U.S. patent application number 10/640252 was filed with the patent office on 2004-02-19 for isolated human receptor proteins, nucleic acid molecules encoding human receptor proteins, and uses thereof.
This patent application is currently assigned to APPLERA CORPORATION. Invention is credited to Beasley, Ellen M., Di Francesco, Valentina, Gong, Fangcheng, Yan, Chunhua.
Application Number | 20040033227 10/640252 |
Document ID | / |
Family ID | 25312211 |
Filed Date | 2004-02-19 |
United States Patent
Application |
20040033227 |
Kind Code |
A1 |
Gong, Fangcheng ; et
al. |
February 19, 2004 |
Isolated human receptor proteins, nucleic acid molecules encoding
human receptor proteins, and uses thereof
Abstract
The present invention provides amino acid sequences of peptides
that are encoded by genes within the human genome, the receptor
peptides of the present invention. The present invention
specifically provides isolated peptide and nucleic acid molecules,
methods of identifying orthologs and paralogs of the receptor
peptides, and methods of identifying modulators of the receptor
peptides.
Inventors: |
Gong, Fangcheng;
(Germantown, MD) ; Yan, Chunhua; (Boyds, MD)
; Di Francesco, Valentina; (Rockville, MD) ;
Beasley, Ellen M.; (Darnestown, MD) |
Correspondence
Address: |
CELERA GENOMICS CORP.
ATTN: WAYNE MONTGOMERY, VICE PRES, INTEL PROPERTY
45 WEST GUDE DRIVE
C2-4#20
ROCKVILLE
MD
20850
US
|
Assignee: |
APPLERA CORPORATION
Norwalk
CT
|
Family ID: |
25312211 |
Appl. No.: |
10/640252 |
Filed: |
August 14, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10640252 |
Aug 14, 2003 |
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09851985 |
May 10, 2001 |
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6670150 |
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Current U.S.
Class: |
424/143.1 ;
435/320.1; 435/325; 435/6.16; 435/69.1; 530/350; 530/388.22;
536/23.5 |
Current CPC
Class: |
C07K 14/705
20130101 |
Class at
Publication: |
424/143.1 ;
435/6; 435/69.1; 435/320.1; 435/325; 530/350; 530/388.22;
536/23.5 |
International
Class: |
C12Q 001/68; C07H
021/04; A61K 039/395; C12P 021/02; C12N 005/06; C07K 014/705; C07K
016/28 |
Claims
That which is claimed is:
1. An isolated peptide consisting of an amino acid sequence
selected from the group consisting of: (a) an amino acid sequence
shown in SEQ ID NO:2; (b) an amino acid sequence of an allelic
variant of an amino acid sequence shown in SEQ ID NO:2, wherein
said allelic variant is encoded by a nucleic acid molecule that
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid
sequence of an ortholog of an amino acid sequence shown in SEQ ID
NO:2, wherein said ortholog is encoded by a nucleic acid molecule
that hybridizes under stringent conditions to the opposite strand
of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; and (d) a
fragment of an amino acid sequence shown in SEQ ID NO:2, wherein
said fragment comprises at least 10 contiguous amino acids.
2. An isolated peptide comprising an amino acid sequence selected
from the group consisting of: (a) an amino acid sequence shown in
SEQ ID NO:2; (b) an amino acid sequence of an allelic variant of an
amino acid sequence shown in SEQ ID NO:2, wherein said allelic
variant is encoded by a nucleic acid molecule that hybridizes under
stringent conditions to the opposite strand of a nucleic acid
molecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of
an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein
said ortholog is encoded by a nucleic acid molecule that hybridizes
under stringent conditions to the opposite strand of a nucleic acid
molecule shown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino
acid sequence shown in SEQ ID NO:2, wherein said fragment comprises
at least 10 contiguous amino acids.
3. An isolated antibody that selectively binds to a peptide of
claim 2.
4. A method for producing any of the peptides of claim 1 comprising
introducing a nucleotide sequence encoding any of the amino acid
sequences in (a)-(d) into a host cell, and culturing the host cell
under conditions in which the peptides are expressed from the
nucleotide sequence.
5. A method for producing any of the peptides of claim 2 comprising
introducing a nucleotide sequence encoding any of the amino acid
sequences in (a)-(d) into a host cell, and culturing the host cell
under conditions in which the peptides are expressed from the
nucleotide sequence.
6. A method for detecting the presence of any of the peptides of
claim 2 in a sample, said method comprising contacting said sample
with a detection agent that specifically allows detection of the
presence of the peptide in the sample and then detecting the
presence of the peptide.
7. A method for identifying a modulator of a peptide of claim 2,
said method comprising contacting said peptide with an agent and
determining if said agent has modulated the function or activity of
said peptide.
8. The method of claim 7, wherein said agent is administered to a
host cell comprising an expression vector that expresses said
peptide.
9. A method for identifying an agent that binds to any of the
peptides of claim 2, said method comprising contacting the peptide
with an agent and assaying the contacted mixture to determine
whether a complex is formed with the agent bound to the
peptide.
10. A pharmaceutical composition comprising an agent identified by
the method of claim 9 and a pharmaceutically acceptable carrier
therefor.
11. A method for treating a disease or condition mediated by a
human receptor protein, said method comprising administering to a
patient a pharmaceutically effective amount of an agent identified
by the method of claim 9.
12. A method for identifying a modulator of the expression of a
peptide of claim 2, said method comprising contacting a cell
expressing said peptide with an agent, and determining if said
agent has modulated the expression of said peptide.
13. An isolated human receptor peptide having an amino acid
sequence that shares at least 70% homology with an amino acid
sequence shown in SEQ ID NO:2.
14. A peptide according to claim 13 that shares at least 90 percent
homology with an amino acid sequence shown in SEQ ID NO:2.
Description
FIELD OF THE INVENTION
[0001] The present invention is in the field of receptor proteins
that are related to the cytokine receptor subfamily, recombinant
DNA molecules, and protein production. The present invention
specifically provides novel receptor peptides and proteins and
nucleic acid molecules encoding such peptide and protein molecules,
all of which are useful in the development of human therapeutics
and diagnostic compositions and methods.
BACKGROUND OF THE INVENTION
[0002] Receptor Proteins
[0003] Many receptor proteins serve as targets for the action of
pharmaceutically active compounds. Additionally, many
pharmaceutical/therapeutic agents act by competing with and/or
blocking the binding of the receptor with it's natural ligand. It
is, therefore, important in developing new pharmaceutical compounds
to identify receptor proteins that can be put into high-throughput
screening formats. The present invention advances the state of the
art by providing novel human receptor proteins that can serve as
drug targets.
[0004] Cytokine Receptors
[0005] The novel human protein, and encoding gene, provided by the
present invention is related to the family of cytokine receptors,
particularly the hematopoietic and inflammatory cytokines, and
shows the greatest degree of similarity to granulocyte-macrophage
colony-stimulating factor 2 receptor, beta chain (GM-CSF2RB); this
beta chain is also shared with the interleukin-5 (IL5RB) (Tavernier
et al., Cell 66: 1175-1184, 1991) and interleukin-3 receptors
(IL3RB) (Kitamura et al., Cell 66: 1165-1174, 1991). Thus,
references herein to CSF2RB include GM-CSF2B, IL5RB, and IL3RB.
[0006] Mutations in CSF2RB are associated with pulmonary alveolar
proteinosis (Dirksen et al., J. Clin. Invest. 100: 2211-2217, 1997)
and acute myeloid leukemia (Dirksen et al., Blood 92: 1097-1103,
1998). Mutations in CSF2RB may also lead to cancer (D'Andrea et
al., Blood 83: 2802-2808, 1994) and mutations that lead to
over-active CSF2RB may cause hematopoietic disorders, neurological
disorders, and myeloproliferative disorders such as polycythema
vera (D'Andrea et al., J. Clin. Invest. 102: 1951-1960, 1998).
Furthermore, CSF2RB-mediated cytokine signaling plays an important
role in regulating cell fate decisions and it has been suggested
that a key step in lymphoid commitment is down-regulation of the
cytokine receptors that modulate myeloid cell development (Kondo et
al., Nature 407: 383-386, 2000).
[0007] For further information regarding cytokine receptors such as
CSF2RB, see Hayashida et al., Proc Natl Acad Sci USA December 1990;
87(24):9655-9; Tu et al., Blood Aug. 1, 2000; 96(3):794-9; Herman
et al., J Biol Chem Mar. 3, 2000; 275(9):6295-301; Sayani et al.,
Blood Jan. 15, 2000; 95(2):461-9; Gorman et al., J. Biol. Chem.
267: 15842-15848, 1992; Jenkins et al., EMBO J. 14: 4276-4287,
1995; Robb et al., Proc. Nat. Acad. Sci. 92: 9565-9569, 1995; and
Shen et al., Cytogenet. Cell Genet. 61: 175-177, 1992.
[0008] Due to their importance in hematopoietic, inflammatory, and
neurological disorders, as well as cancer, leukemia, and
myeloproliferative disorders, novel human cytokine receptor
proteins/genes, such as provided by the present invention, are
valuable as potential targets for the development of therapeutics
to treat these and other diseases/disorders. Furthermore, SNPs in
cytokine receptor genes, such as provided by the present invention,
may serve as valuable markers for the diagnosis, prognosis,
prevention, and/or treatment of such diseases/disorders.
[0009] Using the information provided by the present invention,
reagents such as probes/primers for detecting the SNPs or the
expression of the protein/gene provided herein may be readily
developed and, if desired, incorporated into kit formats such as
nucleic acid arrays, primer extension reactions coupled with mass
spec detection (for SNP detection), or TaqMan PCR assays (Applied
Biosystems, Foster City, Calif.).
[0010] Receptor proteins, particularly members of the cytokine
receptor subfamily, are a major target for drug action and
development. Accordingly, it is valuable to the field of
pharmaceutical development to identify and characterize previously
unknown members of this subfamily of receptor proteins. The present
invention advances the state of the art by providing previously
unidentified human receptor proteins that have homology to members
of the cytokine receptor subfamily.
SUMMARY OF THE INVENTION
[0011] The present invention is based in part on the identification
of amino acid sequences of human receptor peptides and proteins
that are related to the cytokine receptor subfamily, as well as
allelic variants and other mammalian orthologs thereof. These
unique peptide sequences, and nucleic acid sequences that encode
these peptides, can be used as models for the development of human
therapeutic targets, aid in the identification of therapeutic
proteins, and serve as targets for the development of human
therapeutic agents that modulate receptor activity in cells and
tissues that express the receptor. Experimental data as provided in
FIG. 1 indicates expression in the placenta, marrow, liver/spleen,
and in leukocytes.
DESCRIPTION OF THE FIGURE SHEETS
[0012] FIG. 1 provides the nucleotide sequence of a cDNA molecule
that encodes the receptor protein of the present invention. (SEQ ID
NO:1) In addition, structure and functional information is
provided, such as ATG start, stop and tissue distribution, where
available, that allows one to readily determine specific uses of
inventions based on this molecular sequence. Experimental data as
provided in FIG. 1 indicates expression in the placenta, marrow,
liver/spleen, and in leukocytes.
[0013] FIG. 2 provides the predicted amino acid sequence of the
receptor of the present invention. (SEQ ID NO:2) In addition
structure and functional information such as protein family,
function, and modification sites is provided where available,
allowing one to readily determine specific uses of inventions based
on this molecular sequence.
[0014] FIG. 3 provides genomic sequences that span the gene
encoding the receptor protein of the present invention. (SEQ ID
NO:3) In addition structure and functional information, such as
intron/exon structure, promoter location, etc., is provided where
available, allowing one to readily determine specific uses of
inventions based on this molecular sequence. As illustrated in FIG.
3, SNPs were identified at 25 different nucleotide positions.
DETAILED DESCRIPTION OF THE INVENTION
[0015] General Description
[0016] The present invention is based on the sequencing of the
human genome. During the sequencing and assembly of the human
genome, analysis of the sequence information revealed previously
unidentified fragments of the human genome that encode peptides
that share structural and/or sequence homology to
protein/peptide/domains identified and characterized within the art
as being a receptor protein or part of a receptor protein and are
related to the cytokine receptor subfamily. Utilizing these
sequences, additional genomic sequences were assembled and
transcript and/or cDNA sequences were isolated and characterized.
Based on this analysis, the present invention provides amino acid
sequences of human receptor peptides and proteins that are related
to the cytokine receptor subfamily, nucleic acid sequences in the
form of transcript sequences, cDNA sequences and/or genomic
sequences that encode these receptor peptides and proteins, nucleic
acid variation (allelic information), tissue distribution of
expression, and information about the closest art known
protein/peptide/domain that has structural or sequence homology to
the receptor of the present invention.
[0017] In addition to being previously unknown, the peptides that
are provided in the present invention are selected based on their
ability to be used for the development of commercially important
products and services. Specifically, the present peptides are
selected based on homology and/or structural relatedness to known
receptor proteins of the cytokine receptor subfamily and the
expression pattern observed. Experimental data as provided in FIG.
1 indicates expression in the placenta, marrow, liver/spleen, and
in leukocytes. The art has clearly established the commercial
importance of members of this family of proteins and proteins that
have expression patterns similar to that of the present gene. Some
of the more specific features of the peptides of the present
invention, and the uses thereof, are described herein, particularly
in the Background of the Invention and in the annotation provided
in the Figures, and/or are known within the art for each of the
known cytokine family or subfamily of receptor proteins.
[0018] Specific Embodiments
[0019] Peptide Molecules
[0020] The present invention provides nucleic acid sequences that
encode protein molecules that have been identified as being members
of the receptor family of proteins and are related to the cytokine
receptor subfamily (protein sequences are provided in FIG. 2,
transcript/cDNA sequences are provided in FIG. 1 and genomic
sequences are provided in FIG. 3). The peptide sequences provided
in FIG. 2, as well as the obvious variants described herein,
particularly allelic variants as identified herein and using the
information in FIG. 3, will be referred herein as the receptor
peptides of the present invention, receptor peptides, or
peptides/proteins of the present invention.
[0021] The present invention provides isolated peptide and protein
molecules that consist of, consist essentially of, or comprise the
amino acid sequences of the receptor peptides disclosed in the FIG.
2, (encoded by the nucleic acid molecule shown in FIG. 1,
transcript/cDNA or FIG. 3, genomic sequence), as well as all
obvious variants of these peptides that are within the art to make
and use. Some of these variants are described in detail below.
[0022] As used herein, a peptide is said to be "isolated" or
"purified" when it is substantially free of cellular material or
free of chemical precursors or other chemicals. The peptides of the
present invention can be purified to homogeneity or other degrees
of purity. The level of purification will be based on the intended
use. The critical feature is that the preparation allows for the
desired function of the peptide, even if in the presence of
considerable amounts of other components (the features of an
isolated nucleic acid molecule is discussed below).
[0023] In some uses, "substantially free of cellular material"
includes preparations of the peptide having less than about 30% (by
dry weight) other proteins (i.e., contaminating protein), less than
about 20% other proteins, less than about 10% other proteins, or
less than about 5% other proteins. When the peptide is
recombinantly produced, it can also be substantially free of
culture medium, i.e., culture medium represents less than about 20%
of the volume of the protein preparation.
[0024] The language "substantially free of chemical precursors or
other chemicals" includes preparations of the peptide in which it
is separated from chemical precursors or other chemicals that are
involved in its synthesis. In one embodiment, the language
"substantially free of chemical precursors or other chemicals"
includes preparations of the receptor peptide having less than
about 30% (by dry weight) chemical precursors or other chemicals,
less than about 20% chemical precursors or other chemicals, less
than about 10% chemical precursors or other chemicals, or less than
about 5% chemical precursors or other chemicals.
[0025] The isolated receptor peptide can be purified from cells
that naturally express it, purified from cells that have been
altered to express it (recombinant), or synthesized using known
protein synthesis methods. Experimental data as provided in FIG. 1
indicates expression in the placenta, marrow, liver/spleen, and in
leukocytes. For example, a nucleic acid molecule encoding the
receptor peptide is cloned into an expression vector, the
expression vector introduced into a host cell and the protein
expressed in the host cell. The protein can then be isolated from
the cells by an appropriate purification scheme using standard
protein purification techniques. Many of these techniques are
described in detail below.
[0026] Accordingly, the present invention provides proteins that
consist of the amino acid sequences provided in FIG. 2 (SEQ ID
NO:2), for example, proteins encoded by the transcript/cDNA nucleic
acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic
sequences provided in FIG. 3 (SEQ ID NO:3). The amino acid sequence
of such a protein is provided in FIG. 2. A protein consists of an
amino acid sequence when the amino acid sequence is the final amino
acid sequence of the protein.
[0027] The present invention further provides proteins that consist
essentially of the amino acid sequences provided in FIG. 2 (SEQ ID
NO:2), for example, proteins encoded by the transcript/cDNA nucleic
acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic
sequences provided in FIG. 3 (SEQ ID NO:3). A protein consists
essentially of an amino acid sequence when such an amino acid
sequence is present with only a few additional amino acid residues,
for example from about 1 to about 100 or so additional residues,
typically from 1 to about 20 additional residues in the final
protein.
[0028] The present invention further provides proteins that
comprise the amino acid sequences provided in FIG. 2 (SEQ ID NO:2),
for example, proteins encoded by the transcript/cDNA nucleic acid
sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences
provided in FIG. 3 (SEQ ID NO:3). A protein comprises an amino acid
sequence when the amino acid sequence is at least part of the final
amino acid sequence of the protein. In such a fashion, the protein
can be only the peptide or have additional amino acid molecules,
such as amino acid residues (contiguous encoded sequence) that are
naturally associated with it or heterologous amino acid
residues/peptide sequences. Such a protein can have a few
additional amino acid residues or can comprise several hundred or
more additional amino acids. The preferred classes of proteins that
are comprised of the receptor peptides of the present invention are
the naturally occurring mature proteins. A brief description of how
various types of these proteins can be made/isolated is provided
below.
[0029] The receptor peptides of the present invention can be
attached to heterologous sequences to form chimeric or fusion
proteins. Such chimeric and fusion proteins comprise a receptor
peptide operatively linked to a heterologous protein having an
amino acid sequence not substantially homologous to the receptor
peptide. "Operatively linked" indicates that the receptor peptide
and the heterologous protein are fused in-frame. The heterologous
protein can be fused to the N-terminus or C-terminus of the
receptor peptide.
[0030] In some uses, the fusion protein does not affect the
activity of the receptor peptide per se. For example, the fusion
protein can include, but is not limited to, enzymatic fusion
proteins, for example beta-galactosidase fusions, yeast two-hybrid
GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig
fusions. Such fusion proteins, particularly poly-His fusions, can
facilitate the purification of recombinant receptor peptide. In
certain host cells (e.g., mammalian host cells), expression and/or
secretion of a protein can be increased by using a heterologous
signal sequence.
[0031] A chimeric or fusion protein can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for
the different protein sequences are ligated together in-frame in
accordance with conventional techniques. In another embodiment, the
fusion gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and re-amplified to
generate a chimeric gene sequence (see Ausubel et al., Current
Protocols in Molecular Biology, 1992). Moreover, many expression
vectors are commercially available that already encode a fusion
moiety (e.g., a GST protein). A receptor peptide-encoding nucleic
acid can be cloned into such an expression vector such that the
fusion moiety is linked in-frame to the receptor peptide.
[0032] As mentioned above, the present invention also provides and
enables obvious variants of the amino acid sequence of the proteins
of the present invention, such as naturally occurring mature forms
of the peptide, allelic/sequence variants of the peptides,
non-naturally occurring recombinantly derived variants of the
peptides, and orthologs and paralogs of the peptides. Such variants
can readily be generated using art-known techniques in the fields
of recombinant nucleic acid technology and protein biochemistry. It
is understood, however, that variants exclude any amino acid
sequences disclosed prior to the invention.
[0033] Such variants can readily be identified/made using molecular
techniques and the sequence information disclosed herein. Further,
such variants can readily be distinguished from other peptides
based on sequence and/or structural homology to the receptor
peptides of the present invention. The degree of homology/identity
present will be based primarily on whether the peptide is a
functional variant or non-functional variant, the amount of
divergence present in the paralog family and the evolutionary
distance between the orthologs.
[0034] To determine the percent identity of two amino acid
sequences or two nucleic acid sequences, the sequences are aligned
for optimal comparison purposes (e.g., gaps can be introduced in
one or both of a first and a second amino acid or nucleic acid
sequence for optimal alignment and non-homologous sequences can be
disregarded for comparison purposes). In a preferred embodiment, at
least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of
a reference sequence is aligned for comparison purposes. The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position (as used herein
amino acid or nucleic acid "identity" is equivalent to amino acid
or nucleic acid "homology"). The percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences, taking into account the number of gaps, and the
length of each gap, which need to be introduced for optimal
alignment of the two sequences.
[0035] The comparison of sequences and determination of percent
identity and similarity between two sequences can be accomplished
using a mathematical algorithm. (Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a
preferred embodiment, the percent identity between two amino acid
sequences is determined using the Needleman and Wunsch (J. Mol.
Biol. (48):444-453 (1970)) algorithm which has been incorporated
into the GAP program in the GCG software package (available at
http://www.gcg.com), using either a Blossom 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment,
the percent identity between two nucleotide sequences is determined
using the GAP program in the GCG software package (Devereux, J., et
al., Nucleic Acids Res. 12(1):387 (1984)) (available at
http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight
of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or
6. In another embodiment, the percent identity between two amino
acid or nucleotide sequences is determined using the algorithm of
E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been
incorporated into the ALIGN program (version 2.0), using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4.
[0036] The nucleic acid and protein sequences of the present
invention can further be used as a "query sequence" to perform a
search against sequence databases to, for example, identify other
family members or related sequences. Such searches can be performed
using the NBLAST and XBLAST programs (version 2.0) of Altschul, et
al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches
can be performed with the NBLAST program, score=100, wordlength=12
to obtain nucleotide sequences homologous to the nucleic acid
molecules of the invention. BLAST protein searches can be performed
with the XBLAST program, score=50, wordlength=3 to obtain amino
acid sequences homologous to the proteins of the invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al. (Nucleic Acids Res.
25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used.
[0037] Full-length pre-processed forms, as well as mature processed
forms, of proteins that comprise one of the peptides of the present
invention can readily be identified as having complete sequence
identity to one of the receptor peptides of the present invention
as well as being encoded by the same genetic locus as the receptor
peptide provided herein. The gene encoding the novel receptor
protein of the present invention is located on a genome component
that has been mapped to human chromosome 22 (as indicated in FIG.
3), which is supported by multiple lines of evidence, such as STS
and BAC map data.
[0038] Allelic variants of a receptor peptide can readily be
identified as being a human protein having a high degree
(significant) of sequence homology/identity to at least a portion
of the receptor peptide as well as being encoded by the same
genetic locus as the receptor peptide provided herein. Genetic
locus can readily be determined based on the genomic information
provided in FIG. 3, such as the genomic sequence mapped to the
reference human. The gene encoding the novel receptor protein of
the present invention is located on a genome component that has
been mapped to human chromosome 22 (as indicated in FIG. 3), which
is supported by multiple lines of evidence, such as STS and BAC map
data. As used herein, two proteins (or a region of the proteins)
have significant homology when the amino acid sequences are
typically at least about 70-80%, 80-90%, and more typically at
least about 90-95% or more homologous. A significantly homologous
amino acid sequence, according to the present invention, will be
encoded by a nucleic acid sequence that will hybridize to a
receptor peptide encoding nucleic acid molecule under stringent
conditions as more fully described below.
[0039] FIG. 3 provides information on SNPs that have been found in
the gene encoding the receptor protein of the present invention.
SNPs were identified at 25 different nucleotide positions. Some of
these SNPs that are located outside the ORF and in introns may
affect gene expression.
[0040] Paralogs of a receptor peptide can readily be identified as
having some degree of significant sequence homology/identity to at
least a portion of the receptor peptide, as being encoded by a gene
from humans, and as having similar activity or function. Two
proteins will typically be considered paralogs when the amino acid
sequences are typically at least about 60% or greater, and more
typically at least about 70% or greater homology through a given
region or domain. Such paralogs will be encoded by a nucleic acid
sequence that will hybridize to a receptor peptide encoding nucleic
acid molecule under moderate to stringent conditions as more fully
described below.
[0041] Orthologs of a receptor peptide can readily be identified as
having some degree of significant sequence homology/identity to at
least a portion of the receptor peptide as well as being encoded by
a gene from another organism. Preferred orthologs will be isolated
from mammals, preferably primates, for the development of human
therapeutic targets and agents. Such orthologs will be encoded by a
nucleic acid sequence that will hybridize to a receptor peptide
encoding nucleic acid molecule under moderate to stringent
conditions, as more fully described below, depending on the degree
of relatedness of the two organisms yielding the proteins.
[0042] Non-naturally occurring variants of the receptor peptides of
the present invention can readily be generated using recombinant
techniques. Such variants include, but are not limited to
deletions, additions and substitutions in the amino acid sequence
of the receptor peptide. For example, one class of substitutions
are conserved amino acid substitution. Such substitutions are those
that substitute a given amino acid in a receptor peptide by another
amino acid of like characteristics. Typically seen as conservative
substitutions are the replacements, one for another, among the
aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the
hydroxyl residues Ser and Thr; exchange of the acidic residues Asp
and Glu; substitution between the amide residues Asn and Gln;
exchange of the basic residues Lys and Arg; and replacements among
the aromatic residues Phe and Tyr. Guidance concerning which amino
acid changes are likely to be phenotypically silent are found in
Bowie et al., Science 247:1306-1310 (1990).
[0043] Variant receptor peptides can be fully functional or can
lack function in one or more activities, e.g. ability to bind
substrate, ability to phosphorylate substrate, ability to mediate
signaling, etc. Fully functional variants typically contain only
conservative variation or variation in non-critical residues or in
non-critical regions. FIG. 2 provides the result of protein
analysis and can be used to identify critical domains/regions.
Functional variants can also contain substitution of similar amino
acids that result in no change or an insignificant change in
function. Alternatively, such substitutions may positively or
negatively affect function to some degree.
[0044] Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, or truncation or a substitution, insertion, inversion,
or deletion in a critical residue or critical region.
[0045] Amino acids that are essential for function can be
identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham et al.,
Science 244:1081-1085 (1989)), particularly using the results
provided in FIG. 2. The latter procedure introduces single alanine
mutations at every residue in the molecule. The resulting mutant
molecules are then tested for biological activity such as receptor
activity or in assays such as an in vitro proliferative activity.
Sites that are critical for binding partner/substrate binding can
also be determined by structural analysis such as crystallization,
nuclear magnetic resonance or photoaffinity labeling (Smith et al.,
J. Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312
(1992)).
[0046] The present invention further provides fragments of the
receptor peptides, in addition to proteins and peptides that
comprise and consist of such fragments, particularly those
comprising the residues identified in FIG. 2. The fragments to
which the invention pertains, however, are not to be construed as
encompassing fragments that may be disclosed publicly prior to the
present invention.
[0047] As used herein, a fragment comprises at least 8, 10, 12, 14,
16, or more contiguous amino acid residues from a receptor peptide.
Such fragments can be chosen based on the ability to retain one or
more of the biological activities of the receptor peptide or could
be chosen for the ability to perform a function, e.g. bind a
substrate or act as an immunogen. Particularly important fragments
are biologically active fragments, peptides that are, for example,
about 8 or more amino acids in length. Such fragments will
typically comprise a domain or motif of the receptor peptide, e.g.,
active site, a transmembrane domain or a substrate-binding domain.
Further, possible fragments include, but are not limited to, domain
or motif containing fragments, soluble peptide fragments, and
fragments containing immunogenic structures. Predicted domains and
functional sites are readily identifiable by computer programs well
known and readily available to those of skill in the art (e.g.,
PROSITE analysis). The results of one such analysis are provided in
FIG. 2.
[0048] Polypeptides often contain amino acids other than the 20
amino acids commonly referred to as the 20 naturally occurring
amino acids. Further, many amino acids, including the terminal
amino acids, may be modified by natural processes, such as
processing and other post-translational modifications, or by
chemical modification techniques well known in the art. Common
modifications that occur naturally in receptor peptides are
described in basic texts, detailed monographs, and the research
literature, and they are well known to those of skill in the art
(some of these features are identified in FIG. 2).
[0049] Known modifications include, but are not limited to,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
crosslinks, formation of cystine, formation of pyroglutamate,
formylation, gamma carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
[0050] Such modifications are well known to those of skill in the
art and have been described in great detail in the scientific
literature. Several particularly common modifications,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation, for
instance, are described in most basic texts, such as
Proteins--Structure and Molecular Properties, 2nd Ed., T. E.
Creighton, W. H. Freeman and Company, New York (1993). Many
detailed reviews are available on this subject, such as by Wold,
F., Posttranslational Covalent Modification of Proteins, B. C.
Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al.
(Meth. Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N. Y
Acad. Sci. 663:48-62 (1992)).
[0051] Accordingly, the receptor peptides of the present invention
also encompass derivatives or analogs in which a substituted amino
acid residue is not one encoded by the genetic code, in which a
substituent group is included, in which the mature receptor peptide
is fused with another compound, such as a compound to increase the
half-life of the receptor peptide (for example, polyethylene
glycol), or in which the additional amino acids are fused to the
mature receptor peptide, such as a leader or secretory sequence or
a sequence for purification of the mature receptor peptide or a
pro-protein sequence.
[0052] Protein/Peptide Uses
[0053] The proteins of the present invention can be used in
substantial and specific assays related to the functional
information provided in the Figures; to raise antibodies or to
elicit another immune response; as a reagent (including the labeled
reagent) in assays designed to quantitatively determine levels of
the protein (or its binding partner or ligand) in biological
fluids; and as markers for tissues in which the corresponding
protein is preferentially expressed (either constitutively or at a
particular stage of tissue differentiation or development or in a
disease state). Where the protein binds or potentially binds to
another protein or ligand (such as, for example, in a
receptor-effector protein interaction or receptor-ligand
interaction), the protein can be used to identify the binding
partner/ligand so as to develop a system to identify inhibitors of
the binding interaction. Any or all of these uses are capable of
being developed into reagent grade or kit format for
commercialization as commercial products.
[0054] Methods for performing the uses listed above are well known
to those skilled in the art. References disclosing such methods
include "Molecular Cloning: A Laboratory Manual", 2d ed., Cold
Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T.
Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular
Cloning Techniques", Academic Press, Berger, S. L. and A. R. Kimmel
eds., 1987.
[0055] The potential uses of the peptides of the present invention
are based primarily on the source of the protein as well as the
class/action of the protein. For example, receptors isolated from
humans and their human/mammalian orthologs serve as targets for
identifying agents for use in mammalian therapeutic applications,
e.g. a human drug, particularly in modulating a biological or
pathological response in a cell or tissue that expresses the
receptor. Experimental data as provided in FIG. 1 indicates that
receptor proteins of the present invention are expressed in the
placenta, marrow, and liver/spleen, as indicated by virtual
northern blot analysis. In addition, PCR-based tissue screening
panels indicates expression in human leukocytes. A large percentage
of pharmaceutical agents are being developed that modulate the
activity of receptor proteins, particularly members of the cytokine
subfamily (see Background of the Invention). The structural and
functional information provided in the Background and Figures
provide specific and substantial uses for the molecules of the
present invention, particularly in combination with the expression
information provided in FIG. 1. Experimental data as provided in
FIG. 1 indicates expression in the placenta, marrow, liver/spleen,
and in leukocytes. Such uses can readily be determined using the
information provided herein, that which is known in the art, and
routine experimentation.
[0056] The proteins of the present invention (including variants
and fragments that may have been disclosed prior to the present
invention) are useful for biological assays related to receptors
that are related to members of the cytokine subfamily. Such assays
involve any of the known receptor functions or activities or
properties useful for diagnosis and treatment of receptor-related
conditions that are specific for the subfamily of receptors that
the one of the present invention belongs to, particularly in cells
and tissues that express the receptor. Experimental data as
provided in FIG. 1 indicates that receptor proteins of the present
invention are expressed in the placenta, marrow, and liver/spleen,
as indicated by virtual northern blot analysis. In addition,
PCR-based tissue screening panels indicates expression in human
leukocytes.
[0057] The proteins of the present invention are also useful in
drug screening assays, in cell-based or cell-free systems.
Cell-based systems can be native, i.e., cells that normally express
the receptor, as a biopsy or expanded in cell culture. Experimental
data as provided in FIG. 1 indicates expression in the placenta,
marrow, liver/spleen, and in leukocytes. In an alternate
embodiment, cell-based assays involve recombinant host cells
expressing the receptor protein.
[0058] The polypeptides can be used to identify compounds that
modulate receptor activity of the protein in its natural state or
an altered form that causes a specific disease or pathology
associated with the receptor. Both the receptors of the present
invention and appropriate variants and fragments can be used in
high-throughput screens to assay candidate compounds for the
ability to bind to the receptor. These compounds can be further
screened against a functional receptor to determine the effect of
the compound on the receptor activity. Further, these compounds can
be tested in animal or invertebrate systems to determine
activity/effectiveness. Compounds can be identified that activate
(agonist) or inactivate (antagonist) the receptor to a desired
degree.
[0059] Further, the proteins of the present invention can be used
to screen a compound for the ability to stimulate or inhibit
interaction between the receptor protein and a molecule that
normally interacts with the receptor protein, e.g. an extracellular
binding ligand or a component of the signal pathway that the
receptor protein normally interacts (for example, a cytosolic
signal protein). Such assays typically include the steps of
combining the receptor protein with a candidate compound under
conditions that allow the receptor protein, or fragment, to
interact with the target molecule, and to detect the formation of a
complex between the protein and the target or to detect the
biochemical consequence of the interaction with the receptor
protein and the target, such as any of the associated effects of
signal transduction such as protein phosphorylation, cAMP turnover,
and adenylate cyclase activation, etc.
[0060] Candidate compounds include, for example, 1) peptides such
as soluble peptides, including Ig-tailed fusion peptides and
members of random peptide libraries (see, e.g., Lam et al, Nature
354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and
combinatorial chemistry-derived molecular libraries made of D-
and/or L-configuration amino acids; 2) phosphopeptides (e.g.,
members of random and partially degenerate, directed phosphopeptide
libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3)
antibodies (e.g., polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric, and single chain antibodies as well as
Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[0061] One candidate compound is a soluble fragment of the receptor
that competes for substrate binding. Other candidate compounds
include mutant receptors or appropriate fragments containing
mutations that affect receptor function and thus compete for
substrate. Accordingly, a fragment that competes for substrate, for
example with a higher affinity, or a fragment that binds substrate
but does not allow release, is encompassed by the invention.
[0062] The invention further includes other end point assays to
identify compounds that modulate (stimulate or inhibit) receptor
activity. The assays typically involve an assay of events in the
signal transduction pathway that indicate receptor activity. Thus,
the phosphorylation of a substrate, activation of a protein, a
change in the expression of genes that are up- or down-regulated in
response to the receptor protein dependent signal cascade can be
assayed.
[0063] Any of the biological or biochemical functions mediated by
the receptor can be used as an endpoint assay. These include all of
the biochemical or biochemical/biological events described herein,
in the references cited herein, incorporated by reference for these
endpoint assay targets, and other functions known to those of
ordinary skill in the art or that can be readily identified using
the information provided in the Figures, particularly FIG. 2.
Specifically, a biological function of a cell or tissues that
expresses the receptor can be assayed. Experimental data as
provided in FIG. 1 indicates that receptor proteins of the present
invention are expressed in the placenta, marrow, and liver/spleen,
as indicated by virtual northern blot analysis. In addition,
PCR-based tissue screening panels indicates expression in human
leukocytes.
[0064] Binding and/or activating compounds can also be screened by
using chimeric receptor proteins in which the amino terminal
extracellular domain, or parts thereof, the entire transmembrane
domain or subregions, such as any of the seven transmembrane
segments or any of the intracellular or extracellular loops and the
carboxy terminal intracellular domain, or parts thereof, can be
replaced by heterologous domains or subregions. For example, a
substrate-binding region can be used that interacts with a
different substrate then that which is recognized by the native
receptor. Accordingly, a different set of signal transduction
components is available as an end-point assay for activation. This
allows for assays to be performed in other than the specific host
cell from which the receptor is derived.
[0065] The proteins of the present invention are also useful in
competition binding assays in methods designed to discover
compounds that interact with the receptor (e.g. binding partners
and/or ligands). Thus, a compound is exposed to a receptor
polypeptide under conditions that allow the compound to bind or to
otherwise interact with the polypeptide. Soluble receptor
polypeptide is also added to the mixture. If the test compound
interacts with the soluble receptor polypeptide, it decreases the
amount of complex formed or activity from the receptor target. This
type of assay is particularly useful in cases in which compounds
are sought that interact with specific regions of the receptor.
Thus, the soluble polypeptide that competes with the target
receptor region is designed to contain peptide sequences
corresponding to the region of interest.
[0066] To perform cell free drug screening assays, it is sometimes
desirable to immobilize either the receptor protein, or fragment,
or its target molecule to facilitate separation of complexes from
uncomplexed forms of one or both of the proteins, as well as to
accommodate automation of the assay.
[0067] Techniques for immobilizing proteins on matrices can be used
in the drug screening assays. In one embodiment, a fusion protein
can be provided which adds a domain that allows the protein to be
bound to a matrix. For example, glutathione-S-transferase fusion
proteins can be adsorbed onto glutathione sepharose beads (Sigma
Chemical, St. Louis, Mo.) or glutathione derivatized microtitre
plates, which are then combined with the cell lysates (e.g.,
.sup.35S-labeled) and the candidate compound, and the mixture
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads are washed to remove any unbound label, and the matrix
immobilized and radiolabel determined directly, or in the
supernatant after the complexes are dissociated. Alternatively, the
complexes can be dissociated from the matrix, separated by
SDS-PAGE, and the level of receptor-binding protein found in the
bead fraction quantitated from the gel using standard
electrophoretic techniques. For example, either the polypeptide or
its target molecule can be immobilized utilizing conjugation of
biotin and streptavidin using techniques well known in the art.
Alternatively, antibodies reactive with the protein but which do
not interfere with binding of the protein to its target molecule
can be derivatized to the wells of the plate, and the protein
trapped in the wells by antibody conjugation. Preparations of a
receptor-binding protein and a candidate compound are incubated in
the receptor protein-presenting wells and the amount of complex
trapped in the well can be quantitated. Methods for detecting such
complexes, in addition to those described above for the
GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with the receptor protein target
molecule, or which are reactive with receptor protein and compete
with the target molecule, as well as enzyme-linked assays which
rely on detecting an enzymatic activity associated with the target
molecule.
[0068] Agents that modulate one of the receptors of the present
invention can be identified using one or more of the above assays,
alone or in combination. It is generally preferable to use a
cell-based or cell free system first and then confirm activity in
an animal or other model system. Such model systems are well known
in the art and can readily be employed in this context.
[0069] Modulators of receptor protein activity identified according
to these drug screening assays can be used to treat a subject with
a disorder mediated by the receptor pathway, by treating cells or
tissues that express the receptor. Experimental data as provided in
FIG. 1 indicates expression in the placenta, marrow, liver/spleen,
and in leukocytes. These methods of treatment include the steps of
administering a modulator of receptor activity in a pharmaceutical
composition to a subject in need of such treatment, the modulator
being identified as described herein.
[0070] In yet another aspect of the invention, the receptor
proteins can be used as "bait proteins" in a two-hybrid assay or
three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et
al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300),
to identify other proteins, which bind to or interact with the
receptor and are involved in receptor activity. Such
receptor-binding proteins are also likely to be involved in the
propagation of signals by the receptor proteins or receptor targets
as, for example, downstream elements of a receptor-mediated
signaling pathway. Alternatively, such receptor-binding proteins
are likely to be receptor inhibitors.
[0071] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for a receptor
protein is fused to a gene encoding the DNA binding domain of a
known transcription factor (e.g., GAL-4). In the other construct, a
DNA sequence, from a library of DNA sequences, that encodes an
unidentified protein ("prey" or "sample") is fused to a gene that
codes for the activation domain of the known transcription factor.
If the "bait" and the "prey" proteins are able to interact, in
vivo, forming a receptor-dependent complex, the DNA-binding and
activation domains of the transcription factor are brought into
close proximity. This proximity allows transcription of a reporter
gene (e.g., LacZ) which is operably linked to a transcriptional
regulatory site responsive to the transcription factor. Expression
of the reporter gene can be detected and cell colonies containing
the functional transcription factor can be isolated and used to
obtain the cloned gene which encodes the protein which interacts
with the receptor protein.
[0072] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein in an appropriate animal model. For example, an
agent identified as described herein (e.g., a receptor-modulating
agent, an antisense receptor nucleic acid molecule, a
receptor-specific antibody, or a receptor-binding partner) can be
used in an animal or other model to determine the efficacy,
toxicity, or side effects of treatment with such an agent.
Alternatively, an agent identified as described herein can be used
in an animal or other model to determine the mechanism of action of
such an agent. Furthermore, this invention pertains to uses of
novel agents identified by the above-described screening assays for
treatments as described herein.
[0073] The receptor proteins of the present invention are also
useful to provide a target for diagnosing a disease or
predisposition to disease mediated by the peptide. Accordingly, the
invention provides methods for detecting the presence, or levels
of, the protein (or encoding mRNA) in a cell, tissue, or organism.
Experimental data as provided in FIG. 1 indicates expression in the
placenta, marrow, liver/spleen, and in leukocytes. The method
involves contacting a biological sample with a compound capable of
interacting with the receptor protein such that the interaction can
be detected. Such an assay can be provided in a single detection
format or a multi-detection format such as an antibody chip
array.
[0074] One agent for detecting a protein in a sample is an antibody
capable of selectively binding to protein. A biological sample
includes tissues, cells and biological fluids isolated from a
subject, as well as tissues, cells and fluids present within a
subject.
[0075] The peptides of the present invention also provide targets
for diagnosing active protein activity, disease, or predisposition
to disease, in a patient having a variant peptide, particularly
activities and conditions that are known for other members of the
family of proteins to which the present one belongs. Thus, the
peptide can be isolated from a biological sample and assayed for
the presence of a genetic mutation that results in aberrant
peptide. This includes amino acid substitution, deletion,
insertion, rearrangement, (as the result of aberrant splicing
events), and inappropriate post-translational modification.
Analytic methods include altered electrophoretic mobility, altered
tryptic peptide digest, altered receptor activity in cell-based or
cell-free assay, alteration in substrate or antibody-binding
pattern, altered isoelectric point, direct amino acid sequencing,
and any other of the known assay techniques useful for detecting
mutations in a protein. Such an assay can be provided in a single
detection format or a multi-detection format such as an antibody
chip array.
[0076] In vitro techniques for detection of peptide include enzyme
linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence using a detection
reagent, such as an antibody or protein binding agent.
Alternatively, the peptide can be detected in vivo in a subject by
introducing into the subject a labeled anti-peptide antibody or
other types of detection agent. For example, the antibody can be
labeled with a radioactive marker whose presence and location in a
subject can be detected by standard imaging techniques.
Particularly useful are methods that detect the allelic variant of
a peptide expressed in a subject and methods which detect fragments
of a peptide in a sample.
[0077] The peptides are also useful in pharmacogenomic analysis.
Pharmacogenomics deal with clinically significant hereditary
variations in the response to drugs due to altered drug disposition
and abnormal action in affected persons. See, e.g., Eichelbaum, M.
(Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996)), and
Linder, M. W. (Clin. Chem. 43(2):254-266 (1997)). The clinical
outcomes of these variations result in severe toxicity of
therapeutic drugs in certain individuals or therapeutic failure of
drugs in certain individuals as a result of individual variation in
metabolism. Thus, the genotype of the individual can determine the
way a therapeutic compound acts on the body or the way the body
metabolizes the compound. Further, the activity of drug
metabolizing enzymes effects both the intensity and duration of
drug action. Thus, the pharmacogenomics of the individual permit
the selection of effective compounds and effective dosages of such
compounds for prophylactic or therapeutic treatment based on the
individual's genotype. The discovery of genetic polymorphisms in
some drug metabolizing enzymes has explained why some patients do
not obtain the expected drug effects, show an exaggerated drug
effect, or experience serious toxicity from standard drug dosages.
Polymorphisms can be expressed in the phenotype of the extensive
metabolizer and the phenotype of the poor metabolizer. Accordingly,
genetic polymorphism may lead to allelic protein variants of the
receptor protein in which one or more of the receptor functions in
one population is different from those in another population. The
peptides thus allow a target to ascertain a genetic predisposition
that can affect treatment modality. Thus, in a ligand-based
treatment, polymorphism may give rise to amino terminal
extracellular domains and/or other substrate-binding regions that
are more or less active in substrate binding, and receptor
activation. Accordingly, substrate dosage would necessarily be
modified to maximize the therapeutic effect within a given
population containing a polymorphism. As an alternative to
genotyping, specific polymorphic peptides could be identified.
[0078] The peptides are also useful for treating a disorder
characterized by an absence of, inappropriate, or unwanted
expression of the protein. Experimental data as provided in FIG. 1
indicates expression in the placenta, marrow, liver/spleen, and in
leukocytes. Accordingly, methods for treatment include the use of
the receptor protein or fragments.
[0079] Antibodies
[0080] The invention also provides antibodies that selectively bind
to one of the peptides of the present invention, a protein
comprising such a peptide, as well as variants and fragments
thereof. As used herein, an antibody selectively binds a target
peptide when it binds the target peptide and does not significantly
bind to unrelated proteins. An antibody is still considered to
selectively bind a peptide even if it also binds to other proteins
that are not substantially homologous with the target peptide so
long as such proteins share homology with a fragment or domain of
the peptide target of the antibody. In this case, it would be
understood that antibody binding to the peptide is still selective
despite some degree of cross-reactivity.
[0081] As used herein, an antibody is defined in terms consistent
with that recognized within the art: they are multi-subunit
proteins produced by a mammalian organism in response to an antigen
challenge. The antibodies of the present invention include
polyclonal antibodies and monoclonal antibodies, as well as
fragments of such antibodies, including, but not limited to, Fab or
F(ab').sub.2, and Fv fragments.
[0082] Many methods are known for generating and/or identifying
antibodies to a given target peptide. Several such methods are
described by Harlow, Antibodies, Cold Spring Harbor Press,
(1989).
[0083] In general, to generate antibodies, an isolated peptide is
used as an immunogen and is administered to a mammalian organism,
such as a rat, rabbit or mouse. The full-length protein, an
antigenic peptide fragment or a fusion protein can be used.
Particularly important fragments are those covering functional
domains, such as the domains identified in FIG. 2, and domain of
sequence homology or divergence amongst the family, such as those
that can readily be identified using protein alignment methods and
as presented in the Figures.
[0084] Antibodies are preferably prepared from regions or discrete
fragments of the receptor proteins. Antibodies can be prepared from
any region of the peptide as described herein. However, preferred
regions will include those involved in function/activity and/or
receptor/binding partner interaction. FIG. 2 can be used to
identify particularly important regions while sequence alignment
can be used to identify conserved and unique sequence
fragments.
[0085] An antigenic fragment will typically comprise at least 8
contiguous amino acid residues. The antigenic peptide can comprise,
however, at least 10, 12, 14, 16 or more amino acid residues. Such
fragments can be selected on a physical property, such as fragments
correspond to regions that are located on the surface of the
protein, e.g., hydrophilic regions or can be selected based on
sequence uniqueness (see FIG. 2).
[0086] Detection on an antibody of the present invention can be
facilitated by coupling (i.e., physically linking) the antibody to
a detectable substance. Examples of detectable substances include
various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, and radioactive
materials. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0087] Antibody Uses
[0088] The antibodies can be used to isolate one of the proteins of
the present invention by standard techniques, such as affinity
chromatography or immunoprecipitation. The antibodies can
facilitate the purification of the natural protein from cells and
recombinantly produced protein expressed in host cells. In
addition, such antibodies are useful to detect the presence of one
of the proteins of the present invention in cells or tissues to
determine the pattern of expression of the protein among various
tissues in an organism and over the course of normal development.
Experimental data as provided in FIG. 1 indicates that receptor
proteins of the present invention are expressed in the placenta,
marrow, and liver/spleen, as indicated by virtual northern blot
analysis. In addition, PCR-based tissue screening panels indicates
expression in human leukocytes. Further, such antibodies can be
used to detect protein in situ, in vitro, or in a cell lysate or
supernatant in order to evaluate the abundance and pattern of
expression. Also, such antibodies can be used to assess abnormal
tissue distribution or abnormal expression during development or
progression of a biological condition. Antibody detection of
circulating fragments of the full length protein can be used to
identify turnover.
[0089] Further, the antibodies can be used to assess expression in
disease states such as in active stages of the disease or in an
individual with a predisposition toward disease related to the
protein's function. When a disorder is caused by an inappropriate
tissue distribution, developmental expression, level of expression
of the protein, or expressed/processed form, the antibody can be
prepared against the normal protein. Experimental data as provided
in FIG. 1 indicates expression in the placenta, marrow,
liver/spleen, and in leukocytes. If a disorder is characterized by
a specific mutation in the protein, antibodies specific for this
mutant protein can be used to assay for the presence of the
specific mutant protein.
[0090] The antibodies can also be used to assess normal and
aberrant subcellular localization of cells in the various tissues
in an organism. Experimental data as provided in FIG. 1 indicates
expression in the placenta, marrow, liver/spleen, and in
leukocytes. The diagnostic uses can be applied, not only in genetic
testing, but also in monitoring a treatment modality. Accordingly,
where treatment is ultimately aimed at correcting expression level
or the presence of aberrant sequence and aberrant tissue
distribution or developmental expression, antibodies directed
against the protein or relevant fragments can be used to monitor
therapeutic efficacy.
[0091] Additionally, antibodies are useful in pharmacogenomic
analysis. Thus, antibodies prepared against polymorphic proteins
can be used to identify individuals that require modified treatment
modalities. The antibodies are also useful as diagnostic tools as
an immunological marker for aberrant protein analyzed by
electrophoretic mobility, isoelectric point, tryptic peptide
digest, and other physical assays known to those in the art.
[0092] The antibodies are also useful for tissue typing.
Experimental data as provided in FIG. 1 indicates expression in the
placenta, marrow, liver/spleen, and in leukocytes. Thus, where a
specific protein has been correlated with expression in a specific
tissue, antibodies that are specific for this protein can be used
to identify a tissue type.
[0093] The antibodies are also useful for inhibiting protein
function, for example, blocking the binding of the receptor peptide
to a binding partner such as a substrate. These uses can also be
applied in a therapeutic context in which treatment involves
inhibiting the protein's function. An antibody can be used, for
example, to block binding, thus modulating (agonizing or
antagonizing) the peptides activity. Antibodies can be prepared
against specific fragments containing sites required for function
or against intact protein that is associated with a cell or cell
membrane. See FIG. 2 for structural information relating to the
proteins of the present invention.
[0094] The invention also encompasses kits for using antibodies to
detect the presence of a protein in a biological sample. The kit
can comprise antibodies such as a labeled or labelable antibody and
a compound or agent for detecting protein in a biological sample;
means for determining the amount of protein in the sample; means
for comparing the amount of protein in the sample with a standard;
and instructions for use. Such a kit can be supplied to detect a
single protein or epitope or can be configured to detect one of a
multitude of epitopes, such as in an antibody detection array.
Arrays are described in detail below for nuleic acid arrays and
similar methods have been developed for antibody arrays.
[0095] Nucleic Acid Molecules
[0096] The present invention further provides isolated nucleic acid
molecules that encode a receptor peptide or protein of the present
invention (cDNA, transcript and genomic sequence). Such nucleic
acid molecules will consist of, consist essentially of, or comprise
a nucleotide sequence that encodes one of the receptor peptides of
the present invention, an allelic variant thereof, or an ortholog
or paralog thereof.
[0097] As used herein, an "isolated" nucleic acid molecule is one
that is separated from other nucleic acid present in the natural
source of the nucleic acid. Preferably, an "isolated" nucleic acid
is free of sequences which naturally flank the nucleic acid (i.e.,
sequences located at the 5' and 3' ends of the nucleic acid) in the
genomic DNA of the organism from which the nucleic acid is derived.
However, there can be some flanking nucleotide sequences, for
example up to about 5KB, 4KB, 3KB, 2KB, or 1KB or less,
particularly contiguous peptide encoding sequences and peptide
encoding sequences within the same gene but separated by introns in
the genomic sequence. The important point is that the nucleic acid
is isolated from remote and unimportant flanking sequences such
that it can be subjected to the specific manipulations described
herein such as recombinant expression, preparation of probes and
primers, and other uses specific to the nucleic acid sequences.
[0098] Moreover, an "isolated" nucleic acid molecule, such as a
transcript/cDNA molecule, can be substantially free of other
cellular material, or culture medium when produced by recombinant
techniques, or chemical precursors or other chemicals when
chemically synthesized. However, the nucleic acid molecule can be
fused to other coding or regulatory sequences and still be
considered isolated.
[0099] For example, recombinant DNA molecules contained in a vector
are considered isolated. Further examples of isolated DNA molecules
include recombinant DNA molecules maintained in heterologous host
cells or purified (partially or substantially) DNA molecules in
solution. Isolated RNA molecules include in vivo or in vitro RNA
transcripts of the isolated DNA molecules of the present invention.
Isolated nucleic acid molecules according to the present invention
further include such molecules produced synthetically.
[0100] Accordingly, the present invention provides nucleic acid
molecules that consist of the nucleotide sequence shown in FIG. 1
or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic
sequence), or any nucleic acid molecule that encodes the protein
provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists
of a nucleotide sequence when the nucleotide sequence is the
complete nucleotide sequence of the nucleic acid molecule.
[0101] The present invention further provides nucleic acid
molecules that consist essentially of the nucleotide sequence shown
in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3,
genomic sequence), or any nucleic acid molecule that encodes the
protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule
consists essentially of a nucleotide sequence when such a
nucleotide sequence is present with only a few additional nucleic
acid residues in the final nucleic acid molecule.
[0102] The present invention further provides nucleic acid
molecules that comprise the nucleotide sequences shown in FIG. 1 or
3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic
sequence), or any nucleic acid molecule that encodes the protein
provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule comprises
a nucleotide sequence when the nucleotide sequence is at least part
of the final nucleotide sequence of the nucleic acid molecule. In
such a fashion, the nucleic acid molecule can be only the
nucleotide sequence or have additional nucleic acid residues, such
as nucleic acid residues that are naturally associated with it or
heterologous nucleotide sequences. Such a nucleic acid molecule can
have a few additional nucleotides or can comprises several hundred
or more additional nucleotides. A brief description of how various
types of these nucleic acid molecules can be readily made/isolated
is provided below.
[0103] In FIGS. 1 and 3, both coding and non-coding sequences are
provided. Because of the source of the present invention, humans
genomic sequence (FIG. 3) and cDNA/transcript sequences (FIG. 1),
the nucleic acid molecules in the Figures will contain genomic
intronic sequences, 5' and 3' non-coding sequences, gene regulatory
regions and non-coding intergenic sequences. In general such
sequence features are either noted in FIGS. 1 and 3 or can readily
be identified using computational tools known in the art. As
discussed below, some of the non-coding regions, particularly gene
regulatory elements such as promoters, are useful for a variety of
purposes, e.g. control of heterologous gene expression, target for
identifying gene activity modulating compounds, and are
particularly claimed as fragments of the genomic sequence provided
herein.
[0104] The isolated nucleic acid molecules can encode the mature
protein plus additional amino or carboxyl-terminal amino acids, or
amino acids interior to the mature peptide (when the mature form
has more than one peptide chain, for instance). Such sequences may
play a role in processing of a protein from precursor to a mature
form, facilitate protein trafficking, prolong or shorten protein
half-life or facilitate manipulation of a protein for assay or
production among other things. As generally is the case in situ,
the additional amino acids may be processed away from the mature
protein by cellular enzymes.
[0105] As mentioned above, the isolated nucleic acid molecules
include, but are not limited to, the sequence encoding the receptor
peptide alone, the sequence encoding the mature peptide and
additional coding sequences, such as a leader or secretory sequence
(e.g., a pre-pro or pro-protein sequence), the sequence encoding
the mature peptide, with or without the additional coding
sequences, plus additional non-coding sequences, for example
introns and non-coding 5' and 3' sequences such as transcribed but
non-translated sequences that play a role in transcription, mRNA
processing (including splicing and polyadenylation signals),
ribosome binding and stability of mRNA. In addition, the nucleic
acid molecule may be fused to a marker sequence encoding, for
example, a peptide that facilitates purification.
[0106] Isolated nucleic acid molecules can be in the form of RNA,
such as mRNA, or in the form DNA, including cDNA and genomic DNA
obtained by cloning or produced by chemical synthetic techniques or
by a combination thereof. The nucleic acid, especially DNA, can be
double-stranded or single-stranded. Single-stranded nucleic acid
can be the coding strand (sense strand) or the non-coding strand
(anti-sense strand).
[0107] The invention further provides nucleic acid molecules that
encode fragments of the peptides of the present invention as well
as nucleic acid molecules that encode obvious variants of the
receptor proteins of the present invention that are described
above. Such nucleic acid molecules may be naturally occurring, such
as allelic variants (same locus), paralogs (different locus), and
orthologs (different organism), or may be constructed by
recombinant DNA methods or by chemical synthesis. Such
non-naturally occurring variants may be made by mutagenesis
techniques, including those applied to nucleic acid molecules,
cells, or organisms. Accordingly, as discussed above, the variants
can contain nucleotide substitutions, deletions, inversions and
insertions. Variation can occur in either or both the coding and
non-coding regions. The variations can produce both conservative
and non-conservative amino acid substitutions.
[0108] The present invention further provides non-coding fragments
of the nucleic acid molecules provided in FIGS. 1 and 3. Preferred
non-coding fragments include, but are not limited to, promoter
sequences, enhancer sequences, gene modulating sequences and gene
termination sequences. Such fragments are useful in controlling
heterologous gene expression and in developing screens to identify
gene-modulating agents. A promoter can readily be identified as
being 5' to the ATG start site in the genomic sequence provided in
FIG. 3.
[0109] A fragment comprises a contiguous nucleotide sequence
greater than 12 or more nucleotides. Further, a fragment could at
least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length
of the fragment will be based on its intended use. For example, the
fragment can encode epitope bearing regions of the peptide, or can
be useful as DNA probes and primers. Such fragments can be isolated
using the known nucleotide sequence to synthesize an
oligonucleotide probe. A labeled probe can then be used to screen a
cDNA library, genomic DNA library, or mRNA to isolate nucleic acid
corresponding to the coding region. Further, primers can be used in
PCR reactions to clone specific regions of gene.
[0110] A probe/primer typically comprises substantially a purified
oligonucleotide or oligonucleotide pair. The oligonucleotide
typically comprises a region of nucleotide sequence that hybridizes
under stringent conditions to at least about 12, 20, 25, 40, 50 or
more consecutive nucleotides.
[0111] Orthologs, homologs, and allelic variants can be identified
using methods well known in the art. As described in the Peptide
Section, these variants comprise a nucleotide sequence encoding a
peptide that is typically 60-70%, 70-80%, 80-90%, and more
typically at least about 90-95% or more homologous to the
nucleotide sequence shown in the Figure sheets or a fragment of
this sequence. Such nucleic acid molecules can readily be
identified as being able to hybridize under moderate to stringent
conditions, to the nucleotide sequence shown in the Figure sheets
or a fragment of the sequence. Allelic variants can readily be
determined by genetic locus of the encoding gene. The gene encoding
the novel receptor protein of the present invention is located on a
genome component that has been mapped to human chromosome 22 (as
indicated in FIG. 3), which is supported by multiple lines of
evidence, such as STS and BAC map data.
[0112] FIG. 3 provides information on SNPs that have been found in
the gene encoding the receptor protein of the present invention.
SNPs were identified at 25 different nucleotide positions. Some of
these SNPs that are located outside the ORF and in introns may
affect gene expression.
[0113] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences encoding a peptide at
least 60-70% homologous to each other typically remain hybridized
to each other. The conditions can be such that sequences at least
about 60%, at least about 70%, or at least about 80% or more
homologous to each other typically remain hybridized to each other.
Such stringent conditions are known to those skilled in the art and
can be found in Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent
hybridization conditions are hybridization in 6.times.sodium
chloride/sodium citrate (SSC) at about 45 C., followed by one or
more washes in 0.2.times.SSC, 0.1% SDS at 50-65 C. Examples of
moderate to low stringency hybridization conditions are well known
in the art.
[0114] Nucleic Acid Molecule Uses
[0115] The nucleic acid molecules of the present invention are
useful for probes, primers, chemical intermediates, and in
biological assays. The nucleic acid molecules are useful as a
hybridization probe for messenger RNA, transcript/cDNA and genomic
DNA to isolate full-length cDNA and genomic clones encoding the
peptide described in FIG. 2 and to isolate cDNA and genomic clones
that correspond to variants (alleles, orthologs, etc.) producing
the same or related peptides shown in FIG. 2. As illustrated in
FIG. 3, SNPs were identified at 25 different nucleotide
positions.
[0116] The probe can correspond to any sequence along the entire
length of the nucleic acid molecules provided in the Figures.
Accordingly, it could be derived from 5' noncoding regions, the
coding region, and 3' noncoding regions. However, as discussed,
fragments are not to be construed as encompassing fragments
disclosed prior to the present invention.
[0117] The nucleic acid molecules are also useful as primers for
PCR to amplify any given region of a nucleic acid molecule and are
useful to synthesize antisense molecules of desired length and
sequence.
[0118] The nucleic acid molecules are also useful for constructing
recombinant vectors. Such vectors include expression vectors that
express a portion of, or all of, the peptide sequences. Vectors
also include insertion vectors, used to integrate into another
nucleic acid molecule sequence, such as into the cellular genome,
to alter in situ expression of a gene and/or gene product. For
example, an endogenous coding sequence can be replaced via
homologous recombination with all or part of the coding region
containing one or more specifically introduced mutations.
[0119] The nucleic acid molecules are also useful for expressing
antigenic portions of the proteins.
[0120] The nucleic acid molecules are also useful as probes for
determining the chromosomal positions of the nucleic acid molecules
by means of in situ hybridization methods. The gene encoding the
novel receptor protein of the present invention is located on a
genome component that has been mapped to human chromosome 22 (as
indicated in FIG. 3), which is supported by multiple lines of
evidence, such as STS and BAC map data.
[0121] The nucleic acid molecules are also useful in making vectors
containing the gene regulatory regions of the nucleic acid
molecules of the present invention.
[0122] The nucleic acid molecules are also useful for designing
ribozymes corresponding to all, or a part, of the mRNA produced
from the nucleic acid molecules described herein.
[0123] The nucleic acid molecules are also useful for making
vectors that express part, or all, of the peptides.
[0124] The nucleic acid molecules are also useful for constructing
host cells expressing a part, or all, of the nucleic acid molecules
and peptides.
[0125] The nucleic acid molecules are also useful for constructing
transgenic animals expressing all, or a part, of the nucleic acid
molecules and peptides.
[0126] The nucleic acid molecules are also useful as hybridization
probes for determining the presence, level, form and distribution
of nucleic acid expression. Experimental data as provided in FIG. 1
indicates that receptor proteins of the present invention are
expressed in the placenta, marrow, and liver/spleen, as indicated
by virtual northern blot analysis. In addition, PCR-based tissue
screening panels indicates expression in human leukocytes.
Accordingly, the probes can be used to detect the presence of, or
to determine levels of, a specific nucleic acid molecule in cells,
tissues, and in organisms. The nucleic acid whose level is
determined can be DNA or RNA. Accordingly, probes corresponding to
the peptides described herein can be used to assess expression
and/or gene copy number in a given cell, tissue, or organism. These
uses are relevant for diagnosis of disorders involving an increase
or decrease in receptor protein expression relative to normal
results.
[0127] In vitro techniques for detection of mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detecting DNA includes Southern hybridizations and in situ
hybridization.
[0128] Probes can be used as a part of a diagnostic test kit for
identifying cells or tissues that express a receptor protein, such
as by measuring a level of a receptor-encoding nucleic acid in a
sample of cells from a subject e.g., mRNA or genomic DNA, or
determining if a receptor gene has been mutated. Experimental data
as provided in FIG. 1 indicates that receptor proteins of the
present invention are expressed in the placenta, marrow, and
liver/spleen, as indicated by virtual northern blot analysis. In
addition, PCR-based tissue screening panels indicates expression in
human leukocytes.
[0129] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate receptor nucleic acid
expression.
[0130] The invention thus provides a method for identifying a
compound that can be used to treat a disorder associated with
nucleic acid expression of the receptor gene, particularly
biological and pathological processes that are mediated by the
receptor in cells and tissues that express it. Experimental data as
provided in FIG. 1 indicates expression in the placenta, marrow,
liver/spleen, and in leukocytes. The method typically includes
assaying the ability of the compound to modulate the expression of
the receptor nucleic acid and thus identifying a compound that can
be used to treat a disorder characterized by undesired receptor
nucleic acid expression. The assays can be performed in cell-based
and cell-free systems. Cell-based assays include cells naturally
expressing the receptor nucleic acid or recombinant cells
genetically engineered to express specific nucleic acid
sequences.
[0131] The assay for receptor nucleic acid expression can involve
direct assay of nucleic acid levels, such as mRNA levels, or on
collateral compounds involved in the signal pathway. Further, the
expression of genes that are up- or down-regulated in response to
the receptor protein signal pathway can also be assayed. In this
embodiment the regulatory regions of these genes can be operably
linked to a reporter gene such as luciferase.
[0132] Thus, modulators of receptor gene expression can be
identified in a method wherein a cell is contacted with a candidate
compound and the expression of mRNA determined. The level of
expression of receptor mRNA in the presence of the candidate
compound is compared to the level of expression of receptor mRNA in
the absence of the candidate compound. The candidate compound can
then be identified as a modulator of nucleic acid expression based
on this comparison and be used, for example to treat a disorder
characterized by aberrant nucleic acid expression. When expression
of mRNA is statistically significantly greater in the presence of
the candidate compound than in its absence, the candidate compound
is identified as a stimulator of nucleic acid expression. When
nucleic acid expression is statistically significantly less in the
presence of the candidate compound than in its absence, the
candidate compound is identified as an inhibitor of nucleic acid
expression.
[0133] The invention further provides methods of treatment, with
the nucleic acid as a target, using a compound identified through
drug screening as a gene modulator to modulate receptor nucleic
acid expression in cells and tissues that express the receptor.
Experimental data as provided in FIG. 1 indicates that receptor
proteins of the present invention are expressed in the placenta,
marrow, and liver/spleen, as indicated by virtual northern blot
analysis. In addition, PCR-based tissue screening panels indicates
expression in human leukocytes. Modulation includes both
up-regulation (i.e. activation or agonization) or down-regulation
(suppression or antagonization) or nucleic acid expression.
[0134] Alternatively, a modulator for receptor nucleic acid
expression can be a small molecule or drug identified using the
screening assays described herein as long as the drug or small
molecule inhibits the receptor nucleic acid expression in the cells
and tissues that express the protein. Experimental data as provided
in FIG. 1 indicates expression in the placenta, marrow,
liver/spleen, and in leukocytes.
[0135] The nucleic acid molecules are also useful for monitoring
the effectiveness of modulating compounds on the expression or
activity of the receptor gene in clinical trials or in a treatment
regimen. Thus, the gene expression pattern can serve as a barometer
for the continuing effectiveness of treatment with the compound,
particularly with compounds to which a patient can develop
resistance. The gene expression pattern can also serve as a marker
indicative of a physiological response of the affected cells to the
compound. Accordingly, such monitoring would allow either increased
administration of the compound or the administration of alternative
compounds to which the patient has not become resistant. Similarly,
if the level of nucleic acid expression falls below a desirable
level, administration of the compound could be commensurately
decreased.
[0136] The nucleic acid molecules are also useful in diagnostic
assays for qualitative changes in receptor nucleic acid expression,
and particularly in qualitative changes that lead to pathology. The
nucleic acid molecules can be used to detect mutations in receptor
genes and gene expression products such as mRNA. The nucleic acid
molecules can be used as hybridization probes to detect naturally
occurring genetic mutations in the receptor gene and thereby to
determine whether a subject with the mutation is at risk for a
disorder caused by the mutation. Mutations include deletion,
addition, or substitution of one or more nucleotides in the gene,
chromosomal rearrangement, such as inversion or transposition,
modification of genomic DNA, such as aberrant methylation patterns
or changes in gene copy number, such as amplification. Detection of
a mutated form of the receptor gene associated with a dysfunction
provides a diagnostic tool for an active disease or susceptibility
to disease when the disease results from overexpression,
underexpression, or altered expression of a receptor protein.
[0137] Individuals carrying mutations in the receptor gene can be
detected at the nucleic acid level by a variety of techniques. FIG.
3 provides information on SNPs that have been found in the gene
encoding the receptor protein of the present invention. SNPs were
identified at 25 different nucleotide positions. Some of these SNPs
that are located outside the ORF and in introns may affect gene
expression. The gene encoding the novel receptor protein of the
present invention is located on a genome component that has been
mapped to human chromosome 22 (as indicated in FIG. 3), which is
supported by multiple lines of evidence, such as STS and BAC map
data. Genomic DNA can be analyzed directly or can be amplified by
using PCR prior to analysis. RNA or cDNA can be used in the same
way. In some uses, detection of the mutation involves the use of a
probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S.
Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR,
or, alternatively, in a ligation chain reaction (LCR) (see, e.g.,
Landegran et al., Science 241:1077-1080 (1988); and Nakazawa et
al., PNAS 91:360-364 (1994)), the latter of which can be
particularly useful for detecting point mutations in the gene (see
Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). This method
can include the steps of collecting a sample of cells from a
patient, isolating nucleic acid (e.g., genomic, mRNA or both) from
the cells of the sample, contacting the nucleic acid sample with
one or more primers which specifically hybridize to a gene under
conditions such that hybridization and amplification of the gene
(if present) occurs, and detecting the presence or absence of an
amplification product, or detecting the size of the amplification
product and comparing the length to a control sample. Deletions and
insertions can be detected by a change in size of the amplified
product compared to the normal genotype. Point mutations can be
identified by hybridizing amplified DNA to normal RNA or antisense
DNA sequences.
[0138] Alternatively, mutations in a receptor gene can be directly
identified, for example, by alterations in restriction enzyme
digestion patterns determined by gel electrophoresis.
[0139] Further, sequence-specific ribozymes (U.S. Pat. No.
5,498,531) can be used to score for the presence of specific
mutations by development or loss of a ribozyme cleavage site.
Perfectly matched sequences can be distinguished from mismatched
sequences by nuclease cleavage digestion assays or by differences
in melting temperature.
[0140] Sequence changes at specific locations can also be assessed
by nuclease protection assays such as RNase and S1 protection or
the chemical cleavage method. Furthermore, sequence differences
between a mutant receptor gene and a wild-type gene can be
determined by direct DNA sequencing. A variety of automated
sequencing procedures can be utilized when performing the
diagnostic assays (Naeve, C. W., (1995) Biotechniques 19:448),
including sequencing by mass spectrometry (see, e.g., PCT
International Publication No. WO 94/16101; Cohen et al., Adv.
Chromatogr. 36:127-162 (1996); and Griffin et al, Appl. Biochem.
Biotechnol. 38:147-159 (1993)).
[0141] Other methods for detecting mutations in the gene include
methods in which protection from cleavage agents is used to detect
mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al.,
Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988);
Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et
al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144
(1993); and Hayashi et al., Genet. Anal. Tech. Appl. 9:73-79
(1992)), and movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed
using denaturing gradient gel electrophoresis (Myers et al., Nature
313:495 (1985)). Examples of other techniques for detecting point
mutations include selective oligonucleotide hybridization,
selective amplification, and selective primer extension.
[0142] The nucleic acid molecules are also useful for testing an
individual for a genotype that while not necessarily causing the
disease, nevertheless affects the treatment modality. Thus, the
nucleic acid molecules can be used to study the relationship
between an individual's genotype and the individual's response to a
compound used for treatment (pharmacogenomic relationship).
Accordingly, the nucleic acid molecules described herein can be
used to assess the mutation content of the receptor gene in an
individual in order to select an appropriate compound or dosage
regimen for treatment. FIG. 3 provides information on SNPs that
have been found in the gene encoding the receptor protein of the
present invention. SNPs were identified at 25 different nucleotide
positions. Some of these SNPs that are located outside the ORF and
in introns may affect gene expression.
[0143] Thus nucleic acid molecules displaying genetic variations
that affect treatment provide a diagnostic target that can be used
to tailor treatment in an individual. Accordingly, the production
of recombinant cells and animals containing these polymorphisms
allow effective clinical design of treatment compounds and dosage
regimens.
[0144] The nucleic acid molecules are thus useful as antisense
constructs to control receptor gene expression in cells, tissues,
and organisms. A DNA antisense nucleic acid molecule is designed to
be complementary to a region of the gene involved in transcription,
preventing transcription and hence production of receptor protein.
An antisense RNA or DNA nucleic acid molecule would hybridize to
the mRNA and thus block translation of mRNA into receptor
protein.
[0145] Alternatively, a class of antisense molecules can be used to
inactivate mRNA in order to decrease expression of receptor nucleic
acid. Accordingly, these molecules can treat a disorder
characterized by abnormal or undesired receptor nucleic acid
expression. This technique involves cleavage by means of ribozymes
containing nucleotide sequences complementary to one or more
regions in the mRNA that attenuate the ability of the mRNA to be
translated. Possible regions include coding regions and
particularly coding regions corresponding to the catalytic and
other functional activities of the receptor protein, such as
substrate binding.
[0146] The nucleic acid molecules also provide vectors for gene
therapy in patients containing cells that are aberrant in receptor
gene expression. Thus, recombinant cells, which include the
patient's cells that have been engineered ex vivo and returned to
the patient, are introduced into an individual where the cells
produce the desired receptor protein to treat the individual.
[0147] The invention also encompasses kits for detecting the
presence of a receptor nucleic acid in a biological sample.
Experimental data as provided in FIG. 1 indicates that receptor
proteins of the present invention are expressed in the placenta,
marrow, and liver/spleen, as indicated by virtual northern blot
analysis. In addition, PCR-based tissue screening panels indicates
expression in human leukocytes. For example, the kit can comprise
reagents such as a labeled or labelable nucleic acid or agent
capable of detecting receptor nucleic acid in a biological sample;
means for determining the amount of receptor nucleic acid in the
sample; and means for comparing the amount of receptor nucleic acid
in the sample with a standard. The compound or agent can be
packaged in a suitable container. The kit can further comprise
instructions for using the kit to detect receptor protein mRNA or
DNA.
[0148] Nucleic Acid Arrays
[0149] The present invention further provides nucleic acid
detection kits, such as arrays or microarrays of nucleic acid
molecules that are based on the sequence information provided in
FIGS. 1 and 3 (SEQ ID NOS:1 and 3).
[0150] As used herein "Arrays" or "Microarrays" refers to an array
of distinct polynucleotides or oligonucleotides synthesized on a
substrate, such as paper, nylon or other type of membrane, filter,
chip, glass slide, or any other suitable solid support. In one
embodiment, the microarray is prepared and used according to the
methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT
application W095/11995 (Chee et al.), Lockhart, D. J. et al. (1996;
Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc.
Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated
herein in their entirety by reference. In other embodiments, such
arrays are produced by the methods described by Brown et al., U.S.
Pat. No. 5,807,522.
[0151] The microarray or detection kit is preferably composed of a
large number of unique, single-stranded nucleic acid sequences,
usually either synthetic antisense oligonucleotides or fragments of
cDNAs, fixed to a solid support. The oligonucleotides are
preferably about 6-60 nucleotides in length, more preferably 15-30
nucleotides in length, and most preferably about 20-25 nucleotides
in length. For a certain type of microarray or detection kit, it
may be preferable to use oligonucleotides that are only 7-20
nucleotides in length. The microarray or detection kit may contain
oligonucleotides that cover the known 5', or 3', sequence,
sequential oligonucleotides which cover the full length sequence;
or unique oligonucleotides selected from particular areas along the
length of the sequence. Polynucleotides used in the microarray or
detection kit may be oligonucleotides that are specific to a gene
or genes of interest.
[0152] In order to produce oligonucleotides to a known sequence for
a microarray or detection kit, the gene(s) of interest (or an ORF
identified from the contigs of the present invention) is typically
examined using a computer algorithm which starts at the 5' or at
the 3' end of the nucleotide sequence. Typical algorithms will then
identify oligomers of defined length that are unique to the gene,
have a GC content within a range suitable for hybridization, and
lack predicted secondary structure that may interfere with
hybridization. In certain situations it may be appropriate to use
pairs of oligonucleotides on a microarray or detection kit. The
"pairs" will be identical, except for one nucleotide that
preferably is located in the center of the sequence. The second
oligonucleotide in the pair (mismatched by one) serves as a
control. The number of oligonucleotide pairs may range from two to
one million. The oligomers are synthesized at designated areas on a
substrate using a light-directed chemical process. The substrate
may be paper, nylon or other type of membrane, filter, chip, glass
slide or any other suitable solid support.
[0153] In another aspect, an oligonucleotide may be synthesized on
the surface of the substrate by using a chemical coupling procedure
and an ink jet application apparatus, as described in PCT
application W095/251116 (Baldeschweiler et al.) which is
incorporated herein in its entirety by reference. In another
aspect, a "gridded" array analogous to a dot (or slot) blot may be
used to arrange and link cDNA fragments or oligonucleotides to the
surface of a substrate using a vacuum system, thermal, UV,
mechanical or chemical bonding procedures. An array, such as those
described above, may be produced by hand or by using available
devices (slot blot or dot blot apparatus), materials (any suitable
solid support), and machines (including robotic instruments), and
may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or
any other number between two and one million which lends itself to
the efficient use of commercially available instrumentation.
[0154] In order to conduct sample analysis using a microarray or
detection kit, the RNA or DNA from a biological sample is made into
hybridization probes. The mRNA is isolated, and cDNA is produced
and used as a template to make antisense RNA (aRNA). The aRNA is
amplified in the presence of fluorescent nucleotides, and labeled
probes are incubated with the microarray or detection kit so that
the probe sequences hybridize to complementary oligonucleotides of
the microarray or detection kit. Incubation conditions are adjusted
so that hybridization occurs with precise complementary matches or
with various degrees of less complementarity. After removal of
nonhybridized probes, a scanner is used to determine the levels and
patterns of fluorescence. The scanned images are examined to
determine degree of complementarity and the relative abundance of
each oligonucleotide sequence on the microarray or detection kit.
The biological samples may be obtained from any bodily fluids (such
as blood, urine, saliva, phlegm, gastric juices, etc.), cultured
cells, biopsies, or other tissue preparations. A detection system
may be used to measure the absence, presence, and amount of
hybridization for all of the distinct sequences simultaneously.
This data may be used for large-scale correlation studies on the
sequences, expression patterns, mutations, variants, or
polymorphisms among samples.
[0155] Using such arrays, the present invention provides methods to
identify the expression of the receptor proteins/peptides of the
present invention. In detail, such methods comprise incubating a
test sample with one or more nucleic acid molecules and assaying
for binding of the nucleic acid molecule with components within the
test sample. Such assays will typically involve arrays comprising
many genes, at least one of which is a gene of the present
invention and or alleles of the receptor gene of the present
invention. FIG. 3 provides information on SNPs that have been found
in the gene encoding the receptor protein of the present invention.
SNPs were identified at 25 different nucleotide positions. Some of
these SNPs that are located outside the ORF and in introns may
affect gene expression.
[0156] Conditions for incubating a nucleic acid molecule with a
test sample vary. Incubation conditions depend on the format
employed in the assay, the detection methods employed, and the type
and nature of the nucleic acid molecule used in the assay. One
skilled in the art will recognize that any one of the commonly
available hybridization, amplification or array assay formats can
readily be adapted to employ the novel fragments of the Human
genome disclosed herein. Examples of such assays can be found in
Chard, T, An Introduction to Radioimmunoassay and Related
Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands
(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,
Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3
(1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays:
Laboratory Techniques in Biochemistry and Molecular Biology,
Elsevier Science Publishers, Amsterdam, The Netherlands (1985).
[0157] The test samples of the present invention include cells,
protein or membrane extracts of cells. The test sample used in the
above-described method will vary based on the assay format, nature
of the detection method and the tissues, cells or extracts used as
the sample to be assayed. Methods for preparing nucleic acid
extracts or of cells are well known in the art and can be readily
be adapted in order to obtain a sample that is compatible with the
system utilized.
[0158] In another embodiment of the present invention, kits are
provided which contain the necessary reagents to carry out the
assays of the present invention.
[0159] Specifically, the invention provides a compartmentalized kit
to receive, in close confinement, one or more containers which
comprises: (a) a first container comprising one of the nucleic acid
molecules that can bind to a fragment of the Human genome disclosed
herein; and (b) one or more other containers comprising one or more
of the following: wash reagents, reagents capable of detecting
presence of a bound nucleic acid.
[0160] In detail, a compartmentalized kit includes any kit in which
reagents are contained in separate containers. Such containers
include small glass containers, plastic containers, strips of
plastic, glass or paper, or arraying material such as silica. Such
containers allows one to efficiently transfer reagents from one
compartment to another compartment such that the samples and
reagents are not cross-contaminated, and the agents or solutions of
each container can be added in a quantitative fashion from one
compartment to another. Such containers will include a container
which will accept the test sample, a container which contains the
nucleic acid probe, containers which contain wash reagents (such as
phosphate buffered saline, Tris-buffers, etc.), and containers
which contain the reagents used to detect the bound probe. One
skilled in the art will readily recognize that the previously
unidentified receptor gene of the present invention can be
routinely identified using the sequence information disclosed
herein can be readily incorporated into one of the established kit
formats which are well known in the art, particularly expression
arrays.
[0161] Vectors/host Cells
[0162] The invention also provides vectors containing the nucleic
acid molecules described herein. The term "vector" refers to a
vehicle, preferably a nucleic acid molecule, which can transport
the nucleic acid molecules. When the vector is a nucleic acid
molecule, the nucleic acid molecules are covalently linked to the
vector nucleic acid. With this aspect of the invention, the vector
includes a plasmid, single or double stranded phage, a single or
double stranded RNA or DNA viral vector, or artificial chromosome,
such as a BAC, PAC, YAC, OR MAC.
[0163] A vector can be maintained in the host cell as an
extrachromosomal element where it replicates and produces
additional copies of the nucleic acid molecules. Alternatively, the
vector may integrate into the host cell genome and produce
additional copies of the nucleic acid molecules when the host cell
replicates.
[0164] The invention provides vectors for the maintenance (cloning
vectors) or vectors for expression (expression vectors) of the
nucleic acid molecules. The vectors can function in prokaryotic or
eukaryotic cells or in both (shuttle vectors).
[0165] Expression vectors contain cis-acting regulatory regions
that are operably linked in the vector to the nucleic acid
molecules such that transcription of the nucleic acid molecules is
allowed in a host cell. The nucleic acid molecules can be
introduced into the host cell with a separate nucleic acid molecule
capable of affecting transcription. Thus, the second nucleic acid
molecule may provide a trans-acting factor interacting with the
cis-regulatory control region to allow transcription of the nucleic
acid molecules from the vector. Alternatively, a trans-acting
factor may be supplied by the host cell. Finally, a trans-acting
factor can be produced from the vector itself. It is understood,
however, that in some embodiments, transcription and/or translation
of the nucleic acid molecules can occur in a cell-free system.
[0166] The regulatory sequence to which the nucleic acid molecules
described herein can be operably linked include promoters for
directing mRNA transcription. These include, but are not limited
to, the left promoter from bacteriophage .lambda., the lac, TRP,
and TAC promoters from E. Coli, the early and late promoters from
SV40, the CMV immediate early promoter, the adenovirus early and
late promoters, and retrovirus long-terminal repeats.
[0167] In addition to control regions that promote transcription,
expression vectors may also include regions that modulate
transcription, such as repressor binding sites and enhancers.
Examples include the SV40 enhancer, the cytomegalovirus immediate
early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus LTR enhancers.
[0168] In addition to containing sites for transcription initiation
and control, expression vectors can also contain sequences
necessary for transcription termination and, in the transcribed
region a ribosome binding site for translation. Other regulatory
control elements for expression include initiation and termination
codons as well as polyadenylation signals. The person of ordinary
skill in the art would be aware of the numerous regulatory
sequences that are useful in expression vectors. Such regulatory
sequences are described, for example, in Sambrook et al., Molecular
Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., (1989).
[0169] A variety of expression vectors can be used to express a
nucleic acid molecule. Such vectors include chromosomal, episomal,
and virus-derived vectors, for example vectors derived from
bacterial plasmids, from bacteriophage, from yeast episomes, from
yeast chromosomal elements, including yeast artificial chromosomes,
from viruses such as baculoviruses, papovaviruses such as SV40,
Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses,
and retroviruses. Vectors may also be derived from combinations of
these sources such as those derived from plasmid and bacteriophage
genetic elements, e.g. cosmids and phagemids. Appropriate cloning
and expression vectors for prokaryotic and eukaryotic hosts are
described in Sambrook et al., Molecular Cloning: A Laboratory
Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., (1989).
[0170] The regulatory sequence may provide constitutive expression
in one or more host cells (i.e. tissue specific) or may provide for
inducible expression in one or more cell types such as by
temperature, nutrient additive, or exogenous factor such as a
hormone or other ligand. A variety of vectors providing for
constitutive and inducible expression in prokaryotic and eukaryotic
hosts are well known to those of ordinary skill in the art.
[0171] The nucleic acid molecules can be inserted into the vector
nucleic acid by well-known methodology. Generally, the DNA sequence
that will ultimately be expressed is joined to an expression vector
by cleaving the DNA sequence and the expression vector with one or
more restriction enzymes and then ligating the fragments together.
Procedures for restriction enzyme digestion and ligation are well
known to those of ordinary skill in the art.
[0172] The vector containing the appropriate nucleic acid molecule
can be introduced into an appropriate host cell for propagation or
expression using well-known techniques. Bacterial cells include,
but are not limited to, E. coli, Streptomyces, and Salmonella
typhimurium. Eukaryotic cells include, but are not limited to,
yeast, insect cells such as Drosophila, animal cells such as COS
and CHO cells, and plant cells.
[0173] As described herein, it may be desirable to express the
peptide as a fusion protein. Accordingly, the invention provides
fusion vectors that allow for the production of the peptides.
Fusion vectors can increase the expression of a recombinant
protein, increase the solubility of the recombinant protein, and
aid in the purification of the protein by acting for example as a
ligand for affinity purification. A proteolytic cleavage site may
be introduced at the junction of the fusion moiety so that the
desired peptide can ultimately be separated from the fusion moiety.
Proteolytic enzymes include, but are not limited to, factor Xa,
thrombin, and enteroreceptor. Typical fusion expression vectors
include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New
England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway,
N.J.) which fuse glutathione S-transferase (GST), maltose E binding
protein, or protein A, respectively, to the target recombinant
protein. Examples of suitable inducible non-fusion E. coli
expression vectors include pTrc (Amann et al., Gene 69:301-315
(1988)) and pET 11d (Studier et al., Gene Expression Technology:
Methods in Enzymology 185:60-89 (1990)).
[0174] Recombinant protein expression can be maximized in host
bacteria by providing a genetic background wherein the host cell
has an impaired capacity to proteolytically cleave the recombinant
protein. (Gottesman, S., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128).
Alternatively, the sequence of the nucleic acid molecule of
interest can be altered to provide preferential codon usage for a
specific host cell, for example E. coli. (Wada et al., Nucleic
Acids Res. 20:2111-2118 (1992)).
[0175] The nucleic acid molecules can also be expressed by
expression vectors that are operative in yeast. Examples of vectors
for expression in yeast e.g., S. cerevisiae include pYepSec1
(Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjuan et al.,
Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123
(1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).
[0176] The nucleic acid molecules can also be expressed in insect
cells using, for example, baculovirus expression vectors.
Baculovirus vectors available for expression of proteins in
cultured insect cells (e.g., Sf9 cells) include the pAc series
(Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL
series (Lucklow et al., Virology 170:31-39 (1989)).
[0177] In certain embodiments of the invention, the nucleic acid
molecules described herein are expressed in mammalian cells using
mammalian expression vectors. Examples of mammalian expression
vectors include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC
(Kaufman et al., EMBO J. 6:187-195 (1987)).
[0178] The expression vectors listed herein are provided by way of
example only of the well-known vectors available to those of
ordinary skill in the art that would be useful to express the
nucleic acid molecules. The person of ordinary skill in the art
would be aware of other vectors suitable for maintenance
propagation or expression of the nucleic acid molecules described
herein. These are found for example in Sambrook, J., Fritsh, E. F.,
and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed.,
Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989.
[0179] The invention also encompasses vectors in which the nucleic
acid sequences described herein are cloned into the vector in
reverse orientation, but operably linked to a regulatory sequence
that permits transcription of antisense RNA. Thus, an antisense
transcript can be produced to all, or to a portion, of the nucleic
acid molecule sequences described herein, including both coding and
non-coding regions. Expression of this antisense RNA is subject to
each of the parameters described above in relation to expression of
the sense RNA (regulatory sequences, constitutive or inducible
expression, tissue-specific expression).
[0180] The invention also relates to recombinant host cells
containing the vectors described herein. Host cells therefore
include prokaryotic cells, lower eukaryotic cells such as yeast,
other eukaryotic cells such as insect cells, and higher eukaryotic
cells such as mammalian cells.
[0181] The recombinant host cells are prepared by introducing the
vector constructs described herein into the cells by techniques
readily available to the person of ordinary skill in the art. These
include, but are not limited to, calcium phosphate transfection,
DEAE-dextran-mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection,
lipofection, and other techniques such as those found in Sambrook,
et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold
Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1989).
[0182] Host cells can contain more than one vector. Thus, different
nucleotide sequences can be introduced on different vectors of the
same cell. Similarly, the nucleic acid molecules can be introduced
either alone or with other nucleic acid molecules that are not
related to the nucleic acid molecules such as those providing
trans-acting factors for expression vectors. When more than one
vector is introduced into a cell, the vectors can be introduced
independently, co-introduced or joined to the nucleic acid molecule
vector.
[0183] In the case of bacteriophage and viral vectors, these can be
introduced into cells as packaged or encapsulated virus by standard
procedures for infection and transduction. Viral vectors can be
replication-competent or replication-defective. In the case in
which viral replication is defective, replication will occur in
host cells providing functions that complement the defects.
[0184] Vectors generally include selectable markers that enable the
selection of the subpopulation of cells that contain the
recombinant vector constructs. The marker can be contained in the
same vector that contains the nucleic acid molecules described
herein or may be on a separate vector. Markers include tetracycline
or ampicillin-resistance genes for prokaryotic host cells and
dihydrofolate reductase or neomycin resistance for eukaryotic host
cells. However, any marker that provides selection for a phenotypic
trait will be effective.
[0185] While the mature proteins can be produced in bacteria,
yeast, mammalian cells, and other cells under the control of the
appropriate regulatory sequences, cell-free transcription and
translation systems can also be used to produce these proteins
using RNA derived from the DNA constructs described herein.
[0186] Where secretion of the peptide is desired, which is
difficult to achieve with multi-transmembrane domain containing
proteins such as receptors, appropriate secretion signals are
incorporated into the vector. The signal sequence can be endogenous
to the peptides or heterologous to these peptides.
[0187] Where the peptide is not secreted into the medium, which is
typically the case with receptors, the protein can be isolated from
the host cell by standard disruption procedures, including freeze
thaw, sonication, mechanical disruption, use of lysing agents and
the like. The peptide can then be recovered and purified by
well-known purification methods including ammonium sulfate
precipitation, acid extraction, anion or cationic exchange
chromatography, phosphocellulose chromatography,
hydrophobic-interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography, or high
performance liquid chromatography.
[0188] It is also understood that depending upon the host cell in
recombinant production of the peptides described herein, the
peptides can have various glycosylation patterns, depending upon
the cell, or maybe non-glycosylated as when produced in bacteria.
In addition, the peptides may include an initial modified
methionine in some cases as a result of a host-mediated
process.
[0189] Uses of Vectors and Host Cells
[0190] The recombinant host cells expressing the peptides described
herein have a variety of uses. First, the cells are useful for
producing a receptor protein or peptide that can be further
purified to produce desired amounts of receptor protein or
fragments. Thus, host cells containing expression vectors are
useful for peptide production.
[0191] Host cells are also useful for conducting cell-based assays
involving the receptor protein or receptor protein fragments, such
as those described above as well as other formats known in the art.
Thus, a recombinant host cell expressing a native receptor protein
is useful for assaying compounds that stimulate or inhibit receptor
protein function.
[0192] Host cells are also useful for identifying receptor protein
mutants in which these functions are affected. If the mutants
naturally occur and give rise to a pathology, host cells containing
the mutations are useful to assay compounds that have a desired
effect on the mutant receptor protein (for example, stimulating or
inhibiting function) which may not be indicated by their effect on
the native receptor protein.
[0193] Genetically engineered host cells can be further used to
produce non-human transgenic animals. A transgenic animal is
preferably a mammal, for example a rodent, such as a rat or mouse,
in which one or more of the cells of the animal include a
transgene. A transgene is exogenous DNA which is integrated into
the genome of a cell from which a transgenic animal develops and
which remains in the genome of the mature animal in one or more
cell types or tissues of the transgenic animal. These animals are
useful for studying the function of a receptor protein and
identifying and evaluating modulators of receptor protein activity.
Other examples of transgenic animals include non-human primates,
sheep, dogs, cows, goats, chickens, and amphibians.
[0194] A transgenic animal can be produced by introducing nucleic
acid into the male pronuclei of a fertilized oocyte, e.g., by
microinjection, retroviral infection, and allowing the oocyte to
develop in a pseudopregnant female foster animal. Any of the
receptor protein nucleotide sequences can be introduced as a
transgene into the genome of a non-human animal, such as a
mouse.
[0195] Any of the regulatory or other sequences useful in
expression vectors can form part of the transgenic sequence. This
includes intronic sequences and polyadenylation signals, if not
already included. A tissue-specific regulatory sequence(s) can be
operably linked to the transgene to direct expression of the
receptor protein to particular cells.
[0196] Methods for generating transgenic animals via embryo
manipulation and microinjection, particularly animals such as mice,
have become conventional in the art and are described, for example,
in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al.,
U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used
for production of other transgenic animals. A transgenic founder
animal can be identified based upon the presence of the transgene
in its genome and/or expression of transgenic mRNA in tissues or
cells of the animals. A transgenic founder animal can then be used
to breed additional animals carrying the transgene. Moreover,
transgenic animals carrying a transgene can further be bred to
other transgenic animals carrying other transgenes. A transgenic
animal also includes animals in which the entire animal or tissues
in the animal have been produced using the homologously recombinant
host cells described herein.
[0197] In another embodiment, transgenic non-human animals can be
produced which contain selected systems that allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage P1. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al. PNAS
89:6232-6236 (1992). Another example of a recombinase system is the
FLP recombinase system of S. cerevisiae (O'Gorman et al. Science
251:1351-1355 (1991). If a cre/loxP recombinase system is used to
regulate expression of the transgene, animals containing transgenes
encoding both the Cre recombinase and a selected protein is
required. Such animals can be provided through the construction of
"double" transgenic animals, e.g., by mating two transgenic
animals, one containing a transgene encoding a selected protein and
the other containing a transgene encoding a recombinase.
[0198] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut,
I. et al. Nature 385:810-813 (1997) and PCT International
Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell,
e.g., a somatic cell, from the transgenic animal can be isolated
and induced to exit the growth cycle and enter G.sub.o phase. The
quiescent cell can then be fused, e.g., through the use of
electrical pulses, to an enucleated oocyte from an animal of the
same species from which the quiescent cell is isolated. The
reconstructed oocyte is then cultured such that it develops to
morula or blastocyst and then transferred to pseudopregnant female
foster animal. The offspring born of this female foster animal will
be a clone of the animal from which the cell, e.g., the somatic
cell, is isolated.
[0199] Transgenic animals containing recombinant cells that express
the peptides described herein are useful to conduct the assays
described herein in an in vivo context. Accordingly, the various
physiological factors- that are present in vivo and that could
effect substrate binding, receptor protein activation, and signal
transduction, may not be evident from in vitro cell-free or
cell-based assays. Accordingly, it is useful to provide non-human
transgenic animals to assay in vivo receptor protein function,
including substrate interaction, the effect of specific mutant
receptor proteins on receptor protein function and substrate
interaction, and the effect of chimeric receptor proteins. It is
also possible to assess the effect of null mutations, that is,
mutations that substantially or completely eliminate one or more
receptor protein functions.
[0200] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the above-described modes for carrying out
the invention which are obvious to those skilled in the field of
molecular biology or related fields are intended to be within the
scope of the following claims.
Sequence CWU 1
1
4 1 3394 DNA Homo sapiens 1 ctcccaccac acagaggcct ggaggaggca
gaggccagga gggagaggtc ccaagagcct 60 gtgaaatggg tctggcctgg
ctcccagctg ggcaggaaca caggacttca ggatactaag 120 gaccctgtca
tgcccatggc cagcacccac cagtgctggt gcctgcctgt ccagagctga 180
ccagggagat ggtgctggcc caggggctgc tctccatggc cctgctggcc ctgtgctggg
240 agcgcagcct ggcaggggca gaagaaacca tcccgctgca gaccctgcgc
tgctacaacg 300 actacaccag ccacatcacc tgcaggtggg cagacaccca
ggatgcccag cggctcgtca 360 acgtgaccct cattcgccgg gtgaatgagg
acctcctgga gccagtgtcc tgtgacctca 420 gtgatgacat gccctggtca
gcctgccccc atccccgctg cgtgcccagg agatgtgtca 480 ttccctgcca
gagttttgtc gtcactgacg ttgactactt ctcattccaa ccagacaggc 540
ctctgggcac ccggctcacc gtcactctga cccagcatgt ccagcctcct gagcccaggg
600 acctgcagat cagcaccgac caggaccact tcctgctgac ctggagtgtg
gcccttggga 660 gtccccagag ccactggttg tccccagggg atctggagtt
tgaggtggtc tacaagcggc 720 ttcaggactc ttgggaggac gcagccatcc
tcctctccaa cacctcccag gccaccctgg 780 ggccagagca cctcatgccc
agcagcacct acgtggcccg agtacggacc cgcctggccc 840 caggttctcg
gctctcagga cgtcccagca agtggagccc agaggtttgc tgggactccc 900
agccagggga tgaggcccag ccccagaacc tggagtgctt ctttgacggg gccgccgtgc
960 tcagctgctc ctgggaggtg aggaaggagg tggccagctc ggtctccttt
ggcctattct 1020 acaagcccag cccagatgca ggggaggaag agtgctcccc
agtgctgagg gaggggctcg 1080 gcagcctcca caccaggcac cactgccaga
ttcccgtgcc cgaccccgcg acccacggcc 1140 aatacatcgt ctctgttcag
ccaaggaggg cagagaaaca cataaagagc tcagtgaaca 1200 tccagatggc
ccctccatcc ctcaacgtga ccaaggatgg agacagctac agcctgcgct 1260
gggaaacaat gaaaatgcga tacgaacaca tagaccacac atttgagatc cagtacagga
1320 aagacacggc cacgtggaag gacagcaaga ccgagaccct ccagaacgcc
cacagcatgg 1380 ccctgccagc cctggagccc tccaccaggt actgggccag
ggtgagggtc aggacctccc 1440 gcaccggcta caacgggatc tggagcgagt
ggagtgaggc gcgctcctgg gacaccgagt 1500 cggtgctgcc tatgtgggtg
ctggccctca tcgtgatctt cctcaccatc gctgtgctcc 1560 tggccctccg
cttctgtggc atctacgggt acaggctgcg cagaaagtgg gaggagaaga 1620
tccccaaccc cagcaagagc cacctgttcc agaacgggag cgcagagctt tggcccccag
1680 gcagcatgtc ggccttcact agcgggagtc ccccacacca ggggccgtgg
ggcagccgct 1740 tccctgagct ggagggggtg ttccctgtag gattcgggga
cagcgaggtg tcacctctca 1800 ccatagagga ccccaagcat gtctgtgatc
caccatctgg gcctgacacg actccagctg 1860 cctcagatct acccacagag
cagcccccca gcccccagcc aggcccgcct gccgcctccc 1920 acacacctga
gccttatcag actgagatgc ggctggttgt gttgaggact tgtgtgggct 1980
gcctgtcccc ggcagtcgct gatgcacatg acatgattct catctgggtg cagaggtggg
2040 aggcaccagg tgggcacccg tgggggttag ggcttggaag agtggcacag
gactgggcac 2100 gctcagtgag gctcagggaa ttcagactag cctcgattgt
cactccgaga aatgggcatg 2160 gtattggggg tcgggggggc ggtgcaaggg
acgcacatga gagactgttt gggagcttct 2220 ggggagccct gctagttgtc
tcagtgatgt ctgtgggacc tccagtccct tgagacccca 2280 cgtcatgtag
agaagttaac ggcccaagtg gtgggcaggc tggcgggacc tggggaacat 2340
caggagagga gtccagagcc cacgtctact gcggaaaagt caggggaaac tgccaaacaa
2400 aggaaaatgc cccaaaggca tatatgcttt agggcctttg gtccaaatgg
cccgggtggc 2460 cactcttcca gatagaccag gcaactctcc ctcccaccgg
ccacagatga ggggctgctg 2520 atctatgcct gggcctgcac cagggattat
ggttctttta aatctttgcc tttcagatac 2580 aggaaaaata atggcattaa
attgctttaa tttgcattat tttagttatc cagtttgcac 2640 atatttttat
aggtatctta ggcatcgatt ggtatttttt aactgggcca agcccattaa 2700
ggtctttctt ctgttgggtg ctatcatttt ctgattaagt ctttttgact attgacatac
2760 agtctttcac agatggtgga gtgtttttcc cccaaatctg ttgtttgtct
tataatgttg 2820 tatatgaggt tttatggtgt atgaatatga atgcttctgt
aatgtcaaac agatccctag 2880 taaactcctt cttcactttt actgtcagat
ttacaaaggt cctcccattg caaagcagtg 2940 tttgtcctaa ttcatatatc
gtttttctag tccattttgt gtttccaacc ctttatgtaa 3000 aatcttaatt
atttcttgaa tgtgtggatg tgaaactgag gcggcctttc cggaactgaa 3060
atacttataa gacatgaccc gtgtgagtga cactttttgc ggattcatga acactcctcc
3120 cgttcatctc cgttccccgc ccccccatgc gttaattttc tttttatttt
tagacgttga 3180 cggcagccag cttacccgcc gtcttttatc ttggtcccca
cctcgagttc cgccccgcat 3240 agtgttaacc ggacgcgccc tccagccgtc
cctggaccgt atccatgtac ttgtattcct 3300 acaccgcccc ttctgccgcc
accacaataa agtggctaca aatgtattgc atgcgagcgc 3360 acccttttac
ccccctctgc ctgacgcgcc cccc 3394 2 694 PRT Homo sapiens 2 Met Val
Leu Ala Gln Gly Leu Leu Ser Met Ala Leu Leu Ala Leu Cys 1 5 10 15
Trp Glu Arg Ser Leu Ala Gly Ala Glu Glu Thr Ile Pro Leu Gln Thr 20
25 30 Leu Arg Cys Tyr Asn Asp Tyr Thr Ser His Ile Thr Cys Arg Trp
Ala 35 40 45 Asp Thr Gln Asp Ala Gln Arg Leu Val Asn Val Thr Leu
Ile Arg Arg 50 55 60 Val Asn Glu Asp Leu Leu Glu Pro Val Ser Cys
Asp Leu Ser Asp Asp 65 70 75 80 Met Pro Trp Ser Ala Cys Pro His Pro
Arg Cys Val Pro Arg Arg Cys 85 90 95 Val Ile Pro Cys Gln Ser Phe
Val Val Thr Asp Val Asp Tyr Phe Ser 100 105 110 Phe Gln Pro Asp Arg
Pro Leu Gly Thr Arg Leu Thr Val Thr Leu Thr 115 120 125 Gln His Val
Gln Pro Pro Glu Pro Arg Asp Leu Gln Ile Ser Thr Asp 130 135 140 Gln
Asp His Phe Leu Leu Thr Trp Ser Val Ala Leu Gly Ser Pro Gln 145 150
155 160 Ser His Trp Leu Ser Pro Gly Asp Leu Glu Phe Glu Val Val Tyr
Lys 165 170 175 Arg Leu Gln Asp Ser Trp Glu Asp Ala Ala Ile Leu Leu
Ser Asn Thr 180 185 190 Ser Gln Ala Thr Leu Gly Pro Glu His Leu Met
Pro Ser Ser Thr Tyr 195 200 205 Val Ala Arg Val Arg Thr Arg Leu Ala
Pro Gly Ser Arg Leu Ser Gly 210 215 220 Arg Pro Ser Lys Trp Ser Pro
Glu Val Cys Trp Asp Ser Gln Pro Gly 225 230 235 240 Asp Glu Ala Gln
Pro Gln Asn Leu Glu Cys Phe Phe Asp Gly Ala Ala 245 250 255 Val Leu
Ser Cys Ser Trp Glu Val Arg Lys Glu Val Ala Ser Ser Val 260 265 270
Ser Phe Gly Leu Phe Tyr Lys Pro Ser Pro Asp Ala Gly Glu Glu Glu 275
280 285 Cys Ser Pro Val Leu Arg Glu Gly Leu Gly Ser Leu His Thr Arg
His 290 295 300 His Cys Gln Ile Pro Val Pro Asp Pro Ala Thr His Gly
Gln Tyr Ile 305 310 315 320 Val Ser Val Gln Pro Arg Arg Ala Glu Lys
His Ile Lys Ser Ser Val 325 330 335 Asn Ile Gln Met Ala Pro Pro Ser
Leu Asn Val Thr Lys Asp Gly Asp 340 345 350 Ser Tyr Ser Leu Arg Trp
Glu Thr Met Lys Met Arg Tyr Glu His Ile 355 360 365 Asp His Thr Phe
Glu Ile Gln Tyr Arg Lys Asp Thr Ala Thr Trp Lys 370 375 380 Asp Ser
Lys Thr Glu Thr Leu Gln Asn Ala His Ser Met Ala Leu Pro 385 390 395
400 Ala Leu Glu Pro Ser Thr Arg Tyr Trp Ala Arg Val Arg Val Arg Thr
405 410 415 Ser Arg Thr Gly Tyr Asn Gly Ile Trp Ser Glu Trp Ser Glu
Ala Arg 420 425 430 Ser Trp Asp Thr Glu Ser Val Leu Pro Met Trp Val
Leu Ala Leu Ile 435 440 445 Val Ile Phe Leu Thr Ile Ala Val Leu Leu
Ala Leu Arg Phe Cys Gly 450 455 460 Ile Tyr Gly Tyr Arg Leu Arg Arg
Lys Trp Glu Glu Lys Ile Pro Asn 465 470 475 480 Pro Ser Lys Ser His
Leu Phe Gln Asn Gly Ser Ala Glu Leu Trp Pro 485 490 495 Pro Gly Ser
Met Ser Ala Phe Thr Ser Gly Ser Pro Pro His Gln Gly 500 505 510 Pro
Trp Gly Ser Arg Phe Pro Glu Leu Glu Gly Val Phe Pro Val Gly 515 520
525 Phe Gly Asp Ser Glu Val Ser Pro Leu Thr Ile Glu Asp Pro Lys His
530 535 540 Val Cys Asp Pro Pro Ser Gly Pro Asp Thr Thr Pro Ala Ala
Ser Asp 545 550 555 560 Leu Pro Thr Glu Gln Pro Pro Ser Pro Gln Pro
Gly Pro Pro Ala Ala 565 570 575 Ser His Thr Pro Glu Pro Tyr Gln Thr
Glu Met Arg Leu Val Val Leu 580 585 590 Arg Thr Cys Val Gly Cys Leu
Ser Pro Ala Val Ala Asp Ala His Asp 595 600 605 Met Ile Leu Ile Trp
Val Gln Arg Trp Glu Ala Pro Gly Gly His Pro 610 615 620 Trp Gly Leu
Gly Leu Gly Arg Val Ala Gln Asp Trp Ala Arg Ser Val 625 630 635 640
Arg Leu Arg Glu Phe Arg Leu Ala Ser Ile Val Thr Pro Arg Asn Gly 645
650 655 His Gly Ile Gly Gly Arg Gly Gly Gly Ala Arg Asp Ala His Glu
Arg 660 665 670 Leu Phe Gly Ser Phe Trp Gly Ala Leu Leu Val Val Ser
Val Met Ser 675 680 685 Val Gly Pro Pro Val Pro 690 3 21968 DNA
Homo sapiens misc_feature (1)...(21968) n = A,T,C or G 3 tgactgtcga
ccaattacat aacatcccac ccctgttttc tcatctacaa agtggagctt 60
ctagtggtaa cctatacata gatgtattca tatccgtaaa accttttgaa taatgcctgg
120 ctcatggaaa acactgcaat attgtcagcc actatcatga attctctctg
tgtatgtgag 180 tactgtttgt gctctttgat gatattgatt tgttttaacc
ttttaaagta aaagacacta 240 acctgtcctc cataattctg gttggtatgg
tctatatgtt gaagtgcacc caagtttatg 300 ttgcaaccta agccccaatg
tgagaatatc tggaggtagt gcctgtggca ccctataaaa 360 taccccagcg
agctcccttg cttattccat gtgagattac agggagaaga ctgcaaggaa 420
caggccctca ccagatagtg aatctgcccg caccttgatc ttgaacttcc cagcctccag
480 aactatgaga aataaacttt gttactagta agccactcca tctatggtat
gttgttacag 540 tagcctaacg gatgagtaca ttggtgcaaa gattttctgt
ttgcactccg tcctttcctt 600 ttatggttca gtcagtttga caaattacag
aagatttcac ttttatgtac ttctgccagc 660 ctttgtctct gtgatgtctc
ctatgctttt aagcttaaag agttcaaagg cattcactcc 720 tctggtttgc
agctttcata ggatgtaaca ttttacattt aaatttataa ttaacttgtt 780
atttggggaa tggtgtgaaa tggggtttta gataggcatt tttatgttgt taatcaatga
840 ttttagcatt tttttaaatt taatatttca tcgcttcaca ttgattcctg
attccttctc 900 agtctggggt caagtagagt ttgtttccag ccagcatgtc
cattgataga ttctgtgtgt 960 gtgtatgtgt gtctgtgcat gtatctgtat
gtgtgtgtgt atgtctgtgt gtaggtggta 1020 tatgcgagtg tgtatgtgtg
tgtgtgttgt gcatgtatgt gtatgtgtgt ctgtgtatgt 1080 gtgcctctgt
gtgtgaattt ctgtgtatgt gtgaatgtgt gtgcatgttt gtgtgtatat 1140
gtgtatatat gtgtatatac atgtatgtat ctgtgttgtt catgtgtgta tgtgtgcctg
1200 tgtctctgtg tgtgtgtttg tatgtgtgtg tgcatgtttg tgtgtgtata
tgtgtgtata 1260 ttgtatgtat ctgtgtgtgt ctgtgtctgc ctctgtgtgt
gtgtctgtgt gtgcatttgt 1320 gtgtatacat gtgggtatgt gtgtgcatgt
tttgtgtgta cgtgtatata tgtatgtatc 1380 tgtgtgtgtc tgtgtgttgt
gcgtgtgtgt gcctgcatgt gtccgtgtgc ttgtgtgtgt 1440 gtgtgtgttt
gcgtgggcat cttgagtgaa gcttccaaca atctaacaga agaaaaggag 1500
ccacacttgt ctgttctgct ctcttgggta cttcccagac cagtgaaatg aaagggagga
1560 aacccccggc ctccgaggag aaaagggaac tggcaagcag agggtggggg
gatgacggta 1620 aaaggagcag gggtggggag agcacaggcc ctgtggaagt
gaggacatgt gtgtgtacat 1680 gtgttcatgt ccaggggatg acactgtggc
atccaacagc cgtggaccag cagcccacgg 1740 ggagcttagt aggagtcaaa
tcctaggccc ccgtctcagc tgctctgctg tctagcgcat 1800 tgtgcagtgg
taggtgtcag tgatccaagt ggggacccag tccctcaggc cacacagcgc 1860
atgtccattg ccttcatgcc gcaggagact gaggggtcat gcatgagggc cacttctctg
1920 ggttgtcccc acaacactgg cggtgtccct gggacatgtg gaaaggggag
gggcagctca 1980 ctgctgacat ctccttctgc aggcctggag gaggcagagg
ccaggaggga gaggtcccaa 2040 gagcctgtga aatgggtctg gcctggctcc
cagctgggca ggaacacagg acttcaggac 2100 actaaggacc ctgtcatgcc
catggccagc acccaccagt gctggtgcct gcctgtccag 2160 agctgaccag
ggtagatggt gctggcccag gggctgctct ccatggccct gctggccctg 2220
tgctgggagc gcagcctggc aggggcagaa ggtgagtccc gtggctccca cccacttccc
2280 tgtccctgtc ctcactgctg caccctgggg gagggccgca gcgtatcctc
aggatcctgc 2340 ccgccagccc tcctcctgct cccctccctc tgtctctccc
cctggccttc cctgggcctc 2400 ccccgcttcc ctcctcctgc acattcctgc
tcatcctgtc ttggaaagtc cagctgagcg 2460 tgtctggctt ccttgcccac
atttctcagg gcggcactcc cggcccctag gctccaggat 2520 ggctgctctg
gccgtttccc tgcccctcct tccccagcag acactctctg tgcctcagtg 2580
gttcccacct ccgggacttt gctcctgcag ggccttggct ggggttctct ccctgcttca
2640 gccgctagca ccctccttgt gcctgaagcc cgcactggga tgctcctggg
ctcttgaggt 2700 gaaatggccc ctcccaaggg ctcccagaga cctggcttct
gtgataatgc tgggaccaca 2760 gtccccttaa caaataccag gctcctgagg
acggggacta gagaggaggt gggaggttgc 2820 agggtagaac tctgccacgc
tgcatcccag ggctgagtgt gtgccccctc ccaggctgca 2880 cagtcggtcc
aggggccagg cctgtgcttg atgcatgtcc ctgtcctggg gtggggggag 2940
gggaccgtgc ccaggaacag cacactgcgg aggcccagaa accatgctga gaaccaacag
3000 aatgtcttgc ttctgccagg agaggagggt ccgcaaccag gagcccaccc
cggcagacat 3060 gaacacatgt acatgtgcct ggccacctgg tgcctctgca
gggacctggg agacccctcc 3120 ccagagcggg acatcccaaa gcagctgggg
gtgatggtga caagggtccc tgcaggaaag 3180 agaggtgacc cccttctacc
cctcttgtca gaaaccatcc cgctgcagac cctgcgctgc 3240 tacaacgact
acaccagcca catcacctgc aggtgggcag acacccagga tgcccagcgg 3300
ctcgtcaacg tgaccctcat tcgccgggtg aatgagtgag tgatgctggg gcaggggcca
3360 cgggcnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 3420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 3480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3540 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3600 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3660
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
3720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 3780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 3840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3900 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3960 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4020
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
4080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 4140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 4200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnctcccca ccccctttgc ccaccgccgg 4260 ggcctgggcc tggaccaggg
ctcctgtctg aggcctgaga cacgatgctg tctggtcctg 4320 ttgctcagga
acatcacggc ccccaagtcc ccactctgcc ctgtgccacc caccatgccc 4380
tctcccagga tctttcccaa ttgccttgca aacctgcccc aaccccctct cagatgccca
4440 caggagtgca gcgcctgggc atctcctgac ctggcaaccc tgcggcccct
ctttctctgc 4500 ttcctttgac agcagcattc cctaaagagt tgtccacatt
caccacgtcc agtgtctttc 4560 cagccatttc ctcctgggcc cactccccta
ggatgctgcc cccaccaggc cacccaaact 4620 gctctggtcc aagtcagcaa
atgtcccagg gagcaggatc tcatggttgg gcctccgcct 4680 tcttcctact
tgaccagggg cagcccctga caaggaggac caccgcggcc tccctgcagt 4740
ttctcttgca cctgactctg cccacagctc acccagtgcc cccggggccc tgcccatcct
4800 ctctttctcc ttgtctgctg cctcctgatg gcaagacacc caggcccagt
ccatggacct 4860 cctctctgct gtatccactc tgcagagacc ccctcaggcc
cctggggcat ctcccaaagg 4920 tgacctcaag cccacaattc ccctaagctc
cagccctcca ggcgtactgg cctctccacc 4980 tgtcacttag acaacaggtg
tctccgactc caaacgtcct caaccaaagt cccgctctca 5040 cccgagctgc
gctgctgcct ctgtcttccc catctccctt tctaaatggg agctcctagt 5100
ggctcaaact aaacaacagg gaggattttg ttttgttttt aagtctcttt tgtcaccatt
5160 ccttcctgac tgctacatcc ctccaccagc aaatgctgcc aaccttatct
caagcagaac 5220 atgaatctca cccacgccag cctgaccacc ctgttgcacg
ccacttcctg tcacctgaat 5280 attccagaag cctcgtaact gggctccctg
cttccaacat tgccacattc ccacctcagt 5340 ctattcgcaa ccgaggaccc
tcaggaatcc tttcaaagca tttatcaaaa tctacaacac 5400 ctctgctcat
gactgtttcc cactcactcg cagtgagatc caatcccatc atgtagatgg 5460
atgggtctcc tagacctccc tatcacacgc tctactccct gcgcactggt tttaggccaa
5520 gccacgcagc acaccaaact ctgcctgtcc cagggccttt gcacgtgctg
ttcccatttc 5580 ctgaaatcct cacaatttgc acagtccctc cctcaatgcc
tttaacattc tgcccccagg 5640 tcacctcctt agagaagcct gcttgtccca
tatttcaaat tgcctccctg gcccagccct 5700 tcctgcctcc tcacctgctt
cttgctttat ttttccacat gaagttctct ccactgcaca 5760 cgctacacac
tttgttccat gcaggtcccc ttctcccagg atggcagctc cgtgggcagg 5820
atttttgtgt gttttatcac tgctggcccc tgattctgaa cagagccagg catgtggtga
5880 gcactcgagg aacctttcgt gagtcagtga tacccataca ccctgggcta
agccgtgtcc 5940 tctcccaaca gggacctcct ggagccagtg tcctgtgacc
tcagtgatga catgccctgg 6000 tcagcctgcc cccatccccg ctgcgtgccc
aggagatgtg tcattccctg ccagagtttt 6060 gtcgtcactg acgttgacta
cttctcattc caaccagaca ggcctctggg cacccggctc 6120 accgtcactc
tgacccagca tggtgagggg ctgggggccc tgcccggggc ttggtttcct 6180
gtgtggacag cgggggcacc aggggtggtc cagggagtct tcaaggcaga aggctgtggc
6240 ttggggttgg gtgagggttt cttgagggat gagggtatgg tctggttaca
tggagacttc 6300 agagcagagg ggcccctgac aaaggcttct cctgtgcccc
tggctgctac cacctcccac 6360 taagccatgg gcttccagca ctgcaggctt
gtcttgaaga aaaccgttct caccactcac 6420 ttagaaacct accaagttag
aaaactctgt ccgcttagtt gtttcatctc atctctgaag 6480 caacccattt
cctggatgtg gcccttgagg cccaggaggt cagatgcctc ctctgaagtc 6540
ccacagctga gcagtaatga cagagatggg gtcagccttc ccgggggctt cctgcatggt
6600 agacagggag cttgtctcct cgctgccctg gaggaggggc cccggtgtgg
gccgaaatcc 6660 cacagtggtc catgaagcca tctgtggtct agggtggggt
agatgattat tttatgtagt 6720 taggttttat ttgcctctat attagaaaga
aatataacct gcacttcgaa gtcctggatt 6780 caaggaaatg gttgcttaga
atgaagctaa acctgaaaag ggacacgttt tatgaaagtt 6840 ttcgttgacg
tggcaaaaac ccatgctggt ggcaggctgg taggaggtac tgcaggctgt 6900
cattgggagt ctcagaacca cggcaggctt ggtgctgtta ttggtgcctt tggctgctgc
6960 tgaaacggag acagagggag gtgactagtc caaggcctct ctactgctgc
tggcagagct 7020 gagatccagt gcaggatggt ctgagtcccc ggtcctaacc
accgaaccac ctggtgctct 7080 ttgcaagggt cacacggaag gggctctgac
catggcttcc tgcccttttt tgccacaaca 7140 tcataaagcc actgccagag
gaggggatca gttaggccac caggagtcca tcagaagcaa 7200 catttccaca
catgggtttg atggacacga gagtcccttc cttcccgaat ggagctcagg 7260
gggcccaggc tggagggagg
ggaaacactg tatgccgtcc accgtgagaa cttactgaca 7320 gtggcgggtg
ggtgcctcat gcagggggta aagggcaggg cccctgggag atcagaacag 7380
gcttcccaga gggaagagct ttgcaactca gatctgaagg gtagacagga ggggagggca
7440 gtaagagcct ctctggcagg gtaacagctg tgcagaggct gggggatcag
aagggcatgg 7500 gcagctgggg aacctgctca ccattgtgtc cagggagcat
cagcttcaag ggctggacag 7560 cggtgggaag aagcaggaga cacaggtaag
gacctgtccc tggagaggct tctgtctcct 7620 gcagtctcag gtagagggga
ccctctaggc atggagagca ctgaggggaa atgacatgat 7680 cagatgtgga
tgtgagaatg ctggggacag gggacttaca gggtagattg gaggagcatt 7740
agttgggaaa gaagttgaaa ttcaggctgt ggcctaaagc actcattcct agaaatgatg
7800 aggacacaac tggctaggag atggggacat gagggcatgc aaatcatagt
tcagatatac 7860 acacaattca ttcattcaac cttctttcct tgagcgccta
cataagccaa cgaagcagtc 7920 aacaaaccag ccagcaaccc tgcccttgga
gcttacagcc tggaaacaag gaaggtttct 7980 aaggcgatgg agacaactta
gatataggga gagaagtgtg gcccttggcc tcctggagcc 8040 tgtgttgagt
agcgaggtag ggtccagcct aatgcagatt agcaatgaat caaggatgct 8100
gggaggtact gggcaggcga caggcatgca tctgatctgc tctgagtctc catgtttgga
8160 gaccttggac attttacttc ctctatgagt ttctccgtct gtgaaatgag
ctggtggact 8220 caagaggttt ctcttgagtt ggctgagcag gtttctgatg
gggctcccag tctgcgcagt 8280 ttgtggcagc ttccgagagg gctctgccgg
gaagagctcc ccctccatga cagcctcggg 8340 ggctgggagt gcagtgaccc
atgagggacg cctgtcctgg ctgtgggtga ggaggggcgg 8400 ttcccctgct
gtgtgtctgc cgttctgggt tgatggttcc tgacatgctc tagcatgcca 8460
taacccatgt ccagcagaga gcacttacat cctatgtgtg aggatgtttt cgtttgaaag
8520 ccatccctca gcaagcagac accagaaacc agaaatcagg tgccgtgtct
tcattctgca 8580 ttttcttgaa caacccagag ttcccaggag atagatgctt
gccttgtggc tgcaaggatt 8640 tcatgagaag ccccaaagtt gcttacgcgt
atttgttcat tcattcactc attcaccttg 8700 ccccataatt cactgagaag
ccgcatctca gcctggaggt agggaagggg gtcaggacca 8760 atcccaccca
cccccatctc ctcacacctt aggggaggca gacacagaag cataggaatc 8820
cctcagctgt ggtaaggccc tggtggaggg aattccactg agctatggtg aaatgagaga
8880 aggaatgagg gattccgcct ggagatgcag atccgaggat gttcctagag
ccggaggcat 8940 ttgcccgggg cactgacaac aggaaggacc ctgggcagga
ggaagggagc ttggacagca 9000 ggagggggga ggccgctgaa ccgcaggccc
ctctgctagc aggagccacc caggccgcag 9060 cgtgggcagt ggggagcctc
aggacagagg aggctccaat gagtttcctc gccagcgctt 9120 tttatggagt
tcgggtcacg tgcgcattgc aatctgcacg gctttccatt gctttcatgt 9180
tgaaacccta cagttttgca gatgaagggc tgaggctcac agaggagacg ggtcttgctc
9240 aaggtccctc agctgctggg ggcaggggtg gcctggaacc ccctgtgtcc
acacaaaagg 9300 ccatgcaggc cctgactgcc ccccagcggt ccagccctta
ggtgcccttc acttcctccc 9360 ctccagtcca gcctcctgag cccagggacc
tgcagatcag caccgaccag gaccacttcc 9420 tgctgacctg gagtgtggcc
cttgggagtc cccagagcca ctggttgtcc ccaggggatc 9480 tggagtttga
ggtggtctac aagcggcttc aggactcttg ggaggtagga accacggcca 9540
gctctgcccc agcccgaagg gatgggcagc acccctcctc cagcacccac tgtctcctga
9600 caggacgcag ccatcctcct ctccaacacc tcccaggcca ccctggggcc
agagcacctc 9660 atgcccagca gcacctacgt ggcccgagta cggacccgcc
tggccccagg ttctcggctc 9720 tcaggacgtc ccagcaagtg gagcccagag
gtttgctggg actcccagcc aggtaatgtt 9780 gccagagccc aggaaatgcc
ccgtggtggg agggcaggct catcaggagc tcctggcaca 9840 gcagggttcc
tgggctccac ctgggggctt cccagatctc ctgctgccat ctttccagta 9900
gcgtccctgg gccgtcccac ctctactgtg accactgacc agtaggactc tgcatctgtt
9960 cactttgggt ttccagtttt ctgcacgttc tctgccaatg gcaattacaa
taataacaac 10020 aacagtgcta ttagcagctg tgtgttaatg gaggctacag
gatgctcagg gcttacccac 10080 atttttcagt tcaatcccca aacactgaaa
cttagatact atttccattc tcccgggagg 10140 gcgtgcaggt gcacagaact
ctctctctct ctctcggagc tgttggacac acagctggca 10200 ggttcaggct
gaagtttcag ccctggtctt ttggccccag agctcatgac ctctgtgtga 10260
tgaatcacac ggtgggcacc cactgagagc tatgggaggg atgaatgacg gagtacatga
10320 ggacctgtct ccaacccagg ggatgaggcc cagccccaga acctggagtg
cttctttgac 10380 ggggccgccg tgctcagctg ctcctgggag gtgaggaagg
aggtggccag ctcggtctcc 10440 tttggcctat tctacaagcc cagcccagat
gcagggtgag catctttttt ctccatcccc 10500 tcccctcctc ttggccttgc
tctctccaag cttcctcctg tccctggggc cccagcagaa 10560 gccacagccc
accctaagct ctcctccctc ccgtgtgccc tccctctccc tgccctcagc 10620
tctgctgtgc tcctcaggga ggaagagtgc tccccagtgc tgagggaggg gctcggcagc
10680 ctccacacca ggcaccactg ccagattccc gtgcccgacc ccgcgaccca
cggccaatac 10740 atcgtctctg ttcagccaag gagggcagag aaacacataa
agagctcagt gaacagtgag 10800 tttgctccta gcccgctgtg gggatggtct
gggaccagca caccctcatt gtgtaacccg 10860 aatcagttca gggttcctcc
tggccccgtc ttcatgtttg tcactttcaa agagatgcag 10920 tccagtgacc
aaaagtgaac agagaagcaa tgaaaccacg acggcagtgg ccaaaaacag 10980
gagcagatct ttaaaacctc tgatctcttg tccttttctt ctgcttccct ctcccatcct
11040 gcagctctct aaatctccac tgctagccac accctcctgg tcctgtcacc
aaaaccttcc 11100 ctttcattcc tcattggatt ttcctctttc tgatatccga
aattccccac cgactgatat 11160 tctatcttta atgtaattga tctatgatgt
acttttcaac tggagcctgt ggtgtgtaca 11220 aatagtgttg atccttggag
gttaacatcc ctcgtttctg tgatgtaaca aggaccccag 11280 tgcaatggaa
cctctcacgt tgtttcatga tgaccaagtt cttccatcca gtctttagca 11340
tatgtgaagg gcagaggcca atttgtctta attacctggg tttgaattta ccattaactc
11400 tcttgggatc ctcactggaa aaaggaattc tgattgttca aaagcacagg
atatattaag 11460 ggcctcatat aatgcctggc acataagaga cctcagcaaa
ttacgggcat tcatattatg 11520 tttatacagt gaaggcatca aggttataag
cattctcttt tttcttttgg gtagactgaa 11580 gctcagagag gttgagtggc
ttacttaaag ctgcacagct attagtaggc agatcaagat 11640 tagagttcag
aacttctcac tccctgccca gtttctgctt tctttaccct ttgcctcttt 11700
caagttgtgg gtctgccggc caggtgggag gtgctgtctg caaagggctt ccctttctct
11760 ttggccacta tctggctggg gagaggcctc acctagatgt tgttgaaggc
ctgttacagc 11820 cgctttattg gggattttct ggcgatgaga acgtgtgaat
gtcctggcct ttagtcaact 11880 ccctacacct ctgagagtgt cagacaagag
agcccatcac actggtggga ttgcaatctt 11940 tgcctctacc actgcttagc
tgcctttcct tagggcaagt tacttaatgg ttctgtgact 12000 cagtttccct
gtttgtgaaa agtgaaggtt aatagtaccc accatatagg gctgttagaa 12060
tggagtggaa taattcatgt agaataagta tgtataacag tggctaaaac atagtcaggc
12120 tgggcgcggt ggctcacgcc tgtaatccca gcactttggg aggccaaggc
atgtggatca 12180 cgtgaggtca ggagctcaag accagcctgg ccaacatggt
gaaaccccgt ctccactgaa 12240 aatacaaaaa ttagctgggc ttggtggcgg
gtgcctgtaa tcccagctac tcgggaggct 12300 gaggcaggag aatcacttga
acccaggagg cagaggttgc agttagccaa gatcacacca 12360 ctgcactcca
gcctgggcaa cagagtgaga ctccgtctca aaaaaaaaaa aaaaaaaaat 12420
agccagttgc ctagaataga accaactaac agtggtttta tttttactgc aaaaaataaa
12480 aataaaaata ggagtagtgc aagcactggg ccacatcact acaaaacaag
tgtatctcag 12540 catctcccac gagaatacca ctcaggtcaa aacatgatat
agtgaagtgg ggatgaaaag 12600 gatccaacca tgggcagaac ctggggtctg
gtgccagtgg agacagcccc agtgtctagc 12660 atgagacacg gggaatgttc
cgttggaggg tgggtatgat gactctcctg aaagcttccc 12720 tccctccagt
ccagatggcc cctccatccc tcaacgtgac caaggatgga gacagctaca 12780
gcctgcgctg ggaaacaatg aaaatgcgat acgaacacat agaccacaca tttgagatcc
12840 agtacaggaa agacacggcc acgtggaagg tgagggcctt tgcccaggga
cgggagaaac 12900 actggggagg gcgggagaag ggaaagcaac cagaggcatt
ccacctgcaa ggcgtcgggc 12960 ccttggcagg tgaccagtga gaggtagcca
ctgggacgtg gtgatcacta ggctgtgtgg 13020 tcagcaggtc actgtcctgt
ctcttggtga agtaactgag gtttggaaaa gtggcgtggc 13080 ttggccaacg
tgaacagctg accctgagtc cccaggcaac agaagaccct ctgggcaggg 13140
aggggttgaa aggccactgg gaagaaggtt ttcaaaagtc atgaaagttt ggggttattt
13200 cctcagagga atctcatctg gacacacatg gaggctcaga cagagctgct
tctaatgagt 13260 cgggggtgcg cccaggccag ggctcggtcc cctgcctcca
cagagcccag aacagaaacc 13320 acagaaccaa ccccacacct tcagtctaga
aatggggcaa ctgaggctag gagggaggtg 13380 ggccagtggt ggagccagga
gcgggccctg gggtcctgaa cccccattct cagggtccag 13440 agtccagtcg
gcctgcactg cgttcctgaa aaggccacaa tatgggtgca agctgcccca 13500
gaagggctgg gagctgagaa ggctcaaaat agggtgggac aggtggcttc agggttctgg
13560 gcctcagtgt tgtcaatgtc aggggctgca ctgacaggcg gagtccccgg
tgccatccga 13620 agtgctgtcc gtgggtgggc cctcagggag gatccacggt
ggtgagagag aagccgcagc 13680 aggcctgggg tatggcagga gctaggagcc
agcgaagccg agggtccagg tgggagggat 13740 ttgcagctgc tcccacgggc
accgggccag gcctcaccct cagtgccaac ccacaggaca 13800 gcaagaccga
gaccctccag aacgcccaca gcatggccct gccagccctg gagccctcca 13860
ccaggtactg ggccagggtg agggtcagga cctcccgcac cggctacaac gggatctgga
13920 gtgagtggag tgaggcgcgc tcctgggaca ccgagtcggg taggtgaagg
ctggagtcca 13980 gagcttctgg ccaggaccag ctcatagttt ctcactgcca
gaaaatcccc aatgcagcag 14040 ccgtagcagg cctgcaacaa cttgtaggtg
agccgtctcc ccgattagat ggtggccaaa 14100 gagaaaggga aggcctttgc
agagagcacc tcccacctgg tcatcagacc cgcaagttga 14160 aagaggcaaa
gggtaaagca ggcagaaact gggcgtggag tcagaagttc tccacccact 14220
cttcatgaga cagcacagct gggcagagga tgccacatct ctgggtgggc acagagatgt
14280 ggggctccag accccactcc actcaggcct ccttccctcc cttggaccac
tagtcgttgg 14340 aaagtgacac ctggtgttca gtacagactg caagtgagga
cttgagggag cagaggagag 14400 caggtctcat gatcgctcct cctttaaact
gggacttagg cttctgttaa tatggccctt 14460 ggtttcttag cttttgggac
taccaagttt ttgtggttgc tcagcccgag tttcctgtta 14520 ccatggaagc
aagaaaatct agagccacag actgggtcct tggattggaa ggttttgctg 14580
tgattccttc atcttcccac ctccctgcac ttgctgcgtg cactgacccc tcacctttca
14640 ccatagtgac caccaggcga ctcccgcaga acaagacaag tccaaggggt
ggtttcagag 14700 agagtaagga tacaggagat attgggagca acccaagtcc
ccagccaggc ttggtagcgg 14760 caggggccca gggagagggc cacaaccagg
tagaaaggag gccggaccaa tgggcccaag 14820 agaggaggaa gaggggagtc
ctggaggaac ctggcctggg agctcagggc tgggcagcag 14880 gtgggatcag
gggcctctgg aggggccttt gctcctgctg gtgcctctgc cactatgcct 14940
ttccccatct ccacctctaa tccttcctat tactggaggc ccagctaata tgccacctcc
15000 ttcaggaagt cccctgtgat tgccatgaac agtggttctt gccctccctg
tagcacctcc 15060 tgtgctatag atcctggaaa ggatctgacc ctcctgtgtc
ctcccagcac tttgaacact 15120 gtgggggttc agaccaatgc ccttcccagc
atggatctgg cctctctccc ctaatccccc 15180 aaggcagtaa gttccagagc
cctgggcctg cacagagcgg ggtcttaagg aatactgcac 15240 caaccccact
ccctgccctg aggtcgattt cccgcccaga tgctgacatt cctctttctc 15300
cccggctgct ggaagtgctg cctatgtggg tgctggccct catcgtgatc ttcctcacca
15360 tcgctgtgct cctggccctc cgcttctgtg gcatctacgg gtacaggtga
ggggactctg 15420 tggggctgga ggtggcagcc gagaccccag agggcactgg
ggaatcccac ccagctccct 15480 ccaggccctg ctctgccgct cccttcctgg
gcctcagttt cccctctggg catgagcatg 15540 ggagcagctg gccatgaggt
ctctgatggc tgtcacctcc cgtggtgtct tccaggctgc 15600 gcagaaagtg
ggaggagaag atccccaacc ccagcaagag ccacctgttc caggtaggaa 15660
ctggctgcga ggggcggagt gggggcttct ctgttcctgc ctctcttgtc tctgtcccca
15720 cctccctcct tcccttcctg tgggctttgg gtggtaggga tgtaggtgga
ctgggctttg 15780 ggagctttgg gagtggggcc agcacttgga aattcattcc
cgggattccc tggagtgccc 15840 acacctggcc ctgcctgcca gagctctgca
tggagcccag tggagagaac tgagtgtggg 15900 gatgcctggg acagggtggg
ctcacagagg gggttcctgc taccaggatg gatggggtgt 15960 caggaggcag
aagggctccc caaggacaag atacctgggc tgagctttga gggtcccagt 16020
ggtgccaggt ggcaaaagtt gctctggctc agggcatctt gagggtggag acagaaaccc
16080 ttcaggaagg gagctgaagg ggtgggacag gaggctgggg gccatgttgg
gaggctgaga 16140 tgaccatggg gaagacacca gcgtgtgggt gatgggagcc
ctatagtcag gcaggcaggg 16200 ccagctttgc ctgtggaggg gtctcttggc
tggtgtggag gagaaatggg ggtgagcatg 16260 gagacaggca ctccaggtta
gaaaaatcac aggaaaaggg ccctgtcctc aggtagcagg 16320 gacatggggg
aagtccccgc ccctctctgg gcctcacttt gctggtgtca aaaggccgtg 16380
gccagtcctg aagaagtgga gattcttctc ttgtctgggg gctccttgaa tcctcctgca
16440 cccccgtcac cctctgcctt gcccccacct cggacctcct gatgctcacc
ggcccaaatg 16500 tctctgctct tgcagaacgg gagcgcagag ctttggcccc
caggcagcat gtcggccttc 16560 actagcggga gtcccccaca ccaggggccg
tggggcagcc gcttccctga gctggagggg 16620 tgagtgggct cgtggatcac
tcctgacctt tggggttcat acggggggct gactccattg 16680 tggaaatcag
ggactcaagt gaggctgcct gtggctcact gtgaagggat ccaagggagc 16740
tggtttgcac taaagcagga ggcacctggg ggggatgtga ggaagaactt cctctttcca
16800 ggtaaggaag tgccagacaa ggggagtgac cggaagtgaa agagggaaac
agttggcctt 16860 cccctccctg gctgcctcag aggcgtcaca ctgcagaggc
ctcatggggt agctggggct 16920 gcagactccc gggaactcgg cgcctgagcc
ggcagctgac caccaactcg gcagggagaa 16980 gggggtggca tggttgatag
aatgatggaa gggttgccca gtgcacaggc tgggcatgag 17040 cctggtgctg
aaggaggatg gactttaagg tcaaaaggga gagggtacca gccttgcaga 17100
ggaagacctt gagctcagca tggaggatgg agctcctgag ccacggaaag actgaatcca
17160 tgtcccatgt ccttctctgg ggcccctgct ccctcaaccc tgtcccgttc
aggttctctc 17220 tgtgagatct gggggacatc agggcttcca gagaaccatc
tccaccccac caagaccctt 17280 gtgcctgacc cggatcatct gcccagggtg
gtcccaactc ttctgcccat tttcttccca 17340 cagggtgttc cctgtaggat
tcggggacag cgaggtgtca cctctcacca tagaggaccc 17400 caagcatgtc
tgtgatccac catctgggcc tgacacgact ccagctgcct cagatctacc 17460
cacagagcag ccccccagcc cccagccagg cccgcctgcc gcctcccaca cacctgagaa
17520 acaggcttcc agctttgact tcaatgggcc ctacctgggg ccgccccaca
gccgctccct 17580 acctgacatc ctgggccagc cggagccccc acaggagggt
gggagccaga agtccccacc 17640 tccagggtcc ctggagtacc tgtgtctgcc
tgctgggggg caggtgcaac tggtccctct 17700 ggcccaggcg atgggaccgg
gacaggccgt ggaagtggag agaaggccga gccagggggc 17760 tgcagggagt
ccctccctgg agtccggggg aggccctgcc cctcctgctc ttgggccaag 17820
ggtgggagga caggaccaaa aggacagccc tgtggctata cccatgagct ctggggacac
17880 tgaggaccct ggagtggcct ctggttatgt ctcctctgca gacctggtat
tcaccccaaa 17940 ctcaggggcc tcgtctgtct ccctagttcc ctctctgggc
ctcccctcag accagacccc 18000 cagcttatgt cctgggctgg ccagtggacc
ccctggagcc ccaggccctg tgaagtcagg 18060 gtttgagggc tatgtggagc
tccctccaat tgagggccgg tcccccaggt caccaaggaa 18120 caatcctgtc
ccccctgagg ccaaaagccc tgtcctgaac ccaggggaac gcccggcaga 18180
tgtgtcccca acatccccac agcccgaggg cctccttgtc ctgcagcaag tgggcgacta
18240 ttgcttcctc cccggcctgg ggcccggccc tctctcgctc cggagtaaac
cttcttcccc 18300 gggacccggt cctgagatca agaacctaga ccaggctttt
caagtcaaga agcccccagg 18360 ccaggctgtg ccccaggtgc ccgtcattca
gctcttcaaa gccctgaagc agcaggacta 18420 cctgtctctg cccccttggg
aggtcaacaa gcctggggag gtgtgttgag acccccaggc 18480 ctagacaggc
aaggggatgg agagggcttg ccttccctcc cgcctgacct tcctcagtca 18540
tttctgcaaa gccaaggggc agcctcctgt caaggtagct agaggcctgg gaaaggagat
18600 agccttgctc cggccccctt gaccttcagc aaatcacttc tctccctgcg
ctcacacaga 18660 cacacacaca cacacgtaca tgcacacatt tttcctgtca
ggttaactta tttgtaggtt 18720 ctgcattatt agaactttct agatatactc
attccatctc cccctcattt ttttaatcag 18780 gtttccttgc ttttgccatt
tttcttcctt cttttttcac tgatttatta tgagagtggg 18840 gctgaggtct
gagctgagcc ttatcagact gagatgcagc tggttgtgtt gaggacttgt 18900
gtgggctgcc tgtccccggc agtcgctgat gcacatgaca tgattctcat ctgggtgcag
18960 aggtgggagg caccaggtgg gcacccgtgg gggttagggc ttggaagagt
ggcacaggac 19020 tgggcacgct cagtgaggct cagggaattc agactagcct
cgattgtcac tccgagaaat 19080 gggcatggta ttgggggtcg ggggggcggt
gcaagggacg cacatgagag actgtttggg 19140 agcttctggg gagccctgct
agttgtctca gtgatgtctg tgggacctcc agtcccttga 19200 gaccccacgt
catgtagaga agttaacggc ccaagtggtg ggcaggctgg tgggacctgg 19260
ggaacatcag gagaggagtc cagagcccac gtctactgcg gaaaagtcag gggaaactgc
19320 caaacaaagg aaaatgcccc aaaggcatat atgctttagg gcctttggtc
caaatggccc 19380 gggtggccac tcttccagat agaccaggca actctccctc
ccaccggcca cagatgaggg 19440 gctgctgatc tatgcctggg cctgcaccag
ggattatggt tcttttaaat ctttgccttt 19500 cagatacagg aaaaataatg
gcattaaatt gctttaattt gcattatttt agttatccag 19560 tttgcacata
tttttatagg tatcttaggc atcgattggt attttttaac tgggccaagc 19620
ccattaaggt ctttcttctg ttgggtgcta tcattttcct gattaagtct ttttgactat
19680 tgacatacag tctttcacag atggtggagt gtttttcccc caaatctgtt
gtttgtctta 19740 taatgttgta tatgaggttt tatggtgtat gaatatgaat
gcttctgtaa tgtcaaacag 19800 atccctagta aactccttct tcacttttac
tgtcagattt acaaaggtcc tcccattgca 19860 aagcagtgtt tgtcctaatt
tatatattgt ttttctagtt cattttgtgt ttccaacttt 19920 tcatgtaaaa
ttttaattat ttttgaatgt gtggatgtga gactgaggtg ccttttggta 19980
ctgaaattct ttttccatgt acctgaagtg ttacttttgt gatataggaa atccttgtat
20040 atatacttta ttggtcccta ggcttcctat tttgttacct tgctttctct
atggcatcca 20100 ccattttgat tgttctactt ttatgatatg ttttcataag
tggttaagca agtattctcg 20160 ttacttttgc tcttaaatcc ctattcatta
cagcaatgtt ggtggtcaaa gaaaatgata 20220 aacaacttga atgttcaatg
gtcctgaaat acataacaac attttagtac attgtaaagt 20280 agaatcctct
gttcataatg aacaagatga accaatgtgg attagaaaga agtccgagat 20340
attaattcca aaatatccag acattgttaa agggaaaaaa ttgcaataaa atatttgtaa
20400 cataaaacaa agtgaaaccc tgaatttgtg tgtgcatgtt ggtgtagttg
gaggaagggg 20460 ttgctctttg aaacctcaat tgctattgta agtgatacag
ctccagtgac tggaggaaca 20520 ccagggtcct tagtcttgcg ccgatttaaa
taaaacgaca cggaaacaca tggagtggtt 20580 ttaaggagtg gagagtttaa
taggcaagaa agaaggaaga agctcccctg tacagagaca 20640 gagggagggg
ggatccaaag ctgagagagg aaaccctgag tgccacagaa ataagccagt 20700
tatatgagga ggctagagaa ggcagtatct gatttgcata gggctcaggg gattggtttg
20760 accaggcatg tcattcatgt aacccctgaa aaacctggcc ctttcaccct
agcattttaa 20820 tatgcaaatg cagggcgcca tgatgttcta cacaggtggg
actatgtggg ggtggccata 20880 ttgccaggca aacatgggga caaggaaaag
atggcgggaa tccccatgtt tgggtggacc 20940 cagtttctaa cggtctgcat
ttgcatatca aaggttgcca gcctgattct aagagccggg 21000 gctttcctgc
tagacaagaa acgttttttg gagctgcttt taaaacagaa acgaaaactt 21060
cccaaggacc acttttcctc tttatctgcc tcaaataatt ttttaataat tcctataaca
21120 caaggaaatg aattctgcca aacagaaggg cctttggtct ttggggcact
acagtgggta 21180 catgatggcg tgtggaacac cacgcaatgg agggagacgc
agggcactcc tgggaaaatc 21240 caggagggat gagggaagag gaaagacagt
gagggaaagg aggataaaga acacactttt 21300 aaaaaatcgt tcattattat
ctgaaattcc aacacaactg aacgtcctgt atttttccct 21360 agtcatgaga
gtgaaaaact aaatagagac tgagttcccc atacaaataa ctgagagtga 21420
tcgagagctt attcctggca ggccccattc gagtgacctc taagatcaat tcatttagat
21480 actatggaag tggaagatac tatctttatc atttttgtca ttgaggaaac
taaggcacag 21540 agatgtcata caacttgccc caggtctgcc agcaggtaag
gggcagagcc aagatttgaa 21600 ctgtagccct gtgtctcaga gcctgccctc
aaaaggtaag tctctcacct aaagatccat 21660 acggagatgt aagatgatcc
atgttggttg gcaagacgga aaaatccata aaccaaataa 21720 attgatttgg
tggcaggggg agattgaatc agaggtgacc ctgaaaagtg tctgtttatc 21780
tgggagctga acactgaagg tgcaattctc acccattgga gtccagagag accttccctg
21840 gaggactgca gctccctcat ctgtcttccc ctgagaaccc agcccagagc
catgcatgta 21900 gtaggtgatc attgaggtga gcaaggcctg caggaggctg
tccctaagat ggaggagaac 21960 aaaaaact 21968 4 581 PRT Homo sapiens 4
Met Val Leu Ala Gln Gly Leu Leu Ser Met Ala Leu Leu Ala Leu Cys 1 5
10 15 Trp Glu Arg Ser Leu Ala Gly Ala Glu Glu Thr Ile Pro Leu Gln
Thr 20 25 30 Leu Arg Cys Tyr Asn Asp Tyr Thr Ser His Ile Thr Cys
Arg Trp Ala 35 40 45 Asp
Thr Gln Asp Ala Gln Arg Leu Val Asn Val Thr Leu Ile Arg Arg 50 55
60 Val Asn Glu Asp Leu Leu Glu Pro Val Ser Cys Asp Leu Ser Asp Asp
65 70 75 80 Met Pro Trp Ser Ala Cys Pro His Pro Arg Cys Val Pro Arg
Arg Cys 85 90 95 Val Ile Pro Cys Gln Ser Phe Val Val Thr Asp Val
Asp Tyr Phe Ser 100 105 110 Phe Gln Pro Asp Arg Pro Leu Gly Thr Arg
Leu Thr Val Thr Leu Thr 115 120 125 Gln His Val Gln Pro Pro Glu Pro
Arg Asp Leu Gln Ile Ser Thr Asp 130 135 140 Gln Asp His Phe Leu Leu
Thr Trp Ser Val Ala Leu Gly Ser Pro Gln 145 150 155 160 Ser His Trp
Leu Ser Pro Gly Asp Leu Glu Phe Glu Val Val Tyr Lys 165 170 175 Arg
Leu Gln Asp Ser Trp Glu Asp Ala Ala Ile Leu Leu Ser Asn Thr 180 185
190 Ser Gln Ala Thr Leu Gly Pro Glu His Leu Met Pro Ser Ser Thr Tyr
195 200 205 Val Ala Arg Val Arg Thr Arg Leu Ala Pro Gly Ser Arg Leu
Ser Gly 210 215 220 Arg Pro Ser Lys Trp Ser Pro Glu Val Cys Trp Asp
Ser Gln Pro Gly 225 230 235 240 Asp Glu Ala Gln Pro Gln Asn Leu Glu
Cys Phe Phe Asp Gly Ala Ala 245 250 255 Val Leu Ser Cys Ser Trp Glu
Val Arg Lys Glu Val Ala Ser Ser Val 260 265 270 Ser Phe Gly Leu Phe
Tyr Lys Pro Ser Pro Asp Ala Gly Glu Glu Glu 275 280 285 Cys Ser Pro
Val Leu Arg Glu Gly Leu Gly Ser Leu His Thr Arg His 290 295 300 His
Cys Gln Ile Pro Val Pro Asp Pro Ala Thr His Gly Gln Tyr Ile 305 310
315 320 Val Ser Val Gln Pro Arg Arg Ala Glu Lys His Ile Lys Ser Ser
Val 325 330 335 Asn Ile Gln Met Ala Pro Pro Ser Leu Asn Val Thr Lys
Asp Gly Asp 340 345 350 Ser Tyr Ser Leu Arg Trp Glu Thr Met Lys Met
Arg Tyr Glu His Ile 355 360 365 Asp His Thr Phe Glu Ile Gln Tyr Arg
Lys Asp Thr Ala Thr Trp Lys 370 375 380 Asp Ser Lys Thr Glu Thr Leu
Gln Asn Ala His Ser Met Ala Leu Pro 385 390 395 400 Ala Leu Glu Pro
Ser Thr Arg Tyr Trp Ala Arg Val Arg Val Arg Thr 405 410 415 Ser Arg
Thr Gly Tyr Asn Gly Ile Trp Ser Glu Trp Ser Glu Ala Arg 420 425 430
Ser Trp Asp Thr Glu Ser Val Leu Pro Met Trp Val Leu Ala Leu Ile 435
440 445 Val Ile Phe Leu Thr Ile Ala Val Leu Leu Ala Leu Arg Phe Cys
Gly 450 455 460 Ile Tyr Gly Tyr Arg Leu Arg Arg Lys Trp Glu Glu Lys
Ile Pro Asn 465 470 475 480 Pro Ser Lys Ser His Leu Phe Gln Asn Gly
Ser Ala Glu Leu Trp Pro 485 490 495 Pro Gly Ser Met Ser Ala Phe Thr
Ser Gly Ser Pro Pro His Gln Gly 500 505 510 Pro Trp Gly Ser Arg Phe
Pro Glu Leu Glu Gly Val Phe Pro Val Gly 515 520 525 Phe Gly Asp Ser
Glu Val Ser Pro Leu Thr Ile Glu Asp Pro Lys His 530 535 540 Val Cys
Asp Pro Pro Ser Gly Pro Asp Thr Thr Pro Ala Ala Ser Asp 545 550 555
560 Leu Pro Thr Glu Gln Pro Pro Ser Pro Gln Pro Gly Pro Pro Ala Ala
565 570 575 Ser His Thr Pro Glu 580
* * * * *
References