U.S. patent application number 10/801837 was filed with the patent office on 2004-08-05 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 Beasley, Ellen M., Gong, Fangcheng, Higgins, Maureen E., Ladunga, Istvan I., Wei, Ming-Hui.
Application Number | 20040152167 10/801837 |
Document ID | / |
Family ID | 25284172 |
Filed Date | 2004-08-05 |
United States Patent
Application |
20040152167 |
Kind Code |
A1 |
Beasley, Ellen M. ; et
al. |
August 5, 2004 |
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: |
Beasley, Ellen M.;
(Darnestown, MD) ; Wei, Ming-Hui; (Germantown,
MD) ; Gong, Fangcheng; (Germantown, MD) ;
Ladunga, Istvan I.; (Foster City, CA) ; Higgins,
Maureen E.; (Bethesda, MD) |
Correspondence
Address: |
CELERA GENOMICS CORP.
ATTN: WAYNE MONTGOMERY, VICE PRES, INTEL PROPERTY
45 WEST GUDE DRIVE
C2-4#20
ROCKVILLE
MD
20850
US
|
Assignee: |
APPLERA CORPORATION
Global Headquarters 301 Merritt 7, P.O. Box 5435
Norwalk
CT
06856-5435
|
Family ID: |
25284172 |
Appl. No.: |
10/801837 |
Filed: |
March 17, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10801837 |
Mar 17, 2004 |
|
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09841158 |
Apr 25, 2001 |
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6740504 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/324; 536/23.5 |
Current CPC
Class: |
C07K 14/47 20130101 |
Class at
Publication: |
435/069.1 ;
530/324; 536/023.5; 435/320.1; 435/325 |
International
Class: |
C07K 014/47; C07H
021/04 |
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 NOS:3-4; (b) an amino acid sequence of an allelic
variant of an amino acid sequence shown in SEQ ID NOS:3-4, 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
NOS:3-4, 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 NOS:3-4,
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 NOS:3-4; (b) an amino acid sequence of an allelic variant of
an amino acid sequence shown in SEQ ID NOS:3-4, 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
NOS:3-4, 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 NOS:3-4,
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
NOS:3-4; (b) a nucleotide sequence that encodes of an allelic
variant of an amino acid sequence shown in SEQ ID NOS:3-4, 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 NOS:3-4, 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 NOS:3-4, 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
NOS:3-4; (b) a nucleotide sequence that encodes of an allelic
variant of an amino acid sequence shown in SEQ ID NOS:3-4, 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 NOS:3-4, 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 NOS:3-4, 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 NOS:3-4.
21. A peptide according to claim 20 that shares at least 90 percent
homology with an amino acid sequence shown in SEQ ID NOS:3-4.
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 transcobalamin II 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 1993 Sep. 24;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
Sep.;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
Sep.;6(9):815-36.
[0013] Transcobalamin II
[0014] Many biochemical reactions require the involvement of
cobalamin ("Cbl"), also known as vitamin B12, as coenzyme factors.
Human Cbl-dependent metabolism includes the biosynthesis of
methionine from homocysteine and the isomerization of
methylmalonyl-CoA to succinyl-CoA. Although cobalamin is highly
water-soluble, it is nevertheless impervious to plasma membrane.
Cobalamin is delivered into the designated subcellular locations
through multiple physiological steps.
[0015] The cellular uptake of cobalamin is mediated by
transcobalamin II (TCII), a plasma protein that binds Cbl and is
secreted by human umbilical vein endothelial (HUVE) cells. These
cells synthesize and secrete TC II and, therefore, served as the
source of the library from which the TC II cDNA was isolated. This
full-length cDNA consists of 1866 nucleotides that code for a
leader peptide of 18 amino acids, a secreted protein of 409 amino
acids, a 5'-untranslated segment of 37 nucleotides, and a
3'-untranslated region of 548 nucleotides. A single 1.9-kilobase
species of mRNA corresponding to the size of the cDNA was
identified by Northern blot analysis of the RNA isolated from HUVE
cells. TCII has 20% amino acid homology and greater than 50%
nucleotide homology with human transcobalamin I (TCI) and with rat
intrinsic factor (R-IF). TCII has no homology with the
amino-terminal region of R-IF that has been reported to have
significant primary as well as secondary structural homology with
the nucleotide-binding domain of NAD-dependent oxidoreductases. The
regions of homology that are common to all three proteins are
located in seven domains of the amino acid sequence. One or more of
these conserved domains is likely to be involved in Cbl binding, a
function that is common to all three proteins. However, the
difference in the affinity of TCII, TCI, and R-IF for Cbl and Cbl
analogues indicates, a priori, that structural differences in the
ligand-binding site of these proteins exist and these probably
resulted from divergence of a common ancestral gene. (Platica, et
al., J Biol Chem Apr. 25;266(12):7860-3 (1991))
[0016] Extracellularly secreted cobalamin is continually
transported across cellular space through transcytosis within the
endomembrane-secretory system, within which transcobalamin II ("TC
II") binds to proteolytically-released Cbl. TC II-Cbl-containing
vesicles release their contents into the circulation system. The
uptake of TC II-Cbl from the circulating fluids utilizes similar
pathways, including receptor-mediated translocation,
vesicle-dependent trafficking and targeting, and lysosome-based
proteolytical release.
[0017] TC II is a non-glycosylated secretory protein of molecular
mass 43 kDa in plasma while its homologs IF and haptocorrin are
heavily glycosylated. A conserved Cbl-binding domain (ProSite
pattern: PS000468) exists among the three types of the proteins
(Seetharam and Li, Vitam. Horm. 59:337-366 (2000); Seetharam B, et
al., Annu. Rev. Nutr. 19:173-195 (1999); Hofman, et al., Nuc. Acid
Res. 27: 215-219 (1999)). The affinity toward Cbl is suggested to
be the highest for haptocorrin (Fedosov, et al., Biochim. Biophys.
Acta 1292:113-119 (1996)). Of two TCs, TC I has been identified as
a major protein constituent of secondary granules in neotrophil and
mapped onto chromosome 11q11-q12 (Johnston, et al., J. Biol. Chem.
264:15754-15757 (1989)). IF (Chr 11), TC I and TC II (Chr 22q) are
proposed to be diverged from a common ancestral gene as they are
conserved in the multiple regions, but with different affinity
toward Cbl (Platica, et al., J Biol Chem Apr. 25;266(12):7860-3
(1991)).
[0018] Disorders of transport proteins such as TC II can lead to
abnormal function of methylmalonyl-CoA mutase and methionine
synthase (Fowler, Eur. J. Pediatr. 157(suppl. 2):S60-S66 (1998)).
Clinical evidence has demonstrated that autosomal recessive
mutations of TC II gene can lead to a disorder whose observed
symptoms include megaloblastic anemia, impaired immune response and
neurological manifestations. Li, et al., Hum. Mol. Genet.
3:1835-1840 (1994). Single nucleotide deletions in patients were
reported to cause TC II deficiency disease. (Li, et al., Biochem.
Biophys. Res. Commun. 204:1111-1118 (1994)).
[0019] Cancer cells are commonly characterized by a disturbed
balance of methionine metabolism, resulting in ceased proliferation
of methionine-dependent cells and over-production of
methionine-independent cells. The imbalance between methionine
comsumption and formation is related to methionine synthase and
methylcobalamin cofactor. The lack of cellular methylcobalamin,
resulted from various defects in cobalamin metabolism as depicted
above, causes a low rate of homocysteine remethylation, and thus
methionine production (Fiskerstrand, et al., J. Biol. Chem. 273:
20180-20184(1998)).
[0020] Secreted proteins, particularly members of the
transcobalamin II 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 transcobalamin II secreted protein subfamily.
SUMMARY OF THE INVENTION
[0021] 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 transcobalamin II 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 of
isoform 1 in adult adrenal gland, mammary gland, retinoblastoma,
adenocarcinoma cell line, embryonal carcinoma cell line, adult
uterus, adult head-neck, and leukocytes. Experimental data as
provided in FIG. 1 indicates expression of isoform 2 in adult
adrenal gland, adult uterus, adult head-neck, adult lung tumor,
mammary gland, retinoblastoma, adenocarcinoma, and the
hippocampus.
DESCRIPTION OF THE FIGURE SHEETS
[0022] FIG. 1 provides the nucleotide sequence of a cDNA molecule
or transcript sequence that encodes the secreted protein of the
present invention. (SEQ ID NOS:1-2) 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 of isoform 1 in adult adrenal gland, mammary gland,
retinoblastoma, adenocarcinoma cell line, embryonal carcinoma cell
line, adult uterus, adult head-neck, and leukocytes. Experimental
data as provided in FIG. 1 indicates expression of isoform 2 in
adult adrenal gland, adult uterus, adult head-neck, adult lung
tumor, mammary gland, retinoblastoma, adenocarcinoma, and the
hippocampus.
[0023] FIG. 2 provides the predicted amino acid sequence of the
secreted protein of the present invention. (SEQ ID NOS:3-4) 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.
[0024] FIG. 3 provides genomic sequences that span the gene
encoding the secreted protein of the present invention. (SEQ ID
NO:5) 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 for isoform 1 at 36 different nucleotide
positions and for isoform 2 at 34 different nucleotide
positions.
DETAILED DESCRIPTION OF THE INVENTION
[0025] General Description
[0026] 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 transcobalamin II 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 transcobalamin II 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.
[0027] 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 transcobalamin II secreted protein
subfamily and the expression pattern observed. Experimental data as
provided in FIG. 1 indicates expression of isoform 1 in adult
adrenal gland, mammary gland, retinoblastoma, adenocarcinoma cell
line, embryonal carcinoma cell line, adult uterus, adult head-neck,
and leukocytes. Experimental data as provided in FIG. 1 indicates
expression of isoform 2 in adult adrenal gland, adult uterus, adult
head-neck, adult lung tumor, mammary gland, retinoblastoma,
adenocarcinoma, and the hippocampus. 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 transcobalamin II family or
subfamily of secreted proteins.
[0028] Specific Embodiments
[0029] Peptide Molecules
[0030] 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
transcobalamin II 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.
[0031] 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.
[0032] 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).
[0033] 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.
[0034] 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.
[0035] 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 of isoform 1 in adult adrenal gland, mammary
gland, retinoblastoma, adenocarcinoma cell line, embryonal
carcinoma cell line, adult uterus, adult head-neck, and leukocytes.
Experimental data as provided in FIG. 1 indicates expression of
isoform 2 in adult adrenal gland, adult uterus, adult head-neck,
adult lung tumor, mammary gland, retinoblastoma, adenocarcinoma,
and the hippocampus. 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.
[0036] Accordingly, the present invention provides proteins that
consist of the amino acid sequences provided in FIG. 2 (SEQ ID
NOS:3-4), for example, proteins encoded by the transcript/cDNA
nucleic acid sequences shown in FIG. 1 (SEQ ID NOS:1-2) and the
genomic sequences provided in FIG. 3 (SEQ ID NO:5). 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.
[0037] The present invention further provides proteins that consist
essentially of the amino acid sequences provided in FIG. 2 (SEQ ID
NOS:3-4), for example, proteins encoded by the transcript/cDNA
nucleic acid sequences shown in FIG. 1 (SEQ ID NOS:1-2) and the
genomic sequences provided in FIG. 3 (SEQ ID NO:5). 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.
[0038] The present invention further provides proteins that
comprise the amino acid sequences provided in FIG. 2 (SEQ ID
NOS:3-4), for example, proteins encoded by the transcript/cDNA
nucleic acid sequences shown in FIG. 1 (SEQ ID NOS:1-2) and the
genomic sequences provided in FIG. 3 (SEQ ID NO:5). 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] FIG. 3 provides information on SNPs that have been found in
the gene encoding the enzyme of the present invention. SNPs were
identified for isoform 1 at 36 different nucleotide positions, and
for isoform 2 at 34 different nucleotide positions. 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.
[0050] 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.
[0051] 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.
[0052] 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).
[0053] 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.
[0054] 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.
[0055] 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)).
[0056] 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.
[0057] 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.
[0058] 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).
[0059] 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.
[0060] 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)).
[0061] 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.
[0062] Protein/Peptide Uses
[0063] 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.
[0064] 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.
[0065] 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 isoform 1 of secreted proteins of the present
invention are expressed in adult adrenal gland, mammary gland,
retinoblastoma, adenocarcinoma cell line, embryonal carcinoma cell
line, adult uterus, adult head-neck, and leukocytes. Specifically,
a virtual northern blot shows expression in adult adrenal gland,
mammary gland, retinoblastoma, adenocarcinoma cell line, embryonal
carcinoma cell line, adult uterus, and adult head-neck. In
addition, PCR-based tissue screening panel indicates expression in
leukocytes. Experimental data as provided in FIG. 1 indicates that
isoform 2 of secreted proteins of the present invention are
expressed in adult adrenal gland, adult uterus, adult head-neck,
adult lung tumor, mammary gland, retinoblastoma, adenocarcinoma,
and the hippocampus. Specifically, a virtual northern blot shows
expression in adult adrenal gland, adult uterus, adult head-neck,
adult lung tumor, mammary gland, retinoblastoma, and
adenocarcinoma. In addition, PCR-based tissue screening panel
indicates expression in the hippocampus. A large percentage of
pharmaceutical agents are being developed that modulate the
activity of secreted proteins, particularly members of the
transcobalamin II 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 of isoform 1 in adult
adrenal gland, mammary gland, retinoblastoma, adenocarcinoma cell
line, embryonal carcinoma cell line, adult uterus, adult head-neck,
and leukocytes. Experimental data as provided in FIG. 1 indicates
expression of isoform 2 in adult adrenal gland, adult uterus, adult
head-neck, adult lung tumor, mammary gland, retinoblastoma,
adenocarcinoma, and the hippocampus. Such uses can readily be
determined using the information provided herein, that which is
known in the art, and routine experimentation.
[0066] 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 transcobalamin II
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 isoform 1 of secreted proteins of the present
invention are expressed in adult adrenal gland, mammary gland,
retinoblastoma, adenocarcinoma cell line, embryonal carcinoma cell
line, adult uterus, adult head-neck, and leukocytes. Specifically,
a virtual northern blot shows expression in adult adrenal gland,
mammary gland, retinoblastoma, adenocarcinoma cell line, embryonal
carcinoma cell line, adult uterus, and adult head-neck. In
addition, PCR-based tissue screening panel indicates expression in
leukocytes. Experimental data as provided in FIG. 1 indicates that
isoform 2 of secreted proteins of the present invention are
expressed in adult adrenal gland, adult uterus, adult head-neck,
adult lung tumor, mammary gland, retinoblastoma, adenocarcinoma,
and the hippocampus. Specifically, a virtual northern blot shows
expression in adult adrenal gland, adult uterus, adult head-neck,
adult lung tumor, mammary gland, retinoblastoma, and
adenocarcinoma. In addition, PCR-based tissue screening panel
indicates expression in the hippocampus.
[0067] 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 of
isoform 1 in adult adrenal gland, mammary gland, retinoblastoma,
adenocarcinoma cell line, embryonal carcinoma cell line, adult
uterus, adult head-neck, and leukocytes. Experimental data as
provided in FIG. 1 indicates expression of isoform 2 in adult
adrenal gland, adult uterus, adult head-neck, adult lung tumor,
mammary gland, retinoblastoma, adenocarcinoma, and the hippocampus.
In an alternate embodiment, cell-based assays involve recombinant
host cells expressing the secreted protein.
[0068] 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.
[0069] 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.
[0070] 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).
[0071] 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.
[0072] 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 isoform 1 of
secreted proteins of the present invention are expressed in adult
adrenal gland, mammary gland, retinoblastoma, adenocarcinoma cell
line, embryonal carcinoma cell line, adult uterus, adult head-neck,
and leukocytes. Specifically, a virtual northern blot shows
expression in adult adrenal gland, mammary gland, retinoblastoma,
adenocarcinoma cell line, embryonal carcinoma cell line, adult
uterus, and adult head-neck. In addition, PCR-based tissue
screening panel indicates expression in leukocytes. Experimental
data as provided in FIG. 1 indicates that isoform 2 of secreted
proteins of the present invention are expressed in adult adrenal
gland, adult uterus, adult head-neck, adult lung tumor, mammary
gland, retinoblastoma, adenocarcinoma, and the hippocampus.
Specifically, a virtual northern blot shows expression in adult
adrenal gland, adult uterus, adult head-neck, adult lung tumor,
mammary gland, retinoblastoma, and adenocarcinoma. In addition,
PCR-based tissue screening panel indicates expression in the
hippocampus.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] Techniques for immobilizing proteins-on matrices can be used
in the drug screening assays. In one embodiment, a fusion protein
can be provided which adds a domain that allows the protein to be
bound to a matrix. For example, glutathione-S-transferase fusion
proteins can be adsorbed onto glutathione sepharose beads (Sigma
Chemical, St. Louis, Mo.) or glutathione derivatized microtitre
plates, which are then combined with the cell lysates (e.g.,
.sup.35S-labeled) and the candidate compound, and the mixture
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads are washed to remove any unbound label, and the matrix
immobilized and radiolabel determined directly, or in the
supernatant after the complexes are dissociated. Alternatively, the
complexes can be dissociated from the matrix, separated by
SDS-PAGE, and the level of 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.
[0077] 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
an animal or other model system. Such model systems are well known
in the art and can readily be employed in this context.
[0078] 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 of isoform 1 in
adult adrenal gland, mammary gland, retinoblastoma, adenocarcinoma
cell line, embryonal carcinoma cell line, adult uterus, adult
head-neck, and leukocytes. Experimental data as provided in FIG. 1
indicates expression of isoform 2 in adult adrenal gland, adult
uterus, adult head-neck, adult lung tumor, mammary gland,
retinoblastoma, adenocarcinoma, and the hippocampus. 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.
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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 of
isoform 1 in adult adrenal gland, mammary gland, retinoblastoma,
adenocarcinoma cell line, embryonal carcinoma cell line, adult
uterus, adult head-neck, and leukocytes. Experimental data as
provided in FIG. 1 indicates expression of isoform 2 in adult
adrenal gland, adult uterus, adult head-neck, adult lung tumor,
mammary gland, retinoblastoma, adenocarcinoma, and the hippocampus.
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.
[0083] 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.
[0084] 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.
[0085] 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.
[0086] 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.
[0087] 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 of isoform 1 in adult adrenal gland, mammary
gland, retinoblastoma, adenocarcinoma cell line, embryonal
carcinoma cell line, adult uterus, adult head-neck, and leukocytes.
Experimental data as provided in FIG. 1 indicates expression of
isoform 2 in adult adrenal gland, adult uterus, adult head-neck,
adult lung tumor, mammary gland, retinoblastoma, adenocarcinoma,
and the hippocampus. Accordingly, methods for treatment include the
use of the secreted protein or fragments.
[0088] Antibodies
[0089] 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.
[0090] 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.
[0091] 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).
[0092] 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.
[0093] 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.
[0094] 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).
[0095] 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.
[0096] Antibody Uses
[0097] 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 isoform 1 of
secreted proteins of the present invention are expressed in adult
adrenal gland, mammary gland, retinoblastoma, adenocarcinoma cell
line, embryonal carcinoma cell line, adult uterus, adult head-neck,
and leukocytes. Specifically, a virtual northern blot shows
expression in adult adrenal gland, mammary gland, retinoblastoma,
adenocarcinoma cell line, embryonal carcinoma cell line, adult
uterus, and adult head-neck. In addition, PCR-based tissue
screening panel indicates expression in leukocytes. Experimental
data as provided in FIG. 1 indicates that isoform 2 of secreted
proteins of the present invention are expressed in adult adrenal
gland, adult uterus, adult head-neck, adult lung tumor, mammary
gland, retinoblastoma, adenocarcinoma, and the hippocampus.
Specifically, a virtual northern blot shows expression in adult
adrenal gland, adult uterus, adult head-neck, adult lung tumor,
mammary gland, retinoblastoma, and adenocarcinoma. In addition,
PCR-based tissue screening panel indicates expression in the
hippocampus. 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.
[0098] 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 of isoform 1 in adult adrenal gland,
mammary gland, retinoblastoma, adenocarcinoma cell line, embryonal
carcinoma cell line, adult uterus, adult head-neck, and leukocytes.
Experimental data as provided in FIG. 1 indicates expression of
isoform 2 in adult adrenal gland, adult uterus, adult head-neck,
adult lung tumor, mammary gland, retinoblastoma, adenocarcinoma,
and the hippocampus. 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.
[0099] 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 of isoform 1 in adult adrenal gland, mammary gland,
retinoblastoma, adenocarcinoma cell line, embryonal carcinoma cell
line, adult uterus, adult head-neck, and leukocytes. Experimental
data as provided in FIG. 1 indicates expression of isoform 2 in
adult adrenal gland, adult uterus, adult head-neck, adult lung
tumor, mammary gland, retinoblastoma, adenocarcinoma, and the
hippocampus. 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.
[0100] 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.
[0101] The antibodies are also useful for tissue typing.
Experimental data as provided in FIG. 1 indicates expression of
isoform 1 in adult adrenal gland, mammary gland, retinoblastoma,
adenocarcinoma cell line, embryonal carcinoma cell line, adult
uterus, adult head-neck, and leukocytes. Experimental data as
provided in FIG. 1 indicates expression of isoform 2 in adult
adrenal gland, adult uterus, adult head-neck, adult lung tumor,
mammary gland, retinoblastoma, adenocarcinoma, and the hippocampus.
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.
[0102] 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.
[0103] 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 nucleic acid arrays and
similar methods have been developed for antibody arrays.
[0104] Nucleic Acid Molecules
[0105] 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.
[0106] As used herein, an "isolated" nucleic acid molecule is one
that is separated from other nucleic acid present in the natural
source of the nucleic acid. Preferably, an "isolated" nucleic acid
is free of sequences which naturally flank the nucleic acid (i.e.,
sequences located at the 5' and 3' ends of the nucleic acid) in the
genomic DNA of the organism from which the nucleic acid is derived.
However, there can be some flanking nucleotide sequences, for
example up to about 5KB, 4KB, 3KB, 2KB, or 1KB or less,
particularly contiguous peptide encoding sequences and peptide
encoding sequences within the same gene but separated by introns in
the genomic sequence. The important point is that the nucleic acid
is isolated from remote and unimportant flanking sequences such
that it can be subjected to the specific manipulations described
herein such as recombinant expression, preparation of probes and
primers, and other uses specific to the nucleic acid sequences.
[0107] 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.
[0108] 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.
[0109] Accordingly, the present invention provides nucleic acid
molecules that consist of the nucleotide sequence shown in FIG. 1
or 3 (SEQ ID NOS:1-2, transcript sequence and SEQ ID NO:5, genomic
sequence), or any nucleic acid molecule that encodes the protein
provided in FIG. 2, SEQ ID NOS:3-4. A nucleic acid molecule
consists of a nucleotide sequence when the nucleotide sequence is
the complete nucleotide sequence of the nucleic acid molecule.
[0110] The present invention further provides nucleic acid
molecules that consist essentially of the nucleotide sequence shown
in FIG. 1 or 3 (SEQ ID NOS:1-2, transcript sequence and SEQ ID
NO:5, genomic sequence), or any nucleic acid molecule that encodes
the protein provided in FIG. 2, SEQ ID NOS:3-4. 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.
[0111] The present invention further provides nucleic acid
molecules that comprise the nucleotide sequences shown in FIG. 1 or
3 (SEQ ID NOS:1-2, transcript sequence and SEQ ID NO:5, genomic
sequence), or any nucleic acid molecule that encodes the protein
provided in FIG. 2, SEQ ID NOS:3-4. 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.
[0112] 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.
[0113] 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.
[0114] 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.
[0115] 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).
[0116] 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.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] 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.
[0121] FIG. 3 provides information on SNPs that have been found in
the gene encoding the enzyme of the present invention. SNPs were
identified for isoform 1 at 36 different nucleotide positions, and
for isoform 2 at 34 different nucleotide positions. 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.
[0122] 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.
[0123] Nucleic Acid Molecule Uses
[0124] 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 for isoform 1 at 36 different
nucleotide positions and for isoform 2 at 34 different nucleotide
positions.
[0125] 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.
[0126] 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.
[0127] 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.
[0128] The nucleic acid molecules are also useful for expressing
antigenic portions of the proteins.
[0129] 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.
[0130] The nucleic acid molecules are also useful in making vectors
containing the gene regulatory regions of the nucleic acid
molecules of the present invention.
[0131] 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.
[0132] The nucleic acid molecules are also useful for making
vectors that express part, or all, of the peptides.
[0133] The nucleic acid molecules are also useful for constructing
host cells expressing a part, or all, of the nucleic acid molecules
and peptides.
[0134] The nucleic acid molecules are also useful for constructing
transgenic animals expressing all, or a part, of the nucleic acid
molecules and peptides.
[0135] 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 isoform 1 of secreted proteins of the present
invention are expressed in adult adrenal gland, mammary gland,
retinoblastoma, adenocarcinoma cell line, embryonal carcinoma cell
line, adult uterus, adult head-neck, and leukocytes. Specifically,
a virtual northern blot shows expression in adult adrenal gland,
mammary gland, retinoblastoma, adenocarcinoma cell line, embryonal
carcinoma cell line, adult uterus, and adult head-neck. In
addition, PCR-based tissue screening panel indicates expression in
leukocytes. Experimental data as provided in FIG. 1 indicates that
isoform 2 of secreted proteins of the present invention are
expressed in adult adrenal gland, adult uterus, adult head-neck,
adult lung tumor, mammary gland, retinoblastoma, adenocarcinoma,
and the hippocampus. Specifically, a virtual northern blot shows
expression in adult adrenal gland, adult uterus, adult head-neck,
adult lung tumor, mammary gland, retinoblastoma, and
adenocarcinoma. In addition, PCR-based tissue screening panel
indicates expression in the hippocampus. 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.
[0136] 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.
[0137] 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 isoform 1 of
secreted proteins of the present invention are expressed in adult
adrenal gland, mammary gland, retinoblastoma, adenocarcinoma cell
line, embryonal carcinoma cell line, adult uterus, adult head-neck,
and leukocytes. Specifically, a virtual northern blot shows
expression in adult adrenal gland, mammary gland, retinoblastoma,
adenocarcinoma cell line, embryonal carcinoma cell line, adult
uterus, and adult head-neck. In addition, PCR-based tissue
screening panel indicates expression in leukocytes. Experimental
data as provided in FIG. 1 indicates that isoform 2 of secreted
proteins of the present invention are expressed in adult adrenal
gland, adult uterus, adult head-neck, adult lung tumor, mammary
gland, retinoblastoma, adenocarcinoma, and the hippocampus.
Specifically, a virtual northern blot shows expression in adult
adrenal gland, adult uterus, adult head-neck, adult lung tumor,
mammary gland, retinoblastoma, and adenocarcinoma. In addition,
PCR-based tissue screening panel indicates expression in the
hippocampus.
[0138] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate secreted protein nucleic acid
expression.
[0139] 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 of isoform 1 in
adult adrenal gland, mammary gland, retinoblastoma, adenocarcinoma
cell line, embryonal carcinoma cell line, adult uterus, adult
head-neck, and leukocytes. Experimental data as provided in FIG. 1
indicates expression of isoform 2 in adult adrenal gland, adult
uterus, adult head-neck, adult lung tumor, mammary gland,
retinoblastoma, adenocarcinoma, and the hippocampus. 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.
[0140] 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.
[0141] 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 isoform 1 of secreted proteins of the present invention are
expressed in adult adrenal gland, mammary gland, retinoblastoma,
adenocarcinoma cell line, embryonal carcinoma cell line, adult
uterus, adult head-neck, and leukocytes. Specifically, a virtual
northern blot shows expression in adult adrenal gland, mammary
gland, retinoblastoma, adenocarcinoma cell line, embryonal
carcinoma cell line, adult uterus, and adult head-neck. In
addition, PCR-based tissue screening panel indicates expression in
leukocytes. Experimental data as provided in FIG. 1 indicates that
isoform 2 of secreted proteins of the present invention are
expressed in adult adrenal gland, adult uterus, adult head-neck,
adult lung tumor, mammary gland, retinoblastoma, adenocarcinoma,
and the hippocampus. Specifically, a virtual northern blot shows
expression in adult adrenal gland, adult uterus, adult head-neck,
adult lung tumor, mammary gland, retinoblastoma, and
adenocarcinoma. In addition, PCR-based tissue screening panel
indicates expression in the hippocampus. Modulation includes both
up-regulation (i.e. activation or agonization) or down-regulation
(suppression or antagonization) or nucleic acid expression.
[0142] 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 of isoform 1 in adult
adrenal gland, mammary gland, retinoblastoma, adenocarcinoma cell
line, embryonal carcinoma cell line, adult uterus, adult head-neck,
and leukocytes. Experimental data as provided in FIG. 1 indicates
expression of isoform 2 in adult adrenal gland, adult uterus, adult
head-neck, adult lung tumor, mammary gland, retinoblastoma,
adenocarcinoma, and the hippocampus.
[0143] 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.
[0144] 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.
[0145] 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 for isoform 1 at 36 different nucleotide
positions, and for isoform 2 at 34 different nucleotide positions.
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 anti sense DNA
sequences.
[0146] Alternatively, mutations in a secreted protein gene can be
directly identified, for example, by alterations in restriction
enzyme digestion patterns determined by gel electrophoresis.
[0147] 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.
[0148] 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)).
[0149] 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.
[0150] 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 for isoform 1 at 36 different
nucleotide positions, and for isoform 2 at 34 different nucleotide
positions. 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.
[0151] 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.
[0152] 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.
[0153] 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.
[0154] 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.
[0155] 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 isoform 1 of
secreted proteins of the present invention are expressed in adult
adrenal gland, mammary gland, retinoblastoma, adenocarcinoma cell
line, embryonal carcinoma cell line, adult uterus, adult head-neck,
and leukocytes. Specifically, a virtual northern blot shows
expression in adult adrenal gland, mammary gland, retinoblastoma,
adenocarcinoma cell line, embryonal carcinoma cell line, adult
uterus, and adult head-neck. In addition, PCR-based tissue
screening panel indicates expression in leukocytes. Experimental
data as provided in FIG. 1 indicates that isoform 2 of secreted
proteins of the present invention are expressed in adult adrenal
gland, adult uterus, adult head-neck, adult lung tumor, mammary
gland, retinoblastoma, adenocarcinoma, and the hippocampus.
Specifically, a virtual northern blot shows expression in adult
adrenal gland, adult uterus, adult head-neck, adult lung tumor,
mammary gland, retinoblastoma, and adenocarcinoma. In addition,
PCR-based tissue screening panel indicates expression in the
hippocampus. 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.
[0156] Nucleic Acid Arrays
[0157] 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).
[0158] 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.
[0159] 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.
[0160] 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.
[0161] In another aspect, an oligonucleotide may be synthesized on
the surface of the substrate by using a chemical coupling procedure
and an ink jet application apparatus, as described in PCT
application W095/251116 (Baldeschweiler et al.) which is
incorporated herein in its entirety by reference. In another
aspect, a "gridded" array analogous to a dot (or slot) blot may be
used to arrange and link cDNA fragments or oligonucleotides to the
surface of a substrate using a vacuum system, thermal, UV,
mechanical or chemical bonding procedures. An array, such as those
described above, may be produced by hand or by using available
devices (slot blot or dot blot apparatus), materials (any suitable
solid support), and machines (including robotic instruments), and
may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or
any other number between two and one million which lends itself to
the efficient use of commercially available instrumentation.
[0162] 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.
[0163] 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 for isoform 1 at 36 different
nucleotide positions, and for isoform 2 at 34 different nucleotide
positions. 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.
[0164] 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 (1 982), 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).
[0165] 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.
[0166] In another embodiment of the present invention, kits are
provided which contain the necessary reagents to carry out the
assays of the present invention.
[0167] 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.
[0168] 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.
[0169] Vectors/host Cells
[0170] 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.
[0171] 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.
[0172] 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).
[0173] 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.
[0174] The regulatory sequence to which the nucleic acid molecules
described herein can be operably linked include promoters for
directing mRNA transcription. These include, but are not limited
to, the left promoter from bacteriophage .lambda., the lac, TRP,
and TAC promoters from E. coli, the early and late promoters from
SV40, the CMV immediate early promoter, the adenovirus early and
late promoters, and retrovirus long-terminal repeats.
[0175] 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.
[0176] 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).
[0177] 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).
[0178] 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.
[0179] 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.
[0180] 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.
[0181] 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 67:3140 (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)).
[0182] 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)).
[0183] The nucleic acid molecules can also be expressed by
expression vectors that are operative in yeast. Examples of vectors
for expression in yeast e.g., S. cerevisiae include pYepSec1
(Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al.,
Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123
(1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).
[0184] 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., Sf 9 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)).
[0185] 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)).
[0186] 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.
[0187] 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).
[0188] 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.
[0189] 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).
[0190] 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.
[0191] 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.
[0192] 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.
[0193] 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.
[0194] 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.
[0195] 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.
[0196] 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.
[0197] Uses of Vectors and Host Cells
[0198] 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.
[0199] 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.
[0200] 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.
[0201] 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.
[0202] 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.
[0203] 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.
[0204] 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.
[0205] 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.
[0206] 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.
[0207] 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.
[0208] 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
7 1 1722 DNA Homo sapiens 1 ttgctcactg ctcacccacc tgctgctgcc
atgaggcacc ttggggcctt cctcttcctt 60 ctgggggtcc tgggggccct
cactgagatg tgtgaaatac cagagatgga cagccatctg 120 gtagagaagt
tgggccagca cctcttacct tggatggacc ggctttccct ggagcacttg 180
aaccccagca tctatgtggg cctacgcctc tccagtctgc aggctgggac caaggaagac
240 ctctacctgc acagcctcaa gcttggttac cagcagtgcc tcctagggtc
tgccttcagc 300 gaggatgacg gtgactgcca gggcaagcct tccatgggcc
agctggccct ctacctgctc 360 gctctcagag ccaactgtga gtttgtcagg
ggccacaagg gggacaggct ggtctcacag 420 ctcaaatggt tcctggagga
tgagaagaga gccattgaca cagcagccat ggcaggcttg 480 gcattcacct
gtctgaagcg ctcaaacttc aaccctggtc ggagacaacg gatcaccatg 540
gccatcagaa cagtgcgaga ggagatcttg aaggcccaga cccccgaggg ccactttggg
600 aatgtctaca gcaccccatt ggcattacag ttcctcatga cttcccccat
gcgtggggca 660 gaactgggaa cagcatgtct caaggcgagg gttgctttgc
tggccagtct gcaggatgga 720 gccttccaga atgctctcat gatttcccag
ctgctgcccg ttctgaacca caagacctac 780 attgatctga tcttcccaga
ctgtctggca ccacgagtca tgttggaacc agctgctgag 840 accattcctc
agacccaaga gatcatcagt gtcacgctgc aggtgcttag tctcttgccg 900
ccgtacagac agtccatctc tgttctggcc gggtccaccg tggaagatgt cctgaagaag
960 gcccatgagt taggaggatt cacatatgaa acacaggcct ccttgtcagg
cccctactta 1020 acctccgtga tggggaaagc ggccggagaa agggagttct
ggcagcttct ccgagacccc 1080 aacaccccac tgttgcaagg tattgctgac
tacagaccca aggatggaga aaccattgag 1140 ctgaggctgg ttagctggta
gcccctgagc tccctcatcc cagcagcctc gcacactccc 1200 taggcttcta
ccctccctcc tgatgtccct ggaacaggaa ctcgcctgac cctgctgcca 1260
cctcctgtgc actttgagca atgccccctg ggatcacccc agccacaagc ccttcgaggg
1320 ccctatacca tggcccacct tggagcagag agccaagcat cttccctggg
aagtctttct 1380 ggccaagtct ggccagcctg gccctgcagg tctcccatga
aggccacccc atggtctgat 1440 gggcatgaag catctcagac tccttggcaa
aaaacggagt ccgcaggccg caggtgttgt 1500 gaagaccact cgttctgtgg
ttggggtcct gcaagaaggc ctcctcagcc cgggggctat 1560 ggccctgacc
ccagctctcc actctgctgt tagagtggca gctccgagct ggttgtggca 1620
cagtagctgg ggagacctca gcagggctgc tcagtgcctg cctctgacaa aattaaagca
1680 ttgatggcct gtgaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 1722 2 1896 DNA
Homo sapiens 2 ggaggattaa tcagtgacag gaagctgcgt ctctcggagc
ggtgaccagc tgtggtcagg 60 agagcctcag cagggccagc cccaggagtc
tttcccgatt cttgctcact gctcacccac 120 ctgctgctgc catgaggcac
cttggggcct tcctcttcct tctgggggtc ctgggggccc 180 tcactgagat
gtgtgaaata ccagagatgg acagccatct ggtagagaag ttgggccagc 240
acctcttacc ttggatggac cggctttccc tggagcactt gaaccccagc atctatgtgg
300 gcctacgcct ctccagtctg caggctggga ccaaggaaga cctctacctg
cacagcctca 360 tgcttggtta ccagcagtgc ctcctagggt ctgccttcag
cgaggatgac ggtgactgcc 420 agggcaagcc ttccatgggc cagctggccc
tctacctgct cgctctcaga gccaactggc 480 atgatcacaa gggccacccc
cacactagct actaccagta tggcctgggc attctggccc 540 tgtgtctcca
ccagaagcgg gtccatgaca gcgtggtgga caaacttctg tatgctgtgg 600
aacctttcca ccagggccac cattctgtgg acacagcagc catggcaggc ttggcattca
660 cctgtctgaa gcgctcaaac ttcaaccctg gtcggagaca acggatcacc
atggccatca 720 gaacagtgcg agaggagatc ttgaaggccc agacccccga
gggccacttt gggaatgtct 780 acagcacccc attggcatta cagttcctca
tgacttcccc catgcgtggg gcagaactgg 840 gaacagcatg tctcaaggcg
agggttgctt tgctggccag tctgcaggat ggagccttcc 900 agaatgctct
catgatttcc cagctgctgc ccgttctgaa ccacaagacc tacattgatc 960
tgatcttccc agactgtctg gcaccacgag tcatgttgga accagctgct gagaccattc
1020 ctcagaccca agagatcatc agtgtcacgc tgcaggtgct tagtctcttg
ccgccgtaca 1080 gacagtccat ctctgttctg gccgggtcca ccgtggaaga
tgtcctgaag aaggcccatg 1140 agttaggagg attcacatat gaaacacagg
cctccttgtc aggcccctac ttaacctccg 1200 tgatggggaa agcggccgga
gaaagggagt tctggcagct tctccgagac cccaacaccc 1260 cactgttgca
aggtattgct gactacagac ccaaggatgg agaaaccatt gagctgaggc 1320
tggttagctg gtagcccctg agctccctca tcccagcagc ctcgcacact ccctaggctt
1380 ctaccctccc tcctgatgtc cctggaacag gaactcgcct gaccctgctg
ccacctcctg 1440 tgcactttga gcaatgcccc ctgggatcac cccagccaca
agcccttcga gggccctata 1500 ccatggccca ccttggagca gagagccaag
catcttccct gggaagtctt tctggccaag 1560 tctggccagc ctggccctgc
aggtctccca tgaaggccac cccatggtct gatgggcatg 1620 aagcatctca
gactccttgg caaaaaacgg agtccgcagg ccgcaggtgt tgtgaagacc 1680
actcgttctg tggttggggt cctgcaagaa ggcctcctca gcccgggggc tatggccctg
1740 accccagctc tccactctgc tgttagagtg gcagctccga gctggttgtg
gcacagtagc 1800 tggggagacc tcagcagggc tgctcagtgc ctgcctctga
caaaattaaa gcattgatgg 1860 cctgtgaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa
1896 3 376 PRT Homo sapiens 3 Met Arg His Leu Gly Ala Phe Leu Phe
Leu Leu Gly Val Leu Gly Ala 1 5 10 15 Leu Thr Glu Met Cys Glu Ile
Pro Glu Met Asp Ser His Leu Val Glu 20 25 30 Lys Leu Gly Gln His
Leu Leu Pro Trp Met Asp Arg Leu Ser Leu Glu 35 40 45 His Leu Asn
Pro Ser Ile Tyr Val Gly Leu Arg Leu Ser Ser Leu Gln 50 55 60 Ala
Gly Thr Lys Glu Asp Leu Tyr Leu His Ser Leu Lys Leu Gly Tyr 65 70
75 80 Gln Gln Cys Leu Leu Gly Ser Ala Phe Ser Glu Asp Asp Gly Asp
Cys 85 90 95 Gln Gly Lys Pro Ser Met Gly Gln Leu Ala Leu Tyr Leu
Leu Ala Leu 100 105 110 Arg Ala Asn Cys Glu Phe Val Arg Gly His Lys
Gly Asp Arg Leu Val 115 120 125 Ser Gln Leu Lys Trp Phe Leu Glu Asp
Glu Lys Arg Ala Ile Asp Thr 130 135 140 Ala Ala Met Ala Gly Leu Ala
Phe Thr Cys Leu Lys Arg Ser Asn Phe 145 150 155 160 Asn Pro Gly Arg
Arg Gln Arg Ile Thr Met Ala Ile Arg Thr Val Arg 165 170 175 Glu Glu
Ile Leu Lys Ala Gln Thr Pro Glu Gly His Phe Gly Asn Val 180 185 190
Tyr Ser Thr Pro Leu Ala Leu Gln Phe Leu Met Thr Ser Pro Met Arg 195
200 205 Gly Ala Glu Leu Gly Thr Ala Cys Leu Lys Ala Arg Val Ala Leu
Leu 210 215 220 Ala Ser Leu Gln Asp Gly Ala Phe Gln Asn Ala Leu Met
Ile Ser Gln 225 230 235 240 Leu Leu Pro Val Leu Asn His Lys Thr Tyr
Ile Asp Leu Ile Phe Pro 245 250 255 Asp Cys Leu Ala Pro Arg Val Met
Leu Glu Pro Ala Ala Glu Thr Ile 260 265 270 Pro Gln Thr Gln Glu Ile
Ile Ser Val Thr Leu Gln Val Leu Ser Leu 275 280 285 Leu Pro Pro Tyr
Arg Gln Ser Ile Ser Val Leu Ala Gly Ser Thr Val 290 295 300 Glu Asp
Val Leu Lys Lys Ala His Glu Leu Gly Gly Phe Thr Tyr Glu 305 310 315
320 Thr Gln Ala Ser Leu Ser Gly Pro Tyr Leu Thr Ser Val Met Gly Lys
325 330 335 Ala Ala Gly Glu Arg Glu Phe Trp Gln Leu Leu Arg Asp Pro
Asn Thr 340 345 350 Pro Leu Leu Gln Gly Ile Ala Asp Tyr Arg Pro Lys
Asp Gly Glu Thr 355 360 365 Ile Glu Leu Arg Leu Val Ser Trp 370 375
4 400 PRT Homo sapiens 4 Met Arg His Leu Gly Ala Phe Leu Phe Leu
Leu Gly Val Leu Gly Ala 1 5 10 15 Leu Thr Glu Met Cys Glu Ile Pro
Glu Met Asp Ser His Leu Val Glu 20 25 30 Lys Leu Gly Gln His Leu
Leu Pro Trp Met Asp Arg Leu Ser Leu Glu 35 40 45 His Leu Asn Pro
Ser Ile Tyr Val Gly Leu Arg Leu Ser Ser Leu Gln 50 55 60 Ala Gly
Thr Lys Glu Asp Leu Tyr Leu His Ser Leu Met Leu Gly Tyr 65 70 75 80
Gln Gln Cys Leu Leu Gly Ser Ala Phe Ser Glu Asp Asp Gly Asp Cys 85
90 95 Gln Gly Lys Pro Ser Met Gly Gln Leu Ala Leu Tyr Leu Leu Ala
Leu 100 105 110 Arg Ala Asn Trp His Asp His Lys Gly His Pro His Thr
Ser Tyr Tyr 115 120 125 Gln Tyr Gly Leu Gly Ile Leu Ala Leu Cys Leu
His Gln Lys Arg Val 130 135 140 His Asp Ser Val Val Asp Lys Leu Leu
Tyr Ala Val Glu Pro Phe His 145 150 155 160 Gln Gly His His Ser Val
Asp Thr Ala Ala Met Ala Gly Leu Ala Phe 165 170 175 Thr Cys Leu Lys
Arg Ser Asn Phe Asn Pro Gly Arg Arg Gln Arg Ile 180 185 190 Thr Met
Ala Ile Arg Thr Val Arg Glu Glu Ile Leu Lys Ala Gln Thr 195 200 205
Pro Glu Gly His Phe Gly Asn Val Tyr Ser Thr Pro Leu Ala Leu Gln 210
215 220 Phe Leu Met Thr Ser Pro Met Arg Gly Ala Glu Leu Gly Thr Ala
Cys 225 230 235 240 Leu Lys Ala Arg Val Ala Leu Leu Ala Ser Leu Gln
Asp Gly Ala Phe 245 250 255 Gln Asn Ala Leu Met Ile Ser Gln Leu Leu
Pro Val Leu Asn His Lys 260 265 270 Thr Tyr Ile Asp Leu Ile Phe Pro
Asp Cys Leu Ala Pro Arg Val Met 275 280 285 Leu Glu Pro Ala Ala Glu
Thr Ile Pro Gln Thr Gln Glu Ile Ile Ser 290 295 300 Val Thr Leu Gln
Val Leu Ser Leu Leu Pro Pro Tyr Arg Gln Ser Ile 305 310 315 320 Ser
Val Leu Ala Gly Ser Thr Val Glu Asp Val Leu Lys Lys Ala His 325 330
335 Glu Leu Gly Gly Phe Thr Tyr Glu Thr Gln Ala Ser Leu Ser Gly Pro
340 345 350 Tyr Leu Thr Ser Val Met Gly Lys Ala Ala Gly Glu Arg Glu
Phe Trp 355 360 365 Gln Leu Leu Arg Asp Pro Asn Thr Pro Leu Leu Gln
Gly Ile Ala Asp 370 375 380 Tyr Arg Pro Lys Asp Gly Glu Thr Ile Glu
Leu Arg Leu Val Ser Trp 385 390 395 400 5 27067 DNA Homo sapiens
misc_feature (1)...(27067) n = A,T,C or G 5 atatgtatgg gaaatatgct
gtcttcctat tcctactccc ccaccctcta gcactgagtc 60 caggtaggta
ggcagggggg tgtctccctc ctttacttcg acaccctaac taccttgggg 120
atcagaagtg actctctgga aggatgctgc tgcttctcac cagaggctga cgataacgaa
180 ggctatcctc catggccacc tcctccaggc tgccttcctg gaaataggaa
tcataatagt 240 tgttactgga aacaggcaga gggttggggg agccaaggca
gtcccaccca ggaccaaggt 300 ggctccattg cacacacttc accatgactc
ccctgaaggt ccaaacgtgc ggttctgcgg 360 aagttgggct ccccactggc
ctccctcctt cctcagaacc tccaggggtg ctcctcctag 420 tggccacatc
cagcctttct gactggacaa cctatcattt aaaattttca agtagttccg 480
taaacagaca cacgttgctg tatttattta tgtcaagggc ttggtttgtg ataagtcagg
540 ctcaaaaaga ttgtcttaaa agagtgaacc ttggcaattt accataaaat
aattgcaatg 600 cagattgtgc atggaaatga ttggagatat tttaaggtca
tagtgtcttc acaaattgag 660 ctgaaaggga actgttagga tgatcttgcc
taaccctctc atctcacaca ggaagaacta 720 ttttaaactc gagaggttaa
gtgacctggc caaagtcaca cagccaccac tagttaactc 780 gtatacattg
attctcctgt ggggctgggc agatgaggaa tcttttgttc tcttccctgt 840
ttgcagagat tttttttgag gttactttcc gagttctggc aagtacccct gcttctggta
900 gctttgtgtc tcgattcaat ctcattcttt ttattttatt ttatttttga
gacagggtct 960 cactttgtca cccaagctgg agtgcagtgg tgtaatcttg
gctcactgta gcctccacct 1020 cttgggttca agcgatcctc ctgcctcagc
cccccaagta gctgggatta cagacgtctg 1080 ccaccacgcc aggctaattt
atggtttttt gtatgtgttt tttgtgtttt tgtagagaca 1140 gtgtttcccc
atgttgccca ggctggtctc caactcctga gctcaagtga tctgcccgcc 1200
tcagcctttc aaagtgctag gattacaggt gtgagccacc gtgcccggac ttaatcccat
1260 tctttaactt gttttgtttt gtcctctcca ggaggctccc agccctttcg
gattggttga 1320 gaaaagtggc ctggctggtc tggggccagc agcacccacc
ctcccctcaa ttgcccaact 1380 ccccccccca ccgaactgcc caactccccc
tccccaactg cccaactccc ccacccccac 1440 aatcccctcc cgccacaact
gagggaggcg gtgctgaaaa acagctgact ccagcaatgc 1500 tgctcacgtg
accactgcag ctgcagctcc cgttccactc cttgtcctgg gctaggtggg 1560
cactaccagg ggctcctttg gtaaggagta ccgggtaggc acccggtcct gccaatccac
1620 cactggaaca gctgggggga cagcagacag gcacggtcgg acagacttga
cagatcaggc 1680 atcaggccct ctgcgctggt cccgggctct ttaagcagga
acgtgaatgg cctcaagatg 1740 tctcacatgg tcccactagc cctcctcctc
cctttgttcc ctacctccag gagggctgct 1800 ctgcccttcc ttcctctgtt
ctttggcctt atgttccccg ccaccacagg ccttcccccg 1860 ccccacccct
ctgcagactt agccgtgcat tgcaggcatg gaggattaat cagtgacagg 1920
aagctgcgtc tctcggagcg gtgaccagct gtggtcagga gagcctcagc agggccagcc
1980 ccaggagtct ttcccgattc ttgctcactg ctcacccacc tgctgctgcc
atgaggcacc 2040 ttggggcctt cctcttcctt ctgggggtcc tgggggccct
cactgagatg tgtggtgagt 2100 aactcgcctc tatcctgtgc ctctttcctc
ctgggtcctt agtggggtgg ctagggcata 2160 ggatgaggga acttacctgc
ccttctaagc tcccatagca gtttgggctt agctggacct 2220 cagcatttaa
cacatcctat tgtgattgat tatatgtttg actcctcacc agacaagatc 2280
tccgttaatt cagtcattcg ttcacacatt cattcagcgc atactgagcc ttttctgtgt
2340 caggcccagt gttagccttt ggggaacgtg caaagcatga gacaagtcta
atccctgcca 2400 tcctagagct tatgttctag ggaaggggga cagacaaaag
aaatggttag gtgctcccac 2460 ctgaaatctc agcattttgg aaggctgagg
cgggagggga ggatcgcttg agctcaacag 2520 ttcaaggtca gcctgggcaa
catagggaga ccccatctct acaaaaaata aaaaaaatta 2580 aaaaatagct
gggcatgggg aagactttct gaagaccaag aggacacatg ggagctgaaa 2640
ctcgaaggaa gaaaaggagc tggcaggaaa ggagtggggg acacacattc taggcagcag
2700 gaagtgagcc ttcggaggtc ctgcctgctc cagctctgtg ccccaagggg
tctcttggag 2760 cacagtctcc tgggacctgt ctatgagtct gagcttagag
gctcagggct gctccttcag 2820 acaggaggca gaaggcagac tttgggaact
ttgggccgcc cacgcgcctt ttctcctcct 2880 ctgcacctag gattacgttg
agcaatacac tttcaccccc atggtctctt gagaccctgg 2940 ggaaaccctg
agaggtgggt gcagtcatgt ccaggtgtca agtgaagaag tcgagggttg 3000
gaggggctga gtgacccact cagggtgctc caccttttcc agagctttgc tgaacttagt
3060 ttttagaact tgaagcctcg tttgttttcg ttttgttttt tgttgagaga
ggttctccct 3120 ctgttgccca ggctggagtg cagtggcacg atcttggctc
actgcagcct ctgccttgtg 3180 ggttcaagtg attcccccac ctcagcctcc
caagtagctg gagactgcat gtgcatacta 3240 ccatgcttgg ctaatttttg
tatttttttg tagagacagg gtttcgccat gttgcccagg 3300 ctggtctcga
actcctgggc tcaagtgaaa ctcttgcctc ggcctcccaa attgctgaga 3360
ttacaggcgt gagccaccgt gcccggccag aactccaagc ctctcatctg tgttccataa
3420 atgcaatcag acacctcagg tctgggccca ggaaccccag ctcttggttc
atgtccggac 3480 agtccccagg ggagttctgg gttcaaccag caagagctct
tcctcctggc tgatctggtc 3540 ctcagccttg gacagttagt ccattaacct
gaccccacag gagccccaat cccttggggt 3600 ctggggaatc ttgaactggg
gtttggggtg caaatatctg cactgagtca cttaattgca 3660 cccagcctca
ttcctttatc tgtaaagtgg gctaagaatg ctcccctgcc ttcctcctcg 3720
gtgtagtacg aggaaggatc ccatgacacc tgctctccca gtttaaagct ctatatgtat
3780 gttgtgaaat tgacagggat cgctgcacaa acgctaatgc aaagtgggct
cctgtgcttc 3840 cttttctctt tcttcttctt tttttttttt ttaattttct
tctagagatg aggtctcact 3900 atattgccca gggttggttt caaactccta
gggtcaagcg atcctcccac cttggcctcc 3960 caaactgctg gtattacagg
cgtgagccac tctgtctggc tcctatgctt gtgaatgtca 4020 acagcaatca
gcccttagct ggcagggctg ggttggtagg gcgagagctc acccaaggct 4080
gcttttatta ccctgcgtga atctgcctgg ccccttcctt ctaaggaggt tgctctgtgg
4140 ttgtcagtct ctccctttac agctggatcc tgatctttca gtttctaacc
ctgtgctgac 4200 tcatcgtgct ggaagtgaga gcccggggtg aggtcaggga
actcccttgc gcgtttcaag 4260 aaaagggaaa aggaaagaga ggtgaggagg
ggggcagatg accagagaga cacaggctga 4320 gagagactga gacagaccca
gagagcctca cacattgagt gacagagacg gagaaatgga 4380 gataggcacc
aaaaaatggt tctcagtgac agaaagggaa aaaagcaacc ccccagtctc 4440
tcttaacatc tggtgagaaa ccagccatgt gctttggtct gggcccacac agcaaaggat
4500 tatgtagggt ttcatgctgg tggatggtca ccttatagca acaggtatct
ggggctgtcg 4560 ggaaaacaga cacgaggttg tgggacccag acccacagag
atggagctgt tctaggagct 4620 ctggtcctcg ttctggtccc ctgggatatg
gcacagtgaa ggccaccatc aggcagctgg 4680 agcccagcag caactgggag
gcagtaaaca gggaccgaaa gtgcaaggtt acctccgagg 4740 caaactactc
taagctaccc tgtgctgagc tcaagtccct tggaactatc cctaaggctt 4800
ccgcttccag agtgtttgag tattttcgtt gcacagcttc gaataaatcc cacagcaaca
4860 ggtaaacggc tgcaagctgt gactgttttc taagagctca tctcacaatc
tcaggtcctc 4920 ttcatttaaa cagagatggc aggaaaggcg ttattttgag
atctgcatgg aggaagttca 4980 ccaggcagcc tcaattcacc agctggaagt
ttgcgttgtt tggaaatttg atgtgtaaca 5040 cgttctgcat gtgggctgat
gtttttgtaa acgggtagca cacacattca gcagggcacc 5100 aaagagcggg
ggctttgcag ttaggtccat ccttggctct gcagccttgt gtaagacatg 5160
acacgacttt gaacttctgt ttcctcttct gtgcaaagca atgatgacag tatctacatc
5220 acaggactgg catgaggacc aagtgagatt gggcaaggtg cccgggcaca
ccagtctcac 5280 tgtcactgct gatgggcaga gtggttgcct ggcagtagca
tcctctatct tcagcccacc 5340 acctctcttg ctggctcact ccaactgctc
tttagagata cacgcttccc ctcttttctc 5400 ctcccactgc ctttcagtat
ggctgcattt ccccctgcaa gttggtgtgt gctgggtgga 5460 ggtgggggtg
aggacatgta ttctctggag aaggccctgg taacgtcaaa gcacttcttt 5520
gctggtggcc tggccctgtg acctcatttg taccattttc ttttctaaga aataccagag
5580 atggacagcc atctggtaga gaagttgggc cagcacctct taccttggat
ggaccggctt 5640 tccctggagc acttgaaccc cagcatctat gtgggcctac
gcctctccag tctgcaggct 5700 gggaccaagg aagacctcta cctgcacagc
ctcaagcttg gttaccagca gtgcctccta 5760 gggtattgcc acactctctt
tttccatgtc ttgctccaca tactaagaga tgggaaactt 5820 gggtactagt
ttgggcctgt caccactttg tgggcagacc ttaggcaaat tttctccatc 5880
tatagaatgg aggacctttg tccatctata gaatgaaggg gttggttgga ttagatcaga
5940 gatgctaatg caaggctcct tttgctacta ctgtccatca tgtgtctgag
gcagacataa 6000 ctaatccgtg actatactct ttgatgatga gcccaggagc
agcatctgac tctatgctcc 6060 cttagtgtgc ctgaggcaga tatcactaat
cgatgactgc agtcttctac attgagctta 6120 gaagcagcat ctgactctgt
atgctcccct cccatgcatg aggcagacat cagtaatcca 6180 tgaccgcatt
ctttcatact gagcccagaa gcagcatctt ttcttttctt tcctctcact 6240
ctgttgccca ggctagagtg cagtggcaca atcttggctt gccccaacct ccaattcccg
6300 ggttcaagtg attctcgtgc ctcagccacc tgaatagctg ggattacagg
cgtgtgccac 6360 catgcccagc tgatttttgt atttttggta gagatagggt
ttcaccatgt tggccaggct 6420 ggtcttgaac tcctgacctc aggtgatccg
cctgtcttgg cttcccaaag
tgttgggatt 6480 ataggcatga gccactgcac caatccaaaa gcagcatctt
tgtgctccct tttcaagagg 6540 catcacagag aggcctgttt tggggtttga
atgagaggcg aagaatcagc catggagtgc 6600 ctctttctca gactccctct
tgagaagtgg gtgcaggggt ggagagaaaa gaagactagg 6660 catagtggct
catacctgta atcccaacat tttgggaggc tgaggcagga agattgcttg 6720
agctcaggag tttgagacca gcctaggcaa catagtgaga ccacatctct taaaaaaaag
6780 aaaaagaaaa aaaatgagcc aggtgtagtg actcatgcct gtggtcccca
cttctccgga 6840 ggcaaaggtg ggaggatctt ttgaggctga gaaatcgagg
ctacagtgag ccatggtggc 6900 accactgcac tccagcctgg gagacagaga
gaccctatct cagtaaaaaa aaaaaataaa 6960 aatatggctg ggtgtggtgg
ctcacgcctg taatcccagc actttgggag gccaaggtag 7020 gtagatcaca
tgaggttagg agttcgaaac cagtctggcc aacatagtga aaccctgtct 7080
ctactgaaaa tacaaaaaat tagccaaggg tggtggtggg caactgtaat cccagctact
7140 tgggaggccg aggcagaaga atcgcttgaa ctcgggaggc ggaggttgca
gtgagctgag 7200 aacatgccac tgcactccag cctgggcaac aagagcgaaa
ctctgtctca aagaaaataa 7260 ataaataaaa taaaaaaata aaaaaggagg
gggcatatgg gtgaagtatg gacaaaatag 7320 tggggcaggc acagatgatc
tggacacagg agcccttgga gtttattctt gaatctaact 7380 gttcatcttt
attaaatatt tgtggcatac acctcacaac aacatagcca acacacctcc 7440
ttttggagct tttatcgaag tttcccactg ttaagatttt ttcccgcttt gtgatgcggg
7500 tggggtgggt gctgtaagca ggcttacggg gtggcagttt ctcacaaagg
cattaactgg 7560 ccttgtccta ggtctgcctt cagcgaggat gacggtgact
gccagggcaa gccttccatg 7620 ggccagctgg ccctctacct gctcgctctc
agagccaact gtgagtttgt caggggccac 7680 aagggggaca ggctggtctc
acagctcaaa tggttcctgg aggatgagaa gagagccatt 7740 ggtgagcaga
caccatccgc tgggggtggg gagcagctgg gagggctcat cagatgatat 7800
tctccaatga gaatcagaac tttgggtttt ctccccaggc gtctttccca ccatccattc
7860 tgcccatctc actgcctacg tagaggctcg aacctgtccc catagccatc
cttgacccag 7920 cttttcccgc gctgcacaca tactattgac aggtgtgttt
cgtggttttt tgttttttgt 7980 ttgtttgttt gttttgagtt ggaggtttgc
tcttgctgcc caggctggag tacaatggcg 8040 caatctcagc tcaccgcaat
ctctgcctcc tgggttcaag caattctctt gcctcagcct 8100 cctgagtagc
tgggattaca ggcatgcgcc accacaccca gctaattttg tatttttagt 8160
agacgtgggg tttctccatg ttggtcaggc tggtctcgaa ctcctgacct caggtgatcc
8220 gcttgcctta gcctccgaaa gtgctgggat tacaggcatg agccactgcg
ttaggcccac 8280 tgacaagcct tgtattggct agccaccaag attgacttga
ttatccacct tcgggacaac 8340 tggacagcct gcttatgact tacgccatag
tctgtctcta ctagctctcc tgccctgact 8400 tgacccagca tacaacagcc
agagccagcc ttttcaatat aaacctgatc ttgctggcac 8460 tgcttaaacc
ctgcaggggc ctcgcactgc tccatggccc agcctgtcta cccttacctt 8520
ctgcccaggc tctgctcatc cattctctgc ctcccacaca cctgccctct gtgggctcca
8580 gccataccat ctctcaactc ataagccagt tttttcatac aggctccctc
catctggact 8640 ggcttccctg cgtgcagttc actcctgctc tacctttggc
tctgcctcca cccatcctca 8700 gccgtctcca gcattacctc cttggagaat
cctgccttga cttcccagcc acccaaatat 8760 cactacttgg tctgcattct
cgttgcaatt gcagtcgcat gagcaattgc tgtggttgag 8820 gcccgaactg
cgcaagtgcc tgtctgccat gggtctcctg cttcctctaa gcacagtgcc 8880
tgacacacag tgagacctca gcacgtatgg gctgaggcaa tgaaggaatg aaggatccca
8940 tgacccaaaa gagcctgttg gaaagtgcag gccagggtcc caggtgctgg
cggggctggc 9000 tgctgggtgg gggcagagag gcaacccctc tgtttttttc
cctctcaggg catgatcaca 9060 agggccaccc ccacactagc tactaccagt
atggcctggg cattctggcc ctgtgtctcc 9120 accagaagcg ggtccatgac
agcgtggtgg acaaacttct gtatgctgtg gaacctttcc 9180 accagggcca
ccattctgtg ggtgagtagg tcagaccgtg ccaaggccag gctggcactc 9240
cctcagtccc caggtctgca ctgatgacgt ccataccctg gcccccacac tcacctttcc
9300 ttggggctcc tccgaatcaa gtcctttagg gacgaattgg cgagggctca
tgggtgatgc 9360 tccagctgtg agccagcttt ggagctggta ggtggatctc
ttgaggccag gagttcaaga 9420 caacgtggtg aaaccccatc tctactaaaa
ataaaaaagt tagccgggca tggtggcaca 9480 tgcctgtagt cccagctact
cgggaggctg aggcaggaga atcacttgaa cctgggaggc 9540 ggaggctgca
gtgagtggag atcgcaccac tgccctccag cctgggcaac agagtgagtg 9600
agactctgtc tcaaaaaata aaaaataaaa taaaactccc ctagtgattc caatgtgcag
9660 ctaagtttgg aaataggtgg tatggggtca agtcctcttg ggcctccctc
ctccagtcct 9720 tctccctaac ctctagccct caagttgcag agtgatcagc
caaaccagtt tgcccagaaa 9780 tgagcagttt cctgggacac aggattttca
gagtccagac aaggaaagtc ttgggcagac 9840 caggttgagt tggtgccctt
agctgatctg accatgttgc ccttcttctc caagccctcc 9900 tgtggttgtc
catagctaca agggcctgac cctcaagccc ctgcctgtcc tggccccttt 9960
ggctctccag ctcattgcat gttctgtccc ccacttcaag acacagcagc catggcaggc
10020 ttggcattca cctgtctgaa gcgctcaaac ttcaaccctg gtcggagaca
acggatcacc 10080 atggccatca gaacagtgcg agaggagatc ttgaaggccc
agacccccga gggccacttt 10140 gggaatgtct acagcacccc attggcatta
caggtgggaa agagaccctg gagccatggc 10200 caccctgggg aacagtcggg
tggagtggtc aggtgctgga acacctagcc cctccctgcc 10260 ggctgacctc
ctctctctct tcctcactct atcaccagtt cctcatgact tcccccatgc 10320
ctggggcaga actgggaaca gcatgtctca aggcgagggt tgctttgctg gccagtctgc
10380 aggatggagc cttccagaat gctctcatga tttcccagct gctgcccgtt
ctgaaccaca 10440 agacctacat tgatctgatc ttcccagact gtctggcacc
acgaggtagc ccaacttttt 10500 gtggaagcac agccctttac aatctgctgc
gcacccattg acgtcccagt gaggggaggt 10560 tgcttcatcc tgatttgctg
agtcagcaca agtttgtggg tgtgcatggg acacagtagc 10620 caaaatgtgg
tcatagcttc tagaagctca cagtgtgggg aggaagacag taaatggaga 10680
tccctgggca tatcgcttgt gtgataccca gtacagaaat gtttggatgg atggatggat
10740 ggatggatgg atggatggat ggatggatgg atgaggagag acacattttg
gttaactcta 10800 atacaacatg ataagcccca gtagcagcat gatccaggct
ttctctgaga gagggtctga 10860 ggacgtgact gggatttgcc aattaagaat
ggagaaagag gccaggtgca gtgactcatg 10920 cctgtaatcc caacactttg
ggaggccgag gcgggtggct cacctgaggt caggagttcg 10980 agaccagcct
ggctaacatg gcgaaactcc atctattaaa aatacaaaaa agtagctggg 11040
tgtggtggcg agtgcctgta accccagcta agctactcag gaggctgagg caagagaatc
11100 acttgaacct cagaggtgga ggttgcagtg agccaagatc atgccactgc
actccagtct 11160 gggtgacaga gtaagactat gtctcaaaaa aaaaaaaaaa
aaatggagaa gaaggaagct 11220 ggacatggtg gctcgtgctt ataatcctag
cactctggga agctgaggca gatggattgc 11280 ctgagcccag gagtttgaga
ccagcctggg caacatggtg aaaccctgtc tttactaaaa 11340 tacgaaagat
tagccaggca tggtggtaga cacctataat cccagctact agggaggctg 11400
agccacaaga atcacttgaa cctgggagac agaggttgca gtgagccgag atcgcgccat
11460 tgcactccag cctgggcgac agtgtgagac tctgtctcca gaaaaaacaa
gaatggatag 11520 agtggagcca agaagaggca ggaagaacaa agacacagag
gtgcacagag tttgggggaa 11580 ttttgaggaa tggtcttgca aaagagtggg
atctgggaga atgagtggga gtggaaagca 11640 gatgaatgaa gagaaggtga
gcgcatcagg gtaacagaga tgcgttgtga acaaatgcat 11700 gttctaggaa
gagccctctg gagtgctagg tgccagagag gtgggaggaa ggatactgga 11760
agcagagaaa ccagtgaggg gcctgatctt gggtggtggg gaatgaggga caggggaggc
11820 cgggatggaa gccaggtggt ggggaatgag ggacagggga ggccgggatg
gaagccaggt 11880 ttcagctgag caggtggcgg tggcattgat ggagatgagg
acatggggaa ggacaaagtc 11940 caggtgtcct tgagggaaga caagaagaca
aataatccag gctctctgtc ctcacaccag 12000 ctgcccgccc ctttcttcct
ggcacagtca tgttggaacc agctgctgag accattcctc 12060 agacccaaga
gatcatcagt gtcacgctgc aggtgcttag tctcttgccg ccgtacagac 12120
agtccatctc tgttctggcc gggtccaccg tggaagatgt cctgaagaag gcccatgagt
12180 taggaggatt cacgtgagac tcccacctcc cagtcctcac cccacccaac
ctcacatgcc 12240 tgataacagg gtcacagaaa agacggggaa cagaggagag
ggttccctcg ggagagacac 12300 tggccctgct tctgcttcta cctgctcagc
tcctttcttg cccacggtgt tatggaaaca 12360 gggagccata ggccagcatt
gtcactgaga gagcaggctt tggaggcaga gccccccagt 12420 tggaatccca
actctaacca gctaggttcc aggtaggcac ccacaattca ccgaggagaa 12480
cagttgtgcc ccttccctgc agggccagtg tgaagagtcc aggagttagt acacatagag
12540 atagtggcat gtgcttttta tatgtgcaag gtccagcaca tagcaagcgc
tcaacacagc 12600 gttgctttca tcagagtaag aactgttttt tgtttgtttg
tttgtttgtt tttaagagac 12660 agggtctcaa tcttatcacc caggctggag
tgtaattgtg caatcacgtc tcactgcagt 12720 ctcgaactct ggggatgaag
caaccctact gtcctgcctc agcctcccaa atagctgaga 12780 ctataggcac
gtgccacaca accctgggta attttttttt tttttttttt gagatagggt 12840
ctctgtctgt tgcccaggct ggtctcaaat tcctggcctc aaaccatcct cacacctgag
12900 gcgctcaaaa tattgggatt ataggtgcga gccatcatgc tcagccagaa
taataactgg 12960 ttttttttgt tttttttttg agacagagtc tcactctatt
acccaggctc tggaggccca 13020 actcgtgttt gtgtatttgt ttatttttat
ttatttattt atttcgagac agagcctctc 13080 tctttcacct aggctggagt
gcagtggcgc aatctcggct cactgcaacc tccgtctcct 13140 gggttcaagt
gattgtcctg cctcagcctc ctgagtagct ggtgctacag gcgcgtgcca 13200
ccatgcccag ctaatttttg tatttttagt agagacaggg ttttactatg ttggccagct
13260 ggtttctaac tcctgaactc gggtgatctg cctgcctcgg cctcccaaag
tgctgggatt 13320 acaggcatgg gcctccgtgc ccggccatgt atttatttag
gcaaggtctc tctctgttat 13380 ccaggctgaa gtgcagtggc acattcatag
ctcactgcag cctcaaatta tccaagtaac 13440 agggactaca ggcatgcacc
accacaccca tctacttttt tttgagatgg agtctccctc 13500 tgtcgcccag
actgggttgc agtggcacaa tttcagctca tggcagcatc tacctcccag 13560
gttcaagcga ttctccttcc tcagtctccc gagtagctgg gactatgggc atgcaccacc
13620 atacctggct aatgtttata ttttgagtag agatggaatt ttgccatttt
ggccaggctg 13680 gtcttgagct cttgacctca agtgatatgt ctgcctcagn
nnnnnnnnnn nnnnnnnnnn 13740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13800 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13860 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13920
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
13980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 14040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 14100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14160 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14220 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14280
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
14340 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 14400 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 14460 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14520 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14580 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14640
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
14700 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 14760 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 14820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14880 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14940 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15000
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
15060 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 15120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 15180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15240 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15300 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15360
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
15420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 15480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 15540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15600 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15660 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15720
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
15780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 15840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 15900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15960 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16020 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16080
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
16140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 16200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 16260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16320 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16380 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16440
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
16500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 16560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 16620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16680 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16740 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16800
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
16860 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 16920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 16980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17040 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17100 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17160
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
17220 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 17280 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 17340 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17400 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17460 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17520
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
17580 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 17640 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 17700 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17760 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17820 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17880
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
17940 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 18000 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 18060 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18120 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18180 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18240
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
18300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 18360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 18420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18480 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18540 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18600
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
18660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 18720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 18780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18840 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18900 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18960
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
19020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 19080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 19140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19200 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19260 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19320
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
19380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 19440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 19500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19560 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19620 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19680
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
19740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 19800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 19860 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19920 nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19980 nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20040
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
20100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 20160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnccaaatc
aaccagttgc ataaatcact 20220 cctctatctt ccttggggtg gaaagtggat
gggagttata atttgagttc tcttttgtct 20280 tagtccattg aagctgctat
tacaaaatac cataaactgg gtggcttata aacagcagaa 20340 atgaggccgg
gtgcggtggc tcatgcctat aattccagca ctttgggagg ccaaggcagg 20400
tggatcacct gagatcagta gttcaagact agcctgacca acatggtgaa accctgtctc
20460 tactaaaaat acaaaaaatt agctgggggt ggtggcgggc acctgtaatc
ccagctactc 20520 aggaggctga ggcaggagaa tcgcttgaac ccaggaggcg
gaggttgccg tgagctgaga 20580 tcacgccatt gcatttcagc ctgggcacaa
agagtgaaac tccatctcaa aatgaaataa 20640 aataacagaa atgtatttct
taacagttct ggaggttggg tgggcagtcc cagatcagga 20700 cactgacaga
ttcagtgtct gatgggggcc cactttctgg tgttacctgc tggctgtgtt 20760
ctcacatggt ggaaggaaca tggcaacttt ctggggcctt gttttttaat ttaaaaaaaa
20820 aaaatatttt cctggccctt gcctgctgaa ggaacctctt ttataatggt
acttaaaaat 20880 tttttttttt gagatggggg tctcactctg tcacccacgc
tgagtgcagt atcacaatct 20940 cagctcactg caacctctgc ctccctggct
taagcgatcc tcccacctca gcctcctgag 21000 tacgtgtgac cataggccca
tggcacaaag cccagctaat tttttgtatt tttagtagaa 21060 atgtggtttc
accatgttgc ataggctggt ctcgaacttc tgaactcaag tgatctgcct 21120
gccttggcct cccaaagtgc tgggattcta ggtatgagcc accctgctcg gcctataatg
21180 gcactttcct atcccattga tgaggctcta ctctcatgac ctaatcatct
cccaaaggcc 21240 ctaaggcctc ctgataccat cacctttggg gttaggtttt
aacatataca ttttgggggg 21300 acacagacat tttagaccat agcacctcca
ttgaaaggaa acatttctga cacctggcta 21360 tctcaaaggg ccctttcagt
tcccctgcag gctgcattcc cacatcacca acaagagcag 21420 cgacactcac
tcagaggtta aataacttgt ccagagtcac agcagtaatg aatgacagag 21480
ctggggcttg aatccaggcg tcctcctaga gcctggattc tgtgtagtga
gtgaaagctg 21540 actcctggga gacttctgcg tggtcctggt tctctctcca
gactgcactg cgcaagtttc 21600 tcttcctgat ggtccctagg gtattacaaa
gacagtggcc ctgcctgtca ggtgttttta 21660 ttaccagatg aggtcatggc
ctcaggaacc ctgtaggaag ctgagttcag agtctttgag 21720 caggctttag
ggaggttcca gcttcccacc accaagcccc aggtggattc ttacagactc 21780
tagcctcagg gtggggggtc tggaagatga ggttgcgggg tgcgatattc tgcccaattc
21840 gcccctcctt gctcaatctg tttctgcagg tattgctgac tacagaccca
aggatggaga 21900 aaccattgag ctgaggctgg ttagctggta gcccctgagc
tccctcatcc cagcagcctc 21960 gcacactccc taggcttcta ccctccctcc
tgatgtccct ggaacaggaa ctcgcctgac 22020 cctgctgcca cctcctgtgc
actttgagca atgccccctg ggatcacccc agccacaagc 22080 ccttcgaggg
ccctatacca tggcccacct tggagcagag agccaagcat cttccctggg 22140
aagtctttct ggccaagtct ggccagcctg gccctgcagg tctcccatga aggccacccc
22200 atggtctgat gggcatgaag catctcagac tccttggcaa aaaacggagt
ccgcaggccg 22260 caggtgttgt gaagaccact cgttctgtgg ttggggtcct
gcaagaaggc ctcctcagcc 22320 cgggggctat ggccctgacc ccagctctcc
actctgctgt tagagtggca gctccgagct 22380 ggttgtggca cagtagctgg
ggagacctca gcagggctgc tcagtgcctg cctctgacaa 22440 aattaaagca
ttgatggcct gtggacctgc tacagtggcc tggtgcctca tactcctcag 22500
gtgcaggggc agggacaaga gaagggggaa gtaaccccat cagggaggag tggagggtgc
22560 ctgagccgcc atgtgggcat tgggggagtg atgggaatgc cagcagtgat
gacgttgact 22620 actgactgag cacccactac tatgactgag cactcactcg
ctagatacta tcttgaactg 22680 ctctgtgagg ttgttgatat tttcattttt
atctgtgctt tacaaatcag gaaactggga 22740 ggccgggcgt ggtggctcac
gcctgtaatc ccagcacttt aggaggccaa ggcaggtgga 22800 tcacaaggtc
aggagtttga gatcagcctg gccaacatgg tgaaactcca tctttactaa 22860
aaatacaaaa aattagccag gcatggtgtt gcatgcctgc atgcctgtaa tcccagttac
22920 ttgggaagct gaggcaggag aattgcttga accctggagg cggaggttgt
agtgagccga 22980 gatcacgcca ttgcactcca gcttgggcaa gaagagaaac
actctcaaaa aaaaaaaaaa 23040 atcaggaaac tggtgctcaa aaaggaaaag
tgactcacca aggtcacaga ctaggcagtg 23100 atgctggggg aacctggctc
aggggacaca gacctggcct ggggcagcct tgcagctcct 23160 ccactaaaat
actgaaaatg aggggcttcg atgatggtta taatcgtatg gcagagcccc 23220
aactcaactg gagccctggg acccagaagc tagggtctca ctccctgctt ttccacaagg
23280 caccattagg gcatcacccc aggcctcggc agccacgacg cagggatcct
gcctctcatt 23340 ggttgggggc ttaggggctc tgggctgccc tcttgaagag
ggggttcagc ccagcgaggc 23400 accccctatg ctgcacccca ccaaggttag
gaagaggtcc tgtcctcagt ggggccctct 23460 gatgaacagc ccatcaggtc
tgcgtccaca tgccttggaa gagatggtga catactcaaa 23520 gtccttgaag
ccgcatatta aaccacctag agcaccatct tcaaacattt agggtctgag 23580
aagatagggg aagtaagcaa tttaaaacat ttctttatat tgggccaggt gcaatggctc
23640 acgtctgtaa tcccagcgct ttgggaggac gaggatcacc tgaggtcagg
agttcaagat 23700 cagcctggcc aacatggaga aaccccatct ctactaaaaa
tacaaaaatt agctcaggcg 23760 tggtgatgtg cacctgtaat cctagctatt
caggaggctg aggcacaaga attgcttgag 23820 tcaatattgc accactgcac
tccagcctgg gcaacagcga gactcttgtc tcaaaaaaaa 23880 aaaaagatat
ttgctgaaaa gacccagcct gccaaactca ggggcagcca agggaggtag 23940
tgaaatggaa gttggagctc agcgctccca cacctccact gccctcaggc cttctctgcc
24000 tctttcccat cagtcagctg cttctgggca tggtcctggc agagacttgg
cctccttcca 24060 gttcaagctc cctcttagat tgtgtcccac gccactgagt
ctttgggaca ctgggtcaga 24120 tgtctagtct ggcacaattg gcaggaatcc
caagaaacag tgtgagtgag gggacagtcg 24180 tgttgagtgc cctccatctg
ggactgggag gcaggtctat gtcaggcctg catttagatc 24240 tctaatggct
ccagacaagc cccttcagct cactaagcct gtttcctaac acagctgtgg 24300
gatggtgctt tggtttacat agcacgcgat accatcatag atcacatggg gaaactgagg
24360 ccccaggagt gatctgctgg cacatgcagt gacaagagga gaggcccatc
tcagccttgc 24420 agcaaggttg ccagaaatcg attctcgccc ccatcccgta
aagatagctg ggattacagg 24480 tgtgcaccac catgcccagc ctaatttttg
tattattagt agagatgggg tttcaccatg 24540 ttgtccaggc tggtcatgaa
ctcctgacct caagtgatcc acccgctttg gcctcccaaa 24600 gtgctgggat
tacaagcatg agccacagtg cctggcctga ccctgctctt ttgaaagacc 24660
attcccccaa attctgtgca cctgtgtgcc tttcttctct ctgcctcctc tcagctctgc
24720 cccgctctcc tcccttctcc tctggcaaat cccactcatc tcttgaagcc
cttcttccag 24780 gggaagccct gatcatgctg ctttctcctg tgggagggat
gaaggacgtg gcccacggag 24840 tttgttttgt tttgttttga gatggagttt
tgctcatgtt gcccaggctg gggtacaatg 24900 gtacgatctc agctcactgc
aacctctacg tcccgggttc aagcggttct cctgccttag 24960 cctccccagt
agctgggatt actggcatga accaccacac ctggctaatt ttgtgttttt 25020
agtagagatg gggtttcttc atgttggtca ggctggtctc gaactcccaa cctcaggtga
25080 tctgcctgcc tcggcctccc aaagtactgg gattacaggg ttgagccact
gtgcctggcc 25140 caggcccacg gagttttaag aggcttcctg tggcagtggc
atccagacgg agtgcagaaa 25200 ctcaaagttg aaggccagaa gctcagggaa
gggggagtgt gagttgagga gtctcttggc 25260 tgccagggcc agaaaccgaa
ctccaagcct ctccacaaca gcgggtgtag agcatgtaga 25320 atcagagagg
aggctgagcc atgcagcccc gagaagaggg gaatgccact gagccacaga 25380
gacccagtgc cactgccagg tgtctctgcc tccacttccc atgacccggc ctgtctctgt
25440 atgcaggctt caccctctct cgttgtacat tgtacacatt ctaggtgaca
ccagcagctt 25500 ctgattctca tctcccataa catcagcccc ccagagaggg
gacaactgct gagctgataa 25560 cataatagat gcccctttcc tggaggccat
ggtcatggtc agcgtggaga ggatgaagcc 25620 tgagcaggca ggatcggggg
tctagagggg aaggaggtgg aagttgagat cacagacctg 25680 tggtcaggtg
gcctgggaag ggtttgacga gtgtcggccc aaagagcttg gaagggattt 25740
tgctgctgtg ggtgagcact gcctctcccc ttagggacaa cagccacctc ttctctcccc
25800 atttgccttt cccttctgta gatatgaaac acaggcctcc ttgtcaggcc
cctacttaac 25860 ctccgtgatg gggaaagcgg ccggagaaag ggagttctgg
cagcttctcc gagaccccaa 25920 caccccactg ttgcaaggtg agtcatggcc
tgacactctg gatgtgtccc ctaccccaag 25980 cttactcagc caagaggctt
catcaactca ccccagcttt ccctagcacc ctcctgggcc 26040 acaccttcac
aaaatcactg atgctcaaag ttggatataa tatattgaac tgaagcctta 26100
gcatttttat gcaagttact gtggaaattc taggaaacca gacagattac aaaaaaaaaa
26160 aaaaactaga agaaaattaa catcacctag gatatactac ctaggaataa
cgtcttttat 26220 tttgagatgg agtttcgctc ttgttgccca ggctggagtg
cagcggtatg atctcggctc 26280 gctgcaacct ccgcctcctg ggttcatgtg
attcttccac ctcggccttc ctagagccca 26340 agtggtctgc ctgcctctgc
ctcccaaagt tctgggatta caggcatgag ccaccgcacc 26400 cagccaaaat
tacttaactt ttcttctaga tactttttaa aaatatggca gtaagttttt 26460
cataaaaaat ggagccatgc tatccagtgg aaatttaatg ttgcccacat gtataactta
26520 aaaatttcat atatgtgtat acatatatat gaaatatata tatacagaca
cacatatata 26580 tgtatacata tatatacaca tatatatgta tacatatata
cacacatata tgtatacata 26640 tatatacaca catatacaca tatatacaca
cacatacata tatacacaca catatataca 26700 cacatatata cacacatgca
cacatatata tgtatacata tatacacaca tgtatacgta 26760 tatatacaca
catatataca cacatatata tacacacata tacacacata cacacacata 26820
tatacacaca tatatacaca catatataca cacatatata tgtatacata tatatacaca
26880 catatataca catacacaca tacatatata cacatataca catatacaca
cacatataca 26940 cacatgtata catatatata cacacatgta tacatatgta
tacacacaca tatatgtata 27000 catatataca cacatacata tgtgtacata
tatacacaca tacatatgta tacatatata 27060 cacacat 27067 6 427 PRT Homo
sapiens 6 Met Arg His Leu Gly Ala Phe Leu Phe Leu Leu Gly Val Leu
Gly Ala 1 5 10 15 Leu Thr Glu Met Cys Glu Ile Pro Glu Met Asp Ser
His Leu Val Glu 20 25 30 Lys Leu Gly Gln His Leu Leu Pro Trp Met
Asp Arg Leu Ser Leu Glu 35 40 45 His Leu Asn Pro Ser Ile Tyr Val
Gly Leu Arg Leu Ser Ser Leu Gln 50 55 60 Ala Gly Thr Lys Glu Asp
Leu Tyr Leu His Ser Leu Lys Leu Gly Tyr 65 70 75 80 Gln Gln Cys Leu
Leu Gly Ser Ala Phe Ser Glu Asp Asp Gly Asp Cys 85 90 95 Gln Gly
Lys Pro Ser Met Gly Gln Leu Ala Leu Tyr Leu Leu Ala Leu 100 105 110
Arg Ala Asn Cys Glu Phe Val Arg Gly His Lys Gly Asp Arg Leu Val 115
120 125 Ser Gln Leu Lys Trp Phe Leu Glu Asp Glu Lys Arg Ala Ile Gly
His 130 135 140 Asp His Lys Gly His Pro His Thr Ser Tyr Tyr Gln Tyr
Gly Leu Gly 145 150 155 160 Ile Leu Ala Leu Cys Leu His Gln Lys Arg
Val His Asp Ser Val Val 165 170 175 Asp Lys Leu Leu Tyr Ala Val Glu
Pro Phe His Gln Gly His His Ser 180 185 190 Val Asp Thr Ala Ala Met
Ala Gly Leu Ala Phe Thr Cys Leu Lys Arg 195 200 205 Ser Asn Phe Asn
Pro Gly Arg Arg Gln Arg Ile Thr Met Ala Ile Arg 210 215 220 Thr Val
Arg Glu Glu Ile Leu Lys Ala Gln Thr Pro Glu Gly His Phe 225 230 235
240 Gly Asn Val Tyr Ser Thr Pro Leu Ala Leu Gln Phe Leu Met Thr Ser
245 250 255 Pro Met Arg Gly Ala Glu Leu Gly Thr Ala Cys Leu Lys Ala
Arg Val 260 265 270 Ala Leu Leu Ala Ser Leu Gln Asp Gly Ala Phe Gln
Asn Ala Leu Met 275 280 285 Ile Ser Gln Leu Leu Pro Val Leu Asn His
Lys Thr Tyr Ile Asp Leu 290 295 300 Ile Phe Pro Asp Cys Leu Ala Pro
Arg Val Met Leu Glu Pro Ala Ala 305 310 315 320 Glu Thr Ile Pro Gln
Thr Gln Glu Ile Ile Ser Val Thr Leu Gln Val 325 330 335 Leu Ser Leu
Leu Pro Pro Tyr Arg Gln Ser Ile Ser Val Leu Ala Gly 340 345 350 Ser
Thr Val Glu Asp Val Leu Lys Lys Ala His Glu Leu Gly Gly Phe 355 360
365 Thr Tyr Glu Thr Gln Ala Ser Leu Ser Gly Pro Tyr Leu Thr Ser Val
370 375 380 Met Gly Lys Ala Ala Gly Glu Arg Glu Phe Trp Gln Leu Leu
Arg Asp 385 390 395 400 Pro Asn Thr Pro Leu Leu Gln Gly Ile Ala Asp
Tyr Arg Pro Lys Asp 405 410 415 Gly Glu Thr Ile Glu Leu Arg Leu Val
Ser Trp 420 425 7 427 PRT Homo sapiens 7 Met Arg His Leu Gly Ala
Phe Leu Phe Leu Leu Gly Val Leu Gly Ala 1 5 10 15 Leu Thr Glu Met
Cys Glu Ile Pro Glu Met Asp Ser His Leu Val Glu 20 25 30 Lys Leu
Gly Gln His Leu Leu Pro Trp Met Asp Arg Leu Ser Leu Glu 35 40 45
His Leu Asn Pro Ser Ile Tyr Val Gly Leu Arg Leu Ser Ser Leu Gln 50
55 60 Ala Gly Thr Lys Glu Asp Leu Tyr Leu His Ser Leu Lys Leu Gly
Tyr 65 70 75 80 Gln Gln Cys Leu Leu Gly Ser Ala Phe Ser Glu Asp Asp
Gly Asp Cys 85 90 95 Gln Gly Lys Pro Ser Met Gly Gln Leu Ala Leu
Tyr Leu Leu Ala Leu 100 105 110 Arg Ala Asn Cys Glu Phe Val Arg Gly
His Lys Gly Asp Arg Leu Val 115 120 125 Ser Gln Leu Lys Trp Phe Leu
Glu Asp Glu Lys Arg Ala Ile Gly His 130 135 140 Asp His Lys Gly His
Pro His Thr Ser Tyr Tyr Gln Tyr Gly Leu Gly 145 150 155 160 Ile Leu
Ala Leu Cys Leu His Gln Lys Arg Val His Asp Ser Val Val 165 170 175
Asp Lys Leu Leu Tyr Ala Val Glu Pro Phe His Gln Gly His His Ser 180
185 190 Val Asp Thr Ala Ala Met Ala Gly Leu Ala Phe Thr Cys Leu Lys
Arg 195 200 205 Ser Asn Phe Asn Pro Gly Arg Arg Gln Arg Ile Thr Met
Ala Ile Arg 210 215 220 Thr Val Arg Glu Glu Ile Leu Lys Ala Gln Thr
Pro Glu Gly His Phe 225 230 235 240 Gly Asn Val Tyr Ser Thr Pro Leu
Ala Leu Gln Phe Leu Met Thr Ser 245 250 255 Pro Met Arg Gly Ala Glu
Leu Gly Thr Ala Cys Leu Lys Ala Arg Val 260 265 270 Ala Leu Leu Ala
Ser Leu Gln Asp Gly Ala Phe Gln Asn Ala Leu Met 275 280 285 Ile Ser
Gln Leu Leu Pro Val Leu Asn His Lys Thr Tyr Ile Asp Leu 290 295 300
Ile Phe Pro Asp Cys Leu Ala Pro Arg Val Met Leu Glu Pro Ala Ala 305
310 315 320 Glu Thr Ile Pro Gln Thr Gln Glu Ile Ile Ser Val Thr Leu
Gln Val 325 330 335 Leu Ser Leu Leu Pro Pro Tyr Arg Gln Ser Ile Ser
Val Leu Ala Gly 340 345 350 Ser Thr Val Glu Asp Val Leu Lys Lys Ala
His Glu Leu Gly Gly Phe 355 360 365 Thr Tyr Glu Thr Gln Ala Ser Leu
Ser Gly Pro Tyr Leu Thr Ser Val 370 375 380 Met Gly Lys Ala Ala Gly
Glu Arg Glu Phe Trp Gln Leu Leu Arg Asp 385 390 395 400 Pro Asn Thr
Pro Leu Leu Gln Gly Ile Ala Asp Tyr Arg Pro Lys Asp 405 410 415 Gly
Glu Thr Ile Glu Leu Arg Leu Val Ser Trp 420 425
* * * * *
References