U.S. patent application number 10/274971 was filed with the patent office on 2003-03-20 for isolated human secreted proteins, nucleic acid molecules encoding secreted proteins and uses thereof.
This patent application is currently assigned to APPLERA CORPORATION. Invention is credited to Brandenberger, Ralph, Gong, Fangcheng, Higgins, Maureen E., Hu, Song, Ketchum, Karen A., Ladunga, Istvan I..
Application Number | 20030055237 10/274971 |
Document ID | / |
Family ID | 25270524 |
Filed Date | 2003-03-20 |
United States Patent
Application |
20030055237 |
Kind Code |
A1 |
Hu, Song ; et al. |
March 20, 2003 |
Isolated human secreted proteins, nucleic acid molecules encoding
secreted proteins and uses thereof
Abstract
The present invention provides amino acid sequences of peptides
that are encoded by genes within the human genome, the secreted
peptides of the present invention. The present invention
specifically provides isolated peptide and nucleic acid molecules,
methods of identifying orthologs and paralogs of the secreted
peptides, and methods of identifying modulators of the secreted
peptides.
Inventors: |
Hu, Song; (Pleasanton,
CA) ; Ketchum, Karen A.; (Germantown, MD) ;
Gong, Fangcheng; (Germantown, MD) ; Ladunga, Istvan
I.; (Foster City, CA) ; Higgins, Maureen E.;
(Bethesda, MD) ; Brandenberger, Ralph; (Menlo
Park, CA) |
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: |
25270524 |
Appl. No.: |
10/274971 |
Filed: |
October 22, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10274971 |
Oct 22, 2002 |
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09835811 |
Apr 17, 2001 |
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6482936 |
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Current U.S.
Class: |
536/23.2 ;
435/183; 435/320.1; 435/325; 435/69.1; 530/350 |
Current CPC
Class: |
A61K 38/00 20130101;
A01K 2217/05 20130101; C07K 14/47 20130101 |
Class at
Publication: |
536/23.2 ;
435/69.1; 435/320.1; 435/325; 435/183; 530/350 |
International
Class: |
C07H 021/04; C12N
009/00; C12P 021/02; C12N 005/06; C07K 014/435 |
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. An isolated nucleic acid molecule consisting of a nucleotide
sequence selected from the group consisting of: (a) a nucleotide
sequence that encodes an amino acid sequence shown in SEQ ID NO:2;
(b) a nucleotide sequence that encodes of an allelic variant of an
amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide
sequence hybridizes under stringent conditions to the opposite
strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a
nucleotide sequence that encodes an ortholog of an amino acid
sequence shown in SEQ ID NO:2, wherein said nucleotide sequence
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotide
sequence that encodes a fragment of an amino acid sequence shown in
SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous
amino acids; and (e) a nucleotide sequence that is the complement
of a nucleotide sequence of (a)-(d).
5. An isolated nucleic acid molecule comprising a nucleotide
sequence selected from the group consisting of: (a) a nucleotide
sequence that encodes an amino acid sequence shown in SEQ ID NO:2;
(b) a nucleotide sequence that encodes of an allelic variant of an
amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide
sequence hybridizes under stringent conditions to the opposite
strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a
nucleotide sequence that encodes an ortholog of an amino acid
sequence shown in SEQ ID NO:2, wherein said nucleotide sequence
hybridizes under stringent conditions to the opposite strand of a
nucleic acid molecule shown in SEQ ID NOS:1 or 3; (d) a nucleotide
sequence that encodes a fragment of an amino acid sequence shown in
SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous
amino acids; and (e) a nucleotide sequence that is the complement
of a nucleotide sequence of (a)-(d).
6. A gene chip comprising a nucleic acid molecule of claim 5.
7. A transgenic non-human animal comprising a nucleic acid molecule
of claim 5.
8. A nucleic acid vector comprising a nucleic acid molecule of
claim 5.
9. A host cell containing the vector of claim 8.
10. 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.
11. 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.
12. 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.
13. A method for detecting the presence of a nucleic acid molecule
of claim 5 in a sample, said method comprising contacting the
sample with an oligonucleotide that hybridizes to said nucleic acid
molecule under stringent conditions and determining whether the
oligonucleotide binds to said nucleic acid molecule in the
sample.
14. 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.
15. The method of claim 14, wherein said agent is administered to a
host cell comprising an expression vector that expresses said
peptide.
16. 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.
17. A pharmaceutical composition comprising an agent identified by
the method of claim 16 and a pharmaceutically acceptable carrier
therefor.
18. A method for treating a disease or condition mediated by a
human secreted protein, said method comprising administering to a
patient a pharmaceutically effective amount of an agent identified
by the method of claim 16.
19. 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.
20. An isolated human secreted peptide having an amino acid
sequence that shares at least 70% homology with an amino acid
sequence shown in SEQ ID NO:2.
21. A peptide according to claim 20 that shares at least 90 percent
homology with an amino acid sequence shown in SEQ ID NO:2.
22. An isolated nucleic acid molecule encoding a human secreted
peptide, said nucleic acid molecule sharing at least 80 percent
homology with a nucleic acid molecule shown in SEQ ID NOS:1 or
3.
23. A nucleic acid molecule according to claim 22 that shares at
least 90 percent homology with a nucleic acid molecule shown in SEQ
ID NOS:1 or 3.
Description
FIELD OF THE INVENTION
[0001] The present invention is in the field of secreted proteins
that are related to the nodal-related secreted subfamily,
recombinant DNA molecules, and protein production. The present
invention specifically provides novel peptides and proteins that
effect protein phosphorylation 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] Secreted Proteins
[0003] Many human proteins serve as pharmaceutically active
compounds. Several classes of human proteins that serve as such
active compounds include hormones, cytokines, cell growth factors,
and cell differentiation factors. Most proteins that can be used as
a pharmaceutically active compound fall within the family of
secreted proteins. It is, therefore, important in developing new
pharmaceutical compounds to identify secreted proteins that can be
tested for activity in a variety of animal models. The present
invention advances the state of the art by providing many novel
human secreted proteins.
[0004] Secreted proteins are generally produced within cells at
rough endoplasmic reticulum, are then exported to the golgi
complex, and then move to secretory vesicles or granules, where
they are secreted to the exterior of the cell via exocytosis.
[0005] Secreted proteins are particularly useful as diagnostic
markers. Many secreted proteins are found, and can easily be
measured, in serum. For example, a `signal sequence trap` technique
can often be utilized because many secreted proteins, such as
certain secretory breast cancer proteins, contain a molecular
signal sequence for cellular export. Additionally, antibodies
against particular secreted serum proteins can serve as potential
diagnostic agents, such as for diagnosing cancer.
[0006] Secreted proteins play a critical role in a wide array of
important biological processes in humans and have numerous
utilities; several illustrative examples are discussed herein. For
example, fibroblast secreted proteins participate in extracellular
matrix formation. Extracellular matrix affects growth factor
action, cell adhesion, and cell growth. Structural and quantitative
characteristics of fibroblast secreted proteins are modified during
the course of cellular aging and such aging related modifications
may lead to increased inhibition of cell adhesion, inhibited cell
stimulation by growth factors, and inhibited cell proliferative
ability (Eleftheriou et al., Mutat Res 1991
March-November;256(2-6): 127-38).
[0007] The secreted form of amyloid beta/A4 protein precursor (APP)
functions as a growth and/or differentiation factor. The secreted
form of APP can stimulate neurite extension of cultured
neuroblastoma cells, presumably through binding to a cell surface
receptor and thereby triggering intracellular transduction
mechanisms. (Roch et al., Ann N Y Acad Sci Sep. 24,
1993;695:149-57). Secreted APPs modulate neuronal excitability,
counteract effects of glutamate on growth cone behaviors, and
increase synaptic complexity. The prominent effects of secreted
APPs on synaptogenesis and neuronal survival suggest that secreted
APPs play a major role in the process of natural cell death and,
furthermore, may play a role in the development of a wide variety
of neurological disorders, such as stroke, epilepsy, and
Alzheimer's disease (Mattson et al., Perspect Dev Neurobiol 1998;
5(4):337-52).
[0008] Breast cancer cells secrete a 52K estrogen-regulated protein
(see Rochefort et al., Ann N Y Acad Sci 1986;464:190-201). This
secreted protein is therefore useful in breast cancer
diagnosis.
[0009] Two secreted proteins released by platelets, platelet factor
4 (PF4) and beta-thromboglobulin (betaTG), are accurate indicators
of platelet involvement in hemostasis and thrombosis and assays
that measure these secreted proteins are useful for studying the
pathogenesis and course of thromboembolic disorders (Kaplan, Adv
Exp Med Biol 1978;102:105-19).
[0010] Vascular endothelial growth factor (VEGF) is another example
of a naturally secreted protein. VEGF binds to cell-surface heparan
sulfates, is generated by hypoxic endothelial cells, reduces
apoptosis, and binds to high-affinity receptors that are
up-regulated by hypoxia (Asahara et al., Semin Interv Cardiol 1996
September;1(3):225-32).
[0011] Many critical components of the immune system are secreted
proteins, such as antibodies, and many important functions of the
immune system are dependent upon the action of secreted proteins.
For example, Saxon et al., Biochem Soc Trans 1997 May;25(2):383-7,
discusses secreted IgE proteins.
[0012] For a further review of secreted proteins, see
Nilsen-Hamilton et al., Cell Biol Int Rep 1982
September;6(9):815-36.
[0013] Nodal Proteins
[0014] Nodal and Nodal-related proteins have functions in mesoderm
and endoderm induction and formation, as well as subsequent
organization of axial structures such as heart and stomach in early
embryogenesis. It has been demonstrated that dorsal tissue in a
developing vertebrate embryo contributes predominantly to the axial
structures of the notochord and pre-chordal plate while it recruits
surrounding cells to form non-axial embryonic structures. Recent
genetic and molecular studies in mouse, zebrafish and Xenopus
reveal the molecular evidences of Nodal signaling (Zhou et al,
Nature 361: 543-547 (1993); Feldman et al, Nature 395: 181-185,
(1998); Rebagliati et al, Dev. Biol. 199: 261-272 (1998);
Rebagliati MR, et al., Proc. Natl. Acad. Sci. USA. 95: 9932-9937
(1998); Sampath et al, Nature 395: 185-189 (1998); Ezal et al, J.
Biol. Chem. 275: 14124-14131 (2000); Takahashi et al, Development
127: 5319-5329 (2000)). When squint and cyclops of zebrafish
Nodal-related genes become simultaneously disrupted, embryos fail
to form mesoderm and endoderm. Nodal-related genes Xnr5 and Xnr6
have been reported to initiate inductive events in Xenopus
Nierwkoop center. Mouse Nodal gene has been identified through the
insertional mutation. One mouse Nodal mutant (a
retrovirally-induced recessive prenatal lethal mutation) fails to
form mesoderm, and its embryonic ectoderm over-proliferates and
subsequently degenerates.
[0015] The Nodal and Nodal-related proteins have been identified to
use extracellular factors of transforming growth factors beta
(TGF-beta) for signaling relay. Nodal signaling pathway is
suggested as following (Stemple, Curr. Biol. 10:R843-846 (2000);
Alexander and Stainier, Curr. Biol. 9:1147-1157 (1999)). Nodal
ligands interact with their co-factors to activate activin type I
and type II or related receptors, which phosphorylate Smad2. The
phosphorylated Smad2 forms a complex with Smad4 for nuclear import.
In the nucleus, the Smad2/Smad4 complex serves as an effector of
TGF-beta signaling by regulating transcription factors Fast-1 and
Mixer, which control the expression of the genes for the
development of dorsal axial structures and left-right
asymmetry.
[0016] During gastrulation, the three germ layers of the embryo are
formed and organized along the anterior-posterior body axis. In the
mouse, gastrulation involves the delamination of ectodermal cells
through the primitive streak and their differentiation into
mesoderm. These processes do not occur in embryos homozygous for a
retrovirally induced recessive prenatal lethal mutation, the strain
413-d insertional mutation. Instead of giving rise to mesoderm,
embryonic ectoderm in 413-d mutants overproliferates and then
rapidly degenerates, although extraembryonic lineages remain
viable. For more information, see Dubois, et al., J Biol Chem. May
5, 1995;270(18):10618-24.
[0017] Nodal, a member of the transforming growth factor beta
(TGF-beta) superfamily, is implicated in many events critical to
the early vertebrate embryo, including mesoderm formation, anterior
patterning, and left-right axis specification. Experimental
evidence has demonstrated that nodal signaling activates pAR3-Lux,
a luciferase reporter previously shown to respond specifically to
activin and TGF-beta. However, nodal is unable to induce pT1x2-Lux,
a reporter specifically responsive to bone morphogenetic proteins.
Furthermore, it has also been demonstrated that nodal induces
p(CAGA)(12), a reporter previously shown to be specifically
activated by Smad3. Expression of a dominant negative Smad2
significantly reduces the level of luciferase reporter activity
induced by nodal treatment. Also, experimental evidence has shown
that nodal signaling rapidly leads to the phosphorylation of Smad2.
These results provide direct biochemical evidence that nodal
signaling is mediated by both activin-TGF-beta pathway Smads, Smad2
and Smad3. Further evidence has shown that the extracellular cripto
protein is required for nodal signaling, making it distinct from
activin or TGF-beta signaling. For more information, see Sirotkin,
et al., Curr Biol. Sep. 7, 2000;10(17):10514; and Kumar A, et al.,
J Biol Chem. January 5;276(1):656-61.
[0018] Nodal-related signals comprise a subclass of the
transforming growth factor-beta (TGF-beta) superfamily and regulate
key events in vertebrate embryogenesis, including mesoderm
formation, establishment of left-right asymmetry and neural
patterning. Nodal ligands are thought to act with EGF-CFC protein
co-factors to activate activin type I and II or related receptors,
which phosphorylate Smad2 and trigger nuclear translocation of a
Smad2/4 complex. The winged-helix transcription factor forkhead
activin signal transducer-1 (Fast-1) acts as a co-factor for Smad2.
Xenopus Fast-1 is thought to function as a transcriptional effector
of Nodal signals during mesoderm formation, but no mutations in the
Fast-i gene have been identified. We report the identification of
the zebrafish fast1 gene and show that it is disrupted in
schmalspur (sur) mutants, which have defects in the development of
dorsal midline cell types and establishment of left-right
asymmetry. We find that prechordal plate and notochord are strongly
reduced in maternal-zygotic sur mutants, whereas other
mesendodermal structures are present--a less severe phenotype than
that caused by complete loss of Nodal signaling. These results show
that fast1 is required for development of dorsal axial structures
and left-right asymmetry, and suggest that Nodal signals act
through Fast1-dependent and independent pathways. For more
information, see Sirotkin, et al., Curr Biol. Sep. 7,
2000;10(17):1051-4.
[0019] Secreted proteins, particularly members of the nodal-related
secreted protein 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 secreted proteins. The present
invention advances the state of the art by providing previously
unidentified human secreted proteins that have homology to members
of the nodal-related secreted protein subfamily.
SUMMARY OF THE INVENTION
[0020] The present invention is based in part on the identification
of amino acid sequences of human secreted peptides and proteins
that are related to the nodal-related secreted protein 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 secreted protein activity in cells
and tissues that express the secreted protein. Experimental data as
provided in FIG. 1 indicates expression in hypothalamus,
teratocarcinoma, fetal whole brain, pooled glioblastoma, and pooled
fetal lung, testis, and B-cell.
DESCRIPTION OF THE FIGURE SHEETS
[0021] FIG. 1 provides the nucleotide sequence of a cDNA molecule
or transcript sequence that encodes the secreted 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 hypothalamus, teratocarcinoma, fetal whole brain,
pooled glioblastoma, and pooled fetal lung, testis, and B-cell.
[0022] FIG. 2 provides the predicted amino acid sequence of the
secreted protein 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.
[0023] FIG. 3 provides genomic sequences that span the gene
encoding the secreted 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 12 different nucleotide positions.
DETAILED DESCRIPTION OF THE INVENTION
[0024] General Description
[0025] 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 secreted protein or part of a secreted protein and are
related to the nodal-related secreted protein 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 secreted peptides and proteins that are related
to the nodal-related secreted protein subfamily, nucleic acid
sequences in the form of transcript sequences, cDNA sequences
and/or genomic sequences that encode these secreted 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 secreted protein of the present invention.
[0026] 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
secreted proteins of the nodal-related secreted protein subfamily
and the expression pattern observed. Experimental data as provided
in FIG. 1 indicates expression in hypothalamus, teratocarcinoma,
fetal whole brain, pooled glioblastoma, and pooled fetal lung,
testis, and B-cell. 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 nodal-related family or subfamily of secreted proteins.
[0027] Specific Embodiments
[0028] Peptide Molecules
[0029] The present invention provides nucleic acid sequences that
encode protein molecules that have been identified as being members
of the secreted protein family of proteins and are related to the
nodal-related secreted protein 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 secreted peptides of the present invention, secreted
peptides, or peptides/proteins of the present invention.
[0030] The present invention provides isolated peptide and protein
molecules that consist of, consist essentially of, or comprise the
amino acid sequences of the secreted 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.
[0031] 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).
[0032] 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.
[0033] 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 secreted 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.
[0034] The isolated secreted 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 hypothalamus, teratocarcinoma, fetal whole
brain, pooled glioblastoma, and pooled fetal lung, testis, and
B-cell. For example, a nucleic acid molecule encoding the secreted
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.
[0035] 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.
[0036] 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.
[0037] 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 secreted 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.
[0038] The secreted peptides of the present invention can be
attached to heterologous sequences to form chimeric or fusion
proteins. Such chimeric and fusion proteins comprise a secreted
peptide operatively linked to a heterologous protein having an
amino acid sequence not substantially homologous to the secreted
peptide. "Operatively linked" indicates that the secreted peptide
and the heterologous protein are fused in-frame. The heterologous
protein can be fused to the N-terminus or C-terminus of the
secreted peptide.
[0039] In some uses, the fusion protein does not affect the
activity of the secreted 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 secreted 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.
[0040] 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 secreted peptide-encoding nucleic
acid can be cloned into such an expression vector such that the
fusion moiety is linked in-frame to the secreted peptide.
[0041] 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.
[0042] 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 secreted
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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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 secreted peptides of the present invention
as well as being encoded by the same genetic locus as the secreted
peptide provided herein.
[0047] Allelic variants of a secreted 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 secreted peptide as well as being encoded by the same
genetic locus as the secreted 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. 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
secreted peptide encoding nucleic acid molecule under stringent
conditions as more fully described below.
[0048] FIG. 3 provides information on SNPs that have been found in
the gene encoding the enzyme of the present invention. SNPs were
identified at 12 different nucleotide positions, including a
non-synonymous coding SNP at position 7990. Changes in the amino
acid sequence caused by these SNPs is indicated in FIG. 3 and can
readily be determined using the universal genetic code and the
protein sequence provided in FIG. 2 as a reference. Some of these
SNPs that are located outside the ORF and in introns may affect
gene expression. Positioning of each SNP in an exon, intron, or
outside the ORF can readily be determined using the DNA position
given for each SNP and the start/stop, exon, and intron genomic
coordinates given in FIG. 3.
[0049] Paralogs of a secreted peptide can readily be identified as
having some degree of significant sequence homology/identity to at
least a portion of the secreted 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 secreted peptide encoding nucleic
acid molecule under moderate to stringent conditions as more fully
described below.
[0050] Orthologs of a secreted peptide can readily be identified as
having some degree of significant sequence homology/identity to at
least a portion of the secreted 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 secreted 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.
[0051] Non-naturally occurring variants of the secreted 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 secreted peptide. For example, one class of substitutions
are conserved amino acid substitution. Such substitutions are those
that substitute a given amino acid in a secreted 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).
[0052] Variant secreted 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.
[0053] 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.
[0054] 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 secreted
protein 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)).
[0055] The present invention further provides fragments of the
secreted 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.
[0056] As used herein, a fragment comprises at least 8, 10, 12, 14,
16, or more contiguous amino acid residues from a secreted peptide.
Such fragments can be chosen based on the ability to retain one or
more of the biological activities of the secreted 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 secreted peptide, e.g.,
active site 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.
[0057] 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 secreted 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).
[0058] 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.
[0059] 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)).
[0060] Accordingly, the secreted 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 secreted peptide
is fused with another compound, such as a compound to increase the
half-life of the secreted peptide (for example, polyethylene
glycol), or in which the additional amino acids are fused to the
mature secreted peptide, such as a leader or secretory sequence or
a sequence for purification of the mature secreted peptide or a
pro-protein sequence.
[0061] Protein/Peptide Uses
[0062] 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 secreted
protein-effector protein interaction or secreted protein-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.
[0063] 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.
[0064] 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, secreted proteins
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 secreted protein. Experimental data as provided in
FIG. 1 indicates that secreted proteins of the present invention
are expressed in hypothalamus, teratocarcinoma, fetal whole brain,
pooled glioblastoma, and pooled fetal lung, testis, and B-cell.
Specifically, a virtual northern blot shows expression in
hypothalamus, teratocarcinoma, pooled glioblastoma, and pooled
fetal lung, testis, and B-cell. In addition, PCR-based tissue
screening panel indicates expression in fetal whole brain. A large
percentage of pharmaceutical agents are being developed that
modulate the activity of secreted proteins, particularly members of
the nodal-related 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 hypothalamus,
teratocarcinoma, fetal whole brain, pooled glioblastoma, and pooled
fetal lung, testis, and B-cell. Such uses can readily be determined
using the information provided herein, that which is known in the
art, and routine experimentation.
[0065] 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 secreted
proteins that are related to members of the nodal-related
subfamily. Such assays involve any of the known secreted protein
functions or activities or properties useful for diagnosis and
treatment of secreted protein-related conditions that are specific
for the subfamily of secreted proteins that the one of the present
invention belongs to, particularly in cells and tissues that
express the secreted protein. Experimental data as provided in FIG.
1 indicates that secreted proteins of the present invention are
expressed in hypothalamus, teratocarcinoma, fetal whole brain,
pooled glioblastoma, and pooled fetal lung, testis, and B-cell.
Specifically, a virtual northern blot shows expression in
hypothalamus, teratocarcinoma, pooled glioblastoma, and pooled
fetal lung, testis, and B-cell. In addition, PCR-based tissue
screening panel indicates expression in fetal whole brain.
[0066] 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 secreted protein, as a biopsy or expanded in cell culture.
Experimental data as provided in FIG. 1 indicates expression in
hypothalamus, teratocarcinoma, fetal whole brain, pooled
glioblastoma, and pooled fetal lung, testis, and B-cell. In an
alternate embodiment, cell-based assays involve recombinant host
cells expressing the secreted protein.
[0067] The polypeptides can be used to identify compounds that
modulate secreted protein activity of the protein in its natural
state or an altered form that causes a specific disease or
pathology associated with the secreted protein. Both the secreted
proteins 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 secreted protein. These
compounds can be further screened against a functional secreted
protein to determine the effect of the compound on the secreted
protein 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 secreted protein to a desired degree.
[0068] Further, the proteins of the present invention can be used
to screen a compound for the ability to stimulate or inhibit
interaction between the secreted protein and a molecule that
normally interacts with the secreted protein, e.g. a substrate or a
component of the signal pathway that the secreted protein normally
interacts (for example, another secreted protein). Such assays
typically include the steps of combining the secreted protein with
a candidate compound under conditions that allow the secreted
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 secreted protein and the target.
[0069] 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).
[0070] One candidate compound is a soluble fragment of the receptor
that competes for substrate binding. Other candidate compounds
include mutant secreted proteins or appropriate fragments
containing mutations that affect secreted protein 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.
[0071] Any of the biological or biochemical functions mediated by
the secreted protein 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 secreted protein can be assayed.
Experimental data as provided in FIG. 1 indicates that secreted
proteins of the present invention are expressed in hypothalamus,
teratocarcinoma, fetal whole brain, pooled glioblastoma, and pooled
fetal lung, testis, and B-cell. Specifically, a virtual northern
blot shows expression in hypothalamus, teratocarcinoma, pooled
glioblastoma, and pooled fetal lung, testis, and B-cell. In
addition, PCR-based tissue screening panel indicates expression in
fetal whole brain.
[0072] Binding and/or activating compounds can also be screened by
using chimeric secreted 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
secreted protein. 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 secreted protein is
derived.
[0073] The proteins of the present invention are also useful in
competition binding assays in methods designed to discover
compounds that interact with the secreted protein (e.g. binding
partners and/or ligands). Thus, a compound is exposed to a secreted
protein polypeptide under conditions that allow the compound to
bind or to otherwise interact with the polypeptide. Soluble
secreted protein polypeptide is also added to the mixture. If the
test compound interacts with the soluble secreted protein
polypeptide, it decreases the amount of complex formed or activity
from the secreted protein target. This type of assay is
particularly useful in cases in which compounds are sought that
interact with specific regions of the secreted protein. Thus, the
soluble polypeptide that competes with the target secreted protein
region is designed to contain peptide sequences corresponding to
the region of interest.
[0074] To perform cell free drug screening assays, it is sometimes
desirable to immobilize either the secreted 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.
[0075] 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.35-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 secreted protein-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
secreted protein-binding protein and a candidate compound are
incubated in the secreted 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 secreted protein target
molecule, or which are reactive with secreted 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.
[0076] Agents that modulate one of the secreted proteins 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
animal or other model system. Such model systems are well known in
the art and can readily be employed in this context.
[0077] Modulators of secreted protein activity identified according
to these drug screening assays can be used to treat a subject with
a disorder mediated by the secreted protein pathway, by treating
cells or tissues that express the secreted protein. Experimental
data as provided in FIG. 1 indicates expression in hypothalamus,
teratocarcinoma, fetal whole brain, pooled glioblastoma, and pooled
fetal lung, testis, and B-cell. These methods of treatment include
the steps of administering a modulator of secreted protein activity
in a pharmaceutical composition to a subject in need of such
treatment, the modulator being identified as described herein.
[0078] In yet another aspect of the invention, the secreted
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
secreted protein and are involved in secreted protein activity.
[0079] 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 secreted
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 secreted protein-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 secreted protein.
[0080] 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 secreted
protein-modulating agent, an antisense secreted protein nucleic
acid molecule, a secreted protein-specific antibody, or a secreted
protein-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.
[0081] The secreted 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
hypothalamus, teratocarcinoma, fetal whole brain, pooled
glioblastoma, and pooled fetal lung, testis, and B-cell. The method
involves contacting a biological sample with a compound capable of
interacting with the secreted 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.
[0082] 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.
[0083] 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 secreted protein 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.
[0084] 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.
[0085] 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
secreted protein in which one or more of the secreted protein
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
secreted protein 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.
[0086] 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 hypothalamus, teratocarcinoma, fetal whole
brain, pooled glioblastoma, and pooled fetal lung, testis, and
B-cell. Accordingly, methods for treatment include the use of the
secreted protein or fragments.
[0087] Antibodies
[0088] 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.
[0089] 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.
[0090] 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).
[0091] 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.
[0092] Antibodies are preferably prepared from regions or discrete
fragments of the secreted 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
secreted protein/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.
[0093] 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).
[0094] 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.
[0095] Antibody Uses
[0096] 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 secreted
proteins of the present invention are expressed in hypothalamus,
teratocarcinoma, fetal whole brain, pooled glioblastoma, and pooled
fetal lung, testis, and B-cell. Specifically, a virtual northern
blot shows expression in hypothalamus, teratocarcinoma, pooled
glioblastoma, and pooled fetal lung, testis, and B-cell. In
addition, PCR-based tissue screening panel indicates expression in
fetal whole brain. 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.
[0097] 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 hypothalamus, teratocarcinoma,
fetal whole brain, pooled glioblastoma, and pooled fetal lung,
testis, and B-cell. 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.
[0098] 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 hypothalamus, teratocarcinoma, fetal whole brain,
pooled glioblastoma, and pooled fetal lung, testis, and B-cell. 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.
[0099] 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.
[0100] The antibodies are also useful for tissue typing.
Experimental data as provided in FIG. 1 indicates expression in
hypothalamus, teratocarcinoma, fetal whole brain, pooled
glioblastoma, and pooled fetal lung, testis, and B-cell. 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.
[0101] The antibodies are also useful for inhibiting protein
function, for example, blocking the binding of the secreted 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.
[0102] 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.
[0103] Nucleic Acid Molecules
[0104] The present invention further provides isolated nucleic acid
molecules that encode a secreted 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 secreted peptides of
the present invention, an allelic variant thereof, or an ortholog
or paralog thereof.
[0105] 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 5 KB, 4 KB, 3 KB, 2 KB, or 1 KB 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.
[0106] 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.
[0107] 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.
[0108] 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.
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] As mentioned above, the isolated nucleic acid molecules
include, but are not limited to, the sequence encoding the secreted
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.
[0114] 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).
[0115] 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
secreted 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.
[0116] 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.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] FIG. 3 provides information on SNPs that have been found in
the gene encoding the enzyme of the present invention. SNPs were
identified at 12 different nucleotide positions, including a
non-synonymous coding SNP at position 7990. Changes in the amino
acid sequence caused by these SNPs is indicated in FIG. 3 and can
readily be determined using the universal genetic code and the
protein sequence provided in FIG. 2 as a reference. Some of these
SNPs that are located outside the ORF and in introns may affect
gene expression. Positioning of each SNP in an exon, intron, or
outside the ORF can readily be determined using the DNA position
given for each SNP and the start/stop, exon, and intron genomic
coordinates given in FIG. 3.
[0121] 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 45C, followed by one or more
washes in 0.2.times.SSC, 0.1% SDS at 50-65C. Examples of moderate
to low stringency hybridization conditions are well known in the
art.
[0122] Nucleic Acid Molecule Uses
[0123] 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 12 different nucleotide
positions.
[0124] 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.
[0125] 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.
[0126] 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.
[0127] The nucleic acid molecules are also useful for expressing
antigenic portions of the proteins.
[0128] 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.
[0129] The nucleic acid molecules are also useful in making vectors
containing the gene regulatory regions of the nucleic acid
molecules of the present invention.
[0130] 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.
[0131] The nucleic acid molecules are also useful for making
vectors that express part, or all, of the peptides.
[0132] The nucleic acid molecules are also useful for constructing
host cells expressing a part, or all, of the nucleic acid molecules
and peptides.
[0133] The nucleic acid molecules are also useful for constructing
transgenic animals expressing all, or a part, of the nucleic acid
molecules and peptides.
[0134] 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 secreted proteins of the present invention are
expressed in hypothalamus, teratocarcinoma, fetal whole brain,
pooled glioblastoma, and pooled fetal lung, testis, and B-cell.
Specifically, a virtual northern blot shows expression in
hypothalamus, teratocarcinoma, pooled glioblastoma, and pooled
fetal lung, testis, and B-cell. In addition, PCR-based tissue
screening panel indicates expression in fetal whole brain.
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 secreted protein expression relative to normal
results.
[0135] In vitro techniques for detection of mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detecting DNA include Southern hybridizations and in situ
hybridization.
[0136] Probes can be used as a part of a diagnostic test kit for
identifying cells or tissues that express a secreted protein, such
as by measuring a level of a secreted protein-encoding nucleic acid
in a sample of cells from a subject e.g., mRNA or genomic DNA, or
determining if a secreted protein gene has been mutated.
Experimental data as provided in FIG. 1 indicates that secreted
proteins of the present invention are expressed in hypothalamus,
teratocarcinoma, fetal whole brain, pooled glioblastoma, and pooled
fetal lung, testis, and B-cell. Specifically, a virtual northern
blot shows expression in hypothalamus, teratocarcinoma, pooled
glioblastoma, and pooled fetal lung, testis, and B-cell. In
addition, PCR-based tissue screening panel indicates expression in
fetal whole brain.
[0137] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate secreted protein nucleic acid
expression.
[0138] 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 secreted protein gene, particularly
biological and pathological processes that are mediated by the
secreted protein in cells and tissues that express it. Experimental
data as provided in FIG. 1 indicates expression in hypothalamus,
teratocarcinoma, fetal whole brain, pooled glioblastoma, and pooled
fetal lung, testis, and B-cell. The method typically includes
assaying the ability of the compound to modulate the expression of
the secreted protein nucleic acid and thus identifying a compound
that can be used to treat a disorder characterized by undesired
secreted protein nucleic acid expression. The assays can be
performed in cell-based and cell-free systems. Cell-based assays
include cells naturally expressing the secreted protein nucleic
acid or recombinant cells genetically engineered to express
specific nucleic acid sequences.
[0139] Thus, modulators of secreted protein 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 secreted protein mRNA in the presence of the
candidate compound is compared to the level of expression of
secreted protein 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.
[0140] 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 secreted protein
nucleic acid expression in cells and tissues that express the
secreted protein. Experimental data as provided in FIG. 1 indicates
that secreted proteins of the present invention are expressed in
hypothalamus, teratocarcinoma, fetal whole brain, pooled
glioblastoma, and pooled fetal lung, testis, and B-cell.
Specifically, a virtual northern blot shows expression in
hypothalamus, teratocarcinoma, pooled glioblastoma, and pooled
fetal lung, testis, and B-cell. In addition, PCR-based tissue
screening panel indicates expression in fetal whole brain.
Modulation includes both up-regulation (i.e. activation or
agonization) or down-regulation (suppression or antagonization) or
nucleic acid expression.
[0141] Alternatively, a modulator for secreted protein 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 secreted protein nucleic acid expression in
the cells and tissues that express the protein. Experimental data
as provided in FIG. 1 indicates expression in hypothalamus,
teratocarcinoma, fetal whole brain, pooled glioblastoma, and pooled
fetal lung, testis, and B-cell.
[0142] The nucleic acid molecules are also useful for monitoring
the effectiveness of modulating compounds on the expression or
activity of the secreted protein 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.
[0143] The nucleic acid molecules are also useful in diagnostic
assays for qualitative changes in secreted protein nucleic acid
expression, and particularly in qualitative changes that lead to
pathology. The nucleic acid molecules can be used to detect
mutations in secreted protein 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 secreted protein 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 secreted protein 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 secreted protein.
[0144] Individuals carrying mutations in the secreted protein 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 enzyme of the present invention.
SNPs were identified at 12 different nucleotide positions,
including a non-synonymous coding SNP at position 7990. Changes in
the amino acid sequence caused by these SNPs is indicated in FIG. 3
and can readily be determined using the universal genetic code and
the protein sequence provided in FIG. 2 as a reference. Some of
these SNPs that are located outside the ORF and in introns may
affect gene expression. Positioning of each SNP in an exon, intron,
or outside the ORF can readily be determined using the DNA position
given for each SNP and the start/stop, exon, and intron genomic
coordinates given in FIG. 3. 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.
[0145] Alternatively, mutations in a secreted protein gene can be
directly identified, for example, by alterations in restriction
enzyme digestion patterns determined by gel electrophoresis.
[0146] 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.
[0147] 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 secreted protein 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)).
[0148] 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.
[0149] 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 secreted protein 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 enzyme of the present
invention. SNPs were identified at 12 different nucleotide
positions, including a non-synonymous coding SNP at position 7990.
Changes in the amino acid sequence caused by these SNPs is
indicated in FIG. 3 and can readily be determined using the
universal genetic code and the protein sequence provided in FIG. 2
as a reference. Some of these SNPs that are located outside the ORF
and in introns may affect gene expression. Positioning of each SNP
in an exon, intron, or outside the ORF can readily be determined
using the DNA position given for each SNP and the start/stop, exon,
and intron genomic coordinates given in FIG. 3.
[0150] 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.
[0151] The nucleic acid molecules are thus useful as antisense
constructs to control secreted protein 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
secreted protein. An antisense RNA or DNA nucleic acid molecule
would hybridize to the mRNA and thus block translation of mRNA into
secreted protein.
[0152] Alternatively, a class of antisense molecules can be used to
inactivate mRNA in order to decrease expression of secreted protein
nucleic acid. Accordingly, these molecules can treat a disorder
characterized by abnormal or undesired secreted protein 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 secreted protein, such as
substrate binding.
[0153] The nucleic acid molecules also provide vectors for gene
therapy in patients containing cells that are aberrant in secreted
protein 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 secreted protein to treat the individual.
[0154] The invention also encompasses kits for detecting the
presence of a secreted protein nucleic acid in a biological sample.
Experimental data as provided in FIG. 1 indicates that secreted
proteins of the present invention are expressed in hypothalamus,
teratocarcinoma, fetal whole brain, pooled glioblastoma, and pooled
fetal lung, testis, and B-cell. Specifically, a virtual northern
blot shows expression in hypothalamus, teratocarcinoma, pooled
glioblastoma, and pooled fetal lung, testis, and B-cell. In
addition, PCR-based tissue screening panel indicates expression in
fetal whole brain. For example, the kit can comprise reagents such
as a labeled or labelable nucleic acid or agent capable of
detecting secreted protein nucleic acid in a biological sample;
means for determining the amount of secreted protein nucleic acid
in the sample; and means for comparing the amount of secreted
protein 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 secreted protein
mRNA or DNA.
[0155] Nucleic Acid Arrays
[0156] 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).
[0157] 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 WO95/11995 (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.
[0158] 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.
[0159] 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.
[0160] 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 WO95/251116 (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.
[0161] 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.
[0162] Using such arrays, the present invention provides methods to
identify the expression of the secreted 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 secreted protein gene of the
present invention. FIG. 3 provides information on SNPs that have
been found in the gene encoding the enzyme of the present
invention. SNPs were identified at 12 different nucleotide
positions, including a non-synonymous coding SNP at position 7990.
Changes in the amino acid sequence caused by these SNPs is
indicated in FIG. 3 and can readily be determined using the
universal genetic code and the protein sequence provided in FIG. 2
as a reference. Some of these SNPs that are located outside the ORF
and in introns may affect gene expression. Positioning of each SNP
in an exon, intron, or outside the ORF can readily be determined
using the DNA position given for each SNP and the start/stop, exon,
and intron genomic coordinates given in FIG. 3.
[0163] 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).
[0164] 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.
[0165] In another embodiment of the present invention, kits are
provided which contain the necessary reagents to carry out the
assays of the present invention.
[0166] 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.
[0167] 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 secreted protein 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.
[0168] Vectors/Host Cells
[0169] 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.
[0170] 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.
[0171] 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).
[0172] 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.
[0173] 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 X, 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.
[0174] 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.
[0175] 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).
[0176] 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).
[0177] 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.
[0178] 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.
[0179] 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.
[0180] 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 enterokinase. 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)).
[0181] 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)).
[0182] 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 pYepSecl
(Baldari, et al., EMBO J 6:229-234 (1987)), pMFa (Kurjan et al.,
Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123
(1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).
[0183] 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)).
[0184] 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)).
[0185] 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.
[0186] 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).
[0187] 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.
[0188] 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).
[0189] 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.
[0190] 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.
[0191] 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.
[0192] 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.
[0193] Where secretion of the peptide is desired, which is
difficult to achieve with multi-transmembrane domain containing
proteins such as kinases, appropriate secretion signals are
incorporated into the vector. The signal sequence can be endogenous
to the peptides or heterologous to these peptides.
[0194] Where the peptide is not secreted into the medium, which is
typically the case with kinases, 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.
[0195] 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.
[0196] Uses of Vectors and Host Cells
[0197] The recombinant host cells expressing the peptides described
herein have a variety of uses. First, the cells are useful for
producing a secreted protein or peptide that can be further
purified to produce desired amounts of secreted protein or
fragments. Thus, host cells containing expression vectors are
useful for peptide production.
[0198] Host cells are also useful for conducting cell-based assays
involving the secreted protein or secreted protein fragments, such
as those described above as well as other formats known in the art.
Thus, a recombinant host cell expressing a native secreted protein
is useful for assaying compounds that stimulate or inhibit secreted
protein function.
[0199] Host cells are also useful for identifying secreted 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 secreted protein (for example, stimulating or
inhibiting function) which may not be indicated by their effect on
the native secreted protein.
[0200] 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 secreted protein and
identifying and evaluating modulators of secreted protein activity.
Other examples of transgenic animals include non-human primates,
sheep, dogs, cows, goats, chickens, and amphibians.
[0201] 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
secreted protein nucleotide sequences can be introduced as a
transgene into the genome of a non-human animal, such as a
mouse.
[0202] 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
secreted protein to particular cells.
[0203] 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.
[0204] 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.
[0205] 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.
[0206] 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, secreted 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 secreted protein function,
including substrate interaction, the effect of specific mutant
secreted proteins on secreted protein function and substrate
interaction, and the effect of chimeric secreted proteins. It is
also possible to assess the effect of null mutations, that is,
mutations that substantially or completely eliminate one or more
secreted protein functions.
[0207] 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
5 1 1696 DNA Human 1 catgcacgcc cactgcctgc ccttccttct gcacgcctgg
tgggccctac tccaggcggg 60 tgctgcgacg gtggccactg cgctcctgcg
tacgcggggg cagccctcgt cgccatcccc 120 tctggcgtac atgctgagcc
tctaccgcga cccgctgccg agggcagaca tcatccgcag 180 cctacaggca
gaagatgtgg cagtggatgg gcagaactgg acgtttgctt ttgacttctc 240
cttcctgagc caacaagagg atctggcatg ggctgagctc cggctgcagc tgtccagccc
300 tgtggacctc cccactgagg gctcacttgc cattgagatt ttccaccagc
caaagcccga 360 cacagagcag gcttcagaca gctgcttaga gcggtttcag
atggacctat tcactgtcac 420 tttgtcccag gtcacctttt ccttgggcag
catggttttg gaggtgacca ggcctctctc 480 caagtggctg aagcgccctg
gggccctgga gaagcagatg tccagggtag ctggagagtg 540 ctggccgcgg
ccccccacac cgcctgccac caatgtgctc cttatgctct actccaacct 600
ctcgcaggag cagaggcagc tgggtgggtc caccttgctg tgggaagccg agagctcctg
660 gcgggcccag gagggacagc tgtcctggga gtggggcaag aggcaccgtc
gacatcactt 720 gccagacaga agtcaactgt gtcggaaggt caagttccag
gtggacttca acctgatcgg 780 atggggctcc tggatcatct accccaagca
gtacaacgcc tatcgctgtg agggcgagtg 840 tcctaatcct gttggggagg
agtttcatcc gaccaaccat gcatacatcc agagtctgct 900 gaaacgttac
cagccccacc gagtcccttc cacttgttgt gccccagtga agaccaagcc 960
gctgagcatg ctgtatgtgg ataatggcag agtgctccta gatcaccata aagacatgat
1020 cgtggaagaa tgtgggtgcc tctgatgaca tcctggaggg agactggatt
tgcctgcact 1080 ctggaaggct gggaaactcc tggaagacat gataaccatc
taatccagta aggagaaaca 1140 gagaggggca aagttgctct gcccaccaga
actgaagagg aggggctgcc cactctgtaa 1200 atgaagggct cagtggagtc
tggccaagca cagaggctgc tgtcaggaag agggaggaag 1260 aagcctgtgc
agggggctgg ctggatgttc tctttactga aaagacagtg gcaaggaaaa 1320
gcacaagtgc atgagttctt tactggattt tttaaaaacc tgtgaacccc ccgaaactgt
1380 atgtgaaagt tgagacatat gtgcatgtat tttggaggtg ggatgaagtc
acctatagct 1440 ttcatgtatt ctccaaagta gtctgtgtgt gacctgtccc
cctccccaaa gattaaggat 1500 cactgtatag attaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1560 aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1620 aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1680
aaaaaaaaaa aaaaaa 1696 2 347 PRT Human 2 Met His Ala His Cys Leu
Pro Phe Leu Leu His Ala Trp Trp Ala Leu 1 5 10 15 Leu Gln Ala Gly
Ala Ala Thr Val Ala Thr Ala Leu Leu Arg Thr Arg 20 25 30 Gly Gln
Pro Ser Ser Pro Ser Pro Leu Ala Tyr Met Leu Ser Leu Tyr 35 40 45
Arg Asp Pro Leu Pro Arg Ala Asp Ile Ile Arg Ser Leu Gln Ala Glu 50
55 60 Asp Val Ala Val Asp Gly Gln Asn Trp Thr Phe Ala Phe Asp Phe
Ser 65 70 75 80 Phe Leu Ser Gln Gln Glu Asp Leu Ala Trp Ala Glu Leu
Arg Leu Gln 85 90 95 Leu Ser Ser Pro Val Asp Leu Pro Thr Glu Gly
Ser Leu Ala Ile Glu 100 105 110 Ile Phe His Gln Pro Lys Pro Asp Thr
Glu Gln Ala Ser Asp Ser Cys 115 120 125 Leu Glu Arg Phe Gln Met Asp
Leu Phe Thr Val Thr Leu Ser Gln Val 130 135 140 Thr Phe Ser Leu Gly
Ser Met Val Leu Glu Val Thr Arg Pro Leu Ser 145 150 155 160 Lys Trp
Leu Lys Arg Pro Gly Ala Leu Glu Lys Gln Met Ser Arg Val 165 170 175
Ala Gly Glu Cys Trp Pro Arg Pro Pro Thr Pro Pro Ala Thr Asn Val 180
185 190 Leu Leu Met Leu Tyr Ser Asn Leu Ser Gln Glu Gln Arg Gln Leu
Gly 195 200 205 Gly Ser Thr Leu Leu Trp Glu Ala Glu Ser Ser Trp Arg
Ala Gln Glu 210 215 220 Gly Gln Leu Ser Trp Glu Trp Gly Lys Arg His
Arg Arg His His Leu 225 230 235 240 Pro Asp Arg Ser Gln Leu Cys Arg
Lys Val Lys Phe Gln Val Asp Phe 245 250 255 Asn Leu Ile Gly Trp Gly
Ser Trp Ile Ile Tyr Pro Lys Gln Tyr Asn 260 265 270 Ala Tyr Arg Cys
Glu Gly Glu Cys Pro Asn Pro Val Gly Glu Glu Phe 275 280 285 His Pro
Thr Asn His Ala Tyr Ile Gln Ser Leu Leu Lys Arg Tyr Gln 290 295 300
Pro His Arg Val Pro Ser Thr Cys Cys Ala Pro Val Lys Thr Lys Pro 305
310 315 320 Leu Ser Met Leu Tyr Val Asp Asn Gly Arg Val Leu Leu Asp
His His 325 330 335 Lys Asp Met Ile Val Glu Glu Cys Gly Cys Leu 340
345 3 13205 DNA Human 3 gacagggttt ctccatgttg gtcaagctgg tctcaaaccc
ccgacctcag gtgatcttcc 60 cacctcagcc tcccaaagtg ctaggattac
aggcgtgagc cactgcgtct ggcctcactg 120 aattatttac ttgtttttat
tttttttttt taaagagggt cttgctatgt tgaccagact 180 ggtctcaaac
tcctggcctc aagctatcct ccagccttgg cctcccatgt gttgggatta 240
caggcatgaa ccaccacacc tggcctgaat tatttactta acacagattt tttgggttcc
300 aacgatgatg atggtatata ctgtgtgaca gcaggatgaa caagacattt
ccagctttag 360 gaaactaaca gactgtccag ggctcatgtg gtggacagcc
caggtctcct gaatcttaga 420 tgaggcctcc agctatgtgg gacagcctcc
tcccctaaga cagaggacaa aggcttggag 480 ctcctagttg agcctgctgg
gcctttctcc attttctaac aacctgctca ggtcgcagcc 540 tatggttctt
ttgttgttgt tgttgttgtt gttttgtttt ttgagacagg gactcactcg 600
gttgcccaga ctggagtgca gtggcccgat cttggcttat tgcaaccttt gcctcccagg
660 ctcaagcaat cctcccactt cagcctccag aggagctggg actacaggcg
cacaccacaa 720 aaatgcctgg ctcatttttg tatttttggg tagagacagg
gtttcaccat gttgaccagg 780 ctggtcttga actcctgagc tcaggtatct
gccagcctca tcctccctaa gtgctgaaat 840 tacaggtgtg agccaccgtg
cctggccagc agcctatggt tcttttgagc tagaggcatc 900 ctgtgaaaga
agtcaggtgt atagagcact tagcactatg cctggcgctt agtaagtgtt 960
cagtaagtgt tggccagcag ctaagtgctt gtcaaggcta ggtaggtggg gggagctaag
1020 aagagaaaga gccctcaggt gtaaaggaaa acggatgggg ccagacactg
gggcagggat 1080 ttacatttct ttaaaagtca ttccagccct gacttgactt
tgcacctgga agggaggagg 1140 ccagacaaga tcactcttgt gcaaataagg
aaagctgagc ccagagatca aagtgagtgg 1200 ccctgagccg acacccaggt
cttctgtggc cctgccacac caggcaggga ttgggggatg 1260 tgggcaacaa
gggtaacacc tttcctcctg aaatcactgg ggtgatgtct gcctgtcggg 1320
gaagagcctg atgaaccagg atcaccccgt tggctccatc tggctccagc ttctgctgaa
1380 cactcccttt tcaggtggag aagtcatcgt cctggtccct cctgccttct
gtcttcccca 1440 ggaaagcact tcagtatttg gtagggccag gggtgctccc
cgaggccatc tagacagtgg 1500 ccagggaggc tgcgccggcc agattgtgtc
aaagtctgcc tgagagccac gaggatccct 1560 cggcattctc ttcctgcttc
ttgaggagcc tgcggtccat ttgcagcctg agtggagagg 1620 atggggcagg
gatcccaggt gaggtcgagt aaggaagtcg cagttctgac ctgggaatct 1680
gccgggacaa gcctgctctg tctcagcatc aaggcgtttg gtacctagag gagcagggtg
1740 ccagggtgtc cagaccgggc aaaccaggag ccctagatcc cgtccctgga
actaggtcct 1800 ccgccagctt tgcgggcctc ctccgcacca ggctcaggtc
tcgcccatcc ccagcaggag 1860 gtctctctgc cccccaccct ccggaggggg
gttatatgat cttaaagctt ccccagaggg 1920 aggaaaggtg ggggcggggc
ggctgctgag gcccaggata taagggctgg aggtgctgct 1980 ttcaggcctg
gccagcccac catgcacgcc cactgcctgc ccttccttct gcacgcctgg 2040
tgggccctac tccaggcggg tgctgcgacg gtggccactg cgctcctgcg tacgcggggg
2100 cagccctcgt cgccatcccc tctggcgtac atgctgagcc tctaccgcga
cccgctgccg 2160 agggcagaca tcatccgcag cctacaggca gaaggtaggc
agtgccgcgt gccgcgccct 2220 gctgggcacc cccggggcgc ctccgccgcg
tccagccagc ggactcggga agtgctgtgg 2280 gttgggggct gcggctccga
gccgggtttg cagccgcccg ggcgtcccga gcccagggcc 2340 tagctctgcg
ggtgtctccg cgtcagcagg ctcggggtgc agcgttggtg gctgggggcg 2400
tatccacggc cgagtcggga agggattcta gcgttcaggg tgtgtcctcg acggggacca
2460 ttgtctctgg gttttggttt gggattgcgc ggagcgcagc gcggaagggt
gggagcttct 2520 aatctccagt cttgtgaagt tgcttatccc ggagcctggg
tctgcgcatc tgtaggatag 2580 gtgtaataaa taacacctcg cctatcagac
tgtggaaagc gcgagatgac aatgcgcgcg 2640 aaacgctcag cgcagtaccc
ggcacagcca cagtcaacgg tcgttggtat tactgtaatg 2700 gtttggtctt
ggcgattttt ttttctttct gcgagtgagg gtgaatgggt cccggggtgt 2760
gacgtcggga gtatcggcag ctgagctggt aacatcgggg attcgggctc acggcccgga
2820 gatcagggat gggctgtccc gaagtcgcga actgtggcag ccttgggtcc
tccagccgcg 2880 ccggggaagt gtcaagtgtc tcgcttaacc ccgggttcgg
ggccatgatt tgcagggtag 2940 tgggtgtcaa ggacggcagg gatctgaggg
tatcgccctc gaggacctgg cagcgcgttc 3000 tgggcaccca gcgcggcgag
caggtgggtg ctgcggagag ggagcccctt ccgcgcctca 3060 atccacattc
tgccgcctgg gcagccgcgg ccgcccacgc ctccctccgc ctgggggggc 3120
cagacggccc tccctggggc cggggcgcaa tccacaaacg ctaatctgat ccgacctgcc
3180 gcctgcccgc cccttgtgac ctggtgccgg gggcccttcg ctcccgcgcc
tggggtcaga 3240 cagccggtga ccctctccgg aagggtcatc tggggaccag
ccagaccagg ggacaccctc 3300 gggggcgggg caatgagaaa tttgctggag
tgctcggccc ctcaaccgaa aagcggccgg 3360 ggatgggagg gggcaaagaa
gggagggagc gcttttccag ttcactccct tctggaaagt 3420 tcgagatgtg
tgcggtgatg gacaggcatc tggaaactgg gttctaggcc tcaaatcctg 3480
cttcctgatt ccctgttgtc cctggcgcgt cttggtttat ctgccttccc ttctgtaaat
3540 taggagactg ctctctttca gctaaacact gtttttttgt ttgtttgttt
tttctttgaa 3600 acagagtctc cctctgttgc ccaggctgga gtgcagtgac
acgatttcgg ctcactgcaa 3660 cctccgcctc ccaggttcaa ggaattctcc
tgcctcagcc tcccgagtaa ctgagactac 3720 aggcgcgcgc cactgcgccc
agctattttt taatattttt agtagacaca gagtttcacc 3780 atattggcca
ggctggtctc gcctggtctc gtggtccgcc tgcctcggcc tcccaaggtg 3840
ttgggattac aggcgtgagc cactgtgacc ggcctaaaca ctgtttttta gtgtacagcg
3900 gcatgtggca cagttacgtt gtgaattggc ctcattttct aagagcagtg
ggaagagccc 3960 gcctggaggg agactggagg agaagggatg tcctggcccc
actccttcca tcttgggttt 4020 atttagaata agctgcaccg cagttgcatg
aaagggatgt tggagtccaa ttgggtttac 4080 atctgtatct gttgccgttt
aaccttcacc ttggaccttc agttttctca tctgcaaatg 4140 gtgagatgag
aggaacagtg aggagattta gagaatgcct gtgaaacact tggcacacgg 4200
gaagccactg atggtaacga cccttataat ttatgaaaat gtagagtggg tgctcagtgt
4260 caggtcctta cagccctgcc tcacagccct gtctggggac ggcggtgtcg
gtgactggaa 4320 gggaacccaa gggtgtgagc cactttcttc ttcagaactg
gaggtggggc cgggcacagt 4380 ggcttacgcc tgtaacccag cactttcggg
aggccgaggt ggtcggatca tgaggtcaag 4440 agatcaagac cgtcctgccc
aacatgatga aaccctgtct ctactaaaaa tacaaaaatt 4500 agctgggcat
ggtggcgcgc acctgtagtc ccagctactc gggagggtga ggcaggagaa 4560
tcacttgaac ccaggaggtg gaggctgcag tgagctgaga tcgcaccact gcactcgaag
4620 cctggccaca gagcgagact ccatctcaaa aaaaagaact ggaggtgggc
caggaggaca 4680 tcaggggcat ctgtgtggct aagtctgcat tcagcatatg
aatgggtttt tgggtgtcca 4740 tgaacccctt cctcataccc accttaaaat
cacatttgtt gtatttggat gtgtgtttct 4800 ttctgataac attcctacct
tgcatcaggc accagtgaca cccacaatat tagaaccctt 4860 attctaagat
gaaccagaga acctatcagc cctgttccag ggcaccctgg cagcagtggg 4920
agaaggtagg aagggtcaca gacttctttc agcaggaagg agcagcctct tccctaccca
4980 gcctttccca ctgcccatcc tgttcatagg aaactttgca cccctgtcca
ggtccttgtg 5040 gcctgtccgg acaccttcat ttctaactct cggacctggg
caataatgag gcagctgatg 5100 atggccaatg tttattgaac tttggttgtg
ctgggcatgg cacattacat gaattatctc 5160 agttaatcct aacacccctg
tgaggtggct acttttacca tctccctttt acagagaggg 5220 aaactgaggc
tcagagaagg aaagtagcct gaccaaagtc aaacagcaag tgacagaatt 5280
tgagccatca tggggagact tgggggcccc tgctcttaac cattaagcag gagtgccttc
5340 ttctaaaaga gttgagataa agtttttctt ttttcgagac agagttttgc
ttttcgtcac 5400 ccaggctgga gtgcaatggc gcaatctcag ctcactgcaa
cctccacctc ctgggttcaa 5460 gcgattctcc tgcctctgcc tctcaagtag
ctgggattac aggtgtgcac caccatgcct 5520 ggctaatttt gtattttttt
tttagtagag acagggtttc accatgttga tcaggctggt 5580 ctcgaacttc
tgacctcaag tgatccaccc acctcagcct cccaaagtgc tgggattata 5640
ggagtgaccc accgcacctg gccaagataa agtgttttta aactcaccgc ccgaatcctc
5700 caggtacagg tggcagcagt gagtgctgag taatgttagc ctcctcccca
cacttctggg 5760 gaaagctgct agtcaggggc cggttattgc ctcctgtagg
tgctaatgag gagggaagtg 5820 gaaattaagt gaaaaagagc tgctggagat
aggctgggag aagctgccat gaggctgaga 5880 ttgggagtga cccctttaca
atacaagctg gatttcccag agaaattcac ttcctctctg 5940 accaaagatg
ctcaacgaga cagtcaatga ataaactgtt atgcagccag cctggaccta 6000
catcagagat catcactacc tccagagtct attcacaaag aattataaat gccctgtctt
6060 atttctaatg aataacccca acttttattt agcaataaac cttcaaacct
ctttatttct 6120 ctgggtcctt gtgataatca gagagggtgg tacagcatta
ttcccatcgc aaagaagagg 6180 aaactgagct tgaagaaagt tcattactat
gtttgttgtt gttgttgttg agacagagtt 6240 tcgctcttgt tgcccaggct
agagttcagt ggcgcgatcc cagctcacca caacctccac 6300 ctcctgggtt
caagcaattc tcctgcctca gccttcctga gtagctggga ttaggcgtga 6360
gccaccacac ccggctaatt ttgtattttt agtagagacg gggtttctcc atgttggtca
6420 ggctggtttc gaactcctga cctcaggtga tccacccgcc ttggcctccc
aaagtgctgg 6480 gattacaggc gtgagccacc gcgcccggca tcactattaa
gtcacctgaa aatccacaaa 6540 tctgtgctat tcccagacat cagggaaaat
caatccagga ccagaataaa tggagtgtgc 6600 tcccctactc cttgtcatcc
agcttgggtt tggttgtctt gggtgtgtgg aacagggctg 6660 ggtgtgtgtg
tcttctgggt gatgatctct gatgtaggtc cagtctgggc atctaacagt 6720
ttattcatct gttgtctcat agagcatctt tgatcagaga gaaagtgaat ttctttggga
6780 aataatccac cttgtattgt aaaggggcaa ctcccacttt cagccttctg
gaagcttctg 6840 ggtgcatgtg attgctgtgt gccccaggtg tgtgaatgac
ctgggacagg aatgggcgag 6900 catcctgtag gtgggtagac atgcctagaa
agcctttaga aacctatttg taaggttgcc 6960 aaaaactctt aggaactggg
gggtgggtag agggtgacca gtctttggct tctattgcct 7020 gtggctccct
gagagctctg agcttttcca tgatattcac atctagctca atgtgtctgt 7080
aaaacaagtc actctttttt tttttttttt ttttgagaca gagtttcact cttgttgtgc
7140 aggctggagt gcaatggcac tgtcttggct cactgcaacc tctgcctccc
gggttcaaat 7200 gattctcgag tggctgggat tacaggcgca aaccaccacg
cccagctaat tttgtatttt 7260 tagtagagac agggtttctc catgttggtc
agacttcatg tatgggagga gacaggggag 7320 ggggagatgc aactcatcac
atttttactg actgtcctct ggctgtgcaa gttatcttgg 7380 aagggggact
ggaagaacag taattcggag tctgggcttg gcagttgggc aaatccaggt 7440
ttactcttgg ctctgccacc ttccaagaat gacaccttgg tcagatcttt taaccacact
7500 gagcctcagt tttcctcatc tctaaaaggg actcgaaaat cttaccaact
catagagttg 7560 gggtgagaat tcgaaggtaa ttctctataa ggtaaggcct
ccagcaagag ctatggtggt 7620 tgtgacactg actgaggctg ggggaggccc
tcactcaccc tccttccttc ttggttttct 7680 cctacccaga tgtggcagtg
gatgggcaga actggacgtt tgcttttgac ttctccttcc 7740 tgagccaaca
agaggatctg gcatgggctg agctccggct gcagctgtcc agccctgtgg 7800
acctccccac tgagggctca cttgccattg agattttcca ccagccaaag cccgacacag
7860 agcaggcttc agacagctgc ttagagcggt ttcagatgga cctattcact
gtcactttgt 7920 cccaggtcac cttttccttg ggcagcatgg ttttggaggt
gaccaggcct ctctccaagt 7980 ggctgaagca ccctggggcc ctggagaagc
agatgtccag ggtagctgga gagtgctggc 8040 cgcggccccc cacaccgcct
gccaccaatg tgctccttat gctctactcc aacctctcgc 8100 aggagcagag
gcagctgggt gggtccacct tgctgtggga agccgagagc tcctggcggg 8160
cccaggaggg acagctgtcc tgggagtggg gcaagaggca ccgtcgacat cacttgccag
8220 acagaagtca actgtgtcgg aaggtcaagt tccaggtgga cttcaacctg
atcggatggg 8280 gctcctggat catctacccc aagcagtaca acgcctatcg
ctgtgagggc gagtgtccta 8340 atcctgttgg ggaggagttt catccgacca
accatgcata catccaggtg ggatgccagg 8400 cgtgaggggg aggggaggca
gtaagctggc cttgggggac agggctctag ctttgctatt 8460 aaatgactat
gttcctgtat tcacagttac tcaagcacct ccaatgtacc aggatcctgt 8520
gctaggtacc aggcatgtag agatgaccga gtgtaacatc cttatagagc ctgtatgggg
8580 tcatgcgtca ctgggtagcc ttgaacacat tgtttccctg ggactttatc
cttgtctttc 8640 ataaaattag ggggtggggg tagaccagag ggtctcagca
cttccctagc cctgacattc 8700 taggaaagga agaatctgga ctttggggac
aggcaggcct gcatttgact cctcattagc 8760 tgtgtgatac tgtttaggtt
gcttattctc tcaagcttca gtttttccac ctgtaagatg 8820 aggagggcaa
tgccatattt cagagggatg ctgtagggtt aatgagatga catgcacatg 8880
gcatgtaaag cagttggtag gtaacaggca tgtttggctg cattatgggg ccacggttgg
8940 aatcacactg ttccagccac agtattttgg ttgagaattg actacccatt
ctacagaagt 9000 agacaaggcc agccaggtgc ggtggcacat gcctgaaatc
ccaacacttt gggaggctga 9060 ggtgggcgga tcacctgagg tcaggagttt
gagaccagcc tgaccaacat ggtgacaccc 9120 cgtctctact aaaaatacaa
aaatcagccg ggtgttgtgg cacatgcctg taatcccagc 9180 tactcgggag
gctgaggcag gagaatcact tgaacccggg aggcagagat tgcagtgagc 9240
cgagatcatg ccattgcact ccagcctggg caacaggagc gaaactccat ctcaaaaaca
9300 agaaaaagta gacaaagcca gaggctgcca cttggtgcca ctagtcagcc
taaggtctag 9360 gaggtggtgg aggaagaaca gagctggtga gttaggaaag
agaagaagat ggagagaaca 9420 gaaaatataa cctaatatgg tggatctgat
gttataggtg gccctgccct gctgtccaag 9480 gtcatatggg accaaaatgt
tttcatttta ctccatgaag tctggaatga gaatttcttg 9540 gacttgcaat
atatcctcca tgccaagcct caggcgggat tcagggtctc tggggagcat 9600
gagcacatgg gctgaggtca tgcaagggag tccctggggg tactagagct tccgaggatc
9660 tgtcctgagc ctcacccagg cctgctgatt ttcatcgccc acctggagca
ccacaaagac 9720 ccagacagtc ccaactgcca actctgacac tctgatctaa
aaagacgtta ctagaccaca 9780 gggacaggcc ctggttccaa aacgccaagg
gattgttcct ttcctcctat tgactctctc 9840 tgtccttctc tggctgacag
caaatcccat aagaatttcc acatgactaa ctattctgct 9900 tcatatttct
agaactaaac caggaatttc ttggaatatt ttaaaaattg gtattcacag 9960
ccgggcacgg tggctcactc atgtaattcc agcattttgg gaggctgagg tgggtggatc
10020 gcttgagccc aggaggtcaa gaccagcctg ggcaacatgc cgaaacccta
tttctacaaa 10080 aaatacaaaa attagccagg catggtggca tgtacctgta
gtcccagcta ctcaggaggc 10140 tgaggtggga ggatcacctg agcccaggag
gttgaggatg cagtgagctg tgatcacacc 10200 actgcactcc agcctgggca
acagagcaag accatctcaa aaaagaaaaa aaaaatggta 10260 ttccctttag
actagaataa taggagaaca taaaaattag ctaccactta aaaaaaaaaa 10320
tagctactac tctacccaga tacatgtaat tttttgaatc tgtttaaagg ctgttttcac
10380 aaaacaagca cagagctaaa ggtagtttag acattagagt tcatgatata
ttggcaagct 10440 aattccttta tgaaaataat tttctgctat ttttgcactc
aggaactgac tttaactccg 10500 taactttttt actctttacc cagaaatact
attctgacct gcccatcagg atgtgaattg 10560 acacaccctt cctttccttt
acagagtctg ctgaaacgtt accagcccca ccgagtccct 10620 tccacttgtt
gtgccccagt gaagaccaag ccgctgagca tgctgtatgt ggataatggc 10680
agagtgctcc tagatcacca taaagacatg atcgtggaag aatgtgggtg cctctgatga
10740 catcctggag ggagactgga tttgcctgca ctctggaagg ctgggaaact
cctggaagac 10800 atgataacca tctaatccag taaggagaaa cagagagggg
caaagttgct ctgcccacca 10860 gaactgaaga ggaggggctg cccactctgt
aaatgaaggg ctcagtggag tctggccaag 10920 cacagaggct gctgtcagga
agagggagga agaagcctgt gcagggggct ggctggatgt 10980 tctctttact
gaaaagacag tggcaaggaa aagcacaagt gcatgagttc tttactggat 11040
tttttaaaaa cctgtgaacc ccccgaaact gtatgtgaaa gttgagacat atgtgcatgt
11100 attttggagg tgggatgaag tcacctatag
ctttcatgta ttctccaaag tagtctgtgt 11160 gtgacctgtc cccctcccca
aagattaagg atcactgtat agattaaaaa gagtccgtca 11220 atctcattgc
ctcaggctgg gttgggggag ccccacagct ttctggctgg ccagtggcaa 11280
tctactggcc ttgtccagag gctcactgga gtggttctct gctaatgagc tgtacaacaa
11340 taaagccatt gtctagttct cctgggccag ctggtgcctg tgaaggcaga
ggcaggaact 11400 catccaagag gaccggccat gttgggttac agaagacatc
cctgcgtcag tctgcttcgg 11460 cagacacagc ctgagtttgt taaagttggt
gacaatccac ctcagtctct caatgtgtgc 11520 tattaatgag gcctctgagc
ttcctatcca gcagtggtga aggccttgcc ctgggtggca 11580 agatacttgc
tctatggtca cagctcagcc actggaagct gtgcgacctc aggtgagcaa 11640
ttcactgtcc agtctccact tgtaaaagga acgctggtga atcctaatgc attcatatta
11700 aatgtctgtt gtcaggctca gaagagccat gagctttaag ctgtagactg
aataggaaga 11760 gctaccttat ccctacctcc ctctaaagct tataaagctt
gctaaaccca gtccctgcca 11820 catgccctta atctaacctt tatcggctct
tttgttactc agggttctaa gacagactta 11880 atttcaggtt ccacatggga
agcacagcaa aaggagcctg agtgaggcag ctctatcccc 11940 aaaaacccct
gaccacagtt atcttccagg taaaaagacc tcaaaggcag caattctggc 12000
tgggtgcagt ggctcatgcc tgtaatccca gcactttggg aggctgagat gggaagatca
12060 cttgagtcca gtagttcaag accagcctgg gcaacatagt gagaacctgt
ctcctgttta 12120 aataaaacaa acaaaagaag caattctgct cttaacagct
tgatctcatt gagcattgcc 12180 aaagcagggg cacttttttt gtagtttcat
ggagcaaagg ggccacttta ttgggaatgt 12240 gagcccacag tatatctacc
ccattctgct ttcccaagaa gcctacccac ttatgctaaa 12300 gaaaatgatt
ccaatcattt gtgaaccctg ttgagctcag acgaactcaa ctgaggcttt 12360
ggttgagtca acaccatagt catggcccag aaaggggctg gatttgctgg agccaggcct
12420 gggcttgaga aacctcagtg ccttgattcc acaacaaagc tgatccagct
gttcactgcc 12480 tggactagaa atagactgcc cagatccaac aattcagaat
caacaggatt ctgcatctgc 12540 ccatatacag aacagcagag cagggacaac
cttgtgaggt cagcttgtct gatccccatt 12600 cctaagtgag accccaggga
ggcctcaggg cctgaccaca cagctaggag tggcagggcc 12660 aagagctgag
ctctggtctc ttgagcccta cgggcactta ctgcttaaca gcaactagag 12720
gtttgtgctc acctatcttg ggatccaagc cttagccttt agaaccaaac atttgaaccc
12780 aattacacta ctgaactagt tcttagaagt gaacatccca ccccaggact
ttcttacatg 12840 gctggtgcca gagagcgaaa ggtcaagcta tttgtagcta
cagcttgtgg gagtgtttac 12900 agtcagctgt attgactcca agaacaaacc
cttgacgaca gggcattgcc tcatgtcaaa 12960 taaaagtagt gcacaaagta
gtgaaactct gtgttttgct gacccctgct gaaagctcag 13020 tgtttgctac
gtcagcagca gagtcattct gttgcctgga cagagtcact ggggcaaagc 13080
ctacctaccc gagcctaggg aatagctcca tgctctaggc catgtgcttg ctgaaggctc
13140 tataagtcac tttaaatact accttggaga attgtgtcct gtctgaacct
gggatgtttt 13200 ctgtt 13205 4 354 PRT Mus musculus 4 Met Ser Ala
His Ser Leu Arg Ile Leu Leu Leu Gln Ala Cys Trp Ala 1 5 10 15 Leu
Leu His Pro Arg Ala Pro Thr Ala Ala Ala Leu Pro Leu Trp Thr 20 25
30 Arg Gly Gln Pro Ser Ser Pro Ser Pro Leu Ala Tyr Met Leu Ser Leu
35 40 45 Tyr Arg Asp Pro Leu Pro Arg Ala Asp Ile Ile Arg Ser Leu
Gln Ala 50 55 60 Gln Asp Val Asp Val Thr Gly Gln Asn Trp Thr Phe
Thr Phe Asp Phe 65 70 75 80 Ser Phe Leu Ser Gln Glu Glu Asp Leu Val
Trp Ala Asp Val Arg Leu 85 90 95 Gln Leu Pro Gly Pro Met Asp Ile
Pro Thr Glu Gly Pro Leu Thr Ile 100 105 110 Asp Ile Phe His Gln Ala
Lys Gly Asp Pro Glu Arg Asp Pro Ala Asp 115 120 125 Cys Leu Glu Arg
Ile Trp Met Glu Thr Phe Thr Val Ile Pro Ser Gln 130 135 140 Val Thr
Phe Ala Ser Gly Ser Thr Val Leu Glu Val Thr Lys Pro Leu 145 150 155
160 Ser Lys Trp Leu Lys Asp Pro Arg Ala Leu Glu Lys Gln Val Ser Ser
165 170 175 Arg Ala Glu Lys Cys Trp His Gln Pro Tyr Thr Pro Pro Val
Pro Val 180 185 190 Ala Ser Thr Asn Val Leu Met Leu Tyr Ser Asn Arg
Pro Gln Glu Gln 195 200 205 Arg Gln Leu Gly Gly Ala Thr Leu Leu Trp
Glu Ala Glu Ser Ser Trp 210 215 220 Arg Ala Gln Glu Gly Gln Leu Ser
Val Glu Arg Gly Gly Trp Gly Arg 225 230 235 240 Arg Gln Arg Arg His
His Leu Pro Asp Arg Ser Gln Leu Cys Arg Arg 245 250 255 Val Lys Phe
Gln Val Asp Phe Asn Leu Ile Gly Trp Gly Ser Trp Ile 260 265 270 Ile
Tyr Pro Lys Gln Tyr Asn Ala Tyr Arg Cys Glu Gly Glu Cys Pro 275 280
285 Asn Pro Val Gly Glu Glu Phe His Pro Thr Asn His Ala Tyr Ile Gln
290 295 300 Ser Leu Leu Lys Arg Tyr Gln Pro His Arg Val Pro Ser Thr
Cys Cys 305 310 315 320 Ala Pro Val Lys Thr Lys Pro Leu Ser Met Leu
Tyr Val Asp Asn Gly 325 330 335 Arg Val Leu Leu Glu His His Lys Asp
Met Ile Val Glu Glu Cys Gly 340 345 350 Cys Leu 5 157 PRT Human 5
Met Val Gly Arg Met Lys Leu Leu Pro Asn Arg Ile Arg Thr Leu Ala 1 5
10 15 Leu Thr Ala Ile Gly Val Val Leu Leu Gly Val Asp Asp Pro Gly
Ala 20 25 30 Pro Ser Asp Gln Val Glu Val His Leu Glu Leu Asp Leu
Pro Thr Gln 35 40 45 Leu Thr Ser Val Trp Gln Val Met Ser Thr Val
Pro Leu Ala Pro Leu 50 55 60 Pro Gly Gln Leu Ser Leu Leu Gly Pro
Pro Gly Ala Leu Gly Phe Pro 65 70 75 80 Gln Gln Gly Gly Pro Thr Gln
Leu Pro Leu Leu Leu Arg Glu Val Gly 85 90 95 Val Glu His Lys Glu
His Ile Gly Gly Arg Arg Cys Gly Gly Pro Arg 100 105 110 Pro Ala Leu
Ser Ser Tyr Pro Gly His Leu Leu Leu Gln Gly Pro Arg 115 120 125 Ala
Leu Gln Pro Leu Gly Glu Arg Pro Gly His Leu Gln Asn His Ala 130 135
140 Ala Gln Gly Lys Gly Asp Leu Gly Gln Ser Asp Ser Glu 145 150
155
* * * * *
References