ser Cys Ala Asn Pro 260
265 270 275 atc att tac ttc ctt gtt ggc tcc att agg cac cat cgg ttt
caa cgg 920 Ile Ile Tyr Phe Leu Val Gly Ser Ile Arg His His Arg Phe
Gln Arg 280 285 290 aag act ctc aag ctt ctt ctg cag aga gcc atg caa
gac tct cct gag 968 Lys Thr Leu Lys Leu Leu Leu Gln Arg Ala Met Gln
Asp Ser Pro Glu 295 300 305 gag gaa gaa tgt gga gag atg ggt tcc tca
aga aga cct aga gaa ata 1016 Glu Glu Glu Cys Gly Glu Met Gly Ser
Ser Arg Arg Pro Arg Glu Ile 310 315 320 aaa act gtc tgg aag gga ctg
aga gct gct ttg atc agg cat aaa tag 1064 Lys Thr Val Trp Lys Gly
Leu Arg Ala Ala Leu Ile Arg His Lys * 325 330 335 ctttgaagag
aactatgttt ttatcacttt gtggcatttt cataatgttg tttagttgat 1124
gacccaaggt taactcagtt ggggaagtag tcaatgttgt agaagttgat tgatattgaa
1184 cttgttataa atactgagta cagtattttt gcagctatct tgctcagagc
tttaccaact 1244 ccatttgatg ggactcctta taagctctat ggggtccagg
agaggtgttg accacaattg 1304 acaaatccct cttcagaaga aaactcaaga
aagtgcaatg aaaagttata tttcttt 1361 39 338 PRT Mus musculus 39 Met
Asp Leu Val Ile Gln Asp Trp Thr Ile Asn Ile Thr Ala Leu Lys 1 5 10
15 Glu Ser Asn Asp Asn Gly Ile Ser Phe Cys Glu Val Val Ser Arg Thr
20 25 30 Met Thr Phe Leu Ser Leu Ile Ile Ala Leu Val Gly Leu Val
Gly Asn 35 40 45 Ala Thr Val Leu Trp Phe Leu Gly Phe Gln Met Ser
Arg Asn Ala Phe 50 55 60 Ser Val Tyr Ile Leu Asn Leu Ala Gly Ala
Asp Phe Val Phe Met Cys 65 70 75 80 Phe Gln Ile Val His Cys Phe Tyr
Ile Ile Leu Asp Ile Tyr Phe Ile 85 90 95 Pro Thr Asn Phe Phe Ser
Ser Tyr Thr Met Val Leu Asn Ile Ala Tyr 100 105 110 Leu Ser Gly Leu
Ser Ile Leu Thr Val Ile Ser Thr Glu Arg Phe Leu 115 120 125 Ser Val
Met Trp Pro Ile Trp Tyr Arg Cys Gln Arg Pro Arg His Thr 130 135 140
Ser Ala Val Ile Cys Thr Val Leu Trp Val Leu Ser Leu Val Leu Ser 145
150 155 160 Leu Leu Glu Gly Lys Glu Cys Gly Phe Leu Tyr Tyr Thr Ser
Gly Pro 165 170 175 Gly Leu Cys Lys Thr Phe Asp Leu Ile Thr Thr Ala
Trp Leu Ile Val 180 185 190 Leu Phe Val Val Leu Leu Gly Ser Ser Leu
Ala Leu Val Leu Thr Ile 195 200 205 Phe Cys Gly Leu His Lys Val Pro
Val Thr Arg Leu Tyr Val Thr Ile 210 215 220 Val Phe Thr Val Leu Val
Phe Leu Ile Phe Gly Leu Pro Tyr Gly Ile 225 230 235 240 Tyr Trp Phe
Leu Leu Glu Trp Ile Arg Glu Phe His Asp Asn Lys Pro 245 250 255 Cys
Gly Phe Arg Asn Val Thr Ile Phe Leu Ser Cys Ile Asn Ser Cys 260 265
270 Ala Asn Pro Ile Ile Tyr Phe Leu Val Gly Ser Ile Arg His His Arg
275 280 285 Phe Gln Arg Lys Thr Leu Lys Leu Leu Leu Gln Arg Ala Met
Gln Asp 290 295 300 Ser Pro Glu Glu Glu Glu Cys Gly Glu Met Gly Ser
Ser Arg Arg Pro 305 310 315 320 Arg Glu Ile Lys Thr Val Trp Lys Gly
Leu Arg Ala Ala Leu Ile Arg 325 330 335 His Lys 40 1278 DNA Mus
musculus 40 atttcctaat caagaatcta agcacctcag cctggaaaac gaacatcaca
gtgctgaatg 60 gaagctacta catcgatact tcagtttgtg tcaccaggaa
ccaagccatg attttgcttt 120 ccatcatcat ttccctggtt gggatgggac
taaatgccat agtgctgtgg ttcctgggca 180 tccgtatgca cacgaatgcc
ttcactgtct acattctcaa cctggctatg gctgactttc 240 tttacctgtg
ctctcagttt gtaatttgtc ttcttattgc cttttatatc ttctactcaa 300
ttgacatcaa catccctttg gttctttatg ttgtgccaat atttgcttat ctttcaggtc
360 tgagcattct cagcaccatt agcattgagc gctgcttgtc tgtaatatgg
cccatttggt 420 atcgctgtaa acgtccaaga cacacatcag ctatcacatg
ttttgtgctt tgggttatgt 480 ccttattgtt gggtctcctg gaagggaagg
catgtggctt actgtttaat agctttgact 540 cttattggtg tgaaacattt
gatgttatca ctaatatatg gtcagttgtt ttttttggtg 600 ttctctgtgg
gtctagcctc accctgcttg tcaggatctt ctgtggctca cagcgaattc 660
ctatgaccag gctgtatgtg actattacac tcacagtctt ggtcttcctg atctttggtc
720 ttccctttgg gatctattgg atactctatc agtggattag caatttttat
tatgttgaaa 780 tttgtaattt ttatcttgag atactattcc tatcctgtgt
taacagctgt atgaacccca 840 tcatttattt ccttgttggc tccattaggc
accgaaggtt caggcggaag actctcaagc 900 tacttctgca gagagccatg
caagacaccc ctgaggagga acaaagtgga aataagagtt 960 cttcagaaca
ccctgaagaa ctggaaactg ttcagagctg cagctgacaa ctgcttgatc 1020
agacaaaaat ggttttgatg gaaatacttt ttcttatccg tgtggaccat ttttacaacc
1080 tttattcagt ttgttatctc atcttcaatt gtttaattag gacaataatt
tttgtaaaag 1140 ttgagagaaa tgggtcttgt catactaata ctgaatgtag
catttctgaa gctgtgttac 1200 ttagggattt accatctcct tttcatggga
ctccttgtaa gtattctgtg gtagagaact 1260 tctcctattg ttgacaaa 1278 41
338 PRT Mus musculus 41 Met Ser Gly Asp Phe Leu Ile Lys Asn Leu Ser
Thr Ser Ala Trp Lys 1 5 10 15 Thr Asn Ile Thr Val Leu Asn Gly Ser
Tyr Tyr Ile Asp Thr Ser Val 20 25 30 Cys Val Thr Arg Asn Gln Ala
Met Ile Leu Leu Ser Ile Ile Ile Ser 35 40 45 Leu Val Gly Met Gly
Leu Asn Ala Ile Val Leu Trp Phe Leu Gly Ile 50 55 60 Arg Met His
Thr Asn Ala Phe Thr Val Tyr Ile Leu Asn Leu Ala Met 65 70 75 80 Ala
Asp Phe Leu Tyr Leu Cys Ser Gln Phe Val Ile Cys Leu Leu Ile 85 90
95 Ala Phe Tyr Ile Phe Tyr Ser Ile Asp Ile Asn Ile Pro Leu Val Leu
100 105 110 Tyr Val Val Pro Ile Phe Ala Tyr Leu Ser Gly Leu Ser Ile
Leu Ser 115 120 125 Thr Ile Ser Ile Glu Arg Cys Leu Ser Val Ile Trp
Pro Ile Trp Tyr 130 135 140 Arg Cys Lys Arg Pro Arg His Thr Ser Ala
Ile Thr Cys Phe Val Leu 145 150 155 160 Trp Val Met Ser Leu Leu Leu
Gly Leu Leu Glu Gly Lys Ala Cys Gly 165 170 175 Leu Leu Phe Asn Ser
Phe Asp Ser Tyr Trp Cys Glu Thr Phe Asp Val 180 185 190 Ile Thr Asn
Ile Trp Ser Val Val Phe Phe Gly Val Leu Cys Gly Ser 195 200 205 Ser
Leu Thr Leu Leu Val Arg Ile Phe Cys Gly Ser Gln Arg Ile Pro 210 215
220 Met Thr Arg Leu Tyr Val Thr Ile Thr Leu Thr Val Leu Val Phe Leu
225 230 235 240 Ile Phe Gly Leu Pro Phe Gly Ile Tyr Trp Ile Leu Tyr
Gln Trp Ile 245 250 255 Ser Asn Phe Tyr Tyr Val Glu Ile Cys Asn Phe
Tyr Leu Glu Ile Leu 260 265 270 Phe Leu Ser Cys Val Asn Ser Cys Met
Asn Pro Ile Ile Tyr Phe Leu 275 280 285 Val Gly Ser Ile Arg His Arg
Arg Phe Arg Arg Lys Thr Leu Lys Leu 290 295 300 Leu Leu Gln Arg Ala
Met Gln Asp Thr Pro Glu Glu Glu Gln Ser Gly 305 310 315 320 Asn Lys
Ser Ser Ser Glu His Pro Glu Glu Leu Glu Thr Val Gln Ser 325 330 335
Cys Ser 42 1009 DNA Mus musculus 42 ttttctaagc atggctctaa
gaacctcact aataaccacc acagcaccgg ataaaaccag 60 ccttccaatt
tcaatttgta tcatcaagtt ccaagtcatg aatttgcttt ccatcaccat 120
ttcccctgtt gggatggtac tgaatatcat agtgctgtgg ttcctgggct tccagatatg
180 caggaatgcc ttctctgcct acatcctcaa cctggctgtg gctgattttc
tcttcctgtg 240 ttctcattct atattttctt ttcttattgt ctgcaaactg
cactattttt tattctacat 300 tagacagctt ttggatactg tgacaatgtt
tgcttatgtt tttggcctga gcattaccac 360 catcattagc attgagtgct
gcctgtctat catgtggccc atctggtatc actgccaacg 420 tccaagacac
acatcagctg tcatttgtgt cttgctttgg gctctatctc tgctgtttcc 480
tgctctgcag atggaaaaat gtagcgtcct gtttaatact tttgaatatt cttggtgtgg
540 gataatcaat ataatctctg gtgcatggtt agttgtttta tttgtggttc
tctgtgggtt 600 cagcctcatc ctgctcctca ggatctcctg tggatcacag
cagattcctg tgaccaggct 660 gaatgtaact attgcactca gagtgctact
cctcctgatc tttggtattc cctttgggat 720 cttctggata gttgacaaat
ggaatgaaga aaattttttc gttagagctt gtggtttttc 780 acatcatata
ctatacgtat actgtattaa catctgtgtc aatgctacca tatacttcct 840
tgttggctcc attaggcatg gcaagtttca gaagatgact ctgaagctga ttctgcagag
900 agctatacag ggcacccccg aggaagaagg tggagagagg ggtccttaag
gaaatactga 960 agaactggga acagtctagt gcagcaaccg agagctgctt
taataataa 1009 43 312 PRT Mus musculus 43 Met Ala Leu Arg Thr Ser
Leu Ile Thr Thr Thr Ala Pro Asp Lys Thr 1 5 10 15 Ser Leu Pro Ile
Ser Ile Cys Ile Ile Lys Phe Gln Val Met Asn Leu 20 25 30 Leu Ser
Ile Thr Ile Ser Pro Val Gly Met Val Leu Asn Ile Ile Val 35 40 45
Leu Trp Phe Leu Gly Phe Gln Ile Cys Arg Asn Ala Phe Ser Ala Tyr 50
55 60 Ile Leu Asn Leu Ala Val Ala Asp Phe Leu Phe Leu Cys Ser His
Ser 65 70 75 80 Ile Phe Ser Phe Leu Ile Val Cys Lys Leu His Tyr Phe
Leu Phe Tyr 85 90 95 Ile Arg Gln Leu Leu Asp Thr Val Thr Met Phe
Ala Tyr Val Phe Gly 100 105 110 Leu Ser Ile Thr Thr Ile Ile Ser Ile
Glu Cys Cys Leu Ser Ile Met 115 120 125 Trp Pro Ile Trp Tyr His Cys
Gln Arg Pro Arg His Thr Ser Ala Val 130 135 140 Ile Cys Val Leu Leu
Trp Ala Leu Ser Leu Leu Phe Pro Ala Leu Gln 145 150 155 160 Met Glu
Lys Cys Ser Val Leu Phe Asn Thr Phe Glu Tyr Ser Trp Cys 165 170 175
Gly Ile Ile Asn Ile Ile Ser Gly Ala Trp Leu Val Val Leu Phe Val 180
185 190 Val Leu Cys Gly Phe Ser Leu Ile Leu Leu Leu Arg Ile Ser Cys
Gly 195 200 205 Ser Gln Gln Ile Pro Val Thr Arg Leu Asn Val Thr Ile
Ala Leu Arg 210 215 220 Val Leu Leu Leu Leu Ile Phe Gly Ile Pro Phe
Gly Ile Phe Trp Ile 225 230 235 240 Val Asp Lys Trp Asn Glu Glu Asn
Phe Phe Val Arg Ala Cys Gly Phe 245 250 255 Ser His His Ile Leu Tyr
Val Tyr Cys Ile Asn Ile Cys Val Asn Ala 260 265 270 Thr Ile Tyr Phe
Leu Val Gly Ser Ile Arg His Gly Lys Phe Gln Lys 275 280 285 Met Thr
Leu Lys Leu Ile Leu Gln Arg Ala Ile Gln Gly Thr Pro Glu 290 295 300
Glu Glu Gly Gly Glu Arg Gly Pro 305 310 44 1219 DNA Mus musculus 44
tttatggacc tgtgccagat attcctacat aatcacatgg tcctgactga gactatcttg
60 tgttcatatc tcgatttctt tgcaggaatg ccagtggaaa attcctaagc
atgggtacaa 120 ccaccctggc ctggaacatt aacaacaccg ctgaaaatgg
aagttacact gaaatgttct 180 cctgtatcac caagttcaat accctgaatt
ttcttactgt catcatagct gtggttggcc 240 tggcaggaaa cggcatagtg
ctatggcttc tagccttcca cctgcatagg aatgccttct 300 ctgtctatgt
cctcaatctg gctggtgctg atttcttgta ccttttcact caagttgtgc 360
attccctgga atgtgtcctt cagttagata ataactcctt ttatattctc ctcattgtaa
420 caatgtttgc ttaccttgca ggtttgtgta tgattgcagc catcagtgct
gaacgctgcc 480 tatctgttat gtggcctatc tggtatcact gccaaagacc
aagacacaca tcagccatca 540 tgtgtgctct ggtctgggtt tcctctctat
tgttgagcct cgtggtaggg ctaggctgtg 600 gttttctgtt cagttattat
gattattatt tctgtattac tttgaatttt atcactgctg 660 catttttaat
agtgttatct gtggttcttt ctgtatctag cctggccctg ttggtgaaga 720
ttgtgtgggg gtcacacagg attcctgtga ccaggttctt tgtgaccatt gctctcacag
780 tggtggtctt catatacttt ggcatgccct ttggtatctg ctggttcctc
ttatcaagga 840 ttatggagtt tgatagcatt ttctttaaca atgtttatga
aataatagaa ttcctgtcct 900 gtgttaacag ctgtgccaat cccatcattt
acttccttgt tggctccatt agacaacaca 960 ggttgcgatg gcagtctctg
aagctacttc ttcagagagc catgcaggac actcctgagg 1020 aagagagtgg
agagaggggt ccttcgcaaa ggtctgggga actggaaaca gtctagtaca 1080
gtagttgagt gagtccctgg tcaaacatag tttctgtgag agtcaatttt gcctttatct
1140 atataagcaa ttttcataat ttgtttaatc agtagagaat atagtcattt
tatagaaatt 1200 aggagaaatg agcttgtta 1219 45 321 PRT Mus musculus
45 Met Gly Thr Thr Thr Leu Ala Trp Asn Ile Asn Asn Thr Ala Glu Asn
1 5 10 15 Gly Ser Tyr Thr Glu Met Phe Ser Cys Ile Thr Lys Phe Asn
Thr Leu 20 25 30 Asn Phe Leu Thr Val Ile Ile Ala Val Val Gly Leu
Ala Gly Asn Gly 35 40 45 Ile Val Leu Trp Leu Leu Ala Phe His Leu
His Arg Asn Ala Phe Ser 50 55 60 Val Tyr Val Leu Asn Leu Ala Gly
Ala Asp Phe Leu Tyr Leu Phe Thr 65 70 75 80 Gln Val Val His Ser Leu
Glu Cys Val Leu Gln Leu Asp Asn Asn Ser 85 90 95 Phe Tyr Ile Leu
Leu Ile Val Thr Met Phe Ala Tyr Leu Ala Gly Leu 100 105 110 Cys Met
Ile Ala Ala Ile Ser Ala Glu Arg Cys Leu Ser Val Met Trp 115 120 125
Pro Ile Trp Tyr His Cys Gln Arg Pro Arg His Thr Ser Ala Ile Met 130
135 140 Cys Ala Leu Val Trp Val Ser Ser Leu Leu Leu Ser Leu Val Val
Gly 145 150 155 160 Leu Gly Cys Gly Phe Leu Phe Ser Tyr Tyr Asp Tyr
Tyr Phe Cys Ile 165 170 175 Thr Leu Asn Phe Ile Thr Ala Ala Phe Leu
Ile Val Leu Ser Val Val 180 185 190 Leu Ser Val Ser Ser Leu Ala Leu
Leu Val Lys Ile Val Trp Gly Ser 195 200 205 His Arg Ile Pro Val Thr
Arg Phe Phe Val Thr Ile Ala Leu Thr Val 210 215 220 Val Val Phe Ile
Tyr Phe Gly Met Pro Phe Gly Ile Cys Trp Phe Leu 225 230 235 240 Leu
Ser Arg Ile Met Glu Phe Asp Ser Ile Phe Phe Asn Asn Val Tyr 245 250
255 Glu Ile Ile Glu Phe Leu Ser Cys Val Asn Ser Cys Ala Asn Pro Ile
260 265 270 Ile Tyr Phe Leu Val Gly Ser Ile Arg Gln His Arg Leu Arg
Trp Gln 275 280 285 Ser Leu Lys Leu Leu Leu Gln Arg Ala Met Gln Asp
Thr Pro Glu Glu 290 295 300 Glu Ser Gly Glu Arg Gly Pro Ser Gln Arg
Ser Gly Glu Leu Glu Thr 305 310 315 320 Val 46 1281 DNA Mus
musculus 46 atggtcctga cagagagtat catgtgttca tatctctatt tttttgcggg
aacaccactg 60 gaaacttcct aaacatgggt ctaaccactc cagcctggaa
cattaacaac acagtagtga 120 atggaagtaa caatactgaa catttctcct
gtgtcagcaa gttcaatacc ctgaactttc 180 ttactgtcat cattgccatg
tttggcctgg caggaaatgc catagtccta tggcttctag 240 ccttccacct
gcctaggaat gccttctctg tctatgtctg caacttggct tgtgctgatt 300
tcttgcaact ttgcactcag attttaggtt ccctggaatg tttccttcag ttaaatagga
360 gacacacttt ttttctcacc gttgtattta tgtttgctta ccttgcaggt
ttgtgtatga 420 ttgcagccat cagtgttgag cgctctctat ctgttatgtg
gcccatctgg tatcactgcc 480 aaagaccaag acatacatca tccatcatgt
gtgctctgct ctgggctttc tgtctactgt 540 tgaatttcct attaggggaa
ggctgtggcc ttctgttcag tgatcctaaa tattatttct 600 gtattacttg
tgccttaatc actactgcac ttataatatt attaactgtg gttccttctg 660
tgtccagcct ggccctgttg gtcaagatga tctgtggatc acacaggatt cctgtgacca
720 ggttctatgt gaccattgct ctcacattgg tggtcttcat attcttgggt
ctgccctttg 780 ggatttactc atctttcttg ataatgttta aggagtttca
aagcattttc tcttaccatg 840 tccttgaagt gacaatattc ctgtcctgtg
ttaacagctg tgccaatccc atcatttact 900 ttcttgttgg ctccattagg
cagcacaggt tgcaatggca gtctctgaag ctacttcttc 960 agagagccat
gcaggacact cctgaggaag atagtggaga gagggttccc tcacaaaggt 1020
ctggggaact ggaaagtgtt tagtgcagta gttgagtgag tctttgatca gacatggtta
1080 ctctgagagt cagttttgcc tttgtttatg taagcaattt tcacaatctt
gtacaatttg 1140 taaagaaata gtcattttat agaaattggg agaaaggggc
ttgttacaca gaaactgagt 1200 gcaacaccat aaagctgtct tatgtgggtc
tcattacatt ctcttgtgat ataagccttg 1260 taatcacttg ggaacaaaac t 1281
47 322 PRT Mus musculus 47 Met Gly Leu Thr Thr Pro Ala Trp Asn Ile
Asn Asn Thr Val Val Asn 1 5 10 15 Gly Ser Asn Asn Thr Glu His Phe
Ser Cys Val Ser Lys Phe Asn Thr 20 25 30 Leu Asn Phe Leu Thr Val
Ile Ile Ala Met Phe Gly Leu Ala Gly Asn 35 40 45 Ala Ile Val Leu
Trp Leu Leu Ala Phe His Leu Pro Arg Asn Ala Phe 50 55 60 Ser Val
Tyr Val Cys Asn Leu Ala Cys Ala Asp Phe Leu Gln Leu Cys 65 70 75 80
Thr Gln Ile Leu Gly Ser Leu Glu Cys Phe Leu Gln Leu Asn Arg Arg 85
90 95 His Thr Phe Phe Leu Thr Val Val Phe Met Phe Ala Tyr Leu Ala
Gly 100 105 110 Leu Cys Met Ile Ala Ala Ile Ser Val Glu Arg Ser Leu
Ser Val Met 115 120 125 Trp Pro Ile Trp Tyr His Cys Gln Arg Pro Arg
His Thr Ser Ser Ile 130 135 140 Met Cys Ala Leu Leu Trp Ala Phe Cys
Leu Leu Leu Asn Phe Leu Leu 145
150 155 160 Gly Glu Gly Cys Gly Leu Leu Phe Ser Asp Pro Lys Tyr Tyr
Phe Cys 165 170 175 Ile Thr Cys Ala Leu Ile Thr Thr Ala Leu Ile Ile
Leu Leu Thr Val 180 185 190 Val Pro Ser Val Ser Ser Leu Ala Leu Leu
Val Lys Met Ile Cys Gly 195 200 205 Ser His Arg Ile Pro Val Thr Arg
Phe Tyr Val Thr Ile Ala Leu Thr 210 215 220 Leu Val Val Phe Ile Phe
Leu Gly Leu Pro Phe Gly Ile Tyr Ser Ser 225 230 235 240 Phe Leu Ile
Met Phe Lys Glu Phe Gln Ser Ile Phe Ser Tyr His Val 245 250 255 Leu
Glu Val Thr Ile Phe Leu Ser Cys Val Asn Ser Cys Ala Asn Pro 260 265
270 Ile Ile Tyr Phe Leu Val Gly Ser Ile Arg Gln His Arg Leu Gln Trp
275 280 285 Gln Ser Leu Lys Leu Leu Leu Gln Arg Ala Met Gln Asp Thr
Pro Glu 290 295 300 Glu Asp Ser Gly Glu Arg Val Pro Ser Gln Arg Ser
Gly Glu Leu Glu 305 310 315 320 Ser Val 48 1280 DNA Mus musculus 48
ccccactagt tcataacaca gaatttaaca tgggttcttc ttccacccat aggaatgaac
60 tccactcttg acagcagccc agctccaggt ctgaccatca gtcccaccat
ggaccttgtg 120 acctggatct acttttcagt gacattcctc gccatggcca
cgtgtgtggg gggggatggc 180 aggcaactca ttggtgattt ggctcctgag
ctgcaatggc atgcagaggt ctcccttctg 240 tgtctatgtg ctcaacctgg
cggtggctga cttcctcttc ttattctgca tggcctccat 300 gctcagcctg
gaaacagggc ccctgctcat agtcaacatt tctgccaaaa tctatgaagg 360
gatgaggaga atcaagtact ttgcctatac agcaggcctg agcctgctga cagccatcag
420 cacccagcgc tgcctctccg tgcttttccc catctggtat aagtgccacc
ggccccggca 480 cctgtcatca gtggtatctg gtgcactctg ggcactggcc
ttcctgatga acttcctggc 540 ttctttcttc tgcgtccaat tctggcatcc
caacaaacac cagtgcttca aggtggacat 600 tgttttcaac agtcttatcc
tggggatctt catgccggtc atgatcctga ccagcaccat 660 cctcttcatc
cgggtgcgga agaacagcct gatgcagaga cggcggcccc ggcggctgta 720
cgtggtcatc ctgacttcca tccttgtctt cctcacctgt tctctgccct tgggcatcaa
780 ctggttctta ctctactggg tggatgtgaa acgggatgtg aggctacttt
atagctgcgt 840 atcacgcttc tcttcgtctt tgagcagcag tgccaacccg
gtcatttact tcctcgtggg 900 cagccagaag agccaccggc tgcaggagtc
cctgggtgct gtgctggggc gggcactgcg 960 ggatgagcct gagccagagg
gcagagagac gccatccacg tgtactaatg atggggtctg 1020 aagggagccc
aaccaggaac tcctccaaag ccccacccag cccttcccta aaagtaccca 1080
gcaagcctgc aatgcaaagg ccttgcacct caaaatgttt gggtcacgtt cctctctgcc
1140 agggagggtt caccactatc accttgtgtt cctaatctaa actaagaggt
gaggcaatat 1200 atctttctgt tttacctgtt tagacacaga tcctaacttt
gggtcccatc atgggcaagg 1260 ctgtctggga aatggagttt 1280 49 281 PRT
Mus musculus 49 Met Ala Gly Asn Ser Leu Val Ile Trp Leu Leu Ser Cys
Asn Gly Met 1 5 10 15 Gln Arg Ser Pro Phe Cys Val Tyr Val Leu Asn
Leu Ala Val Ala Asp 20 25 30 Phe Leu Phe Leu Phe Cys Met Ala Ser
Met Leu Ser Leu Glu Thr Gly 35 40 45 Pro Leu Leu Ile Val Asn Ile
Ser Ala Lys Ile Tyr Glu Gly Met Arg 50 55 60 Arg Ile Lys Tyr Phe
Ala Tyr Thr Ala Gly Leu Ser Leu Leu Thr Ala 65 70 75 80 Ile Ser Thr
Gln Arg Cys Leu Ser Val Leu Phe Pro Ile Trp Tyr Lys 85 90 95 Cys
His Arg Pro Arg His Leu Ser Ser Val Val Ser Gly Ala Leu Trp 100 105
110 Ala Leu Ala Phe Leu Met Asn Phe Leu Ala Ser Phe Phe Cys Val Gln
115 120 125 Phe Trp His Pro Asn Lys His Gln Cys Phe Lys Val Asp Ile
Val Phe 130 135 140 Asn Ser Leu Ile Leu Gly Ile Phe Met Pro Val Met
Ile Leu Thr Ser 145 150 155 160 Thr Ile Leu Phe Ile Arg Val Arg Lys
Asn Ser Leu Met Gln Arg Arg 165 170 175 Arg Pro Arg Arg Leu Tyr Val
Val Ile Leu Thr Ser Ile Leu Val Phe 180 185 190 Leu Thr Cys Ser Leu
Pro Leu Gly Ile Asn Trp Phe Leu Leu Tyr Trp 195 200 205 Val Asp Val
Lys Arg Asp Val Arg Leu Leu Tyr Ser Cys Val Ser Arg 210 215 220 Phe
Ser Ser Ser Leu Ser Ser Ser Ala Asn Pro Val Ile Tyr Phe Leu 225 230
235 240 Val Gly Ser Gln Lys Ser His Arg Leu Gln Glu Ser Leu Gly Ala
Val 245 250 255 Leu Gly Arg Ala Leu Arg Asp Glu Pro Glu Pro Glu Gly
Arg Glu Thr 260 265 270 Pro Ser Thr Cys Thr Asn Asp Gly Val 275 280
50 1170 DNA Mus musculus 50 gacttctgca gacatcagcc atgacgtccc
tgagcgtgca cacagattct cccagcaccc 60 agggagaaat ggctttcaac
ctgaccatcc tgtccctcac agagctcctc agcctgggcg 120 ggctgctggg
caatggagtg gccctctggc tgctcaacca aaatgtctac aggaacccct 180
tctccatcta tctcttggat gtggcctgcg ccgacctcat cttcctctgc tgccacatgg
240 tggccatcat ccctgagctg ctgcaggacc agctgaactt ccctgaattt
gtacatatca 300 gcctgaccat gctgcggttc ttctgctaca ttgtgggcct
gagcctcctg gcggccatca 360 gcacggagca gtgcctggcc actctcttcc
ctgcctggta cctgtgccgc cgcccacgct 420 acctgaccac ctgtgtgtgt
gcgctcatct gggtgctctg cctgctactg gacctgctgc 480 tgagcggcgc
ctgcacccag ttctttggag cacccagcta ccacctgtgt gacatgctgt 540
ggctggtggt ggcagttctc ctggctgccc tgtgctgcac catgtgtgtg accagcctgc
600 tcctgctgct gcgggtggag cgtggtccag agagacacca gcctcggggc
ttccccaccc 660 tggtcctgct ggccgtcctg ctcttcctct tctgcggcct
gccctttggc atcttctggc 720 tgtccaagaa cctgtcctgg cacatccccc
tctacttcta tcatttcagc ttcttcatgg 780 ccagtgtgca cagtgcagcc
aagcctgcca tctacttttt cttgggcagc acacctggcc 840 agaggtttcg
ggaacccctc cggctggtgc tccagcgggc acttggagat gaggctgagc 900
tgggagctgg gagagaggct tcccaagggg gacttgtgga catgactgtc taagcacagt
960 gggtcacaac tgcagcttca gcccatgggg gtccagggga gctgcctgat
gtaggtaaag 1020 ctgggatcag agctccatca gtaagactct tgagggacat
ctttgctgat gacccagtgc 1080 tgtgtcccct gggaggattc tgggaagggg
caagcagaga gtgatgcttc tgtggagggc 1140 ctggggttgt gtgtgttagg
cagagctcct 1170 51 310 PRT Mus musculus 51 Met Thr Ser Leu Ser Val
His Thr Asp Ser Pro Ser Thr Gln Gly Glu 1 5 10 15 Met Ala Phe Asn
Leu Thr Ile Leu Ser Leu Thr Glu Leu Leu Ser Leu 20 25 30 Gly Gly
Leu Leu Gly Asn Gly Val Ala Leu Trp Leu Leu Asn Gln Asn 35 40 45
Val Tyr Arg Asn Pro Phe Ser Ile Tyr Leu Leu Asp Val Ala Cys Ala 50
55 60 Asp Leu Ile Phe Leu Cys Cys His Met Val Ala Ile Ile Pro Glu
Leu 65 70 75 80 Leu Gln Asp Gln Leu Asn Phe Pro Glu Phe Val His Ile
Ser Leu Thr 85 90 95 Met Leu Arg Phe Phe Cys Tyr Ile Val Gly Leu
Ser Leu Leu Ala Ala 100 105 110 Ile Ser Thr Glu Gln Cys Leu Ala Thr
Leu Phe Pro Ala Trp Tyr Leu 115 120 125 Cys Arg Arg Pro Arg Tyr Leu
Thr Thr Cys Val Cys Ala Leu Ile Trp 130 135 140 Val Leu Cys Leu Leu
Leu Asp Leu Leu Leu Ser Gly Ala Cys Thr Gln 145 150 155 160 Phe Phe
Gly Ala Pro Ser Tyr His Leu Cys Asp Met Leu Trp Leu Val 165 170 175
Val Ala Val Leu Leu Ala Ala Leu Cys Cys Thr Met Cys Val Thr Ser 180
185 190 Leu Leu Leu Leu Leu Arg Val Glu Arg Gly Pro Glu Arg His Gln
Pro 195 200 205 Arg Gly Phe Pro Thr Leu Val Leu Leu Ala Val Leu Leu
Phe Leu Phe 210 215 220 Cys Gly Leu Pro Phe Gly Ile Phe Trp Leu Ser
Lys Asn Leu Ser Trp 225 230 235 240 His Ile Pro Leu Tyr Phe Tyr His
Phe Ser Phe Phe Met Ala Ser Val 245 250 255 His Ser Ala Ala Lys Pro
Ala Ile Tyr Phe Phe Leu Gly Ser Thr Pro 260 265 270 Gly Gln Arg Phe
Arg Glu Pro Leu Arg Leu Val Leu Gln Arg Ala Leu 275 280 285 Gly Asp
Glu Ala Glu Leu Gly Ala Gly Arg Glu Ala Ser Gln Gly Gly 290 295 300
Leu Val Asp Met Thr Val 305 310 52 1519 DNA Mus musculus 52
tgtgttccca gcagcaccca gtgcagggtt tctggcccta aacatytyma gcctccacaa
60 tggcactcac aacaacaaaa tccaatggac gaaacccatc ccctggaagt
accagcatca 120 agattctgat cccaaacttg atgatcatca tctttggact
ggtcgggctg acaggaaacg 180 ccattgtgtt ctggctcctg ggcttccact
tgcgcaggaa tgccttctca gtctacatcc 240 taaacttggc cctggctgac
ttcctcttcc tcctctgtcg catcatagct tccacgcaga 300 aacttctcac
gttctcctca cccaacatta cctttctcat ttgcctttac accttcaggg 360
tgattctcta catcgcaggc ctgagcatgc tcactgccat cagcattgag cgctgcctgt
420 ctgtcctgtg ccccatctgg tatcgctgcc accgcccaga acacacatca
actgtcatgt 480 gtgctgcaat ctgggtcctg tccctgttga tctgcattct
gaataggtat ttctgcggtt 540 tcttagatac caaatatgta aatgactatg
ggtgtatggc atcaaatttc tttaatgctg 600 catacctgat gtttttgttt
gtagtcctct gtgtgtccag cctggctctg ctggccaggt 660 tgttctgtgg
cactgggcgg atgaagctta ccagattgta cgtgaccatc atgctgacca 720
ttttggtttt tctcctctgc gggttgccct gtggcttata ctggttcctg ttattctgga
780 ttaagaatgg ttttgctgta tttgatttta acttttatct agcatcaact
gtcctgagtg 840 ctattaatag ctctgccaac cccatcattt acttcttcgt
gggctcattc aggcatcggt 900 tgaagcacca gaccctcaaa atggttctcc
agagtgcact gcaggatact cctgagacag 960 ctgaaaacat ggtggagatg
tcaagaagca aagcagagcc gtgatgaaga gcctctgcct 1020 ggacctcgga
ggtagctttg gagtgagcac ttccctgctg caattgacca ctgtccactc 1080
tcctctcagc ttactgactc aacatgcctc agtggtccac caacatcttc aacagctctc
1140 cattgattta gtttttctaa ctctcccaag taatagcatt aatcagaaag
tatcatgtct 1200 gcatccttct tgacattaat caaattctca aactaacttc
ctctgaagct ttcttgctga 1260 ttctttggaa cttttgttgc catggaacta
gcccaggtcc agaaccatga ctctcgtatc 1320 tgtgatggtt ctgtacctga
atataaagac aaaggagcct agagatgatc ctgtccattc 1380 ccaaatacca
cctagagagc tggtctccca ggattgcaga caagcctgtg agcacaggta 1440
agaccaccac ttctgctcaa agggacatgc ctggaactac tcaggacaca ggtacagagg
1500 agcattttgg gacaagata 1519 53 303 PRT Mus musculus 53 Asn Pro
Ser Pro Gly Ser Thr Ser Ile Lys Ile Leu Ile Pro Asn Leu 1 5 10 15
Met Ile Ile Ile Phe Gly Leu Val Gly Leu Thr Gly Asn Ala Ile Val 20
25 30 Phe Trp Leu Leu Gly Phe His Leu Arg Arg Asn Ala Phe Ser Val
Tyr 35 40 45 Ile Leu Asn Leu Ala Leu Ala Asp Phe Leu Phe Leu Leu
Cys Arg Ile 50 55 60 Ile Ala Ser Thr Gln Lys Leu Leu Thr Phe Ser
Ser Pro Asn Ile Thr 65 70 75 80 Phe Leu Ile Cys Leu Tyr Thr Phe Arg
Val Ile Leu Tyr Ile Ala Gly 85 90 95 Leu Ser Met Leu Thr Ala Ile
Ser Ile Glu Arg Cys Leu Ser Val Leu 100 105 110 Cys Pro Ile Trp Tyr
Arg Cys His Arg Pro Glu His Thr Ser Thr Val 115 120 125 Met Cys Ala
Ala Ile Trp Val Leu Ser Leu Leu Ile Cys Ile Leu Asn 130 135 140 Arg
Tyr Phe Cys Gly Phe Leu Asp Thr Lys Tyr Val Asn Asp Tyr Gly 145 150
155 160 Cys Met Ala Ser Asn Phe Phe Asn Ala Ala Tyr Leu Met Phe Leu
Phe 165 170 175 Val Val Leu Cys Val Ser Ser Leu Ala Leu Leu Ala Arg
Leu Phe Cys 180 185 190 Gly Thr Gly Arg Met Lys Leu Thr Arg Leu Tyr
Val Thr Ile Met Leu 195 200 205 Thr Ile Leu Val Phe Leu Leu Cys Gly
Leu Pro Cys Gly Leu Tyr Trp 210 215 220 Phe Leu Leu Phe Trp Ile Lys
Asn Gly Phe Ala Val Phe Asp Phe Asn 225 230 235 240 Phe Tyr Leu Ala
Ser Thr Val Leu Ser Ala Ile Asn Ser Ser Ala Asn 245 250 255 Pro Ile
Ile Tyr Phe Phe Val Gly Ser Phe Arg His Arg Leu Lys His 260 265 270
Gln Thr Leu Lys Met Val Leu Gln Ser Ala Leu Gln Asp Thr Pro Glu 275
280 285 Thr Ala Glu Asn Met Val Glu Met Ser Arg Ser Lys Ala Glu Pro
290 295 300 54 2093 DNA Mus musculus 54 tggtatgcac tcactgataa
gcggatatag cccaaaagct gcaaacaacc aggataaaat 60 tcacagacca
catgaagctc aataagaagg aagaacaaag tgtaggtgtt tcagtccttc 120
ttagaaggag aacaaaatac tcacaggagc aaatatggag atacagtata gagcagagac
180 taaaggaaag gtcattcaga gactgtccca actggggatt cattccatat
agagatacca 240 aacccagact ctaaattgga tgcaaacaag tgcatgccaa
aaggagctag ataaggtaac 300 cctgtctcaa aaaaaaaaaa aaggctgtca
cctgaaaggc cctgtcaaag gcttacaaat 360 acagaagcag atgttagtag
tcaacaattg gacagagcat ggggttccta atagaggagt 420 tagaggaagg
aattagggag ttgaagggat ttgcagcccc ataagaacaa caatatcaac 480
caaccggaca ctcccccaga tatcacaggg tctaagccat caacaaagga gtacacatgg
540 ctccagatgc acatatagca gaggacggcc atgtcatgca tcaatggaag
aagagatcct 600 tgtacctatg aaggatcgat agatgaccca gtgtagggga
atcaaggaca gaaaggttgg 660 agtggatgtg tggactggcc ggactgacag
gaaatgccat tgtgttctgg ctcctgctct 720 tccacttgca caggaatgct
ttctcaatct acatcttaaa tttggtcata gctgacttcc 780 ttttcctcct
tggtcacatc atagcttcca caatgcaact tctcaaggtt tcctacctca 840
acattatttt tctttaccgt ttttacacaa tcatgatggt gctctacaac acaggcctga
900 ccatgctcag tgccatcaac actaagcact gcctgtctgt cctgtgtccc
atctggtatc 960 gctcccactg cacaaaacac acatcaactg tcatatgtgc
tgctatacgg gacctgtccc 1020 tgttgatctg ctttctgaat acgtatttct
gtggtctctt agataccaaa tataaaaatg 1080 acaatgggtg tctggcatcg
aatttcttta ttaatgcata ccctgatgtt tttgtttgta 1140 gtcctactgt
ctgtccactc tggctctgct ggccaggttg ttctgtggtg ctgggaagat 1200
gaaatttaca agattattcg tgaccatcat gctgacagtt ttagtttttc tcctctgtgg
1260 gttgccctct gccatctact ggttcctgtt aatctggatt aagattgatt
atggtgtatt 1320 tgcttatgat gtttttctgg catcactcgt cctgagtgct
gttaacagct gtgccaaccc 1380 catcatttac ttcttcgtgg gctctttcag
gcatcggttg aagcaccaaa ccctcaaaat 1440 ggttctccag aatgtactgc
aggacactcc tgagacagct gaaaacatgg tagagatgtc 1500 aagaggcaaa
gcagagccat gatgaagagc ctctgcctgg agctcagagg tggctttgga 1560
gtgagcactg ccctgatgta cttgaccact gtccactctc ctctcagctt actgactaga
1620 catgcctcag tggtccacca tctccaagag ctctccactg actttgtttt
ctacctctcc 1680 tgaataatag cattaatcag aaagtatcat gtctacatcc
ttcttgacat taatcaaatt 1740 ctcatgctat cttcccctga agctttcttg
ctgtttcttt gggacttttt gttgccatgg 1800 aaataacaaa ggtccagaac
catgactctc ttgcctgtga ttgttctgta cctgaatgta 1860 aagataaagg
agccaggaga tgatcctgta tcacggtgct ccataccaaa ataccaccaa 1920
gagagctggt ctcccaggag tgcagacaag cctgtgagca caggtaagac caccatttct
1980 gctcaaaggg acatgcctgg aaccctcagt acacaggaac agaggagcct
ggaactggat 2040 atttccagtt tccatctgca ccccagagct gactctgtac
cacagctctc cat 2093 55 282 PRT Mus musculus 55 Gly Leu Ala Gly Leu
Thr Gly Asn Ala Ile Val Phe Trp Leu Leu Leu 1 5 10 15 Phe His Leu
His Arg Asn Ala Phe Ser Ile Tyr Ile Leu Asn Leu Val 20 25 30 Ile
Ala Asp Phe Leu Phe Leu Leu Gly His Ile Ile Ala Ser Thr Met 35 40
45 Gln Leu Leu Lys Val Ser Tyr Leu Asn Ile Ile Phe Leu Tyr Arg Phe
50 55 60 Tyr Thr Ile Met Met Val Leu Tyr Asn Thr Gly Leu Thr Met
Leu Ser 65 70 75 80 Ala Ile Asn Thr Lys His Cys Leu Ser Val Leu Cys
Pro Ile Trp Tyr 85 90 95 Arg Ser His Cys Thr Lys His Thr Ser Thr
Val Ile Cys Ala Ala Ile 100 105 110 Arg Asp Leu Ser Leu Leu Ile Cys
Phe Leu Asn Thr Tyr Phe Cys Gly 115 120 125 Leu Leu Asp Thr Lys Tyr
Lys Asn Asp Asn Gly Cys Leu Ala Ser Asn 130 135 140 Phe Phe Ile Asn
Ala Tyr Leu Met Phe Leu Phe Val Val Leu Cys Leu 145 150 155 160 Ser
Thr Leu Ala Leu Leu Ala Arg Leu Phe Cys Gly Ala Gly Lys Met 165 170
175 Lys Phe Thr Arg Leu Phe Val Thr Ile Met Leu Thr Val Leu Val Phe
180 185 190 Leu Leu Cys Gly Leu Pro Ser Ala Ile Tyr Trp Phe Leu Leu
Ile Trp 195 200 205 Ile Lys Ile Asp Tyr Gly Val Phe Ala Tyr Asp Val
Phe Leu Ala Ser 210 215 220 Leu Val Leu Ser Ala Val Asn Ser Cys Ala
Asn Pro Ile Ile Tyr Phe 225 230 235 240 Phe Val Gly Ser Phe Arg His
Arg Leu Lys His Gln Thr Leu Lys Met 245 250 255 Val Leu Gln Asn Val
Leu Gln Asp Thr Pro Glu Thr Ala Glu Asn Met 260 265 270 Val Glu Met
Ser Arg Gly Lys Ala Glu Pro 275 280 56 2401 DNA Mus musculus 56
acttgctaac ttctgtaatt gatggccccc aaacaggaaa catcattata tctcacatga
60 ctataattaa tcacccactg tgttcatatc tttgactcaa aatctttccc
ttgtagttaa 120 cttcagacga gcactcgata gattatagta agatctgaga
cttctcagag ttatgaccat 180 gttgggaatt tggttttccc aagctcagga
atctgtccaa atggattgcc acaactacac 240 agagatggaa ggaaaggtag
agaactttcc cagtgccatt acattctaca ggctacagga 300 gccttggctg
gtcagaatgc aactttggtt ggcactcaga acaatgttaa ttttcctttt 360
caattctctc ctatctcttt
ccactctgct catttgttct gttgcagcac atctgtgact 420 tccatgtatg
aaagtagttt ctttttctac tctactctct caattatctt tttaattcta 480
ctatttctac tcacacatta aaatgtgtgt atgtgtgttt gtgttcatac gtgtgtgttg
540 aggctgattt tttccttatt tgctgtatat gaaactctac attctgttgt
acaccccaga 600 tgtcatgtgt taaattgtat ttcatgttct gctctctaaa
acctacattc aggtacagaa 660 caatcacaga caagagagtc atggttttgg
acctgggcta tttccatgrc aacaaaagtt 720 tcaaagaaac agcaagaaag
cttcagagga agttagcacg acaatttgat taatgtcaag 780 aaggatgcag
acatgatact ttctgattaa tgcttttact caggagatgg agaaaaacta 840
agttatggaa gagctgttga aggtgttggt agaccactga ggcatgccaa gtaggtcagc
900 tgaaaggaga gtggacagtg tggtcaagtg cagcagggca gtgctcactc
caaaactacc 960 tctgaaatcc aggcagaggc tcttcatcat ggctctgctt
tgctttttga catctccact 1020 atgttttcag gtgtctcagg aatgtcctgc
agtgcactct ggataaccat tttgagggtc 1080 tggtgctgca atcgatgcct
gaaggagccc acgaagaagt aaatgatggg gttggcacag 1140 ctgttaagag
cagtcaggac acttgatgcc ataaaaagac taaaatcaaa tacaataaaa 1200
acattcttaa tcttggataa caggaaccag tagatgccac agggcaaccc gcagaggaga
1260 aaaaccaaaa tggtcagcat gatggtcacg tacaatctgg taagtttcat
acgcccagcg 1320 ccacagaaca acctggccag cagagccagg ctggatagac
agaggaccac aaacaaaaac 1380 atcaggtatg cagcagtaaa gaagtttgat
gccatacatc catagtcatt tacatatttg 1440 gtatctaaga aaacgcagaa
atacttattc agaatgctga tcaacaggga caggacccag 1500 atcatagcac
acgtgacagt tgatgtgtgt tctgggcggt ggcagcgata ccagatgggg 1560
cacagtacag acagacaccg ttcagtgccg atggcactga gtatgctcag gcctgcaatg
1620 tagagaacca gcatgatgct gaagaagcac ctgcgaaaga taatgttagg
gtaggaaacc 1680 ttgagaagaa acagagtgga agctatgatg tgacagagga
ggaagaggaa gtcagccaga 1740 gccaagttta ggatgtagac tgagaaggca
ttcttgcgca agcggaagcc caggagccag 1800 aacacaatgg catttcctgt
catcccaacc agtccgaaga tgatgatcat caagtgtggg 1860 atcagggtgc
tgatgtcaat acttccaggg atggtttcgt ccattagatt tgttgtcgac 1920
ggtgccattg atgaggcaga ggtgtttagg gccagaaacc ctgcaccggt gctgctggga
1980 acacaaagaa gaaatgaggc tttccctatg aacacacctt ttgtttttct
tttccctttt 2040 ttgtttttgt tgttgttttt aaaaattttt ttctattgga
tattttcttt atttaaattt 2100 caaatgttat cccctttcct gcttttccct
ctccaggaaa tccccatctc atcctccctc 2160 cttctgcttc tatgatggtg
ttcctcaacc cacacaccca cttccacctc tctgccctcg 2220 attcccatac
actggagcat ctattgagcc ttcaaaggtc ctaggacctt tttttccatt 2280
gatgcatgac acagcaattc tctcatacat atacagctgg agccatgttt acttwctttg
2340 ttgatggctt attccatgga ggctggggcc agggggkgtg tctgatttgt
tgatattggt 2400 t 2401 57 305 PRT Mus musculus 57 Met Asp Glu Thr
Ile Pro Gly Ser Ile Asp Ile Ser Thr Leu Ile Pro 1 5 10 15 His Leu
Met Ile Ile Ile Phe Gly Leu Val Gly Met Thr Gly Asn Ala 20 25 30
Ile Val Phe Trp Leu Leu Gly Phe Arg Leu Arg Lys Asn Ala Phe Ser 35
40 45 Val Tyr Ile Leu Asn Leu Ala Leu Ala Asp Phe Leu Phe Leu Leu
Cys 50 55 60 His Ile Ile Ala Ser Thr Leu Phe Leu Leu Lys Val Ser
Tyr Pro Asn 65 70 75 80 Ile Ile Phe Arg Arg Cys Phe Phe Ser Ile Met
Leu Val Leu Tyr Ile 85 90 95 Ala Gly Leu Ser Ile Leu Ser Ala Ile
Gly Thr Glu Arg Cys Leu Ser 100 105 110 Val Leu Cys Pro Ile Trp Tyr
Arg Cys His Arg Pro Glu His Thr Ser 115 120 125 Thr Val Thr Cys Ala
Met Ile Trp Val Leu Ser Leu Leu Ile Ser Ile 130 135 140 Leu Asn Lys
Tyr Phe Cys Val Phe Leu Asp Thr Lys Tyr Val Asn Asp 145 150 155 160
Tyr Gly Cys Met Ala Ser Asn Phe Phe Thr Ala Ala Tyr Leu Met Phe 165
170 175 Leu Phe Val Val Leu Cys Leu Ser Ser Leu Ala Leu Leu Ala Arg
Leu 180 185 190 Phe Cys Gly Ala Gly Arg Met Lys Leu Thr Arg Leu Tyr
Val Thr Ile 195 200 205 Met Leu Thr Ile Leu Val Phe Leu Leu Cys Gly
Leu Pro Cys Gly Ile 210 215 220 Tyr Trp Phe Leu Leu Ser Lys Ile Lys
Asn Val Phe Ile Val Phe Asp 225 230 235 240 Phe Ser Leu Phe Met Ala
Ser Ser Val Leu Thr Ala Leu Asn Ser Cys 245 250 255 Ala Asn Pro Ile
Ile Tyr Phe Phe Val Gly Ser Phe Arg His Arg Leu 260 265 270 Gln His
Gln Thr Leu Lys Met Val Ile Gln Ser Ala Leu Gln Asp Ile 275 280 285
Pro Glu Thr Pro Glu Asn Ile Val Glu Met Ser Lys Ser Lys Ala Glu 290
295 300 Pro 305 58 2110 DNA Mus musculus 58 agaggtgtaa gtgggtatgt
gggttgagga acacccttca tagaagcagg gggagggagg 60 atgagatggg
gttttctggg aaggggcaaa agcaggaaag tggataacat ttgtaattta 120
aataaagaaa atatccaata caaaaaattt aaaaaaaaaa acacaaaacc acacaaaaaa
180 aagacaaaaa aaaagaaatt aaaagttgtg ttcatagtta atgcctcatt
tttctttgtg 240 ttcccagcaa aaccagtgca gggtttctgg ccctaaacac
cttcagcctt ttcaatggca 300 cccaacgaca accaatacaa tggacgaaac
catccctgga cgtattgaca tcgagaccct 360 gatcccaaac ttgatgatca
tcatcttcgg actggtcggg ctgacaggaa atggcattgt 420 gttctggctc
ctgggcttcc gcatgcacag gaatgccttc ttagtctaca tcctaaactt 480
ggccctggct gactttctct tccttctctg tcacatcatt aattccacaa tgcttcttct
540 caaggttctc ccactcaact ggatscttgt tccattgctt taacaccatc
agaacggttc 600 tatacatcac aggcctgagc atgctcagcg ccatcagcac
tgagcgctgc ctgtctgtcc 660 tgtgccccat ctggtatcga tgccgtcgcc
gagaaaacac atcagctgtc atgtgtgctg 720 tgatctgggt cctgtccctg
ttgatctgta ttctgaatag ttatttctgt tattactctg 780 gtcccaaaga
tgtaaataac tctgtgtgtc tggtatcgaa attcttcatc agtacatacc 840
caatgttttt gtttgtagtc ctctgtctgt ccaccctgac tctgctggcc aggttgttct
900 gtggtgctgg gaagaggaaa tttaccagat tattcgtgac catcatactg
accattttgg 960 tttttcttct gtgtgggttg cccctgggct tctactggtt
cctgttacac tgtattaagg 1020 gtagtttcag tgtactacat aatagacttt
ttcaggcatc acttgtccta acttctgtta 1080 acagctgtgc caaccccatc
atttacttct tcgtgggctc cttcagggat cgggtgaagc 1140 accagaccct
caaaatggta ctccagaatg cactgcagga cactcctgag acacctgaaa 1200
acaaggtgga gatgtcaaga agtaaagcag agccatgatg aagagactcg gccaggacct
1260 cagaggtagc tttggagtsa gwactgccct gctrcacttg accactgtcc
actctcctct 1320 cagcttacts acttyggatg cctcagtggt ccaacaacam
cttcaaawgc tctccactga 1380 cttagtattt atacctctcc caagtaatag
cattaatcag aaagtatcat gtctgcatcc 1440 ttcttgacat taatccaatt
ctcatactaa cttcatctga aactttcttg atgttccttt 1500 ggaacttttg
ttgccatggt aatagccyag gtccagcacc atgactctct tgtctgtgat 1560
tkttctgtac ctgaatgtaa agtcaaagga gccaggagat gatcctgtgt cacagtgctc
1620 attacccaaa caccaccaac agagcttgtc tcccaggagt gcagacacgc
ctgtgaacac 1680 aggtaagacc accacttctg cttaaaggga catgcctgga
accctcagaa cacaggaaga 1740 aaagagcagc cttggacagg atacttccag
tttccaactg caccccggag ctgaccctgt 1800 gccacagctc tccataccca
aattcctccc agaaagaacy ggtcwaccaa gagtactgac 1860 acayaggctt
gcaggaggga caagccacmg tcagagatag caaggaccag ctaacaccag 1920
agataaccag atggcaagag gcaagggcaa aaatataagc aatgggaacc aagactattt
1980 ggcatcatca gaacctagtt ctctcaacat ggtgagccat ggctactcca
acagacaaga 2040 aaagcatgac tctgatttaa tgtcacagat gatgatgatg
atgatgatga tgatgatgat 2100 gatgatgatg 2110 59 305 PRT Mus musculus
59 Met Asp Glu Thr Ile Pro Gly Arg Ile Asp Ile Glu Thr Leu Ile Pro
1 5 10 15 Asn Leu Met Ile Ile Ile Phe Gly Leu Val Gly Leu Thr Gly
Asn Gly 20 25 30 Ile Val Phe Trp Leu Leu Gly Phe Arg Met His Arg
Asn Ala Phe Leu 35 40 45 Val Tyr Ile Leu Asn Leu Ala Leu Ala Asp
Phe Leu Phe Leu Leu Cys 50 55 60 His Ile Ile Asn Ser Thr Met Leu
Leu Leu Lys Val Leu Pro Pro Thr 65 70 75 80 Gly Ser Leu Phe His Cys
Phe Asn Thr Ile Arg Thr Val Leu Tyr Ile 85 90 95 Thr Gly Leu Ser
Met Leu Ser Ala Ile Ser Thr Glu Arg Cys Leu Ser 100 105 110 Val Leu
Cys Pro Ile Trp Tyr Arg Cys Arg Arg Arg Glu Asn Thr Ser 115 120 125
Ala Val Met Cys Ala Val Ile Trp Val Leu Ser Leu Leu Ile Cys Ile 130
135 140 Leu Asn Ser Tyr Phe Cys Tyr Tyr Ser Gly Pro Lys Asp Val Asn
Asn 145 150 155 160 Ser Val Cys Leu Val Ser Lys Phe Phe Ile Ser Thr
Tyr Pro Met Phe 165 170 175 Leu Phe Val Val Leu Cys Leu Ser Thr Leu
Thr Leu Leu Ala Arg Leu 180 185 190 Phe Cys Gly Ala Gly Lys Arg Lys
Phe Thr Arg Leu Phe Val Thr Ile 195 200 205 Ile Leu Thr Ile Leu Val
Phe Leu Leu Cys Gly Leu Pro Leu Gly Phe 210 215 220 Tyr Trp Phe Leu
Leu His Cys Ile Lys Gly Ser Phe Ser Val Leu His 225 230 235 240 Asn
Arg Leu Phe Gln Ala Ser Leu Val Leu Thr Ser Val Asn Ser Cys 245 250
255 Ala Asn Pro Ile Ile Tyr Phe Phe Val Gly Ser Phe Arg Asp Arg Val
260 265 270 Lys His Gln Thr Leu Lys Met Val Leu Gln Asn Ala Leu Gln
Asp Thr 275 280 285 Pro Glu Thr Pro Glu Asn Lys Val Glu Met Ser Arg
Ser Lys Ala Glu 290 295 300 Pro 305 60 740 DNA Mus musculus 60
cagggtttct ggccctaaac acctcagcct cggcaatgac acccacgaca aacaattcaa
60 tggacgaaac catccctgga agtattggca ctgagaccct gattcaaaac
ttgatgatca 120 tcatcttcgg actggtcggg ctgacaggaa atgccattgt
gttctggctc ctgggcttcc 180 acttgcacag gaatgccttt ttagtctaca
tcctaaactt ggccctggct gatttcctct 240 tccttctctg tcacatcata
gattccacag tgtttcttct caaggttccc ccacccaacc 300 ggatcttggt
ccattgcttt aacatcatca gaattgtact ctacatcaca ggcttgagca 360
tgctcagtgc catcagcatg gagcgctgcc tgtctgtcct gtgccccatc tggtatcgct
420 gccgccgccc agaaaacaca tcaactgtca tttgtgctgt gatctggatc
ctgtccctgt 480 tgttctgcat tctgaatgga tatttctgtt atttctctgg
tcccaactat gtaaatgact 540 atgtgtgttt tgcatcggac atctttatca
gaacataccc aatgtttttg tttgtagtcc 600 tctgtctgtc cactctggct
ctgctggcca ggttgttctg tggtgctggg aagacgaaat 660 ttaccagatt
attcgtcacc atcatactga ccgttttggt ttttcttctc tgtgggttgc 720
ccctgggctt cttctggttc 740 61 227 PRT Mus musculus 61 Met Asp Glu
Thr Ile Pro Gly Ser Ile Gly Thr Glu Thr Leu Ile Gln 1 5 10 15 Asn
Leu Met Ile Ile Ile Phe Gly Leu Val Gly Leu Thr Gly Asn Ala 20 25
30 Ile Val Phe Trp Leu Leu Gly Phe His Leu His Arg Asn Ala Phe Leu
35 40 45 Val Tyr Ile Leu Asn Leu Ala Leu Ala Asp Phe Leu Phe Leu
Leu Cys 50 55 60 His Ile Ile Asp Ser Thr Val Phe Leu Leu Lys Val
Pro Pro Pro Asn 65 70 75 80 Arg Ile Leu Val His Cys Phe Asn Ile Ile
Arg Ile Val Leu Tyr Ile 85 90 95 Thr Gly Leu Ser Met Leu Ser Ala
Ile Ser Met Glu Arg Cys Leu Ser 100 105 110 Val Leu Cys Pro Ile Trp
Tyr Arg Cys Arg Arg Pro Glu Asn Thr Ser 115 120 125 Thr Val Ile Cys
Ala Val Ile Trp Ile Leu Ser Leu Leu Phe Cys Ile 130 135 140 Leu Asn
Gly Tyr Phe Cys Tyr Phe Ser Gly Pro Asn Tyr Val Asn Asp 145 150 155
160 Tyr Val Cys Phe Ala Ser Asp Ile Phe Ile Arg Thr Tyr Pro Met Phe
165 170 175 Leu Phe Val Val Leu Cys Leu Ser Thr Leu Ala Leu Leu Ala
Arg Leu 180 185 190 Phe Cys Gly Ala Gly Lys Thr Lys Phe Thr Arg Leu
Phe Val Thr Ile 195 200 205 Ile Leu Thr Val Leu Val Phe Leu Leu Cys
Gly Leu Pro Leu Gly Phe 210 215 220 Phe Trp Phe 225 62 1979 DNA Mus
musculus 62 aatacacaaa attaaaaaca acaacaacaa caacacgccc cacaaaaaaa
gaaaacaaaa 60 acaaaaaaga aattaaaagt tgtggtcata gtaaaggcct
cacttcttct ttgtgttccc 120 agcaacacca gtgcagggtt tctggcccga
aacacctcag cctcgacaat gacacccaca 180 acaacaaatc caatgaacga
aaccatccct ggaagtattg acatcgagac cctgatacca 240 aacttgatga
tcatcatctt cggactggtc gggctgacag gaaatgccat tgtgttctgg 300
ctcctgggct tccgcatgca caggactgcc ttctcagtct acatcctaaa cttggccctg
360 gctgacttcc tcttccttct ctgtcacatc ataaattcca cagtgcttct
tctccaggtt 420 tccccaccca acagtacctt ggtccattgc tttgacacca
tcagaatggt tctctacatc 480 gcaggcctga gcatgctcag tgccattagc
actgagcact gcctgtctgt cctgtgcccc 540 atctggtatc gctgccgccg
cccagaacat acttcaactg tcatgtgtgc tgtgatctgg 600 gtcctgtccc
tgttgatctg cattctaagt ggatatttct gtaatttttt tcttcacaaa 660
tatgtatatt actctgtgtg tcgggcattg gaattctgta tcggaacata ccccgatgtt
720 tttgttttgt agtcctctgt ctgtccaccc tggctctgct ggtcaggttg
ttctgtggta 780 ctgggaaggc aaaatttacc agattattcg tgaccatcat
gctgactgtt ttggtttttc 840 ttctctgtgg gttgcccctg tgtttcttct
ggttcctggt agtctggatt aagcgtcctc 900 tcagtgtact aaatattaca
ttttattttg catccattgt cctaactgtt gttaacagct 960 gtgccaaccc
catcatttac ttcttcgtgg gctccttcag gcatcggttg aagcaacaga 1020
acctcaaaat ggttctccag aatgcactgc aggacactgc tgagacacct gaaaacgtgg
1080 cagagatttc aagaagcaaa gcagagccct gatgaggagc ctctgcctgg
acctcagagg 1140 tggctttggc actgagcact gccctgctgc acttgcccac
tgtccactct cctctcagct 1200 tactgactgg caataactca gtggtacaac
aacaccttca aaagctcacc actgacttag 1260 tatttctacc tatcccaagt
aatagcatta atcagaaagt atcatgtctg catccttcta 1320 gacattattc
aaattctcat ccaacttcat ctgaaacttt cttgctattt ctttggaaca 1380
ttttttgcca tggtaatagc ccaggtccag catcatgcct ctcttacctt tgattgttct
1440 gtacctgaat gtaaagaaaa aggagagaga agatgatcct ctgtcacagt
gctcattacc 1500 caagcaccac taagagagct tgtctcccag gagtgcagac
aaacctgtga gcacaggtaa 1560 gactaccact tctgcttaaa ggggcatgcc
tggaacccac aggacacagg taaagaggag 1620 cagcctgaga aaggatactt
tccagtttcc aactgcaccc tggagctgac cctgtgccac 1680 agctctcccc
accttaattc ttcccagaaa gaactggtct mccaggaagt actgacacat 1740
agccttgcag gaggtacaag acactgtcac agatagcaag accagctaac accagagata
1800 accagatggc aagaggcaag ggcaaaaaca taagcaatgg gaaccaaggc
tacttggcat 1860 catcagaacc tagttctctc aacaaagtga gccctggata
ctccaacaca caagaaaagt 1920 atgactgtga ttaaaagtca ccgatgatga
tgatgatgat gatgatgatg atgatgatg 1979 63 305 PRT Mus musculus 63 Met
Asn Glu Thr Ile Pro Gly Ser Ile Asp Ile Glu Thr Leu Ile Pro 1 5 10
15 Asn Leu Met Ile Ile Ile Phe Gly Leu Val Gly Leu Thr Gly Asn Ala
20 25 30 Ile Val Phe Trp Leu Leu Gly Phe Arg Met His Arg Thr Ala
Phe Ser 35 40 45 Val Tyr Ile Leu Asn Leu Ala Leu Ala Asp Phe Leu
Phe Leu Leu Cys 50 55 60 His Ile Ile Asn Ser Thr Val Leu Leu Leu
Gln Val Ser Pro Pro Asn 65 70 75 80 Ser Thr Leu Val His Cys Phe Asp
Thr Ile Arg Met Val Leu Tyr Ile 85 90 95 Ala Gly Leu Ser Met Leu
Ser Ala Ile Ser Thr Glu His Cys Leu Ser 100 105 110 Val Leu Cys Pro
Ile Trp Tyr Arg Cys Arg Arg Pro Glu His Thr Ser 115 120 125 Thr Val
Met Cys Ala Val Ile Trp Val Leu Ser Leu Leu Ile Cys Ile 130 135 140
Leu Ser Gly Tyr Phe Cys Asn Phe Phe Leu His Lys Tyr Val Tyr Tyr 145
150 155 160 Ser Val Cys Arg Ala Leu Glu Phe Cys Ile Gly Thr Tyr Pro
Met Phe 165 170 175 Leu Phe Val Val Leu Cys Leu Ser Thr Leu Ala Leu
Leu Val Arg Leu 180 185 190 Phe Cys Gly Thr Gly Lys Ala Lys Phe Thr
Arg Leu Phe Val Thr Ile 195 200 205 Met Leu Thr Val Leu Val Phe Leu
Leu Cys Gly Leu Pro Leu Cys Phe 210 215 220 Phe Trp Phe Leu Val Val
Trp Ile Lys Arg Pro Leu Ser Val Leu Asn 225 230 235 240 Ile Thr Phe
Tyr Phe Ala Ser Ile Val Leu Thr Val Val Asn Ser Cys 245 250 255 Ala
Asn Pro Ile Ile Tyr Phe Phe Val Gly Ser Phe Arg His Arg Leu 260 265
270 Lys Gln Gln Asn Leu Lys Met Val Leu Gln Asn Ala Leu Gln Asp Thr
275 280 285 Ala Glu Thr Pro Glu Asn Val Ala Glu Ile Ser Arg Ser Lys
Ala Glu 290 295 300 Pro 305 64 1485 DNA Mus musculus 64 aacaacacaa
aaccctgaaa aaaaaaaaga aattaaaagt tttgttcata gtaaaggcct 60
catttcttct ttgtgttcac agcaacatca gtgcacggtt aatggcaata aacacctcag
120 cctcggcaat ggcacccacg acaacaaatc caaagggaag caaacaatcc
ctgggaagta 180 ttgacatcga gaccctgatc tcaaacttga tgatcatcat
tttcgggctg gtagggctgc 240 caggaaatgc cattgtgttc tggctcctgg
gcttctgctt gcacaggaat gccttcttag 300 tctacatcct aaacttggcc
ctggctgacg tcctcttcct tctctgtcac atcataaatt 360 ccacagtgct
tcttctcaag gttcccccac ccaacggtaa tattggtcca ttgcttcaac 420
atcatcagaa ttgttctcta catcacaggc ctgagcatgc tcagtgccat catcactgag
480 cgctgcctgt ctatcctgtg ccccatctgg tatcgctgcc accgcccaga
acacacatca 540 actgccatgt gtgctgtgat ctgggtcctg tctctgttga
tctgcattct tggaagaata 600 tttctgtaat tttttccttc acaaatatgt
aaattactct gtgtgtctgg cattggactc 660 ctttatcgga acatacccaa
tgtttttgct tgtagtcctc tgtctgtcca ccatggctct 720 gctggccagg
ttgttctgtg gttctgggaa gacgaaattt accagattat ttgtgaccat 780
catgcttacc gttttggttt ttcttctctg cttggtttgc ccctgggctt cttctggttc
840 ctgttactct ggattaaggg tgcttacagt gtactaggtt atagatttta
ttttgcatca 900 attgtcctaa ctgctgttaa cagctgtgcc aaccccatca
tttacttctt catgggctca 960 ttcaggcaac gattgcagca caagaccctc
aaaatcgttc tccagagtgc actgcacgac 1020 actcctgaga cacctgaaaa
catggtggag atgtcaagaa gcaaagcaga gccataatga 1080 agagcctctg
cctggacctc agaggtggat ttggagtgag aactgcccta cgcttgacca 1140
ctgtccactc tcctctcagc ttactgactt tggatgccta agtggtccaa caacaacttc
1200 aaaatctctc cactgactta gtatttatac ctctcccaag taatagcatt
aatcagaaag 1260 tatcatgtct gcatccttct tgacattaat ccaattctca
tactaacttc atctgaaact 1320 ttcttgctgt ttctttggaa cttttgttgc
catagtaata gcccagatcc agcaccatga 1380 ctcacttgtc tgtgattatt
ctgtacctga atgtaaagaa aaaggcagga gatgatcctg 1440 tatcacagtg
ctcattacac aaacaccacc aagaaagctc gtctc 1485 65 300 PRT Mus musculus
65 Gly Ser Ile Asp Ile Glu Thr Leu Ile Ser Asn Leu Met Ile Ile Ile
1 5 10 15 Phe Gly Leu Val Gly Leu Pro Gly Asn Ala Ile Val Phe Trp
Leu Leu 20 25 30 Gly Phe Cys Leu His Arg Asn Ala Phe Leu Val Tyr
Ile Leu Asn Leu 35 40 45 Ala Leu Ala Asp Val Leu Phe Leu Leu Cys
His Ile Ile Asn Ser Thr 50 55 60 Val Leu Leu Leu Lys Val Pro His
Pro Thr Val Ile Leu Val His Cys 65 70 75 80 Phe Asn Ile Ile Arg Ile
Val Leu Tyr Ile Thr Gly Leu Ser Met Leu 85 90 95 Ser Ala Ile Ile
Thr Glu Arg Cys Leu Ser Ile Leu Cys Pro Ile Trp 100 105 110 Tyr Arg
Cys His Arg Pro Glu His Thr Ser Thr Ala Met Cys Ala Val 115 120 125
Ile Trp Val Leu Ser Leu Leu Ile Cys Ile Leu Gly Lys Tyr Phe Cys 130
135 140 Asn Phe Phe Leu His Lys Tyr Val Asn Tyr Ser Val Cys Leu Ala
Leu 145 150 155 160 Asp Ser Phe Ile Gly Thr Tyr Pro Met Phe Leu Leu
Val Val Leu Cys 165 170 175 Leu Ser Thr Met Ala Leu Leu Ala Arg Leu
Phe Cys Gly Ser Gly Lys 180 185 190 Thr Lys Phe Thr Arg Leu Phe Val
Thr Ile Met Leu Thr Val Leu Val 195 200 205 Phe Leu Leu Cys Leu Gly
Leu Pro Leu Gly Phe Phe Trp Phe Leu Leu 210 215 220 Leu Trp Ile Lys
Gly Ala Tyr Ser Val Leu Gly Tyr Arg Phe Tyr Phe 225 230 235 240 Ala
Ser Ile Val Leu Thr Ala Val Asn Ser Cys Ala Asn Pro Ile Ile 245 250
255 Tyr Phe Phe Met Gly Ser Phe Arg Gln Arg Leu Gln His Lys Thr Leu
260 265 270 Lys Ile Val Leu Gln Ser Ala Leu His Asp Thr Pro Glu Thr
Pro Glu 275 280 285 Asn Met Val Glu Met Ser Arg Ser Lys Ala Glu Pro
290 295 300 66 1518 DNA Mus musculus 66 aacaacaaaa aaaaaaaaca
gaaaaagaaa ttaaaagttg tgtccatagt gaaggcctca 60 tttcttcttt
gtgtttccag caacaccagt gcagggtttc tggacctaaa cacctcagcc 120
tcggcaatag cacccacaac aaccaaacca atggacgaaa ccatccctgg aagtattgac
180 actgagaccc tgtatccaac acttgatgat catcatcttc ggactggtcg
ggctgacagg 240 aaatggcatt gtgttgtggc tcctgggctt ccacttgcaa
aggaatgcct ttttagtcta 300 catcctaaac ttggccctag ctgacttcct
ctaccttctc tgtcacatca tagattccac 360 aatgcttctt ctcaaggttc
ccccacccaa ctggatcttg gtccattgct ttaggaccat 420 ccaaattttt
ctctacatca caggcctgag catgctcagt gccatcagca cagagcgctg 480
cctgtctgtc ctgtgcccca tctggtatcg ctgccgccgc ccagaaaaca catcaactgt
540 gatgtgtgct gtgatctggg tcctgtcctt gttgatctgc attctgcatg
gatatttttc 600 tgttatttct ctggtctcag ttatgaaaat tactctgtgt
gttttgcatc agcgatcatt 660 atcagttcat acccaacgtt tttgcttgta
gtcctctgtc tgtccaccct ggctctgctg 720 gccaggttgt tctgtggtgc
tgggaagagg aaattttcca gattattcgt gaccatcata 780 cttaccgttt
tggtttttct tctctgtggg ttgccctggg gagccctctg gttcccatta 840
ctctggattc agggtggttt ctggaaaaga ctttttcagg catcaattgt cctatcttct
900 gttaacagct gtgccaaccc catcatttat ttcttcgtgg gctcattcag
gcatcgattg 960 aagcaccaga cccttaaaat ggttctccag aatgcactgc
aggacactcc tgagacaact 1020 gaaaacatgg tggagatgtc aagaagtaaa
gcagagccat gatgaagagc ctctgcctgg 1080 acctcagagg tggatttgga
gtgagcactg ccctgctgca cttgaccact gtccactctc 1140 ctctcagctt
actgacttgg aatgcctcag tggtccaaaa acaccttcaa aagctctcca 1200
ctgactaagt atttctacct atcccaagta atagcattaa tcagaaagta ccatgtctgc
1260 atccttcttg acattaatca aattctctta ctatcttcat ctgaaacttt
cttgttgttt 1320 ctttggaact tttgttgcca tggtaatagc ccaagtccag
caccatgact ttcttatctg 1380 tgattgttct atacctgaat gtaaaggcaa
aggagccagg agatgatcct gtgttacagt 1440 gctcattacc caaacaccac
caagagagct tgtctcccag gagtgcagac acgcctgtga 1500 acacaggtaa
gaccacca 1518 67 303 PRT Mus musculus 67 Met Asp Glu Thr Ile Pro
Gly Ser Ile Asp Thr Glu Thr Leu Tyr Pro 1 5 10 15 Asn Leu Met Ile
Ile Ile Phe Gly Leu Val Gly Leu Thr Gly Asn Gly 20 25 30 Ile Val
Leu Trp Leu Leu Gly Phe His Leu Gln Arg Asn Ala Phe Leu 35 40 45
Val Tyr Ile Leu Asn Leu Ala Leu Ala Asp Phe Leu Tyr Leu Leu Cys 50
55 60 His Ile Ile Asp Ser Thr Met Leu Leu Leu Lys Val Pro Pro Pro
Asn 65 70 75 80 Trp Ile Leu Val His Cys Phe Arg Thr Ile Gln Ile Phe
Leu Tyr Ile 85 90 95 Thr Gly Leu Ser Met Leu Ser Ala Ile Ser Thr
Glu Arg Cys Leu Ser 100 105 110 Val Leu Cys Pro Ile Trp Tyr Arg Cys
Arg Arg Pro Glu Asn Thr Ser 115 120 125 Thr Val Met Cys Ala Val Ile
Trp Val Leu Ser Leu Leu Ile Cys Ile 130 135 140 Leu His Gly Tyr Phe
Cys Cys Tyr Phe Ser Gly Leu Ser Tyr Glu Asn 145 150 155 160 Tyr Ser
Val Cys Phe Ala Ser Ala Ile Ile Ile Ser Ser Tyr Pro Thr 165 170 175
Phe Leu Leu Val Val Leu Cys Leu Ser Thr Leu Ala Leu Leu Ala Arg 180
185 190 Leu Phe Cys Gly Ala Gly Lys Arg Lys Phe Ser Arg Leu Phe Val
Thr 195 200 205 Ile Ile Leu Thr Val Leu Val Phe Leu Leu Cys Gly Leu
Pro Trp Gly 210 215 220 Ala Leu Trp Phe Pro Leu Leu Trp Ile Gln Gly
Gly Phe Trp Lys Arg 225 230 235 240 Leu Phe Gln Ala Ser Ile Val Leu
Ser Ser Val Asn Ser Cys Ala Asn 245 250 255 Pro Ile Ile Tyr Phe Phe
Val Gly Ser Phe Arg His Arg Leu Lys His 260 265 270 Gln Thr Leu Lys
Met Val Leu Gln Asn Ala Leu Gln Asp Thr Pro Glu 275 280 285 Thr Thr
Glu Asn Met Val Glu Met Ser Arg Ser Lys Ala Glu Pro 290 295 300 68
1500 DNA Mus musculus 68 cattttcgga ctggtcgggc tgacaggaaa
taccattgtg ttctggctcc tgggcttctg 60 cttgcacagg aatgcctttt
tagtctacat cctaaacttg gccctggctg acttcctctt 120 ccttctctgc
cacatcataa attccacagt acttcttctc aaggttcccc tacccaactg 180
gatcttgttc cattgcttta acaccatcag aattgttctt tacatcacag gcctgaacat
240 gctcagtgcc atcaacatgg agcactgcct gtctgtcctg tgccccatct
ggtatcactg 300 ctgccgccca gaacacacat caactgtcat gtgtgctgtg
atctgggtcc tgtccctgtt 360 gatctgcatt ctgaatgaat atttctgtga
tttctttggt accaaattgg taaattacta 420 tgtgtgtctg gcatcgaact
tctttatggg agcatacctg ttgtttttgt ttgtagtcct 480 ctgtctgtcc
accctggctc tgctggccag gttgttctgt ggtgctggga atacgaaatt 540
taccagattt cacatgacca tcttgctgac ccctttgttc tttctcctct gcgggttgcc
600 ctttgccatc taatgcttcc tgttattcaa gattaaggat gatttccatg
tattttatat 660 taaccttttt ctagcattag aagtcctgac ttctattaac
agctgtgaca accccatcat 720 ctatttcttc ctggactcct tcagacatca
ggagaagcac cagaccctca aaatggttct 780 ccagagtgca ctgcaggata
ctcytgagac acctgaaaac atggcagaga tgtcaagaag 840 caaagcagag
ccgtgatgaa gagcctctgc ctggatgtca gaggtggctt tggagtgagc 900
actgccctgc tgcacttgac cactgtcaac tctactctca gcttactgac ttgtcatgcc
960 tcagtggttc aacaacacct tcaaaagctc tccactgact tagtattttt
acctctccca 1020 agtagtagca ttaatcagaa agtatcatgt ctgcatcctt
cttgacatta ttcaaattct 1080 catctaactt catctgaaac tttctcccta
tttctttgga acttttgttg ccatggkaat 1140 agcccagatc cagcaccatg
actctcttgt ctgtgattgt tctgaacctg aatgtaaaga 1200 caaaggagag
agaagatgat cctgtgtcac agtgctcatt acccaagcac cgccaagaga 1260
tcttgtctcc caggagtgca gacaagcctg tgcgcactgg taagaccacc acttttgctt
1320 aaagggacat gcctggaact ttcaagacag agtaacagag gagcaccctg
gaacaggata 1380 cttccagttt ccaactgcac accggagctg accctatgca
acagctctcc atacccaact 1440 tcttcccaca aagaactggt gctaccagga
gtactgacac acaggttttc aggaaggaca 1500 69 283 PRT Mus musculus 69
Ile Phe Gly Leu Val Gly Leu Thr Gly Asn Thr Ile Val Phe Trp Leu 1 5
10 15 Leu Gly Phe Cys Leu His Arg Asn Ala Phe Leu Val Tyr Ile Leu
Asn 20 25 30 Leu Ala Leu Ala Asp Phe Leu Phe Leu Leu Cys His Ile
Ile Asn Ser 35 40 45 Thr Val Leu Leu Leu Lys Val Pro Leu Pro Asn
Trp Ile Leu Phe His 50 55 60 Cys Phe Asn Thr Ile Arg Ile Val Leu
Tyr Ile Thr Gly Leu Asn Met 65 70 75 80 Leu Ser Ala Ile Asn Met Glu
His Cys Leu Ser Val Leu Cys Pro Ile 85 90 95 Trp Tyr His Cys Cys
Arg Pro Glu His Thr Ser Thr Val Met Cys Ala 100 105 110 Val Ile Trp
Val Leu Ser Leu Leu Ile Cys Ile Leu Asn Glu Tyr Phe 115 120 125 Cys
Asp Phe Phe Gly Thr Lys Leu Val Asn Tyr Tyr Val Cys Leu Ala 130 135
140 Ser Asn Phe Phe Met Gly Ala Tyr Leu Leu Phe Leu Phe Val Val Leu
145 150 155 160 Cys Leu Ser Thr Leu Ala Leu Leu Ala Arg Leu Phe Cys
Gly Ala Gly 165 170 175 Asn Thr Lys Phe Thr Arg Phe His Met Thr Ile
Leu Leu Thr Pro Leu 180 185 190 Phe Phe Leu Leu Cys Gly Leu Pro Phe
Ala Ile Cys Phe Leu Leu Phe 195 200 205 Lys Ile Lys Asp Asp Phe His
Val Phe Tyr Ile Asn Leu Phe Leu Ala 210 215 220 Leu Glu Val Leu Thr
Ser Ile Asn Ser Cys Asp Asn Pro Ile Ile Tyr 225 230 235 240 Phe Phe
Leu Asp Ser Phe Arg His Gln Glu Lys His Gln Thr Leu Lys 245 250 255
Met Val Leu Gln Ser Ala Leu Gln Asp Thr Pro Glu Thr Pro Glu Asn 260
265 270 Met Ala Glu Met Ser Arg Ser Lys Ala Glu Pro 275 280 70 2504
DNA Mus musculus 70 gtgtgtgcct tggtttttat tgcttatgtt tttgtccttg
catcttgcca tctggttatc 60 tctggtatta gctggtcttg atgtctctga
ttgtccttgt ccctcctgca agcctgtgtg 120 tcatttctcc tgggagacca
gttatttcta gaagaaattt aggtatgggg agttgtggca 180 cagggtcagc
cccagggtgc agatgaaaac tggaaggatc ctgtcccagg tcgctcctct 240
atttctgtgt cctgcgggtt ctgggcatgt ccctttgagc agaagtgttg gtcttacctg
300 tgctcacagg cttgtctgca ctgtggcaca agatcatctc ctggctcctt
tgtctttaca 360 ttcaggtaca gamcaatcmc cagacaagag agtcatgctt
ctggacttgg gctatttcca 420 tggcaacaaa agttccaaag aaacamcaag
aaaggttcag aggaagttag catgagaatt 480 tgattaatgt cataaaggat
gcagacatga tactttctga ttaatgatat tactcgagag 540 aggtagaaaa
tctaagtcag tggagagctt ttgaagatgt tggtggacca ctgaggcatg 600
tcaagtcagt cagcggagag cagagtggac agtgataaag tgcagcaggg cattcttcac
660 tccaaagcca cctctgaggt ccaggcagag gctcttcatc atggctctgc
tttacttctt 720 gacatcccca ccatgttttc aggtgtctca ggagtgtcct
acattgtcct ctggagaacc 780 attttcagtg tctggtgctg caaccgaagc
ctgaaggagc ccgtgaagaa gtaaatgatg 840 gagttggcac aactgttaat
agcagtcatg acaagtgatt ccagataaaa tacaagagta 900 aatacatgaa
aagcatcctt aatcttgcat aacagaaacc agtagatgcc aaagttcaat 960
ctgcaaagga gaaaaccaga gcagtcagca ggatggtcac atactatctg gtaagcttca
1020 tttgcccaac atcacagaac aacctggcca gcagagccag gctggaaaga
cagagatcca 1080 caaacaaaac atcaggtatg cagaagtaaa gaagttcaat
gccagacacc cattgtcatt 1140 ttcatatttg ctatgtaaga aacctcagaa
ataactattc agaatgcaga tcaacaggga 1200 cagtacccag atcacagcac
acatggcagc tgatgtatgt tctgggtggt gacagcaatc 1260 ccagatgggc
acagtacaga caggccgtgc tcagtgctga tggcagtgag catgctcagg 1320
cctgcgatgt agagaaccat catgatgatg taaaagcaca agataaagat aatggggtag
1380 aaaacattga gaagaagcag tatggaatct atggtgtgac ctaggaggaa
gaagaagtca 1440 gccaggtcca agtttaggat gtagaccttg aaagcattcc
tgcgcaaggg gaagtgcagg 1500 atccagaaga caatggaatt tcctgtcagc
ccaaccagtc cgaagatgat ggttatcaag 1560 tttgggatca gaatcctgat
gttgatacct ccagggatgg ttttgtccat tggatttgct 1620 gttgtgggtg
ctgttggtga ggctgatgtg tttagggcca gaaactctgc accagtgctg 1680
ctgggaacac aaagaaaaaa tgaggccttc cctatgaact caccttttgt tttccttttt
1740 gttggatttt taatttcttc tattgcatat tttaaattat ttgctttcct
gtgtcccccc 1800 ccctcccttt cctgaaaacc cctatcccac cctccctcta
ccctgcttac tattgaggat 1860 attcctccac ccactcccac ctctctgccc
tctattgccc tacactgggg caactatcaa 1920 gccttcatag atccatagaa
ctcttctccc atttattcat gacagggcca tcctctgcta 1980 catatgcagc
tggagccatg tgtacttctt tgctgatggc ttgtcccctg ggtgctgggg 2040
gattggtact ggttggttga tattgttttt cttacctatg ggcttgcaaa ccccttcaac
2100 tcccttagtc ctttctctaa ttcttctatt agggaccctg ttctcagtct
aatggctgga 2160 tgctaacatc tgcctctgta tttgtaaggc tctgacagtg
cctctcaaga aacagccata 2220 ttaggctcct gtcagcatgc acttcttgca
atccacaata gtgtctggtt ttggtaactg 2280 tatatggtac gaatccccag
gtgggacagt gtctgtgtga tctttccttt agtctttgct 2340 ctagacttta
tctccataaa aagtattttg ttctccttct aaaaagcact gaagcaccca 2400
ctctttggtc tttcttcttc atggacttca tgtggtctgt gaattttaac ctggttattt
2460 ttcagttttt gagctcctat tcacttatca gtgagtgcat acca 2504 71 301
PRT Mus musculus 71 Met Asp Lys Thr Ile Pro Gly Gly Ile Asn Ile Arg
Ile Leu Ile Pro 1 5 10 15 Asn Leu Ile Thr Ile Ile Phe Gly Leu Val
Gly Leu Thr Gly Asn Ser 20 25 30 Ile Val Phe Trp Ile Leu His Phe
Pro Leu Arg Arg Asn Ala Phe Lys 35 40 45 Val Tyr Ile Leu Asn Leu
Asp Leu Ala Asp Phe Phe Phe Leu Leu Gly 50 55 60 His Thr Ile Asp
Ser Ile Leu Leu Leu Leu Asn Val Phe Tyr Pro Ile 65 70 75 80 Ile Phe
Ile Leu Cys Phe Tyr Ile Ile Met Met Val Leu Tyr Ile Ala 85 90 95
Gly Leu Ser Met Leu Thr Ala Ile Ser Thr Glu His Gly Leu Ser Val 100
105 110 Leu Cys Pro Ile Trp Asp Cys Cys His His Pro Glu His Thr Ser
Ala 115 120 125 Ala Met Cys Ala Val Ile Trp Val Leu Ser Leu Leu Ile
Cys Ile Leu 130 135 140 Asn Ser Tyr Phe Gly Phe Leu His Ser Lys Tyr
Glu Asn Asp Asn Gly 145 150 155 160 Cys Leu Ala Leu Asn Phe Phe Thr
Ser Ala Tyr Leu Met Phe Leu Phe 165 170 175 Val Asp Leu Cys Leu Ser
Ser Leu Ala Leu Leu Ala Arg Leu Phe Cys 180 185 190 Asp Val Gly Gln
Met Lys Leu Thr Arg Tyr Val Thr Ile Leu Leu Thr 195 200 205 Ala Leu
Val Phe Leu Leu Cys Arg Leu Asn Phe Gly Ile Tyr Trp Phe 210 215 220
Leu Leu Cys Lys Ile Lys Asp Ala Phe His Val Phe Thr Leu Val Phe 225
230 235 240 Tyr Leu Glu Ser Leu Val Met Thr Ala Ile Asn Ser Cys Ala
Asn Ser 245 250 255 Ile Ile Tyr Phe Phe Thr Gly Ser Phe Arg Leu Arg
Leu Gln His Gln 260 265 270 Thr Leu Lys Met Val Leu Gln Arg Thr Met
Asp Thr Pro Glu Thr Pro 275 280 285 Glu Asn Met Val Gly Met Ser Arg
Ser Lys Ala Glu Pro 290 295 300 72 2758 DNA Mus musculus 72
aatttttgtg tttcctcttt aagggcttct accaatttat ctgtgttctc ctgtattatt
60 ttaagggagt tatttatgtc tttcttaatg tcctctatca tcatcatcat
catccttatc 120 attttcatca tcatcaccag aggtgacttt aaatcagagt
catgcttttc tggtgtgttg 180 gagtatccag ggctcaccat gttgagagaa
ctaggttctg atgatgccaa gtagccttgg 240 ttcccattgc ttatgttttt
gcccttgcct cttgccatct gattatctct ggagtaagct 300 ggtcttgctc
tctctaactg tggcttgtcc ctcctgcaag cctatgtgtc agtactcctg 360
gtagaccagt tctttctggg agaaatttgg gtatggagag ctgtggcaca gggtcagctc
420 cggggtacag ttggaaactg gaagtatcct gtcccaggct gctcctctgt
tcctgtgtcc 480 tgaggattcc aggcatgtcc atttaagcag aagtggtggt
cttacctatg ttcacaggca 540 tatctgcact cctgggagac aagctttctt
ggtggtgttt gggtaatgag cactgggaca 600 caggaacatc tcctggctcc
tttgtcttta catttgggta cagaacaatc acagacaaga 660 gagtaattgt
gctgaaccta agctattacc atggcaacaa aagttccaaa gaaacagcaa 720
gaatgtttca gatgaagtta gtatgagaat tggattaatg tcaggaagga tgcagacatg
780 gtactttctg attaatgcta ttacttggga gaggtagaaa tactaagtca
gtggagagct 840 tttgaaggtg ttgttggacc actgaggaat gccaagtcag
taagctgaga ggaaagtgga 900 cagtggtcta gtgcagcatg gcagtgctca
ctccaaagcc acctctgagg tccaggcaga 960 ggctcttcat catggctctg
ctttgcttct tgatatatcc accatgtttt caggtgtctc 1020 aggagtgtcc
tgcaatgcac tctggagaac cattttgagg gtcttgtgct tcaacggatg 1080
cctgtatgag cccacgaaga agtaaatgat ggggttggca cagctgttaa cagcagttag
1140 gacaagtgat gccagaaaga atctatagtc tagtatactg aaaccaccct
caatccaggg 1200 taacaggaac cagaggaagc ccaggggcaa cccacagaga
agaaaaacca aaatggtcac 1260 catgatggtc atgaataatc tggtaaattt
cttctttcca gcaccacaga acaacctggc 1320 cagcagagtc agggtagaaa
aacagaggac tacaaacaaa
aaaatagggt atattctgat 1380 gaagaattct gatgcctgac acacagagtt
aatttcatat ttgggaccaa ataaatcaca 1440 gaaatatctg ttcagaaggc
agatcaacag gggacaggac ccagatcacg acacacatga 1500 tggttgatgt
gtgttmtggg cggtggcagc gataccagat ggggcacagg acagacaggc 1560
agcgmtcagt gctgatggca ctgagcatgc tcaggcctgt gatgtagaga accgttctga
1620 tggtgtcaaa gcaatggatg aagatactgt tgtgtgggcg aaccttgaaa
agatgcattg 1680 tggaatttat gatgtgacag agaagaaaga aggaagtcag
ccagggccaa gtttaggatg 1740 tagactaaga tggcattcct gtgaaatcgg
aagcccagga tccagaatac aatggcattt 1800 ccagtcagcc caaccagtcc
gaagatgatg atcatcaagt gtgggataag ggtctcgatt 1860 tcaatacttc
cagagatggt ttcatccatt ggatttgttg tcgtgggtgc cattgctgag 1920
gctgaggtgt ttagggccag aaaccctgca ctggtattgc tggaaacaca aacaagaaat
1980 gaggccttca ctgtgaacac aacttttaat ttctttcttt ttgtttgttt
gtttgtttgt 2040 ttgtggggtt ttgttttttt ttttaatttt tttttgtatt
agatattttc ttcatttaat 2100 tttcaaatgt tatccctttt cctggctttc
ccccctccca gaaaccccct tctgatcctc 2160 ccaccctctt caacccacac
acccacttcc acctctctgc ccctgattcc cttacactgg 2220 agcatctata
gaaccttcat aggttcaagg acctcttctt ccatccatgc aagacatggc 2280
catcatctgc tacatatgca tctggagcca cacgtactcc tttgttgatg gcttagtccc
2340 tgggagttca gggggtgggg gtgggggtgg gggcagtggt ctcttggttc
atactgttgc 2400 tcttcttatg gagcttcaaa ccacttcaac tccctcaggc
ctttctctaa ctcctctatt 2460 agggaccctg tgctcagttt aattgttggc
tgctaacatc agactctgca tttgaaaggc 2520 cctgacatgg cctcttagga
aacagctata tcaggttcct gtcagcattc actccttgac 2580 atccacaata
gtgtctgcat ttggtaactg tgtatgagat gaatccccag gtggaacatt 2640
ctctgggtga cttttccttt agtgtctgtt ctacacatta tctccatatt tgctcttgtg
2700 agtattttgt tcttcttcta agaaggtctg aaacacccac actttcgtct
tccttgtt 2758 73 304 PRT Mus musculus 73 Met Asp Glu Thr Ile Ser
Gly Ser Ile Glu Ile Glu Thr Leu Ile Pro 1 5 10 15 His Leu Met Ile
Ile Ile Phe Gly Leu Val Gly Leu Thr Gly Asn Ala 20 25 30 Ile Val
Phe Trp Ile Leu Gly Phe Arg Phe His Arg Asn Ala Ile Leu 35 40 45
Val Tyr Ile Leu Asn Leu Ala Leu Ala Asp Phe Phe Phe Leu Leu Cys 50
55 60 His Ile Ile Asn Ser Thr Met His Leu Phe Lys Val Arg Pro His
Asn 65 70 75 80 Ser Ile Phe Ile His Cys Phe Asp Thr Ile Arg Thr Val
Leu Tyr Ile 85 90 95 Thr Gly Leu Ser Met Leu Ser Ala Ile Ser Thr
Asp Arg Cys Leu Ser 100 105 110 Val Leu Cys Pro Ile Trp Tyr Arg Cys
His Arg Pro His Thr Ser Thr 115 120 125 Ile Met Cys Val Val Ile Trp
Val Leu Ser Leu Leu Ile Cys Leu Leu 130 135 140 Asn Arg Tyr Phe Cys
Asp Leu Phe Gly Pro Lys Tyr Glu Ile Asn Ser 145 150 155 160 Val Cys
Gln Ala Ser Glu Phe Phe Ile Arg Ile Tyr Pro Ile Phe Leu 165 170 175
Phe Val Val Leu Cys Phe Ser Thr Leu Thr Leu Leu Ala Arg Leu Phe 180
185 190 Cys Gly Ala Gly Lys Lys Lys Phe Thr Arg Leu Phe Met Thr Ile
Met 195 200 205 Val Thr Ile Leu Val Phe Leu Leu Cys Gly Leu Pro Leu
Gly Phe Leu 210 215 220 Trp Phe Leu Leu Pro Trp Ile Glu Gly Gly Phe
Ser Ile Leu Asp Tyr 225 230 235 240 Arg Phe Phe Leu Ala Ser Leu Val
Leu Thr Ala Val Asn Ser Cys Ala 245 250 255 Asn Pro Ile Ile Tyr Phe
Phe Val Gly Ser Tyr Arg His Pro Leu Lys 260 265 270 His Lys Thr Leu
Lys Met Val Leu Gln Ser Ala Leu Gln Asp Thr Pro 275 280 285 Glu Thr
Pro Glu Asn Met Val Asp Ile Ser Arg Ser Lys Ala Glu Pro 290 295 300
74 1738 DNA Mus musculus 74 cacccacaac aaccaaatcc aatggacgaa
accatcccct ggaagtattg acatcaagac 60 cctgatcgca aatttgatga
tcatcatctt cggactggtc gggctgacag aaactgcctt 120 tgtgttctga
ctcctgggct tccacttgca caggaacgcc ttcttagtct acatcctaaa 180
cttggccctg actgacttcc tcttccttct ctgtcacatc ataaattcca cagtgattct
240 tctcaatgtt cccctaccta acatgatctt ggtccattgc tttagcacca
tcagaatatt 300 tctcaacatc acaggcctaa gcattctcag tgccatcagc
actgagcgct gcctgtctgt 360 cctgtgcccc atctggtatc gctgccacca
cccagaacac acatcaactg tcatgtgtgc 420 tgtgatctga gtcctgtccc
tgttgatttg cactctgtat agatatttct gttttttctt 480 tggtcccaaa
tatgtatttg actctgtgtg tctggcaacg acctacttta tcagaacata 540
cccaatgttt ttgtttatgg tcctctgtct gtccactctg gctctgctgg ccaggttgtt
600 ctgtggtgct gggaagamra aatttaccag gattattcgt gaccatcatg
ctgacygttt 660 tggtttttct tctctgtggg atgcccctag gcttcttctg
gttcgtgttc ccatggatta 720 actgtgattt cagtgtacta gattatagac
tttttctggc atcaattgta ctaactgctg 780 ttaacagtta tggcaacccc
atcatttact tcttcgtggg ctccttcagg aatcggttga 840 agcaccagac
cctccaaaag gttctccaga gtgcactgca cgacactcct gagacacctg 900
aaaacatggt agagatgtca agaagcaaag cagagccatg atgaagagtc tctgacagga
960 cttcagaggt ggctttggag tgagcactgc cctgctgcac ttaaccacac
tccactctcc 1020 tctcagctta ctgactatgg atgcctcagt ggtccaacaa
tgccttcaaa agctctccac 1080 tgacttagta tttctacctc tcccaagtaa
tagcattaat cagaaagtac catgtctgca 1140 tccttcttga cattaatcca
attctcatac taacttcatc tgtaactttc ttgctgtttc 1200 tttggaactt
ttgttaccat agtaatagcc taggtccagc accatgattc ccttgtctgt 1260
gattgttctg tacctacctg aatgtaaagc aaagtagcca ggagatgttc ctgtgtycca
1320 gtgctcatta cccaaacacc accaagaaag cttgtctccc aggagtgcag
acaagcctgt 1380 gaacacaggt aagaccacca cttctgctta aaggggcatg
cctggaaccc tcaggacaca 1440 ggaacagagg agcagcctgg gacaggatac
ttccagtttc caactgcact ccagagctga 1500 ccctgtgcca cagctctcca
tacccaaatt cctcccagaa agaattggtg taccaggagt 1560 actgacacac
aggcttgcag aaggaacaag ccacagtcaa agttagcaag acctgctaac 1620
accagagata accagatggc aagacacaag ggcaaaaaca taagcaatgg gaaccaagac
1680 tacttggcat catcagaaac tagttctctc aacatggtga gccatggata
cttcaaca 1738 75 303 PRT Mus musculus 75 Met Asp Glu Thr Ile Pro
Gly Ser Ile Asp Ile Lys Thr Leu Ile Ala 1 5 10 15 Asn Leu Met Ile
Ile Ile Phe Gly Leu Val Gly Leu Thr Glu Thr Ala 20 25 30 Phe Val
Phe Leu Leu Gly Phe His Leu His Arg Asn Ala Phe Leu Val 35 40 45
Tyr Ile Leu Asn Leu Ala Leu Thr Asp Phe Leu Phe Leu Leu Cys His 50
55 60 Ile Ile Asn Ser Thr Val Ile Leu Leu Asn Val Pro Leu Pro Asn
Met 65 70 75 80 Ile Leu Val His Cys Phe Ser Thr Ile Arg Ile Phe Leu
Asn Ile Thr 85 90 95 Gly Leu Ser Ile Leu Ser Ala Ile Ser Thr Glu
Arg Cys Leu Ser Val 100 105 110 Leu Cys Pro Ile Trp Tyr Arg Cys His
His Pro Glu His Thr Ser Thr 115 120 125 Val Met Cys Ala Val Ile Val
Leu Ser Leu Leu Ile Cys Thr Leu Tyr 130 135 140 Arg Tyr Phe Cys Phe
Phe Phe Gly Pro Lys Tyr Val Phe Asp Ser Val 145 150 155 160 Cys Leu
Ala Thr Thr Tyr Phe Ile Arg Thr Tyr Pro Met Phe Leu Phe 165 170 175
Met Val Leu Cys Leu Ser Thr Leu Ala Leu Leu Ala Arg Leu Phe Cys 180
185 190 Gly Ala Gly Lys Lys Lys Phe Thr Arg Leu Phe Val Thr Ile Met
Leu 195 200 205 Thr Val Leu Val Phe Leu Leu Cys Gly Met Pro Leu Gly
Phe Phe Trp 210 215 220 Phe Val Phe Pro Trp Ile Asn Cys Asp Phe Ser
Val Leu Asp Tyr Arg 225 230 235 240 Leu Phe Leu Ala Ser Ile Val Leu
Thr Ala Val Asn Ser Tyr Gly Asn 245 250 255 Pro Ile Ile Tyr Phe Phe
Val Gly Ser Phe Arg Asn Arg Leu Lys His 260 265 270 Gln Thr Leu Gln
Lys Val Leu Gln Ser Ala Leu His Asp Thr Pro Glu 275 280 285 Thr Pro
Glu Asn Met Val Glu Met Ser Arg Ser Lys Ala Glu Pro 290 295 300 76
1011 DNA Mus musculus 76 aagaggaaac acatatattt gggatgttaa
ccaaggtttt ctatagggaa caatggaaaa 60 ctgttcactt caagattaca
gtttagctgc atgattaaac tttaaattga cattaacatt 120 taattactgg
gttttataaa ggtcctgaga tatttaaggt tggattgtct tttatattat 180
gatattaata tgcttagaac aaagaaagaa aagtttattg ttcaatggtg aagtgtcttt
240 taaatagaag tgggcagagt gtcctggcaa acctcaattt ttaccttgac
acagattaaa 300 gtcgtatgag aggagaaatc acaacagcag aaatgacaac
tgaggaattg tctagattat 360 cttggcctgt gggcatgatt atgaggaatt
atctttaaca taaattaatg taagcaaaca 420 tggtctatgg taggttgcac
caataagcta cttaagcagg acctgtaatc atccagaatt 480 ggagcttgga
aggagtgttt cttgtagata ctgttccttg tgttccttga gttcctgaca 540
tgacttccct cactgatgga gtctgtacta agagtataag ccagataacc cattttattt
600 tctaggatgt ttgtggtcaa aatgttttcc catgaaacag aaaaggaaac
tagaacatgc 660 acaaattacc taacagatat ttattaagtt agagaatatt
ctaagttata caaatactaa 720 aggaaactac aaatgtggat ctattaaatt
cttatttaaa caaaatctgt agagatgata 780 aattgttaaa aatgtcataa
attttcaatc actatcaagt tcagttacca atgaaattca 840 gttattaact
gaaaactcct gatctttgga tgaagaaggg gcttgtcaaa aatgggagca 900
gtcttggacc tataattatt acagtgggtc tcatctcaag gggatccagt gaagtgtcat
960 taagaggaga gtaggaaagt tcaacatagt atttctatta aaagtggtgt a 1011
77 274 PRT Mus musculus 77 Leu Leu Ser Ile Ile Ile Ala Phe Ile Gly
Leu Ala Glu Asn Ala Ile 1 5 10 15 Val Leu Trp Leu Leu Gly Phe His
Met His Arg Asn Ala Phe Ser Val 20 25 30 Tyr Ile Leu Asn Ala Gly
Ala Asn Phe Leu Phe Leu Cys Pro Tyr Ile 35 40 45 Val Phe Ser Leu
Val Thr Ile Thr Val Asn Phe His Ser Ile Asn Ser 50 55 60 His Ile
Ile Leu Phe Leu Asn Thr Val Phe Thr Leu Ala Tyr Leu Ala 65 70 75 80
Gly Val Ser Met Ile Thr Ala Ile Ser Val Glu Tyr Trp Leu Ser Val 85
90 95 Ile Trp Ser Asn Trp Tyr His Gly Arg His Pro Lys His Thr Ser
Ala 100 105 110 Phe Ile Cys Thr Leu Leu Trp Ala Val Ser Leu Leu Leu
Ser Leu Pro 115 120 125 His Glu Ile Ile Cys Gly Leu Leu Asp His Ile
Tyr Asn Trp Asp Met 130 135 140 Cys Trp Lys Cys Lys Leu Ile Ile Val
Val Trp Leu Leu Ile Glu Phe 145 150 155 160 Val Val Leu Ser Gln Ser
Asn Gln Ala Met Met Phe Arg Ile Phe Cys 165 170 175 Gly Ser Gln Gln
Thr Pro Met Thr Arg Leu Phe Val Thr Ile Val Leu 180 185 190 Thr Ala
Leu Val Val Leu Ile Cys Gly Phe Pro Leu Gly Ile Tyr Ile 195 200 205
Tyr Phe Leu Tyr Trp Thr Thr Asp Val Tyr Phe Ile Met Pro Cys Asn 210
215 220 Ser Phe His Glu Thr Ile Leu Leu Leu Ser Ala Val Asn Ser Cys
Ala 225 230 235 240 Asn Pro Ile Ile Cys Leu Leu Val Gly Ser Ile Lys
His Cys Gln Phe 245 250 255 Gln Cys Gly Thr Leu Arg Leu Ile Leu Gln
Arg Ala Ile Gln Asp Thr 260 265 270 Pro Glu 78 1358 DNA Mus
musculus 78 taaattactg aatctctgtg atcctgattc cctctcttta tggacctgtg
cctgacatac 60 ccacatagtc acatggtcct gacagaaact atcatgtgtt
catatctcta tgtcttttca 120 ggaatgtcag tggaaaattc ctaagcatgg
gtacaactag cctggcctgg aacattaaca 180 acacagctga aaatggaagc
tacactgaaa tgttctcctg tatcaccacg ttcaataccc 240 tgaattttct
tactgtcatc attgctgtgg ttgtcctggc aggaaattcc atagtgctat 300
ggcttctagc cttccacctg cacaggaatg ccttcttcgt ctatgtcctc aatctggctg
360 gtgctgattt cttgtacctt tgcactcaga ttgtgtattc cctggagtgt
gtcattcagt 420 ttgataaaag ctccttttat attctcctca ttttatcaat
gtttgcttac cttgcaggat 480 tgagtatgat tgcaaccatc agtactgagc
gctgcctatc tgttatgtgg cccatctggt 540 atcactgcca aagaccaaga
cacacatcag ccatcatgtc tgttctgctc tgggttttct 600 ctatactgtt
gagcctcctg gtaggactag gctgtggttt tctgttcaga tattctgaat 660
attatttctg tattactttg aactttatca ctgctgcatt tatcataggg ttatctgtgg
720 ttctttctgt atctagcctg accctgttgg tcaagatcat ctgtggatca
cacaggatac 780 ctgtgaccag gttgtttgtt accatttgct ctcacagtgg
tggtcttcat aatctttggc 840 atgccccttg gaatctgctg gttcctcttt
ccaagtatta ttgagtttca taaaattttc 900 tctaacaatt tttatgaaat
gatagcattc ctgtcatgta ttaatagttg tgccaatccc 960 atcatttact
tccttgttgg ctctattagg caccacaggt tgaaatggca gtctcttaag 1020
ctacttcttc agagagccat gcaggacact cctgaggaag tgagtggaga gaggggtcct
1080 tcagaaaggt ctggggaact ggaaagagtc tagtgcagta gtggagtgag
tccttgatca 1140 gatatagttt ctctgagagt caattttgcc tttatctatt
taggcaattt tcacagtctt 1200 gttcaatcag tagagaaaat agtcatttta
tagaaattag gaggaacagg cttgttacac 1260 agaaactgac ttgcagcacc
ataaagctgc cttatgtggt gctcagtgca tcccctcgtg 1320 atataagcct
tgtaatcact tggggccaga acagctcc 1358 79 268 PRT Mus musculus 79 Phe
Leu Thr Val Ile Ile Ala Val Val Val Leu Ala Gly Asn Ser Ile 1 5 10
15 Val Leu Trp Leu Leu Ala Phe His Leu His Arg Asn Ala Phe Phe Val
20 25 30 Tyr Val Leu Asn Leu Ala Gly Ala Asp Phe Leu Tyr Leu Cys
Thr Gln 35 40 45 Ile Val Tyr Ser Leu Glu Cys Val Ile Gln Phe Asp
Lys Ser Ser Phe 50 55 60 Tyr Ile Leu Leu Ile Leu Ser Met Phe Ala
Tyr Leu Ala Gly Leu Ser 65 70 75 80 Met Ile Ala Thr Ile Ser Thr Glu
Arg Cys Leu Ser Val Met Trp Pro 85 90 95 Ile Trp Tyr His Cys Gln
Arg Pro Arg His Thr Ser Ala Ile Met Ser 100 105 110 Val Leu Leu Trp
Val Phe Ser Ile Leu Leu Ser Leu Leu Val Gly Leu 115 120 125 Gly Cys
Gly Phe Leu Phe Arg Tyr Ser Glu Tyr Tyr Phe Cys Ile Thr 130 135 140
Leu Asn Phe Ile Thr Ala Ala Phe Ile Ile Gly Leu Ser Val Val Leu 145
150 155 160 Ser Val Ser Ser Leu Thr Leu Leu Val Lys Ile Ile Cys Gly
Ser His 165 170 175 Arg Ile Pro Val Thr Arg Leu Phe Val Thr Ile Cys
Phe Thr Val Val 180 185 190 Val Phe Ile Ile Phe Gly Met Pro Leu Gly
Ile Cys Trp Phe Leu Phe 195 200 205 Pro Ser Ile Ile Glu Phe His Lys
Ile Phe Ser Asn Asn Phe Tyr Glu 210 215 220 Met Ile Ala Phe Leu Ser
Cys Ile Asn Ser Cys Ala Asn Pro Ile Ile 225 230 235 240 Tyr Phe Leu
Val Gly Ser Ile Arg His His Arg Leu Lys Trp Gln Ser 245 250 255 Leu
Lys Leu Leu Leu Gln Arg Ala Met Gln Asp Thr 260 265 80 2387 DNA Mus
musculus 80 gggcctgagg cacaaacctc tcgggctggc agatccctgc gcactcacca
tgtaaggtgg 60 ccggttgtct ggacgaggaa ttatctttaa cacatgttaa
tgcaagcaaa catggcctat 120 ggtaagttgc accaaaaagc tacctaagca
ggacctgtaa ccaatccaga attgcagcta 180 ggaaggagag tttcctgtag
acactgttcc ttgtgctgct tgagtttctg acatgacttc 240 cttcactgat
ggactctgta ctgagaggat aagccagata acccatttta tctcctagga 300
tgtttgtggt caaaatgttt tcccatgaaa tagaaaagga aactagaaca ggcacaaatt
360 gcctaaaaga tatttattaa gttagagaat attctaagtc atacaaatac
taaaggaaac 420 tacaaatgtg gatctattaa attcttattt atcatctgta
gagatgataa attgttaaaa 480 atgtcatata cctttcatca ctatcaagtt
cagtgaccaa tgataatcag ttattacctg 540 aagactattg atctttggat
gaagaagggg cttgtcaaaa atgggagcag tcctggaccc 600 ataattatta
cagtgggtct catctcaagg ggatccagtg aagcgtcatt aagaggagag 660
taggaacgtt caacacacta tttctattaa aagtggtgta ctgatctact ttcaagggaa
720 tggttaatat cccaactgat ttcacctcag gccatcaact cagcagggtt
gtagaaatgc 780 cccaaaagga taagggcaaa tttgtcctat aagttctctt
gtgtatcatc acagcagctc 840 tcagttgcat cactagagtg tagtactctc
ttcatcttct tcacctcctt cttgttctac 900 aacttcttca acttcttcat
cttcttcctc agggctctct tgaatggctc tctgaagaat 960 cagcctgaga
gtcccacact ggaattggca gtgcttaatt gagccaacaa ataagcaaat 1020
gataggattg gcacagctgt taacaccgga tagtaggaga attgtctcat aaaaataacc
1080 acaaggcata attgaattct cttctttctt ccagtaaaag aagcatatgc
caatcccaaa 1140 gccacagatc aagacgacca gtgctgtaag cataatggtc
acaagcagcc tggtcacagg 1200 tgtctgctgt gaaccacaga agaccctgaa
cagcagggct tgattggatc tagaaagaac 1260 cacaaataaa acaagtaacc
atacaactat gatgagagca agtttccaac acatatccca 1320 gttataaata
taatccagca ctttacaaat tatccaattc caaagggtca acagaagggg 1380
aaaaaaccca gagcagagta caaatgacag ttgatgtgtg ttttgggcgt tgggcatgat
1440 accaagtggg ccaaaggaca gacaaccagt actccacact aatggctgtg
atcatgctca 1500 cccctgcaag gtatgccagt atggtcacat tgacagaaaa
caacgcccat gtgaatgtcg 1560 atgtagtgaa actgcctaat gagattttcc
agggaaaata caatgtgagt gcagaggaag 1620 aggaagtttg ccccagacag
gttgaagatg tagacagaga aggcattcct gtgcatgtgg 1680 aagcccagaa
gctgcagcac tatgacattt cctgtcagtc caatgatggc aatgataatg 1740
gaaagcaaac tcatggcaag ggacatgtca caagatgaag attccatgaa gtagctttca
1800 ttctgttctc tgaattcaat attccagtct gggaagcttg aatccatgtt
tgggaacact 1860 cctggaataa aaaacaagac ataatcgcat gctttgcatt
ctctaattca caagaccacc 1920 ctgatatttg taagctgata tggcacaaaa
tgatggaaaa tgagcttaag aaatttatca 1980 aaaccagtat gtttagagac
ttcttttaaa accagtctga atttatttgg gttatctaca 2040 atccatgtca
tgtactaaca cgaatgtagt tgatggtcca agtatacacc ccaagtgtct 2100
catgttgtgt ggcagaatga aatggaacac tgaacctgta ggggtttgag tataatggca
2160 tccatcaatc catacatttg aatatacagt cactgtttgg tggaactgtt
tggagaaggg 2220 ttatatgtag gggtaattct gatgctaagg tcctgctccc
caatcagtta
ttgatatgtt 2280 gctaaagaaa gacattggcc ctctgctggt caggggggag
ggcaaagggt gatttacagg 2340 actttgggta cctggagtca agcagagaga
tgcaagagag gaaagga 2387 81 273 PRT Mus musculus 81 Leu Leu Ser Ile
Ile Ile Ala Ile Ile Gly Leu Thr Gly Asn Val Ile 1 5 10 15 Val Leu
Gln Leu Leu Gly Phe His Met His Arg Asn Ala Phe Ser Val 20 25 30
Tyr Ile Phe Asn Leu Ser Gly Ala Asn Phe Leu Phe Leu Cys Thr His 35
40 45 Ile Val Phe Ser Leu Glu Ile Ser Leu Gly Ser Phe Thr Thr Ser
Thr 50 55 60 Phe Thr Trp Ala Leu Phe Ser Val Asn Val Thr Ile Leu
Ala Tyr Leu 65 70 75 80 Ala Gly Val Ser Met Ile Thr Ala Ile Ser Val
Glu Tyr Trp Leu Ser 85 90 95 Val Leu Trp Pro Thr Trp Tyr His Ala
Gln Arg Pro Lys His Thr Ser 100 105 110 Thr Val Ile Cys Thr Leu Leu
Trp Val Phe Ser Leu Leu Leu Thr Leu 115 120 125 Trp Asn Trp Ile Ile
Cys Lys Val Leu Asp Tyr Ile Tyr Asn Trp Asp 130 135 140 Met Cys Trp
Lys Leu Ala Leu Ile Ile Val Val Trp Leu Leu Val Leu 145 150 155 160
Phe Val Val Leu Ser Arg Ser Asn Gln Ala Leu Leu Phe Arg Val Phe 165
170 175 Cys Gly Ser Gln Gln Thr Pro Val Thr Arg Leu Leu Val Thr Ile
Met 180 185 190 Leu Thr Ala Leu Val Val Leu Ile Cys Gly Phe Gly Ile
Gly Ile Cys 195 200 205 Phe Phe Tyr Trp Lys Lys Glu Glu Asn Ser Ile
Met Pro Cys Gly Tyr 210 215 220 Phe Tyr Glu Thr Ile Leu Leu Leu Ser
Gly Val Asn Ser Cys Ala Asn 225 230 235 240 Pro Ile Ile Cys Leu Phe
Val Gly Ser Ile Lys His Cys Gln Phe Gln 245 250 255 Cys Gly Thr Leu
Arg Leu Ile Leu Gln Arg Ala Ile Gln Glu Ser Pro 260 265 270 Glu 82
1319 DNA Mus musculus 82 tttataaacc aggtcagtaa ttaccacata
gcaggatgtt cctgaatcag aaagaacata 60 gcatgtgctc attgttttgt
ttattttgtt ccagaaatag tactggagac ttcctaaaca 120 aggatctaag
catctcaacc ttggaagcta actccagaac atctactgaa cccaatgata 180
cttcaggttg tggcatcaag ttccaaacca agatgttgct ttccctcatt tccctgtttg
240 ggatggtact aaatcccata gtgctgtgat tgctgagctt ccaggtgcac
aggaatgcct 300 tgtttgtcta catcctcaac cttgctgtgg ttgacatttt
cttccggttt gatcagtttg 360 cattttgtgt ttttgttatc atttacacta
tcaagtccat ttccaatgat atcctatcat 420 tttttatttt tgtgccagca
tttctgtatc ttttaagcct gagcattctc ataaccatta 480 gcattgaacg
atgcctgtat gtcatgtggc ccatctggta tcactgtcaa tgtccaagac 540
acacatcagc tgtcatttgt gtcttgcttt gggctctgtc ccttgtgttt atgtttctgg
600 atgggaaggc atatttttta ctgttttctg accctaactc tttttggtat
cagacatttg 660 atatcatcat tactgtatag acaattgttt tatttgtggt
tctctgtggg tccagcttaa 720 tcctacttgt cagaatcttc tgtggctccc
agcagatccc tgtaaccagg ctggatgtga 780 tcattgcact cagagtgctt
ttcttcctga tatttagttt tcccttttgg atctactggc 840 tccttgacca
acggattggg agacgttgta attttttgat gaaatgattt tcttatcctg 900
tattaagagc tgtgtcaact ccatcattta ctttcttgtt gcctccatta tgcacagtag
960 tggattcaag gtgaagagtc tcaaactatt tccagagaga gccatgcagg
acattcctga 1020 agaaggagaa ggtgttgaga atagttctta aggaaatcat
gaagaactgg agaaatctag 1080 tgcagcagac gacagctact ttgattagac
agagtggtcg tttttcttat ctttgtggac 1140 taatttaatg accttattca
gtttgttact taatcttcaa tcagttaaaa atgacaatca 1200 tttttgtaat
agttgaaaga tacagtactt gtcacacaaa tattaactgt gccatttctc 1260
ttgctgtgtt tttgaggcct ttaccatttc cttttgatgg gagtacttgc aagtattct
1319 83 264 PRT Mus musculus 83 Leu Ile Ser Leu Phe Gly Met Val Leu
Asn Pro Ile Val Leu Leu Leu 1 5 10 15 Ser Phe Gln Val His Arg Asn
Ala Leu Phe Val Tyr Ile Leu Asn Leu 20 25 30 Ala Val Val Asp Ile
Phe Phe Arg Phe Asp Gln Phe Ala Phe Cys Val 35 40 45 Phe Val Ile
Ile Tyr Thr Ile Lys Ser Ile Ser Asn Asp Ile Leu Ser 50 55 60 Phe
Phe Ile Phe Val Pro Ala Phe Leu Tyr Leu Leu Ser Leu Ser Ile 65 70
75 80 Leu Ile Thr Ile Ser Ile Glu Arg Cys Leu Tyr Val Met Trp Pro
Ile 85 90 95 Trp Tyr His Cys Gln Cys Pro Arg His Thr Ser Ala Val
Ile Cys Val 100 105 110 Leu Leu Trp Ala Leu Ser Leu Val Phe Met Phe
Leu Asp Gly Lys Ala 115 120 125 Tyr Phe Leu Leu Phe Ser Asp Pro Asn
Ser Phe Trp Tyr Gln Thr Phe 130 135 140 Asp Ile Ile Ile Thr Val Thr
Ile Val Leu Phe Val Val Leu Cys Gly 145 150 155 160 Ser Ser Leu Ile
Leu Leu Phe Arg Ile Phe Cys Gly Ser Gln Gln Ile 165 170 175 Pro Val
Thr Arg Leu Asp Val Ile Ile Ala Leu Arg Val Leu Phe Phe 180 185 190
Leu Ile Phe Ser Phe Pro Phe Trp Ile Tyr Trp Leu Leu Asp Gln Arg 195
200 205 Ile Gly Arg Arg Cys Asn Phe Leu Asn Glu Met Ile Phe Leu Ser
Cys 210 215 220 Ile Lys Ser Cys Val Asn Ser Ile Ile Tyr Phe Leu Val
Ala Ser Ile 225 230 235 240 Met His Ser Ser Gly Phe Lys Val Lys Ser
Leu Lys Leu Phe Pro Glu 245 250 255 Arg Ala Met Gln Asp Thr Pro Glu
260 84 2349 DNA Mus musculus 84 tttctttctg agaaatagtt tgttttaaaa
taggaatttt aaaacagctt gagacactga 60 gagtttatac tggaaccatc
aactactcta atgtcaatac aggatatggg ttgtagataa 120 cccaaatata
tatgaatgat atatttaaat taaggctcca gaaatattga ttttgataaa 180
ttgcttcatg tctaccaccc tgtttcacca ttttaagaac taggtaaacc gttaacatct
240 ataatggtga tcctaagaat cagagaacaa aaagcatgtg ttcatgtctt
gtttttcttt 300 ccagaaacat cagtggaagg gatctaagag tggattcaaa
cataacatac tggggaacaa 360 acatcacagc tgtgaatgaa agcaaccaya
ctggaatgtc attttgtgaa gtcgtgtctt 420 gtaccatgkt ttttctttcc
ctcattgttg ccctagttgg gctggttgga aatgccacag 480 tgctgtggtt
cctgggcttc cagatgcgca ggaatgcatt ctctgtttac atcctcaacc 540
tcgctggtgc tgactttctc ttcatttgct ttcaaattgg atattgtttt cacatgatct
600 tggacattga ttccatcccc attgaaattg atctgtttta ccttgttgtg
ttaaactttc 660 cttatttttg tggcctgagt atcctcagtg ctattagcat
tgaacgttgc ctgtctgtca 720 tgtggcccat ttggtatcac tgccaacgcc
caaggcacac atcagctgtc atatgtaccc 780 tgctttgggt cttgtcccta
gtgtgtagcc tcctggaagg gaaggaatgt ggcttcctat 840 attacactag
tgaccctggt tggtgtaaga catttgattt aatcactgct acatggttaa 900
ttgttttatt tgtagctctc ttgggatcca gtctggcctt agtgattacc atcttctggg
960 gcttacacaa gattcctgtg accaggctgt atgtggccat tgtgttcaca
gtgcttgttt 1020 tcctgctctt tggtctgccc tatgggatct actggttcct
cttagtgtgg attgagaaat 1080 tttattatgt tttaccttgt agtatatatc
cggtcacagt atttctctcc tgtgttaaca 1140 gctctgcaaa acccatcatt
tattgccttg taggctccat taggcatcat cgatttcaac 1200 ggaagactct
caagctattt ctgcagagag ccatgcaaga cactcctgag gaggaagaat 1260
gtggagagat gggttcctca ggaagatcta gagaaataaa aacaatctgg aaaggactga
1320 gagctgcttt gatcaggcat aaagagctct gaagagaact atgtttttat
cactttgttg 1380 cattttcata acgttgttta gttgatgacc caaggttaac
tcagttggga aagtagtcaa 1440 tgttgtagaa gttgattgat attggacttg
ttacaaatac tgggtacaac atttctgcag 1500 ctatcttgct cagggtttta
ccaacttctt ttgatgttac tccttgcaag ctctgtgggg 1560 tccaggaaag
ctgttgacca caattgataa atcccttctt cagaagaaag cttaagaaag 1620
tacaggaaag ggttgcattt cttaactcac ttaacttgat agtggataaa ttcatgttat
1680 attttgcaaa aaaattattc tgtttcaagg caaacttttc ttcagtgttg
aagggttaaa 1740 tagatacatt atataatccc agactttatt aatttctgta
tgttttaaag aatatgtgga 1800 gcaatagttt ttcttataca catttcttaa
taaagaagta aacattctca agagaagtgt 1860 taaacatcca tgtacatagg
aaggtgcagt gtcctctgtg gttctattca cagtttcctt 1920 tttagcatcc
catagttgag tattgtcttt gatatgatcc tcatgctctc tgactgtgta 1980
atccctcatg aaaagtttcc aatgaggtcc tctataaaga ctcccttgaa atacaactta
2040 ttttaaattt ataccatttc aaggagccca cagcatctat taacttagct
atatgcacag 2100 tttagtaaaa ttttctataa aataatattc cttttataaa
gctgcagtaa taatttcaat 2160 ttttctacaa ttaagagaat aaaatatcaa
caaattaaat aaaactaatc agtaggtttt 2220 cttaagttaa tgtagctgca
tgactctgta cctaatcaag acacaaaata ctacactata 2280 tcttttaatt
ttcatttctt ctcctgtcat aattttatat cacagataaa tatgatatcc 2340
atacttctg 2349 85 273 PRT Mus musculus 85 Phe Leu Ser Leu Ile Val
Ala Leu Val Gly Leu Val Gly Asn Ala Thr 1 5 10 15 Val Leu Trp Phe
Leu Gly Phe Gln Met Arg Arg Asn Ala Phe Ser Val 20 25 30 Tyr Ile
Leu Asn Leu Ala Gly Ala Asp Phe Leu Phe Ile Cys Phe Gln 35 40 45
Ile Gly Tyr Cys Phe His Met Ile Leu Asp Ile Asp Ser Ile Pro Ile 50
55 60 Glu Ile Asp Leu Phe Tyr Leu Val Val Leu Asn Phe Pro Tyr Phe
Cys 65 70 75 80 Gly Leu Ser Ile Leu Ser Ala Ile Ser Ile Glu Arg Cys
Leu Ser Val 85 90 95 Met Trp Pro Ile Trp Tyr His Cys Gln Arg Pro
Arg His Thr Ser Ala 100 105 110 Val Ile Cys Thr Leu Leu Trp Val Leu
Ser Leu Val Cys Ser Leu Leu 115 120 125 Glu Gly Lys Glu Cys Gly Phe
Leu Tyr Tyr Thr Ser Asp Pro Gly Trp 130 135 140 Cys Lys Thr Phe Asp
Leu Ile Thr Ala Thr Trp Leu Ile Val Leu Phe 145 150 155 160 Val Ala
Leu Leu Gly Ser Ser Leu Ala Leu Val Ile Thr Ile Phe Trp 165 170 175
Gly Leu His Lys Ile Pro Val Thr Arg Leu Tyr Val Ala Ile Val Phe 180
185 190 Thr Val Leu Val Phe Leu Leu Phe Gly Leu Pro Tyr Gly Ile Tyr
Trp 195 200 205 Phe Leu Leu Val Trp Ile Glu Lys Phe Tyr Tyr Val Leu
Pro Cys Ser 210 215 220 Ile Tyr Pro Val Thr Val Phe Leu Ser Cys Val
Asn Ser Ser Ala Lys 225 230 235 240 Pro Ile Ile Tyr Cys Leu Val Gly
Ser Ile Arg His His Arg Phe Gln 245 250 255 Arg Lys Thr Leu Lys Leu
Phe Leu Gln Arg Ala Met Gln Asp Thr Pro 260 265 270 Glu 86 1313 DNA
Mus musculus 86 tttatttaat tattttgtta ttgttgtttc aggtagcaag
tatttcctaa gcatgggata 60 tagacatttc gagcctgggc atttacatca
tagcaccgaa tggaagcagc tacactaata 120 gtgttgattg tttcttcaaa
atccaagtca tgggttttct ttccctcatc atttcccctg 180 ttgggatggt
attaaattcc acagtgctgt ggtttctggg cttccagata cgtaggaatg 240
ccttctctgt ctacatcctc aacctggccg gggctgactt tctcttcctg cactctcagt
300 ttttatttta ccttcttgct atttttccct ccattcctat ccagatccct
ctcttttttg 360 atatgttgac aaaatttgca tatctttctg ggctgagcat
tctcagcacc attagcattg 420 agcgctgcct gtgtgtcatg tggcccatct
ggtaccgctg tcaaagacca agacacacat 480 catctgtaac ctgttccttg
ctttgggctt tgtccctgtt gtttgctctt ctggatggga 540 tgggatgtgg
cttactgttt aatagttttg accagtcttg gtgtttgaaa tttgatttaa 600
tcatttgtgc gtggtcaatt gttttatttg tggttctctg tgggtccagt ctcatcctac
660 ttgttaggat cttctgtggc tcccagcaga tccctgtgac caggctgtat
gtgaccattg 720 cactcacagt gttattcttc ctaatctgct gtcttccctt
tggaatctcc tggatcatcc 780 aatggagtga aactttgata tatgttggat
tttgtgatta ttttcacgag gaactattcc 840 tatcctgtat taacagctgt
gccaacccta tcatttactt ccttgttggt tttattcgtc 900 agcgaaagtt
ccaacagaag tctctgaagg tgcttcttca aagagcgatg gaggacactc 960
ctgaagaaga aaatgaagac atgggtcctt caagaaatcc agaagaattt gaaacagtct
1020 gtagcaactg agaggttctt tgatcagaca gaaatggttt tttagagaaa
aaaatttttt 1080 ctcatttctg tgggccattt tcacagtttt gyacagtttg
tttcctgata ttcaatcagt 1140 taaaaaataa gcatttttgt gaaagtggat
agatacaaga cttgtcatac aaatactgac 1200 tgtagtattt ttggagctgt
tactcagact ttcatcatct ccttttgatg ggattccatg 1260 taagtgtctg
gagttgagga gatgtgttga ccactattga caaagccctc att 1313 87 270 PRT Mus
musculus 87 Phe Leu Ser Leu Ile Ile Ser Pro Val Gly Met Val Leu Asn
Ser Thr 1 5 10 15 Val Leu Trp Phe Leu Gly Phe Gln Ile Arg Arg Asn
Ala Phe Ser Val 20 25 30 Tyr Ile Leu Asn Leu Ala Gly Ala Asp Phe
Leu Phe Leu His Ser Gln 35 40 45 Phe Leu Phe Tyr Leu Leu Ala Ile
Phe Pro Ser Ile Pro Ile Gln Ile 50 55 60 Pro Leu Phe Phe Asp Met
Leu Thr Lys Phe Ala Tyr Leu Ser Gly Leu 65 70 75 80 Ser Ile Leu Ser
Thr Ile Ser Ile Glu Arg Cys Leu Cys Val Met Trp 85 90 95 Pro Ile
Trp Tyr Arg Cys Gln Arg Pro Arg His Thr Ser Ser Val Thr 100 105 110
Cys Ser Leu Leu Trp Ala Leu Ser Leu Leu Phe Ala Leu Leu Asp Gly 115
120 125 Met Gly Cys Gly Leu Leu Phe Asn Ser Phe Asp Gln Ser Trp Cys
Leu 130 135 140 Lys Phe Asp Leu Ile Ile Cys Ala Trp Ser Ile Val Leu
Phe Val Val 145 150 155 160 Leu Cys Gly Ser Ser Leu Ile Leu Leu Val
Arg Ile Phe Cys Gly Ser 165 170 175 Gln Gln Ile Pro Val Thr Arg Leu
Tyr Val Thr Ile Ala Leu Thr Val 180 185 190 Leu Phe Phe Leu Ile Cys
Cys Leu Pro Phe Gly Ile Ser Trp Ile Ile 195 200 205 Gln Trp Ser Glu
Thr Leu Ile Tyr Val Gly Phe Cys Asp Tyr Phe His 210 215 220 Glu Glu
Leu Phe Leu Ser Cys Ile Asn Ser Cys Ala Asn Pro Ile Ile 225 230 235
240 Tyr Phe Leu Val Gly Phe Ile Arg Gln Arg Lys Phe Gln Gln Lys Ser
245 250 255 Leu Lys Val Leu Leu Gln Arg Ala Met Glu Asp Thr Pro Glu
260 265 270 88 1883 DNA Mus musculus 88 cgtgtgccac caccaccaac
aggtgggaca tttcttaaag tatactattc atttaatctt 60 tatcaagttt
aattaccaaa gcaattctga cacttcttgc actaccttga tccttttcct 120
gagggaggca tttgttccca gtgagagctg ttctgacccc aagagattac aagggttaca
180 tcacaagggg gtgcagtaag gcatacataa ggcagtttga tggtgctgca
gtgaatttct 240 gagtaacaag ctccatttct cctaatttga ataaaatgac
tattttctct accaattaaa 300 caagattgtg aaaactgcct acatagataa
aagcaaaatt gactctcaga gaaactatgt 360 ctcatcaagt actctttcaa
agcctgcact agactctttc cagttcccta gcctttgtga 420 aggacccctc
tctcctctct tttcctcact actgtcctac atggttctct gcagaagttg 480
cttcaaactc tgacattgca acctacggtg cctaacagag ccaaggagag agtaaataat
540 gggattggca cagctgttaa cacaggaatg ctatcacttc aaaaacattg
tatgagaaca 600 tgctatgtaa gtccataaac attgtcaaga ggaatgtgca
gattccaatg ggcataccaa 660 agaatatgaa gaccatcaat gtgagggcaa
tggacacata gaacatggtc acaggaatcc 720 tgagtgatac acagaacatt
tgacaaacag ggccaggcta gacacaaaak aaaccacaga 780 taatactatt
atcaatgcag tagygatata gtggcatrta atacagaaat tgtgttcwta 840
ataacttaac agaaagccac agccttgtrc aaasrgaagg atcarcagta tagagaaaac
900 ccagagcaga gcacacatga cagctgatgt gtgtcttggt cttcagcagt
gataccagat 960 gggacacata acagataggc agtgctcagc actgattgtt
gmaatcatac acaaacctgc 1020 aagttaagca atcataaatc ctgtgaggat
aaaatgatag tagatcataa gtatcttaag 1080 gaaacactgc aggggaatgt
acaaactgtg tgcaaatttg caagaaatca gcacaagaca 1140 ggtttaagac
atagacagag aaggcattcc tatgcaggtg gaaggctaga agccatagca 1200
ctatggcatt tcctgccagg ccaagcacag caatgatgac aataagaaaa ttgaatgtgg
1260 tgaaacagga taaatttttc agtgcattaa cttccattga cttctgtgtt
tttaaatttc 1320 cattccaggg tggttggatc catgcttagg aattttccac
tggcattcct gcaaagaaat 1380 agagatatga atctagggta ctctttgtag
ggactatgtg actatgtagg aatgtatggc 1440 acaggtacat aaggagggag
aaacaggatc acagagatta agtaatttac caacattcca 1500 aaagtgctac
acatttttga aatccatttt gtactattca gtctaactgc agaccagtat 1560
gatgtaaggt agttgatggt cccagtacag ttgctaggca tttatttcag gttatgtgag
1620 gaagagacag aactctgaaa ccaacattct ttttgttcta gggctgagat
ttcttctctg 1680 gtgtaggaaa atggaagttc ttggtgcaag ccatatcttc
cctcagtcac tgggaggaat 1740 ctatcaaaca ggcaaaatag aatcatgaat
gagagtcatg aatgagattc acgaagggaa 1800 tggtacttgc tatgaagacc
tgtaggggaa tagccatgct tcttatgctt gaaagggtag 1860 ttgctcattt
aacaatttta aaa 1883 89 263 PRT Mus musculus 89 Phe Leu Ile Val Ile
Ile Ala Val Leu Gly Leu Ala Gly Asn Ala Ile 1 5 10 15 Val Leu Trp
Leu Leu Ala Phe His Leu His Arg Asn Ala Phe Ser Val 20 25 30 Tyr
Val Leu Asn Leu Ser Cys Ala Asp Phe Leu Gln Ile Cys Thr Gln 35 40
45 Phe Val His Ser Pro Ala Val Phe Leu Lys Ile Leu Met Ile Tyr Tyr
50 55 60 His Phe Ile Leu Thr Gly Phe Met Ile Ala Leu Ala Gly Leu
Cys Met 65 70 75 80 Ile Ser Thr Ile Ser Ala Glu His Cys Leu Ser Val
Met Trp Pro Ile 85 90 95 Trp Tyr His Cys Arg Pro Arg His Thr Ser
Ala Val Met Cys Ala Leu 100 105 110 Leu Trp Val Phe Ser Ile Leu Leu
Ile Leu Leu Phe Val Gln Gly Cys 115 120 125 Gly Phe Leu Leu Ser Tyr
Tyr Glu His Asn Phe Cys Ile Ile Cys His 130 135 140 Tyr Ile Ala Thr
Ala Leu Ile Ile Val Leu Ser Val Val Ser Phe Val 145 150 155 160 Ser
Ser Leu Ala Leu Phe Val Thr Met Phe Cys Val Ser Leu Arg Ile 165 170
175 Pro Val Thr Met
Phe Tyr Val Ser Ile Ala Leu Thr Leu Met Val Phe 180 185 190 Ile Phe
Phe Gly Met Pro Ile Gly Ile Cys Thr Phe Leu Leu Thr Met 195 200 205
Phe Met Asp Leu His Ser Ser Ser His Thr Met Phe Leu Lys His Ser 210
215 220 Cys Val Asn Ser Cys Ala Asn Pro Ile Ile Tyr Ser Leu Leu Gly
Ser 225 230 235 240 Val Arg His Arg Arg Leu Gln Cys Gln Ser Leu Lys
Gln Leu Leu Gln 245 250 255 Arg Thr Met Asp Ser Ser Glu 260 90 1219
DNA Mus musculus 90 ttataaatga ttttattaag ccatattgac aataatatct
atattatatg atgattgcca 60 gaagaagggt aaatgttaag gtgatcaaat
atggtctgtg ttctcagaga caccactgga 120 agatttgtga gcatggatcc
aaccatctca tcccacaaca cagaatctac accactgaat 180 gaaactggtc
attccaaatg cagtccaatc ctgactctgt cctttctggt cctcatcact 240
gtcctggtgg aactaggagg aagcaccatt gtactctggc tcctggaatt cagcatgccc
300 aggaaagcca tctcagtcta tgtcctcaat ctggctctgg cagactcctt
cttcctcggc 360 tgcgatttca ttgaatttct gctacggatc attgacttca
tctatgccca taaattaagc 420 aaagatatct taggcaatac agcaatcatt
ccttatatcg caggacagaa cgttctcagt 480 gctattagca tggagcactg
cctgtctgta ttgtggccaa tctggtacca ctaccaccac 540 ccaagaaaca
tgtcagctat catatgtgcc ctaatctggg ttctgtactt tctcatgggc 600
atcctccatt ggttcttctc agtattcctg ggtgaggctc atcatcattt gaggaaaaag
660 gttgacttta ctataactgc atttctgaat ttttatttat gcttcactct
gtgtccagtc 720 tggccctact gctgaggatc ctctgtggct ccaggaggaa
acccctgtcc aggctgtatg 780 ttaccatcgc tctcacagtg atggtcacct
catctctggc ctgcctcttg ggctttactt 840 gttcctgtta tactggtttg
gggttcattt gcatcatccc tcttgtcaca attaccaagt 900 tacttcagtc
ctgccctgtg taaacagcta taacaacccc atcatttact tcattgtagg 960
ctcctttagg cctcttagaa agcattaatc cctccaaact attcttaaga gggctctgga
1020 ggacactcct gaggagcatg aatatacagc cagccatctt cagaaaacca
ctgagatgtc 1080 agaaagcatt tttgagagtc aaaacaacat taacttaatc
ttctctcaga aacccctcag 1140 tgattgcact gctttcaatt gattattttt
tatccaattt tcttatactt ctcaaagtag 1200 tcataaataa gaatttctc 1219 91
270 PRT Mus musculus 91 Phe Leu Val Leu Ile Thr Val Leu Val Glu Leu
Gly Gly Ser Thr Ile 1 5 10 15 Val Leu Trp Leu Leu Glu Phe Ser Met
Pro Arg Lys Ala Ile Ser Val 20 25 30 Tyr Val Leu Asn Leu Ala Leu
Ala Asp Ser Phe Phe Leu Gly Cys Asp 35 40 45 Phe Ile Glu Phe Leu
Leu Arg Ile Ile Asp Phe Ile Tyr Ala His Lys 50 55 60 Leu Ser Lys
Asp Ile Leu Gly Asn Thr Ala Ile Ile Pro Tyr Ile Ala 65 70 75 80 Gly
Gln Asn Val Leu Ser Ala Ile Ser Met Glu His Cys Leu Ser Val 85 90
95 Leu Trp Pro Ile Trp Tyr His Tyr His His Pro Arg Asn Met Ser Ala
100 105 110 Ile Ile Cys Ala Leu Ile Trp Val Leu Tyr Phe Leu Met Gly
Ile Leu 115 120 125 His Trp Phe Phe Ser Val Phe Leu Gly Glu Ala His
His His Leu Arg 130 135 140 Lys Lys Val Asp Phe Thr Ile Thr Ala Phe
Leu Ile Phe Leu Phe Met 145 150 155 160 Leu His Ser Val Ser Ser Leu
Ala Leu Leu Leu Arg Ile Leu Cys Gly 165 170 175 Ser Arg Arg Lys Pro
Leu Ser Arg Leu Tyr Val Thr Ile Ala Leu Thr 180 185 190 Val Met Val
Tyr Leu Ile Ser Gly Leu Pro Leu Gly Leu Tyr Leu Phe 195 200 205 Leu
Leu Tyr Trp Phe Gly Val His Leu His His Pro Ser Cys His Asn 210 215
220 Tyr Gln Val Thr Ser Val Leu Pro Cys Val Asn Ser Tyr Asn Asn Pro
225 230 235 240 Ile Ile Tyr Phe Ile Val Gly Ser Phe Arg Pro Leu Arg
Lys His Ser 245 250 255 Leu Gln Thr Ile Leu Lys Arg Ala Leu Glu Asp
Thr Pro Glu 260 265 270 92 1178 DNA Mus musculus 92 ttaaggtgat
gaaatatggt ctgtgttctc agggacacca ctggaagatt tgtgagcatg 60
gatccaatca tctcatccca caacagagaa tcacaccact gaatgaaact gcaatcattc
120 caactgcagt ccaatcctga ctctgtcctt tctggtcctc atcactatcc
tggtggaact 180 ggcaggaaac accattgtcc tctggctctt gggattccgc
atgcacagga aagccatctc 240 agtttatgtc ctcaatctgg ctctggcaga
ctccgtattc ctctgctgtc atttcattga 300 ctctctgcta tgcatcattg
acttcatcta tgcccataaa ttaagcagat accttaggca 360 atgcagaaat
cattccctat atcacagggc tgagcatcct cagtgctatt agcatggagg 420
actacctgtc tgtattgtgg ccaatctggt accactgcca tcacccaagg aacatgtcaa
480 ctatcctatg tgccctaatc tgggttctat cctttctcat gggcatcctc
gattggttct 540 tctcaggatt cctgggtgag actcatcatt atttgtgaaa
aaatgttgac tttattataa 600 ctgcatttct gatttttttt tttatttatg
cttctctctg ggtccagtct ggccctactg 660 ctgaggatcc tctgtggctc
caggaggaaa ccactgtcca ggttgtatgc taccatctca 720 ctcacagtga
tggtctacct catctgtggc ctacctcttg ggctttactt gtttctgtta 780
cactcctttg gggttaattt gcatcatccc ttttgtcacc tttacaaagt tactgcagtc
840 ctgtcctgtg taaacatctc taccaacccc atcaatcatt taattcattg
gcatttcttt 900 tttttttaat taggtatttt cctcgtttac attttcaatg
ctatcccaaa ggtcccccat 960 acccaccccc cccaatccct acccacccac
tgcccctttt tggcactggc gttcccctgt 1020 actggggcat ataaagtttg
caagtccaat gggcctctct ttgcagtgat gaccgactag 1080 gccatctttt
gatacatatg cagctaaaga catgagctcc cgggtactgg ttagttcata 1140
ttgttgttcc acctataggg ttgcagttcc ctttagct 1178 93 243 PRT Mus
musculus 93 Phe Leu Val Leu Ile Thr Ile Leu Val Glu Leu Ala Gly Asn
Thr Ile 1 5 10 15 Val Leu Trp Leu Leu Gly Phe Arg Met His Arg Lys
Ala Ile Ser Val 20 25 30 Tyr Val Leu Asn Leu Ala Leu Ala Asp Ser
Val Phe Leu Cys Cys His 35 40 45 Phe Ile Asp Ser Leu Leu Cys Ile
Ile Asp Phe Tyr Leu Cys Pro Asp 50 55 60 Ala Asp Thr Leu Gly Asn
Ala Glu Ile Ile Pro Tyr Ile Thr Gly Leu 65 70 75 80 Ser Ile Leu Ser
Ala Ile Ser Met Glu Asp Tyr Leu Ser Val Leu Trp 85 90 95 Pro Ile
Trp Tyr His Cys His His Pro Arg Asn Met Ser Thr Ile Leu 100 105 110
Cys Ala Leu Ile Trp Val Leu Ser Phe Leu Met Gly Ile Leu Asp Trp 115
120 125 Phe Phe Ser Gly Phe Leu Gly Glu Thr His His Tyr Leu Lys Asn
Val 130 135 140 Asp Phe Ile Ile Thr Ala Phe Leu Ile Phe Phe Phe Ile
Leu Leu Leu 145 150 155 160 Ser Gly Ser Ser Leu Ala Leu Leu Leu Arg
Ile Leu Cys Gly Ser Arg 165 170 175 Arg Lys Pro Leu Ser Arg Leu Tyr
Ala Thr Ile Ser Leu Thr Val Met 180 185 190 Val Tyr Leu Ile Cys Gly
Leu Pro Leu Gly Leu Tyr Leu Phe Leu Leu 195 200 205 His Ser Phe Gly
Val Asn Leu His His Pro Phe Cys His Leu Tyr Lys 210 215 220 Val Thr
Ala Val Leu Ser Cys Val Asn Ile Ser Thr Asn Pro Ile Asn 225 230 235
240 His Leu Ile 94 2416 DNA Mus musculus 94 atggagggac ccatggctcc
agttgcatgt gtagcagagg atggccttgt agctcatcaa 60 tgggaggaga
gacttttggt cctgtgaagg ccctataccc cagtgttggg ggttgccagg 120
gagaagaagt gggagtgggt gggttggtgt acagagggag ggcgataatg ggttttcaaa
180 ggaaaaatca ggaaaaggga taacatttga aatgtaaata aagaaaatat
ttaataaaaa 240 gcaaaaatga aaaaaaagtg caaaaacatg ttctattatg
ggagtgggtg tgttgaggag 300 cagtggggga gggttaaata gagaggggac
tgttggaggg gaaactagga aaggggataa 360 cattggaaat gtaaataaag
aaaatatcta ataaaaaata aaataaaaaa ttttggaaga 420 tatttgaaaa
attcattgac aagggcaaga atgttggaga aattcttatt tttgactact 480
ttgagaagta taagaaaatt agattaaaaa taatcaattg aaagcactgc aatcactgag
540 gcgtttctga gagaagagta agttaatgtt gtcttgactc tcaacatatg
ctttctgaca 600 tctcagtggt tttctgaaga tggctgtctg tatattcatc
ctcttcagga gtgtctttca 660 gagccctatt aagaatagtt tggaaggaac
aacactttct acaatgccta aaggagccta 720 caatgaagta aatgatggga
ttggcagagc tgtttacaca ggacaggact gcagttactt 780 ggtaaatgtg
acaagaggga taatgcaaat gaaccccaaa ccagtgtagc aggaaaaagt 840
aaagcccaag aggcaggcca cagatgagat agaccatcac tgtgagagag atggtaactt
900 acagcctgga caggggtttc ttcctaggac cacagaggat cctcagcagt
agggccagac 960 tggacacaga gagaagcata aataaaaaaa tcagaaatgc
agttataata aaggcaacat 1020 tttccacaaa tgatgattag tctcacccag
gaatcctaag aagaaccaat ccaggatgcc 1080 tatgagaatg gacagaaccc
agattagggc atataggata gctgacatgt tactttggtg 1140 gtggaagtca
taccagattg gccacaatac agacaggcag tgctccatgc taatagcact 1200
gagcaggctg tgccctgcca tatagggaat gattgctgca ttgcctaaga tatctttgtt
1260 taatttatgg gcatagatga agtcaatgat ccatagcaga gagtcaatga
aatggcagca 1320 gaggaagaag gagtcgccca gagccagatt gaggacatag
cctgagatgg gtttcctgtg 1380 cattcagaat cccaggagcc agagaacaat
cgtgtttcct gccagttcca ccaggacagt 1440 gatgaggacc agaaaggacg
gagtcaggat tggactgcag ttgggatgac cagtttcatt 1500 cagtggtatg
attcctgtgt tgtgtgatga gatgattgga tccatgctca caaatctttc 1560
agtggtgtta ctgagaacac agaccacatt taatcacctt aaaattgacc cttcttctgg
1620 aaatcataat ataatataga tatttttgtc aatatgcctt aataaaatca
tttataaata 1680 aaaggaaagt aacatgacca tatggatcaa gaattctggg
ctgtgaattc aaattcagag 1740 cttgtgtata ctctatagtg tgggtcatac
ttcctgtgta taactcagga ctttttaatc 1800 gcgtggaaat ggttccattc
tctcatggac aaggttggat ccatttcctg ctctcctgta 1860 accccagaaa
gggaagcacc agatttgcct ccccagggct taaaataaca caggaaagat 1920
gaagatatca gggtattgtc gaggtacatt aagggaaata tccttctgca tggtcaaaag
1980 aatgtattct gagttatgca cctaactctc ggtcgagaca tgacactggt
ctgtgcaaca 2040 gattacagat cacatgcatt tacctcctcc cttgagatga
ccaagctgca cctatcagtc 2100 acttcaccag gggattgctg aggtggcaga
aggaatgaca actcactcat ctttcacagg 2160 agttatacct tctctgcagc
catctctgac cttccctcag ctggtacagt taagcctgtc 2220 tgcttttctg
aaagcactta aggttccttt ttctttcttt agatctcctt ttcttttgaa 2280
catgggtcaa aagaccaagc aacattttcc tgagagtctg gactctctca atcatttctg
2340 aaacccacat ctctttccac catgaaagtt ttttcccaac ttccattgct
ggacatacca 2400 gctttcttgg ggatgt 2416 95 269 PRT Mus musculus 95
Phe Leu Val Leu Ile Thr Val Leu Val Glu Leu Ala Gly Asn Thr Ile 1 5
10 15 Val Leu Trp Leu Leu Gly Phe Met His Arg Lys Pro Ile Ser Gly
Tyr 20 25 30 Val Leu Asn Leu Ala Leu Gly Asp Ser Phe Phe Leu Cys
Cys His Phe 35 40 45 Ile Asp Ser Leu Leu Trp Ile Ile Asp Phe Ile
Tyr Ala His Lys Leu 50 55 60 Asn Lys Asp Ile Leu Gly Asn Ala Ala
Ile Ile Pro Tyr Met Ala Gly 65 70 75 80 His Ser Leu Leu Ser Ala Ile
Ser Met Glu His Cys Leu Ser Val Leu 85 90 95 Trp Pro Ile Trp Tyr
Asp Phe His His Gln Ser Asn Met Ser Ala Ile 100 105 110 Leu Tyr Ala
Leu Ile Trp Val Leu Ser Ile Leu Ile Gly Ile Leu Asp 115 120 125 Trp
Phe Phe Leu Gly Phe Leu Gly Glu Thr Asn His His Leu Cys Glu 130 135
140 Asn Val Ala Phe Ile Ile Thr Ala Phe Leu Ile Phe Leu Phe Met Leu
145 150 155 160 Leu Ser Val Ser Ser Leu Ala Leu Leu Leu Arg Ile Leu
Cys Gly Pro 165 170 175 Arg Lys Lys Pro Leu Ser Arg Leu Val Thr Ile
Ser Leu Thr Val Met 180 185 190 Val Tyr Leu Ile Cys Gly Leu Pro Leu
Gly Leu Tyr Phe Phe Leu Leu 195 200 205 His Trp Phe Gly Val His Leu
His Tyr Pro Ser Cys His Ile Tyr Gln 210 215 220 Val Thr Ala Val Leu
Ser Cys Val Asn Ser Ser Ala Asn Pro Ile Ile 225 230 235 240 Tyr Phe
Ile Val Gly Ser Phe Arg His Cys Arg Lys Cys Cys Ser Phe 245 250 255
Gln Thr Ile Leu Asn Arg Ala Leu Lys Asp Thr Pro Glu 260 265 96 1954
DNA Mus musculus 96 tggcattcgg tacctgcctc ctggcagaag atgaaggccc
gaaatagggc atgtcccagt 60 aagctgttag cttctgtatt ccaaactctc
acctacacag actagtctca gagggatcgg 120 ggaaccaaga tggcttcccc
atggtactcc agcaaaacac tcccaggtga ggtggacacc 180 tctcctctga
cagggaaggt gcccggatat ctggagcctg aaacggggtc tgcctcagaa 240
gctgttagct tctgtagtcc acactctcac atgtgtaggc tagtctcagc aggatccagg
300 aaccaagatc agaagggtca atgttcaggt gatcaaatgt agtctgtgtt
cacagggata 360 ccactggaag atttgtgagc atggatccaa tcatctcatc
ccacaacaca gaatcacacc 420 actgaatgaa actggtcatc ccaactgcag
tacaatcctg actccatcct ttctggtcct 480 catcactgtc ctggtggaac
tggcaggaaa taccattgta ctctggctcc tgagattcca 540 catgcacagg
atagcccatc tcagactatg tcctcaatct ggctctggca gattccttct 600
tcctctcctg ccagttcatt gactctctgc tatggatcct tgacttcatc taggcccata
660 aattaagcaa agatatctta tggaatgcag caatcattcc caataatgca
gggctgagct 720 acctcagtgc tattagcatg gagcactgcc tgcctgtatt
gtggccaatc tggcaccact 780 gccaccacac aagaaacatg tcagctatca
tatgtgccct aatctgggtt ctgtcctttc 840 tcatgggcat cctcgattag
tacttctcag gattcctggg tgagactcat catcagttgt 900 ggaaaaatgt
tgattttatt ctaactgcat ttctgatttc tttttttttt tatttatgct 960
tctctctggg tccagtctgg ccctacgact gaggatcctc tgtggctcca ggaggaaacc
1020 cctgtccttg ctgtatgtta tcatctctct cacagtgatg gtctacctca
tctgtggcct 1080 acctgttggg ctttacttgt tcctgttaaa ctggtttggg
gttcatttgc atcatcccat 1140 ttgtcacatt tatcaagtta ctgcactcct
gccctttgta aacagctttg ccaaacccat 1200 catttccttc attgtaggct
cctttaggca ttgtagaaag cattggtccc gccaaactat 1260 tattaagagg
gctctggagg acactcctga ggaggatgaa tatacagata gccatcttca 1320
gaaaactact gagatgtcag aaagcagatg ttgagagtca agacaacatt aacttaatct
1380 tctctcagaa acacctcact ggttgcagtg ctttcaattg attatttttt
aatccaattt 1440 tcttataagt ctcaaagtag tcataaataa gaatttctcc
aacattcttg gccttgtcaa 1500 tgaatttctc aaatatcctc caaaacattt
tgtatataat ttaatttttt tagatatttt 1560 ctatatttat atttccaatg
ttatcccctt yccttagttt cccctccaaa agccccctct 1620 ccccttcccc
cccccactgc tcctcaatat actcactccc ataattgaac acctttttgc 1680
acttttttct tttttttcac tttttgtttt ttattagata ttttctttat ttacatttca
1740 aatgttgtcc cttttcctga ttttccctct gaaaacccat tactgtcatc
cccctgtaca 1800 ccatccctcc cacttctact tctatcctag gcattcccct
acactggggt atagggcctt 1860 cacaggacca agagtctctc ctcccattga
tgagctacaa ggccatcctc tgctacacat 1920 ggcaactgga gccatgggtc
cctccatgtg tact 1954 97 272 PRT Mus musculus 97 Phe Leu Val Leu Ile
Thr Val Leu Val Glu Leu Ala Gly Asn Thr Ile 1 5 10 15 Val Leu Trp
Leu Leu Arg Phe His Met His Arg Ile Ala Leu Ser Asp 20 25 30 Tyr
Val Leu Asn Leu Ala Leu Ala Asp Ser Phe Phe Leu Ser Cys Gln 35 40
45 Phe Ile Asp Ser Leu Leu Trp Ile Leu Asp Phe Ile Ala His Lys Leu
50 55 60 Ser Lys Asp Ile Leu Trp Asn Ala Ala Ile Ile Pro Asn Asn
Ala Gly 65 70 75 80 Leu Ser Tyr Leu Ser Ala Ile Ser Met Glu His Cys
Leu Pro Val Leu 85 90 95 Trp Pro Ile Trp His His Cys His His Thr
Arg Asn Met Ser Ala Ile 100 105 110 Ile Cys Ala Leu Ile Trp Val Leu
Ser Phe Leu Met Gly Ile Leu Asp 115 120 125 Tyr Phe Ser Gly Phe Leu
Gly Glu Thr His His Gln Leu Trp Lys Asn 130 135 140 Val Asp Phe Ile
Leu Thr Ala Phe Leu Ile Val Phe Phe Phe Leu Phe 145 150 155 160 Met
Leu Leu Ser Gly Ser Ser Leu Ala Leu Arg Leu Arg Ile Leu Cys 165 170
175 Gly Ser Arg Arg Lys Pro Leu Ser Leu Leu Tyr Val Ile Ile Ser Leu
180 185 190 Thr Val Met Val Tyr Leu Ile Cys Gly Leu Pro Val Gly Leu
Tyr Leu 195 200 205 Phe Leu Leu Asn Trp Phe Gly Val His Leu His His
Pro Ile Cys His 210 215 220 Ile Tyr Gln Val Thr Ala Leu Leu Pro Phe
Val Asn Ser Phe Ala Lys 225 230 235 240 Pro Ile Ile Ser Phe Ile Val
Gly Ser Phe Arg His Cys Arg Lys His 245 250 255 Trp Ser Arg Gln Thr
Ile Ile Lys Arg Ala Leu Glu Asp Thr Pro Glu 260 265 270 98 1893 DNA
Mus musculus 98 ttagcaatcc cctggccagg tgactgacag gtgcagctta
gtctttctca agggatgagg 60 taattgcatg tgatctgtaa tctgttgcac
agaccagtgt catgtctcaa cccagagtta 120 ggtgtataac tcagaatcca
tttttttgac catgcagaag catctttcct ttaatgtact 180 tcaacaaaac
cctgatatct tcatcttttc tgcgttattt taagccctgg ggaggcaaat 240
atgatgcttc ccctttctag gggttacagg ggagcaggaa atggatgcag ccctgaccat
300 gatagtaggg aatcatttcc atgtgattta aaggtcctga gttatacaca
ggaagaatga 360 cccagactag agtatgtaca agctctgaat ttgaatccaa
atccagaatt cttgatccac 420 atggtcatgt tattctcctt tttttataaa
tgattttatt aagccatatt gacaacaata 480 tctatattac attatgattg
ccagaagaag ggtcaatgtt aaggtgatga aatatggtct 540 gtgttcctca
ggcacaacac tggaagattt ttgagcatgg atccaaccat ctcattccac 600
aacacagaat ctacaccact gaatgaaact tgtcatccaa atacagtcca atcctgactc
660 cgtcctttct ggtcctcatc actgtcctgg tggacctggc aggaaacacc
attgttctct 720 ggctcctggg attccgcatg cacaggaaac ccatctcagt
ctatgtcctc aacctggctc 780 tgggcgactc cttcttctgc tgccatttca
ttgactctct gctatggatc attgacttca 840 tctatgccca taaattaagc
aaagatatct taggcaatgt agcaatcgtt ccctatatcg 900 cagggctgag
cgtcctcagt gctattagca tggagaactg actgtttata ttgtggccaa 960
tctggtacca ctgccaccac ccaagaaaca tgtcagctat cctatgtgcc ctaatctggg
1020 ttctgttctt tctcatgggc atcctcggtt ggttcttctt aagatttttg
ggtgaaactc 1080 atcattgact ttattatacc tgcatttctg attttttttt
tatttatgct tctctctggg 1140 tccattctgg ccctactgct gaggatcctc
tatggttcca ggaggaaatc cctgtccagg 1200 ttgtatgtta acatctctct
cacagtgatg gtctacctca tctgtggcct gcctcttgga 1260 ctttacttgg
tcctgttata ctgctttggg gttcatttac atcatccctc tcctcacatt 1320
taccaagtta ctgtggtctt gtcctatgtg gacagctctg ccaaccacat cttttatttc
1380 cttgcaggtt cctttaggta ttgtagaaag cattggtccc tccaaactct
tctaaagagg 1440 actctagagg acactcctgg ggaggatgaa tatacagaca
gccatcttca gaaaaccact 1500 gagatgtcag aaagcagatg ttgagagtca
acacattaac ttactcttct ctaagaaacg 1560 cctcagtgat tgcaatgctt
tcaattggtt tttcttttta atcaaatttt cttatacttc 1620 tcaaagtagt
cagaaatgag aatttctcga aaattcttgg cactgtcaat gaatttttca 1680
aatatcttcc aaaactttct tattttattt tattttattt ttattagaca ttttctttat
1740 ttacatttca aatgttatcc cctttactag tttcccctcc aaaaaagcac
tatcccctca 1800 cccctctacc tgctccccac attacccact cccataattg
aacacttttt tcttttttta 1860 acttattatt tttattagat attttcttta ttt
1893 99 262 PRT Mus musculus 99 Phe Leu Val Leu Ile Thr Val Leu Val
Asp Leu Ala Gly Asn Thr Ile 1 5 10 15 Val Leu Trp Leu Leu Gly Phe
Arg Met His Arg Lys Pro Ile Ser Val 20 25 30 Tyr Val Leu Asn Leu
Ala Leu Gly Asp Ser Phe Phe Cys Cys His Phe 35 40 45 Ile Asp Ser
Leu Leu Trp Ile Ile Asp Phe Ile Tyr Ala His Lys Leu 50 55 60 Ser
Lys Asp Ile Leu Gly Asn Val Ala Ile Val Pro Tyr Ile Ala Gly 65 70
75 80 Leu Ser Val Leu Ser Ala Ile Ser Met Glu Asn Leu Phe Ile Leu
Trp 85 90 95 Pro Ile Trp Tyr His Cys His His Pro Arg Asn Met Ser
Ala Ile Leu 100 105 110 Cys Ala Leu Ile Trp Val Leu Phe Phe Leu Met
Gly Ile Leu Gly Gly 115 120 125 Ser Ser Asp Phe Trp Val Lys Leu Ile
Ile Asp Phe Ile Ile Pro Ala 130 135 140 Phe Leu Ile Phe Phe Leu Phe
Met Leu Leu Ser Gly Ser Ile Leu Ala 145 150 155 160 Leu Leu Leu Arg
Ile Leu Tyr Gly Ser Arg Arg Lys Ser Leu Ser Arg 165 170 175 Leu Tyr
Val Asn Ile Ser Leu Thr Val Met Val Tyr Leu Ile Cys Gly 180 185 190
Leu Pro Leu Gly Leu Tyr Leu Val Leu Leu Tyr Cys Phe Gly Val His 195
200 205 Leu His His Pro Ser Pro His Ile Tyr Gln Val Thr Val Val Leu
Ser 210 215 220 Tyr Val Asp Ser Ser Ala Asn His Ile Phe Tyr Phe Leu
Ala Gly Ser 225 230 235 240 Phe Arg Tyr Cys Arg Lys His Trp Ser Leu
Gln Thr Leu Leu Lys Arg 245 250 255 Thr Leu Glu Asp Thr Pro 260 100
1290 DNA Mus musculus 100 cctctggcta ggtgactgac aggtgcagct
tggtcatctc aagggaggag gttactgcat 60 ttgatctata atctgttgca
cagaccagtg tcttgtctcg acccagagtt aggtgtataa 120 ctcagaatcc
attcttttga ccgtgcaaaa gtatctttct cttgatgtac ctcaacaaaa 180
ccctgatatc ttcatctttc ctgtgttatt ttaagccctg ggggagtaca aatctgatgc
240 ttccctttct gtggttacag gtagagcagg aaatggatcc taccctgacc
atgagagaag 300 ggaatcattt ccatgtgatt aaaaggtcct gagttataca
ctggaagtat gacccagact 360 acagagtata cacaagctct gaatttgaat
ccacagtcca gaattcttga tcaatgtagt 420 catgttactc tccttttttt
tataaatgat tttagcaagc catattgaca acaatatcta 480 tattacatta
tgatcgccag aagaaaggtc aatgttaagg tgatcaaaca tggtcttgtt 540
ctcagggaca ccactggaag atttgtgcgc atggatccaa tcatcttatc ccacaacaca
600 gaatcacact gctgaatgaa actggtcaac ccaacttcag tccaatcctg
actctgtctc 660 tctggtcctc atcactgtcc tgtttgaact ggcaggaaac
accattgtac tctggctcct 720 gggattccac atgcacaagg aaagtcatct
cagtctatgt cctcaatctg gctcttgcag 780 actccttctt cctcagctgc
caattcattg actctctgct ttgaagcatt gacttcctct 840 atgcatataa
attaagcaaa gatatcttag gcaatgcagc aatcgttccc tatatcgcag 900
ggctgagtat cctcagtgct attagcatgg agcactgcct gtctgtatag tggcaaatgc
960 ggtaccactg ccactaccca agaaacatgt cagctatcct atgtgcccta
atctgggttc 1020 tgtcttttct catggacatc ctggattggt tcttctcagg
attcctgggt gagactcatc 1080 atcatttatg gaaaaatatt gacttcatta
taactgcatt tctgattttt ttatttatgc 1140 ttctctctgg ctccagtctg
gccctactgc tgaggattct ttatggcttc aagaggaaac 1200 ccctgtccag
gctatatatt atcatctctc tcacagtgat ggtctacctc atctgggcct 1260
gccccttggg ctttcatttt tcctgttaca 1290 101 207 PRT Mus musculus 101
Leu Val Leu Ile Thr Val Leu Phe Glu Leu Ala Gly Asn Thr Ile Val 1 5
10 15 Leu Trp Leu Leu Gly Phe His Met Thr Arg Lys Val Ile Ser Val
Tyr 20 25 30 Val Leu Asn Leu Ala Leu Ala Asp Ser Phe Phe Leu Ser
Cys Gln Phe 35 40 45 Ile Asp Ser Leu Leu Ser Ile Asp Phe Leu Tyr
Ala Tyr Lys Leu Ser 50 55 60 Lys Asp Ile Leu Gly Asn Ala Ala Ile
Val Pro Tyr Ile Ala Gly Leu 65 70 75 80 Ser Ile Leu Ser Ala Ile Ser
Met Glu His Cys Leu Ser Val Trp Gln 85 90 95 Met Arg Tyr His Cys
His Tyr Pro Arg Asn Met Ser Ala Ile Leu Cys 100 105 110 Ala Leu Ile
Trp Val Leu Ser Phe Leu Met Asp Ile Leu Asp Trp Phe 115 120 125 Phe
Ser Gly Phe Leu Gly Glu Thr His His His Leu Trp Lys Asn Ile 130 135
140 Asp Phe Ile Ile Thr Ala Phe Leu Ile Phe Leu Phe Met Leu Leu Ser
145 150 155 160 Gly Ser Ser Leu Ala Leu Leu Leu Arg Ile Leu Tyr Gly
Phe Lys Arg 165 170 175 Lys Pro Leu Ser Arg Leu Tyr Ile Ile Ile Ser
Leu Thr Val Met Val 180 185 190 Tyr Leu Ile Leu Gly Leu Pro Leu Gly
Leu Ser Phe Phe Leu Leu 195 200 205 102 1389 DNA Mus musculus 102
ttaaggtgat caaatatggc ctgttttctc agggacacca ctggaagatt tttaaacatg
60 gatccaaaca tctcatccca caacacagaa tctactccac tgaatgaaac
tggtcatcca 120 aacttcagta caatactcac gctgtccttt ctggtcctcg
tcactgtcct cgtggaactg 180 gcaggaaaca ccattgtact ctggctcctg
ggattccgca tgcacaggaa agccatctca 240 gtctatgtcc tcaatctggc
tctggcagac tccttcttct gctgccattt cattgactct 300 ctgctatgga
tcactgactt catctatacc cataaattaa gcaaagatat cttacgcaat 360
gcagcaattg ttccctatat cgcaagactg agcgtcctca gtgctattag aatggagcac
420 ttactgttta tattgtggcc aatctggtac cactgccacc acccaagaaa
catatcagct 480 atcctatgtg ccctaatctg ggttctgttc tttctcatgg
gcatccttga ttggttcttc 540 ttaggattcc tgggtgagac tcatcatcat
ttgtggaaaa atattgactt tattatacct 600 gcatttctga tttttttaat
gctgctttct gggtccactc tggccctact gctgaggata 660 ctttgtggtt
ccaggaggaa actcctgtcc aggctgtatg ttaccatctc tctcacagtg 720
atggtctacc tcatctgtgg catgcctctt gggctttact tgttcctgtt atactggttt
780 gggattcatt tacactatcc ctcttgtcac atttaccaag ttactgcact
cttgtcctat 840 gtggacagct ctgccaacca catcttttat ttccttgtag
gctcctttag gcattttaga 900 aagcattggt ccctctaaac tattctaaag
aggaccctgg agaacattcc tgaggaggat 960 gaatatacag acagctatct
tcagaatacc actgagatgt cagaaatcag atgttgagag 1020 tcaacacatt
aacttactct tctctcagaa acgcctcagt gattgcaacg ctttcaattt 1080
ttttgtttgt ttggtttttt tttttttgga ttgttttaaa ttaggtattt tggtatttta
1140 catttccaaa tttatattta tacttccaaa agtcccccat accttcccct
gccaatcccc 1200 tacccacttt ttggccctgg cgtttccctg tactggggca
tataaagttt gcaagtccag 1260 tgggcctctc tttccagtga tggcctacta
agccatcttt tgatacatat gcagctagag 1320 tcaagagctc cagggtactg
attaattcat aatgttgttc cacctatagg gttgcagatc 1380 cctttagca 1389 103
206 PRT Mus musculus 103 Phe Phe Cys Cys His Phe Ile Asp Ser Leu
Leu Trp Ile Thr Asp Phe 1 5 10 15 Ile Tyr Thr His Lys Leu Ser Lys
Val Tyr Leu Thr Gln Cys Ser Asn 20 25 30 Phe Pro Tyr Ile Ala Arg
Leu Ser Val Leu Ser Ala Ile Arg Met Glu 35 40 45 His Leu Leu Phe
Ile Leu Trp Pro Ile Trp Tyr His Cys His His Pro 50 55 60 Arg Asn
Ile Ser Ala Ile Leu Cys Ala Leu Ile Trp Val Leu Phe Phe 65 70 75 80
Leu Met Gly Ile Leu Asp Trp Phe Phe Leu Gly Phe Leu Gly Glu Thr 85
90 95 His His His Leu Trp Lys Asn Ile Asp Phe Ile Ile Pro Ala Phe
Leu 100 105 110 Ile Phe Leu Met Leu Leu Ser Gly Ser Thr Leu Ala Leu
Leu Leu Arg 115 120 125 Ile Leu Cys Gly Ser Arg Arg Lys Leu Leu Ser
Arg Leu Tyr Val Thr 130 135 140 Ile Ser Leu Thr Val Met Val Tyr Leu
Ile Cys Gly Met Pro Leu Gly 145 150 155 160 Leu Tyr Leu Phe Leu Leu
Tyr Trp Phe Gly Ile His Leu His Tyr Pro 165 170 175 Ser Cys His Ile
Tyr Gln Val Thr Ala Leu Leu Ser Tyr Val Asp Ser 180 185 190 Ser Ala
Asn His Ile Phe Tyr Phe Leu Val Gly Ser Phe Arg 195 200 205 104
1420 DNA Mus musculus 104 aaaaaggaac cttacacttt tctgagttag
tgtgcattca gagaatcaga cagtcttaac 60 tgtaccccct gagggaaggt
cagagatggc tgcatagagg gtgcaactcc tgtgaaggat 120 gagtgaattg
tcattccttc tgccatctta gcaatcccct ggccaggtga ctgacaggta 180
caacattgtc aactcaaggg aggakrtaaa tgyrtgtgat ccttaatcta gagcacagac
240 cagagtcaca tmtcaaccca gagttagggg tagaaytcag aatccattct
tttgatgatg 300 aggaagtatc tttcccttaa tatgcctcaa caaaaccctg
atatcatcat cttttctgtg 360 tcattttaag ccctggggag gtaaatgtga
tgcttccctt tctggagtta ccaaggtggc 420 aggaaatgga tccaaccctg
accatgaaaa aaggaaatcg tttccatgtg aattaaagat 480 cctgagttat
acacaggaag aatgatgcag actatagagt aaacacaagc tctaaatttg 540
aatccacagt ccagaattct taatcccatg tggtcatgtt actttccttt tatttataaa
600 tcattttatt taataatgtt gacaagaata tctatattay rttatgattg
ccagaagaag 660 ggtcagtgtt aatgtgctca aatatggtct gtgttctcag
ggacacaact ggaagatttg 720 tgagcatgga ttcaaccatc tcatcccaca
acacaawatc tacacaactg aatgaaactg 780 stratcctaa ctgcagtcca
atcctgacmc tgyccttcct ggccctcatc actgccctgg 840 tttgactggc
agaaaacact attatactct gactcctggg attccccatg cacaggaaag 900
ccatctcagt ctatatcctc aaccaggctc tggcagactc cttcttcctc tgctgtcact
960 tccttgactc tatgctacag atcattgact tctatggcat ctatggccat
aaattaagca 1020 aagatatctt aggcaatgca gcaatcattc cctatatcac
agggctgagc gtcctcagtg 1080 ctattagcac tgcctgtcta tattgtggcc
aatctggtac cattgccacc acccaagaaa 1140 catgtcaggt atcatatgtg
ccctaatctg ggttctgtcc tttctcatgg gcatccttga 1200 ttggttcttc
tcaggattcc tgggtgagac tcattatcat ttgtgggaaa atgttgactt 1260
tattataact gcatttttta tttatgcttc tctctgggtc tactcatgag gatcctctgt
1320 ggaggaaacc cctgtccagg ctgtatgtta ccatctctct cacagtgatg
ggctacctca 1380 tctgtggcct gcctcttggg ctttacttgt ctctgttaca 1420
105 200 PRT Mus musculus 105 Phe Leu Ala Leu Ile Thr Ala Leu Val
Leu Ala Glu Asn Thr Ile Ile 1 5 10 15 Leu Leu Leu Gly Phe Pro Met
His Arg Lys Ala Ile Ser Val Tyr Ile 20 25 30 Leu Asn Gln Ala Leu
Ala Asp Ser Phe Phe Leu Cys Cys His Phe Leu 35 40 45 Asp Ser Met
Leu Gln Ile Ile Asp Phe Tyr Gly Ile Tyr Gly His Lys 50 55 60 Leu
Ser Lys Asp Ile Leu Gly Asn Ala Ala Ile Ile Pro Tyr Ile Thr 65 70
75 80 Gly Leu Ser Val Leu Ser Ala Ile Ser Thr Asp Leu Ser Ile Leu
Trp 85 90 95 Pro Ile Trp Tyr His Cys His His Pro Arg Asn Met Ser
Gly Ile Ile 100 105 110 Cys Ala Leu Ile Trp Val Leu Ser Phe Leu Met
Gly Ile Leu Asp Trp 115 120 125 Phe Phe Ser Gly Phe Leu Gly Glu Thr
His Tyr His Leu Trp Glu Asn 130 135 140 Val Asp Phe Ile Ile Thr Ala
Phe Phe Ile Val Cys Phe Ser Leu Gly 145 150 155 160 Leu Leu Met Arg
Ile Leu Cys Gly Gly Ile Pro Leu Ser Arg Leu Tyr 165 170 175 Val Thr
Ile Ser Leu Thr Val Met Gly Tyr Leu Ile Cys Gly Leu Pro 180 185 190
Leu Gly Leu Tyr Leu Ser Leu Leu 195 200 106 730 DNA Mus musculus
106 tgtgatctgt gttctcaggg acaccgctgg aagcatttgt gagcatggat
ccaatcatct 60 catcccacaa cacagaatca caccactgaa tgaaactggt
catcccaact gcagtccaat 120 cctgacacca ttctttctgg tcctcatcac
tgtactggtg gaattggcag gggaacacca 180 ttatactctg gctcctggga
tttcgcatga acaggaaagc aatctcagtt tatgtcctca 240 atctggctct
ggcagactcc ttcttttcct ctgttgccat ttcattgact ctctgctaca 300
gaacattgac ttcatcaatg cccataaatt aagcaaacat atcttaggaa atgcagcaat
360 cattccctat attgcagggc tgagcctcct cagtgctatt agcatggagc
actgcctgtt 420 tatattatgg ccaatctggt accactgcca ccacatgtca
gctatcatat gtgccctaat 480 ctgggttccg tcctttctca agggcatcct
caatttgttc ttctcaggat tcctgggtga 540 gactcatcat catttgtggg
aaaatattga ctttattata actgcatttc tgattttttt 600 atttatgctt
ctctgtgggt gcactttggc cctagagctg aggatactct gtggctccag 660
gaagaaaccc ctgtccaggc tgtaagttac catctctctc acagcgatgg tctacctcat
720 ctgtggcctg 730 107 198 PRT Mus musculus 107 Phe Leu Val Leu Ile
Thr Val Leu Val Glu Leu Ala Gly Asn Thr Ile 1 5 10 15 Ile Leu Trp
Leu Leu Gly Phe Arg Met Asn Arg Lys Ala Ile Ser Val 20 25 30 Tyr
Val Leu Asn Leu Ala Leu Ala Asp Ser Phe Val Phe Leu Cys Cys 35 40
45 His Phe Ile Asp Ser Leu Leu Gln Asn Ile Asp Phe Ile Asn Ala His
50 55 60 Lys Leu Ser Lys His Ile Leu Gly Asn Ala Ala Ile Ile Pro
Tyr Ile 65 70 75 80 Ala Gly Leu Ser Leu Leu Ser Ala Ile Ser Met Glu
His Cys Leu Phe 85 90 95 Ile Leu Trp Pro Ile Trp Tyr His Cys His
His Met Ser Ala Ile Ile 100 105 110 Cys Ala Leu Ile Trp Val Pro Ser
Phe Leu Lys Gly Ile Leu Asn Leu 115 120 125 Phe Phe Ser Gly Phe Leu
Gly Glu Thr His His His Leu Trp Glu Asn 130 135 140 Ile Asp Phe Ile
Ile Thr Ala Phe Leu Ile Phe Leu Phe Met Leu Leu 145 150 155 160 Cys
Gly Cys Thr Leu Ala Leu Glu Leu Arg Ile Leu Cys Gly Ser Arg 165 170
175 Lys Lys Pro Leu Ser Arg Leu Val Thr Ile Ser Leu Thr Ala Met Val
180 185 190 Tyr Leu Ile Cys Gly Leu 195 108 847 DNA Mus musculus
108 ttcagaattc ttgatccatg tggtcatgtt actccccttt tattaataaa
tgagtacatt 60 aagccatatt gaaaacaata tctatattat attatgattg
cccgaagaag ggtcaatgtt 120 aaggtgatca aatatggcct gttttcctca
gggacaccaa tgggtgattt gtttagcatg 180 gatccaacca tctcatccca
caacacagaa tcacaccact gaatgaacct ggcccatccc 240 gactgcaatc
caatcctggt tctgtccttt ctggtcctca tcgctgtcct ggtggaactg 300
gcaggaaaca ccattgttct ctggctcctg ggattccgca tgcacaggaa acccatctca
360 gtctatgtcc tcaatctggc tctggcagac tccttcttcc tctgctgcca
tttcattgac 420 tctctgctac aaatcattga cttcacctat gcccataaat
taagcaaaga tatcttagac 480 aatgcagcaa ttgttccctt tatcacaggg
ctgagggtcc tcagtgctat tagcatggag 540 cactgcctgt ctgtattgtg
gctaatctgg taccactgcc accacctgag aaatatgtca 600 gctatcctat
gtgccctaat ctgggttctg tcctttctca tgtccatcct ggactagttc 660
ttctcagaat tcctgcatga gactcatcat catttgtggg aaaatgttga ctttattata
720 actgcatttc tgattttttt atttatgctt ctctttaggt ccagtctggc
cctactgcgg 780 aggatcctcc tgtggctcca ggaggaaata cctgtccacg
ctatatgtta tcatttctct 840 cacagtg 847 109 192 PRT Mus musculus 109
Phe Leu Val Leu Ile Ala Val Leu Val Glu Leu Ala Gly Asn Thr Ile 1 5
10 15 Val Leu Trp Leu Leu Gly Phe Arg Met His Arg Lys Pro Ile Ser
Val 20 25 30 Tyr Val Leu Asn Leu Ala Leu Ala Asp Ser Phe Phe Leu
Cys Cys His 35 40 45 Phe Ile Asp Ser Leu Leu Gln Ile Ile Asp Phe
Thr Tyr Ala His Lys 50 55 60 Leu Ser Lys Asp Ile Leu Asp Asn Ala
Ala Ile Val Pro Phe Ile Thr 65 70 75 80 Gly Leu Arg Val Leu Ser Ala
Ile Ser Met Glu His Cys Leu Ser Val 85 90 95 Leu Trp Leu Ile Trp
Tyr His Cys His His Leu Arg Asn Met Ser Ala 100 105 110 Ile Leu Cys
Ala Leu Ile Trp Val Leu Ser Phe Leu Met Ser Ile Leu 115 120 125 Asp
Phe Phe Ser Glu Phe Leu His Glu Thr His His His Leu Trp Glu 130 135
140 Asn Val Asp Phe Ile Ile Thr Ala Phe Leu Ile Phe Leu Phe Met Leu
145 150 155 160 Leu Phe Arg Ser Ser Leu Ala Leu Leu Arg Arg Ile Leu
Cys Gly Ser 165 170 175 Arg Arg Lys Tyr Leu Ser Thr Leu Tyr Val Ile
Ile Ser Leu Thr Val 180 185 190
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