U.S. patent application number 10/172069 was filed with the patent office on 2003-09-04 for ndr2-related proteins.
This patent application is currently assigned to Incyte Genomics, Inc.. Invention is credited to Au-Young, Janice, Hillman, Jennifer L., Shah, Purvi, Stuart, Susan G., Yue, Henry.
Application Number | 20030167480 10/172069 |
Document ID | / |
Family ID | 27807568 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030167480 |
Kind Code |
A1 |
Stuart, Susan G. ; et
al. |
September 4, 2003 |
NDR2-related proteins
Abstract
The invention provides cDNA which encode Ndr2-related proteins.
It also provides for the use of the cDNAs, fragments, complements,
and variants thereof and of the encoded proteins, portions thereof
and antibodies thereto for diagnosis and treatment of cancer,
particularly cancers of the intestine, breast, uterus, liver,
brain, and kidney. The invention additionally provides expression
vectors and host cells for the production of the proteins and a
transgenic model system.
Inventors: |
Stuart, Susan G.; (Montara,
CA) ; Au-Young, Janice; (Brisbane, CA) ;
Hillman, Jennifer L.; (Fremont, CA) ; Shah,
Purvi; (San Jose, CA) ; Yue, Henry;
(Sunnyvale, CA) |
Correspondence
Address: |
INCYTE CORPORATION (formerly known as Incyte
Genomics, Inc.)
3160 PORTER DRIVE
PALO ALTO
CA
94304
US
|
Assignee: |
Incyte Genomics, Inc.
3160 Porter Drive
Palo Alto
CA
94304
|
Family ID: |
27807568 |
Appl. No.: |
10/172069 |
Filed: |
June 13, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10172069 |
Jun 13, 2002 |
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09812484 |
Mar 19, 2001 |
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6444430 |
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09812484 |
Mar 19, 2001 |
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09232160 |
Jan 15, 1999 |
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6368794 |
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Current U.S.
Class: |
800/8 ; 435/226;
435/320.1; 435/325; 435/6.14; 435/69.1; 435/7.23; 435/70.21;
530/388.26 |
Current CPC
Class: |
C12Q 2600/158 20130101;
C12N 2799/026 20130101; C12Q 1/6886 20130101; A61K 38/00 20130101;
C07K 14/47 20130101 |
Class at
Publication: |
800/8 ; 435/226;
435/69.1; 435/325; 435/320.1; 435/70.21; 530/388.26; 435/6;
435/7.23 |
International
Class: |
A01K 067/00; C12Q
001/68; C12P 021/04; C12N 009/64; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. An isolated cDNA comprising a nucleic acid sequence encoding a
protein selected from the amino acid sequence of SEQ ID NO:1 and
SEQ ID NO:2, or the complement thereof.
2. A purified protein or a portion thereof selected from: a) an
amino acid sequence of SEQ ID NO:1 and SEQ ID NO:2; b) an antigenic
epitope of SEQ ID NO:1 or SEQ ID NO:2; and c) a biologically active
portion of SEQ ID NO:1 or SEQ ID NO:2.
3. A composition comprising the protein of claim 2 and a
pharmaceutical carrier.
4. A method for using a protein to screen a plurality of molecules
or compounds to identify at least one ligand, the method
comprising: a) combining the protein of claim 2 with the molecules
or compounds under conditions to allow specific binding; and b)
detecting specific binding, thereby identifying a ligand which
specifically binds the protein.
5. The method of claim 4 wherein the molecules or compounds are
selected from DNA molecules, RNA molecules, peptide nucleic acids,
peptides, proteins, mimetics, agonists, antagonists, antibodies,
immunoglobulins, inhibitors, and drugs.
6. An isolated antibody which specifically binds to a protein of
claim 2.
7. The antibody of claim 6, wherein the antibody is selected from
an intact immunoglobulin molecule, a polyclonal antibody, a
monoclonal antibody, a chimeric antibody, a recombinant antibody, a
humanized antibody, a single chain antibody, a Fab fragment, and a
F(ab').sub.2 fragment.
8. A method of using a protein to prepare and purify a polyclonal
antibody comprising: a) immunizing a animal with the protein of
claim 2 under conditions to elicit an antibody response; b)
isolating animal antibodies; c) attaching the protein to a
substrate; d) contacting the substrate with isolated antibodies
under conditions to allow specific binding to the protein; e)
dissociating the antibodies from the protein, thereby obtaining
purified polyclonal antibodies.
9. A polyclonal antibody produced by the method of claim 8.
10. A method of using a protein to prepare a monoclonal antibody
comprising: a) immunizing a animal with a protein of claim 2 under
conditions to elicit an antibody response; b) isolating
antibody-producing cells from the animal; c) fusing the
antibody-producing cells with immortalized cells in culture to form
monoclonal antibody producing hybridoma cells; d) culturing the
hybridoma cells; and e) isolating monoclonal antibodies from
culture.
11. A monoclonal antibody produced by the method of claim 10.
12. A method for using an antibody to diagnose conditions or
diseases associated with expression of a protein, the method
comprising: a) combining the antibody of claim 6 with a sample,
thereby forming antibody:protein complexes; and b) comparing
complex formation with a standard, wherein the comparison indicates
expression of the protein in the sample.
13. The method of claim 12 wherein expression is diagnostic of a
cancer, particularly intestine cancer, breast cancer, uterine
cancer, liver cancer, brain cancer, and kidney cancer.
14. A composition comprising an antibody of claim 6 and a labeling
moiety.
15. A composition comprising an antibody of claim 6 and a
pharmaceutical agent.
16. A method for using an antibody to immunopurify a protein
comprising: a) attaching the antibody of claim 6 to a substrate, b)
exposing the antibody to a sample containing protein under
conditions to allow antibody:protein complexes to form, c)
dissociating the protein from the complex, and d) collecting the
purified protein.
17. A method of using an antibody to treat a cancer, particularly a
cancer of the intestine, breast, uterus, liver, brain, or kidney
comprising administering to a patient in need of such treatment the
composition of claim 15.
Description
[0001] This application is a divisional application of U.S.
application Ser. No. 09/812,484, filed Mar. 19, 2002, entitled
NDR2-RELATED PROTEINS, which is a continuation-in-part of U.S.
application Ser. No. 09/232,160, filed Jan. 15, 1999, now U.S. Pat.
No. 6,368,794, issued Apr. 9, 2002, entitled DETECTION OF ALTERED
EXPRESSION OF GENES REGULATING CELL PROLIFERATION, all of which
applications and patents are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to cDNAs which encode Ndr2-related
proteins and to the use of the cDNAs and the encoded proteins in
the diagnosis and treatment of cancer, particularly cancers of the
intestine, breast, uterus, liver, brain, and kidney. BACKGROUND OF
THE INVENTION
[0003] Phylogenetic relationships among organisms have been
demonstrated many times, and studies from a diversity of
prokaryotic and eukaryotic organisms suggest a more or less gradual
evolution of molecules, biochemical and physiological mechanisms,
and metabolic pathways. Despite different evolutionary pressures,
the proteins of nematode, fly, rat, and man have common chemical
and structural features and generally perform the same cellular
function. Comparisons of the nucleic acid and protein sequences
from organisms where structure and/or function are known accelerate
the investigation of human sequences and allow the development of
model systems for testing diagnostic and therapeutic agents for
human conditions, diseases, and disorders.
[0004] Reducing agents and tunicamycin-responsive protein (RTP;
also referred to as Drg1, Cap43, and rit43) may play roles in
atherosclerosis, tumorigenesis, differentiation, hypoxia, and
cellular responses to stress (Agarwala et al. (2000) Biochem
Biophys Res Commun 272:641-647). Human RTP is a 43 kDa cytoplasmic
protein of 394 amino acids in length and is expressed ubiquitously.
Although RTP is present predominantly in the cytoplasm of cells,
immunofluorescence studies show that a fraction of RTP is located
in the nucleus, an indication that RTP may be a signaling protein
that shuttles between the cytoplasm and nucleus. The expression
level of RTP increases in response to various chemical compounds,
including homocysteine, cysteine, mercaptoethanol, tunicamycin,
lysophosphatidylcholine, nickel compounds, okadaic acid, calcium
ionophore, DNA-damaging agents, 1,25-(OH).sub.2 vitamin D3,
synthetic retinoids, and phorbol myristate acetate. RTP expression
is decreased in tumor cell lines and in colon, breast and prostate
tumors (Kurdistani et al. (1998) Cancer Res 58:4439-4444; van
Belzen et al. (1997) Lab Invest 77:85-92). Overexpression of RTP
inhibits the growth of cancerous cells. RTP expression is
upregulated in human myelomonocytic cells by retinoids that inhibit
proliferation and promote differentiation (Piquemal et al. (1999)
Biochim Biophys Acta 1450:364-373) and in prostate adenocarcionma
cells by androgens that induce differentiation (Ulrix et al. (1999)
FEBS Lett 455:23-26). The activity of RTP may be regulated by
phosphorylation. RTP is phosphorylated in vivo by an unknown kinase
at multiple sites and can be phosphorylated by protein kinase A in
vitro (Agarwala et al., supra). RTP is dephosphorylated in cells
exposed to homocysteine. RTP expression levels change at different
stages of the cell cycle. RTP expression is highest at the G1 and
G2-M stages and lower at S phase (Kurdistani et al., supra). The
transcription factor p53, a known tumor suppressor, induces
expression of RTP. P53 causes the arrest of cell growth at G1 and
G2, and RTP may play a role in this growth-arrest pathway.
[0005] Mouse Ndr1 is a homolog of RTP that was identified in a
screen for genes regulated by N-myc. Ndr1 shows increased
expression in N-myc deficient mice (Okuda and Kondoh (1999) Mech
Dev 83:39-52). Myc family transcription factors control genes
expressed during embryogenesis, proliferation, differentiation,
apoptosis, and tumorigenesis (Grandori et al. (2000) Annu Rev Cell
Dev Biol 16:653-699). N-myc expression is high during cell
proliferation and decreased during cell differentiation.
Rearrangements and mutations of Myc proteins are found in many
tumors. N-Myc represses transcription of Ndr1. During mouse
embryogenesis, Ndr1 expression is correlated with a decrease in
N-myc expression and Ndr1 levels increase when cells begin to
differentiate. Ndr1 appears, therefore, to be a signaling molecule
involved in cellular differentiation.
[0006] Two Ndr1-related genes, Ndr2 and Ndr3, have recently been
discovered (Okuda and Kondoh (1999) Biochem and Biophys Res Commun
266:208-215). The amino acid sequences of Ndr2 and Ndr3 proteins
show 54% and 64% identity to Ndr1, respectively, and therefore are
grouped with Ndr1 as the Ndr family. All three Ndr genes are
expressed during embryogenesis, but at different times and show
differences in the regulation of their expression levels. Unlike
Ndr1, Ndr2 expression is not upregulated in N-myc deficient mouse
embryos. Ndr1 expression, in mouse embryos, increases after 13.5
dpc whereas Ndr2 expression increases earlier, at 11.5 dpc, and
Ndr3 expression is high at 9.5 dpc and shows little increase at
later developmental stages. RTP, Ndr1, Ndr2, and Ndr3 may represent
a gene family with differentiation-related functions.
[0007] The discovery of cDNAs encoding Ndr2-related proteins
satisfies a need in the art by providing compositions which are
useful in the diagnosis and treatment of cancer, particularly
cancers of the intestine, breast, uterus, liver, brain, and
kidney.
SUMMARY OF THE INVENTION
[0008] The invention is based on the discovery of cDNAs encoding
Ndr2-related proteins (NRP) which are useful in the diagnosis and
treatment of cancer, particularly cancers of the intestine, breast,
uterus, liver, brain, and kidney.
[0009] The invention provides an isolated cDNA comprising a nucleic
acid sequence encoding a protein selected from the group consisting
of the amino acid sequences of SEQ ID NO:1 (NRP1) and SEQ ID NO:2
(NRP2). The invention also provides an isolated cDNA or the
complement thereof selected from the group consisting of the
nucleic acid sequences of SEQ ID NO:3 and SEQ ID NO:11, a fragment
of SEQ ID NO:3 selected from SEQ ID NOs:4-10 or a fragment of SEQ
ID NO:11 selected from SEQ ID NOs:12-15, and a variant of SEQ ID
NO:3 or SEQ ID NO:11 selected from SEQ ID NOs:16-31. The invention
additionally provides a composition, a substrate, and a probe
comprising the cDNA, or the complement of the cDNA, encoding NRP.
The invention further provides a vector containing the cDNA, a host
cell containing the vector and a method for using the cDNA to make
NRP. The invention still further provides a transgenic cell line or
organism comprising the vector containing the cDNA encoding NRP.
The invention additionally provides a fragment, a variant, or the
complement of the cDNA selected from the group consisting of SEQ ID
NOs:2-31. In one aspect, the invention provides a substrate
containing at least one of these fragments or variants or the
complements thereof. In a second aspect, the invention provides a
probe comprising a cDNA or the complement thereof which can be used
in methods of detection, screening, and purification. In a further
aspect, the probe is a single-stranded complementary RNA or DNA
molecule.
[0010] The invention provides a method for using a cDNA to detect
the differential expression of a nucleic acid in a sample
comprising hybridizing a probe to the nucleic acids, thereby
forming hybridization complexes and comparing hybridization complex
formation with a standard, wherein the comparison indicates the
differential expression of the cDNA in the sample. In one aspect,
the method of detection further comprises amplifying the nucleic
acids of the sample prior to hybridization. In another aspect, the
method showing differential expression of the cDNA is used to
diagnose cancer, particularly cancers of the intestine, breast,
uterus, liver, brain, and kidney. In another aspect, the cDNA or a
fragment or a variant or the complements thereof may comprise an
element on an array.
[0011] The invention additionally provides a method for using a
cDNA or a fragment or a variant or the complements thereof to
screen a library or plurality of molecules or compounds to identify
at least one ligand which specifically binds the cDNA, the method
comprising combining the cDNA with the molecules or compounds under
conditions allowing specific binding, and detecting specific
binding to the cDNA, thereby identifying a ligand which
specifically binds the cDNA. In one aspect, the molecules or
compounds are selected from aptamers, DNA molecules, RNA molecules,
peptide nucleic acids, artificial chromosome constructions,
peptides, transcription factors, repressors, and regulatory
molecules.
[0012] The invention provides a purified protein or a portion
thereof selected from the group consisting of an amino acid
sequence of SEQ ID NO:1 or SEQ ID NO:2, a variant having at least
97% identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID
NO:2, an antigenic epitope of SEQ ID NO:1 or SEQ ID NO:2, and a
biologically active portion of SEQ ID NO:1 or SEQ ID NO:2. The
invention also provides a composition comprising the purified
protein in conjunction with a pharmaceutical carrier. The invention
further provides a method of using the NRP to treat a subject with
cancer, particularly cancers of the intestine, breast, uterus,
liver, brain, and kidney comprising administering to a patient in
need of such treatment the composition containing the purified
protein. The invention still further provides a method for using a
protein to screen a library or a plurality of molecules or
compounds to identify at least one ligand, the method comprising
combining the protein with the molecules or compounds under
conditions to allow specific binding and detecting specific
binding, thereby identifying a ligand which specifically binds the
protein. In one aspect, the molecules or compounds are selected
from DNA molecules, RNA molecules, peptide nucleic acids, peptides,
proteins, mimetics, agonists, antagonists, antibodies,
immunoglobulins, inhibitors, and drugs. In another aspect, the
ligand is used to treat a subject with cancer, particularly cancers
of the intestine, breast, uterus, liver, brain, and kidney.
[0013] The invention provides a method of using a protein to screen
a subject sample for antibodies which specifically bind the protein
comprising isolating antibodies from the subject sample, contacting
the isolated antibodies with the protein under conditions that
allow specific binding, dissociating the antibody from the
bound-protein, and comparing the quantity of antibody with known
standards, wherein the presence or quantity of antibody is
diagnostic of cancer, particularly cancers of the intestine,
breast, uterus, liver, brain, and kidney.
[0014] The invention also provides a method of using a protein to
prepare and purify antibodies comprising immunizing a animal with
the protein under conditions to elicit an antibody response,
isolating animal antibodies, attaching the protein to a substrate,
contacting the substrate with isolated antibodies under conditions
to allow specific binding to the protein, dissociating the
antibodies from the protein, thereby obtaining purified
antibodies.
[0015] The invention provides a purified antibody which binds
specifically to a protein which is expressed in cancer,
particularly cancers of the intestine, breast, uterus, liver,
brain, and kidney. The invention also provides a method of using an
antibody to diagnose cancer, particularly cancers of the intestine,
breast, uterus, liver, brain, and kidney comprising combining the
antibody comparing the quantity of bound antibody to known
standards, thereby establishing the presence of cancer,
particularly cancers of the intestine, breast, uterus, liver,
brain, and kidney. The invention further provides a method of using
an antibody to treat cancer, particularly cancers of the intestine,
breast, uterus, liver, brain, and kidney comprising administering
to a patient in need of such treatment a pharmaceutical composition
comprising the purified antibody.
[0016] The invention provides a method for inserting a heterologous
marker gene into the genomic DNA of a mammal to disrupt the
expression of the endogenous polynucleotide. The invention also
provides a method for using a cDNA to produce a mammalian model
system, the method comprising constructing a vector containing the
cDNA selected from SEQ ID NOs:3-31, transforming the vector into an
embryonic stem cell, selecting a transformed embryonic stem,
microinjecting the transformed embryonic stem cell into a mammalian
blastocyst, thereby forming a chimeric blastocyst, transferring the
chimeric blastocyst into a pseudopregnant dam, wherein the dam
gives birth to a chimeric offspring containing the cDNA in its germ
line, and breeding the chimeric mammal to produce a homozygous,
mammalian model system.
BRIEF DESCRIPTION OF THE FIGURES AND TABLE
[0017] FIGS. 1A, 1B, 1C, 1D, 1E, 1F, and 1G show the NRP (SEQ ID
NO:1) encoded by the cDNA (SEQ ID NO:3). The translation was
produced using MACDNASIS PRO software (Hitachi Software
Engineering, South San Francisco Calif.).
[0018] FIGS. 2A, 2B, 2C, 2D, 2E, 2F, and 2G show the NRP (SEQ ID
NO:2) encoded by the cDNA (SEQ ID NO:11). The translation was
produced using MACDNASIS PRO software (Hitachi Software
Engineering).
[0019] FIGS. 3A, 3B, and 3C demonstrate the conserved chemical and
structural similarities among the sequences and domains of NRP1
(2227688; SEQ ID NO:1), NRP2 (3507515; SEQ ID NO:2), mouse Ndr2
(g6141566; SEQ ID NO:32), and human RTP (g1596167; SEQ ID NO:33).
The alignment was produced using the MEGALIGN program of LASERGENE
software (DNASTAR, Madison Wis.).
[0020] Tables 1 and 2 show the northern analysis for NRP produced
using the LIFESEQ Gold database (Incyte Genomics, Palo Alto
Calif.). In Table 1, the first column presents the tissue
categories; the second column, the total number of clones in the
tissue category; the third column, the ratio of the number of
libraries in which at least one transcript was found to the total
number of libraries; the fourth column, absolute clone abundance of
the transcript; and the fifth column, percent abundance of the
transcript. Table 2 shows expression of NRP in intestine, breast,
uterus, liver, brain, and kidney tissues, particularly from
patients with cancer. The first column lists the library name, the
second column, the number of clones sequenced for that library; the
third column, the description of the tissue from which the library
was derived; the fourth column, the absolute abundance of the
transcript; and the fifth column, the percent abundance of the
transcript.
DESCRIPTION OF THE INVENTION
[0021] It is understood that this invention is not limited to the
particular machines, materials and methods described. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments and is not intended to limit
the scope of the present invention which will be limited only by
the appended claims. As used herein, the singular forms "a", "an",
and "the" include plural reference unless the context clearly
dictates otherwise. For example, a reference to "a host cell"
includes a plurality of such host cells known to those skilled in
the art.
[0022] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. All
publications mentioned herein are cited for the purpose of
describing and disclosing the cell lines, protocols, reagents and
vectors which are reported in the publications and which might be
used in connection with the invention. Nothing herein is to be
construed as an admission that the invention is not entitled to
antedate such disclosure by virtue of prior invention.
Definitions
[0023] "NRP" refers to a purified protein obtained from any
mammalian species, including bovine, canine, murine, ovine,
porcine, rodent, simian, and preferably the human species, and from
any source, whether natural, synthetic, semi-synthetic, or
recombinant.
[0024] "Array" refers to an ordered arrangement of at least two
cDNAs on a substrate. At least one of the cDNAs represents a
control or standard, and the other, a cDNA of diagnostic or
therapeutic interest. The arrangement of from about two to about
40,000 cDNAs on the substrate assures that the size and signal
intensity of each labeled hybridization complex formed between each
cDNA and at least one sample nucleic acid is individually
distinguishable.
[0025] The "complement" of a cDNA of the Sequence Listing refers to
a nucleic acid molecule which is completely complementary over its
full length and which will hybridize to the cDNA or an mRNA under
conditions of maximal stringency.
[0026] "cDNA" refers to an isolated polynucleotide, nucleic acid
molecule, or any fragment or complement thereof. It may have
originated recombinantly or synthetically, may be double-stranded
or single-stranded, represents coding and noncoding 3' or 5'
sequence, and generally lacks introns.
[0027] The phrase "cDNA encoding a protein" refers to a nucleotide
sequence that closely aligns with sequences which encode conserved
regions, motifs or domains that were identified by employing
analyses well known in the art. These analyses include BLAST (Basic
Local Alignment Search Tool) which provides identity within the
conserved region (Altschul (1993) J Mol Evol 36: 290-300; Altschul
et al. (1990) J Mol Biol 215:403-410).
[0028] A "composition" comprises the polynucleotide and a labeling
moiety or a purified protein in conjunction with a pharmaceutical
carrier.
[0029] "Derivative" refers to a cDNA or a protein that has been
subjected to a chemical modification. Derivatization of a cDNA can
involve substitution of a nontraditional base such as queosine or
of an analog such as hypoxanthine. These substitutions are well
known in the art. Derivatization of a protein involves the
replacement of a hydrogen by an acetyl, acyl, alkyl, amino, formyl,
or morpholino group. Derivative molecules retain the biological
activities of the naturally occurring molecules but may confer
advantages such as longer lifespan or enhanced activity.
[0030] "Differential expression" refers to an increased,
upregulated or present, or decreased, downregulated or absent, gene
expression as detected by presence, absence or at least two-fold
changes in the amount of transcribed messenger RNA or translated
protein in a sample.
[0031] "Disorder" refers to conditions, diseases or syndromes in
which the cDNAs and NRP are differentially expressed. Such a
disorder includes cancer, particularly cancers of the intestine,
breast, uterus, liver, brain, and kidney.
[0032] "Fragment" refers to a chain of consecutive nucleotides from
about 50 to about 4000 base pairs in length. Fragments may be used
in PCR or hybridization technologies to identify related nucleic
acid molecules and in binding assays to screen for a ligand. Such
ligands are useful as therapeutics to regulate replication,
transcription or translation.
[0033] A "hybridization complex" is formed between a cDNA and a
nucleic acid of a sample when the purines of one molecule hydrogen
bond with the pyrimidines of the complementary molecule, e.g.,
5'-A-G-T-C-3' base pairs with 3'-T-C-A-G-5'. Hybridization
conditions, degree of complementarity and the use of nucleotide
analogs affect the efficiency and stringency of hybridization
reactions.
[0034] "Labeling moiety" refers to any visible or radioactive label
than can be attached to or incorporated into a cDNA or protein.
Visible labels include but are not limited to anthocyanins, green
fluorescent protein (GFP), .beta. glucuronidase, luciferase, Cy3
and Cy5, and the like. Radioactive markers include radioactive
forms of hydrogen, iodine, phosphorous, sulfur, and the like.
[0035] "Ligand" refers to any agent, molecule, or compound which
will bind specifically to a polynucleotide or to an epitope of a
protein. Such ligands stabilize or modulate the activity of
polynucleotides or proteins and may be composed of inorganic and/or
organic substances including minerals, cofactors, nucleic acids,
proteins, carbohydrates, fats, and lipids.
[0036] "Oligonucleotide" refers a single-stranded molecule from
about 18 to about 60 nucleotides in length which may be used in
hybridization or amplification technologies or in regulation of
replication, transcription or translation. Substantially equivalent
terms are amplimer, primer, and oligomer.
[0037] "Portion" refers to any part of a protein used for any
purpose; but especially, to an epitope for the screening of ligands
or for the production of antibodies.
[0038] "Post-translational modification" of a protein can involve
lipidation, glycosylation, phosphorylation, acetylation,
racemization, proteolytic cleavage, and the like. These processes
may occur synthetically or biochemically. Biochemical modifications
will vary by cellular location, cell type, pH, enzymatic milieu,
and the like.
[0039] "Probe" refers to a cDNA that hybridizes to at least one
nucleic acid in a sample. Where targets are single-stranded, probes
are complementary single strands. Probes can be labeled with
reporter molecules for use in hybridization reactions including
Southern, northern, in situ, dot blot, array, and like technologies
or in screening assays.
[0040] "Protein" refers to a polypeptide or any portion thereof. A
"portion" of a protein refers to that length of amino acid sequence
which would retain at least one biological activity, a domain
identified by PFAM or PRINTS analysis or an antigenic epitope of
the protein identified using Kyte-Doolittle algorithms of the
PROTEAN program (DNASTAR, Madison Wis.). An "oligopeptide" is an
amino acid sequence from about five residues to about 15 residues
that is used as part of a fusion protein to produce an
antibody.
[0041] "Purified" refers to any molecule or compound that is
separated from its natural environment and is from about 60% free
to about 90% free from other components with which it is naturally
associated.
[0042] "Sample" is used in its broadest sense as containing nucleic
acids, proteins, antibodies, and the like. A sample may comprise a
bodily fluid; the soluble fraction of a cell preparation, or an
aliquot of media in which cells were grown; a chromosome, an
organelle, or membrane isolated or extracted from a cell; genomic
DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a
tissue; a tissue print; a fingerprint, buccal cells, skin, or hair;
and the like.
[0043] "Specific binding" refers to a special and precise
interaction between two molecules which is dependent upon their
structure, particularly their molecular side groups. For example,
the intercalation of a regulatory protein into the major groove of
a DNA molecule or the binding between an epitope of a protein and
an agonist, antagonist, or antibody.
[0044] "Similarity" as applied to sequences, refers to the
quantification (usually percentage) of nucleotide or residue
matches between at least two sequences aligned using a standardized
algorithm such as Smith-Waterman alignment (Smith and Waterman
(1981) J Mol Biol 147:195-197) or BLAST2 (Altschul et al. (1997)
Nucleic Acids Res 25:3389-3402). BLAST2 may be used in a
standardized and reproducible way to insert gaps in one of the
sequences in order to optimize alignment and to achieve a more
meaningful comparison between them. Particularly in proteins,
similarity is greater than identity in that conservative
substitutions, for example, valine for leucine or isoleucine, are
counted in calculating the reported percentage. Substitutions which
are considered to be conservative are well known in the art.
[0045] "Substrate" refers to any rigid or semi-rigid support to
which cDNAs or proteins are bound and includes membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels,
capillaries or other tubing, plates, polymers, and microparticles
with a variety of surface forms including wells, trenches, pins,
channels and pores.
[0046] "Variant" refers to molecules that are recognized variations
of a cDNA or a protein encoded by the cDNA. Splice variants may be
determined by BLAST score, wherein the score is at least 100, and
most preferably at least 400. Allelic variants have a high percent
identity to the cDNAs and may differ by about three bases per
hundred bases. "Single nucleotide polymorphism" (SNP) refers to a
change in a single base as a result of a substitution, insertion or
deletion. The change may be conservative (purine for purine) or
non-conservative (purine to pyrimidine) and may or may not result
in a change in an encoded amino acid or its secondary, tertiary, or
quaternary structure.
THE INVENTION
[0047] The invention is based on the discovery of cDNAs which
encode NRP and on the use of the cDNAs, or fragments thereof, and
proteins, or portions thereof, directly or as compositions in the
characterization, diagnosis, and treatment of cancer, particularly
cancers of the intestine, breast, uterus, liver, brain, and kidney.
NRP1 and NRP2 of the present invention were discovered as genes
that regulate cell proliferation by their differential expression
in noncancerous, precancerous, and cancerous tissues. The
identification and characterization of the cDNAs and proteins,
fragments or portions thereof, were described in U.S. Ser. No.
09/232,160.
[0048] Nucleic acids encoding the NRP1 of the present invention
were first identified in Incyte Clone 2227688 from the seminal
vesicle cDNA library (SEMVNOT01) using a computer search for
nucleotide and/or amino acid sequence alignments. SEQ ID NO:3 was
derived from the following overlapping and/or extended nucleic acid
sequences (SEQ ID NO:4-10): Incyte Clones 2227688H1 (SEMVNOT01),
3507515H1 (CONCNOT01), 027805X7 (SPLNFET01), 1300824F1 (BRSTNOT07),
1384447F1 (BRAITUT08), 1291929T1 (PGANNOT03), and 1367779H1
(SCORNON02).
[0049] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:1 as shown in FIGS.
1A, 1B, 1C, 1D, 1E, 1F, and 1G. NRP1 is 371 amino acids in length
and has two potential N-glycosylation sites at N136 and N342; four
potential casein kinase II phosphorylation sites at T67, S192,
S203, and S276; two potential protein kinase C phosphorylation
sites at T57 and T252; and one potential tyrosine kinase
phosphorylation site at Y309. PFAM and PRINTS analyses indicate
that the region of NRP1 from F95 to L311 is similar to an
alpha-beta hydrolase fold. PRINTS analysis indicates that the
region of NRP1 from F85 to P103 is similar to an androgen receptor
signature. As shown in FIGS. 3A, 3B, and 3C, NRP1 has chemical and
structural similarity with mouse Ndr2 (g6141566; SEQ ID NO:32), and
human RTP (g1596167; SEQ ID NO:33). In particular, NRP1 and Ndr2
share about 96% identity. NRP1 and RTP share about 52% identity.
NRP1, Ndr2, and RTP share a predicted alpha-beta hydrolase fold and
an androgen receptor signature. Useful antigenic epitopes extend
from about E11 to about R32, from about N238 to about R254, and
from about S335 to about E368; and a biologically active portion of
NRP1 extends from about 85 to about 103. An antibody which
specifically binds NRP1 is useful in a diagnostic assay to identify
cancer, particularly cancers of the intestine, breast, uterus,
liver, brain, and kidney.
[0050] Nucleic acids encoding the NRP2 of the present invention
were first identified in Incyte Clone 3507515 from the chest wall
soft tissue cDNA library (CONCNOT01) using a computer search for
nucleotide and/or amino acid sequence alignments. SEQ ID NO:11 was
derived from the following overlapping and/or extended nucleic acid
sequences (SEQ ID NO:12-15): Incyte Clones 3507515F6 (CONCNOT01),
1214191R1 (BRSTTUT01), 3364735F6 (PROSBPT02), and 1289617F1
(BRAINOT11).
[0051] In another embodiment, the invention encompasses a
polypeptide comprising the amino acid sequence of SEQ ID NO:2 as
shown in FIGS. 2A, 2B, 2C, 2D, 2E, 2F, and 2G. NRP2 is 402 amino
acids in length and has one potential N-glycosylation site at N122;
four potential casein kinase II phosphorylation sites at T53, S178,
S189, and S262; three potential protein kinase C phosphorylation
sites at T43, T238 and S398; and one potential tyrosine kinase
phosphorylation site at Y295. PFAM and PRINTS analyses indicate
that the region of NRP2 from F81 to L297 is similar to an
alpha-beta hydrolase fold. PRINTS analysis indicates that the
regions of NRP2 from F71 to P89 and from R350 to S370 are similar
to an androgen receptor signature. As shown in FIGS. 3A, 3B, and
3C, NRP2 has chemical and structural similarity with mouse Ndr2
(g6141566; SEQ ID NO:32), and human RTP (g1596167; SEQ ID NO:33).
In particular, NRP2 and Ndr2 share about 89% identity. NRP2 and RTP
share about 49% identity. NRP2, Ndr2, and RTP share a predicted
alpha-beta hydrolase fold and an androgen receptor signature.
Useful antigenic epitopes extend from about Q8 to about S28, from
about N221 to about R240, and from about N377 to about G401; and
biologically active portions of NRP2 extend from about 71 to about
89 and from about 350 to about 370. An antibody which specifically
binds NRP2 is useful in an diagnostic assay to identify cancer,
particularly cancers of the intestine, breast, uterus, liver,
brain, and kidney.
[0052] Table 1 shows expression of NRP1 and NRP2 across the tissue
categories. NRP is expressed predominantly in the nervous system,
exocrine glands, female reproductive tissue, and male reproductive
tissue. Table 2 shows expression of NRP in intestine, breast,
uterus, liver, brain, and kidney, particularly from patients with
cancer. Libraries in which the transcript has an absolute abundance
of more than one provide an indication of overexpression in these
tissues. NRP is found in libraries associated with various cancers,
in particular, a small intestine library (SINTTUT01) from a patient
with ileum carcinoid, breast tumor libraries (BRSTTUP03 and
BRSTTUT17) from patients with ductal carcinoma, a uterine tumor
library (UTRSTUE01) from a patient with leiomyoma, a liver tumor
library (LIVRTUT04) from a patient with hepatoma, a brain tumor
library (BRAITUP04) from a patient with oligodendroglioma, a brain
tumor library (BRAITUP06) from a patient with astrocytoma, and
kidney tumor libraries (KIDNTUT13, KIDNTUP05, and KIDNTUE01) from
patients with renal cell carcinoma. Therefore, the cDNAs encoding
NRP1 or NRP2 are useful in assays to diagnose cancer, particularly
cancers of the intestine, breast, uterus, liver, brain, and kidney.
A fragment of the cDNA encoding NRP1 from about nucleotide 1 to
about nucleotide 50 is also useful in diagnostic assays. A fragment
of the cDNA encoding NRP2 from about nucleotide 1 to about
nucleotide 50 is useful in diagnostic assays.
[0053] Mammalian variants of the cDNA encoding NRP were identified
using BLAST2 with default parameters and the ZOOSEQ databases
(Incyte Genomics). These preferred variants have from about 82% to
about 97% identity as shown in the table below. The first column
shows the SEQ ID for the human cDNA (SEQ ID.sub.H); the second
column, the SEQ ID for the variant cDNAs (SEQ ID.sub.var); the
third column, the clone number for the variant cDNAs
(Clone.sub.var); the fourth column, the library name; the fifth
column, the alignment of the variant cDNA to the human cDNA; and
the sixth column, the percent identity to the human cDNA.
1 SEQ SEQ ID.sub.H ID.sub.var Clone.sub.var Library Name Nt.sub.H
Alignment Identity 3 281-545 97% 16 700718124H1 NNBCNOT01 11
362-626 97% 3 1003-1259 96% 17 700710954H1 MNBFNOT02 11 1084-1344
94% 3 1081-1321 95% 18 700707576H1 MNBFNOT01 11 1162-1390 91% 3
228-392 97% 19 700715158H1 MNBCNOT01 11 231-473 97% 3 961-1083 87%
20 700705541H1 MNBFNOT01 11 1042-1164 87% 3 34-103 94% 21
700719804H1 MNBTNOT01 11 158-226 94% 3 502-1143 91% 22 702769047H1
CNLINOT01 11 583-1224 91% 3 376-1023 91% 23 702763624H1 CNLIUNN01
11 457-1104 91% 3 803-1230 91% 24 702776664H1 CNLINOT07 11 884-1262
91% 3 182-409 92% 25 702778279H1 CNLINOT07 11 305-490 91% 3 26
702249907H1 CNBYNOT01 1511-1943 82% 3 569-1173 90% 27 702025443H1
RABRTXT02 11 650-1254 90% 3 316-846 90% 28 702028546H2 RABRTXT02 11
397-927 90% 3 224-728 90% 29 702160576H1 RABRTXT10 11 270-809 90% 3
200-688 89% 30 701938896H1 RALIUNT18 11 305-769 89% 3 31
701646914H1 RALITXT40 103-406 91%
[0054] These cDNAs, SEQ ID NOS:16-31 are particularly useful for
producing transgenic cell lines or organisms.
[0055] It will be appreciated by those skilled in the art that as a
result of the degeneracy of the genetic code, a multitude of cDNAs
encoding NRP, some bearing minimal similarity to the cDNAs of any
known and naturally occurring gene, may be produced. Thus, the
invention contemplates each and every possible variation of cDNA
that could be made by selecting combinations based on possible
codon choices. These combinations are made in accordance with the
standard triplet genetic code as applied to the polynucleotide
encoding naturally occurring NRP, and all such variations are to be
considered as being specifically disclosed.
[0056] The cDNAs of SEQ ID NOs:3-31 may be used in hybridization,
amplification, and screening technologies to identify and
distinguish among SEQ ID NO:3 or SEQ ID NO:11 and related molecules
in a sample. The mammalian cDNAs may be used to produce transgenic
cell lines or organisms which are model systems for human cancer,
particularly cancers of the intestine, breast, uterus, liver,
brain, and kidney and upon which the toxicity and efficacy of
potential therapeutic treatments may be tested. Toxicology studies,
clinical trials, and subject/patient treatment profiles may be
performed and monitored using the cDNAs, proteins, antibodies and
molecules and compounds identified using the cDNAs and proteins of
the present invention.
Characterization and Use of the Invention
[0057] cDNA Libraries
[0058] In a particular embodiment disclosed herein, mRNA is
isolated from mammalian cells and tissues using methods which are
well known to those skilled in the art and used to prepare the cDNA
libraries. The Incyte cDNAs were isolated from mammalian cDNA
libraries a prepared as described in the EXAMPLES. The consensus
sequences are chemically and/or electronically assembled from
fragments including Incyte cDNAs and extension and/or shotgun
sequences using computer programs such as PHRAP (P Green,
University of Washington, Seattle Wash.), and AUTOASSEMBLER
application (Applied Biosystems, Foster City Calif.). After
verification of the 5' and 3' sequence, at least one representative
cDNA which encodes NRP is designated a reagent.
[0059] Sequencing
[0060] Methods for sequencing nucleic acids are well known in the
art and may be used to practice any of the embodiments of the
invention. These methods employ enzymes such as the Klenow fragment
of DNA polymerase I, SEQUENASE, Taq DNA polymerase and thermostable
T7 DNA polymerase (Amersham Pharmacia Biotech (APB), Piscataway
N.J.), or combinations of polymerases and proofreading exonucleases
such as those found in the ELONGASE amplification system (Life
Technologies, Gaithersburg Md.). Preferably, sequence preparation
is automated with machines such as the MICROLAB 2200 system
(Hamilton, Reno Nev.) and the DNA ENGINE thermal cycler (MJ
Research, Watertown Mass.). Machines commonly used for sequencing
include the ABI PRISM 3700, 377 or 373 DNA sequencing systems
(Applied Biosystems), the MEGABACE 1000 DNA sequencing system
(APB), and the like. The sequences may be analyzed using a variety
of algorithms well known in the art and described in Ausubel et al.
(1997; Short Protocols in Molecular Biology, John Wiley & Sons,
New York N.Y., unit 7.7) and in Meyers (1995; Molecular Biology and
Biotechnology, Wiley VCH, New York N.Y., pp. 856-853).
[0061] Shotgun sequencing may also be used to complete the sequence
of a particular cloned insert of interest. Shotgun strategy
involves randomly breaking the original insert into segments of
various sizes and cloning these fragments into vectors. The
fragments are sequenced and reassembled using overlapping ends
until the entire sequence of the original insert is known. Shotgun
sequencing methods are well known in the art and use thermostable
DNA polymerases, heat-labile DNA polymerases, and primers chosen
from representative regions flanking the cDNAs of interest.
Incomplete assembled sequences are inspected for identity using
various algorithms or programs such as CONSED (Gordon (1998) Genome
Res 8:195-202) which are well known in the art. Contaminating
sequences, including vector or chimeric sequences, or deleted
sequences can be removed or restored, respectively, organizing the
incomplete assembled sequences into finished sequences.
[0062] Extension of a Nucleic Acid Sequence
[0063] The sequences of the invention may be extended using various
PCR-based methods known in the art. For example, the XL-PCR kit
(Applied Biosystems), nested primers, and commercially available
cDNA or genomic DNA libraries may be used to extend the nucleic
acid sequence. For all PCR-based methods, primers may be designed
using commercially available software, such as OLIGO primer
analysis software (Molecular Biology Insights, Cascade Colo.) to be
about 22 to 30 nucleotides in length, to have a GC content of about
50% or more, and to anneal to a target molecule at temperatures
from about 55 C. to about 68 C. When extending a sequence to
recover regulatory elements, it is preferable to use genomic,
rather than cDNA libraries.
[0064] Hybridization
[0065] The cDNA and fragments thereof can be used in hybridization
technologies for various purposes. A probe may be designed or
derived from unique regions such as the 5' regulatory region or
from a nonconserved region (i.e., 5' or 3' of the nucleotides
encoding the conserved catalytic domain of the protein) and used in
protocols to identify naturally occurring molecules encoding the
NRP, allelic variants, or related molecules. The probe may be DNA
or RNA, may be single-stranded, and should have at least 50%
sequence identity to any of the nucleic acid sequences, SEQ ID
NOs:3-31. Hybridization probes may be produced using oligolabeling,
nick translation, end-labeling, or PCR amplification in the
presence of a reporter molecule. A vector containing the cDNA or a
fragment thereof may be used to produce an mRNA probe in vitro by
addition of an RNA polymerase and labeled nucleotides. These
procedures may be conducted using commercially available kits such
as those provided by APB.
[0066] The stringency of hybridization is determined by G+C content
of the probe, salt concentration, and temperature. In particular,
stringency can be increased by reducing the concentration of salt
or raising the hybridization temperature. Hybridization can be
performed at low stringency with buffers, such as 5.times.SSC with
1% sodium dodecyl sulfate (SDS) at 60 C., which permits the
formation of a hybridization complex between nucleic acid sequences
that contain some mismatches. Subsequent washes are performed at
higher stringency with buffers such as 0.2.times.SSC with 0.1% SDS
at either 45 C. (medium stringency) or 68 C. (high stringency). At
high stringency, hybridization complexes will remain stable only
where the nucleic acids are completely complementary. In some
membrane-based hybridizations, preferably 35% or most preferably
50%, formamide can be added to the hybridization solution to reduce
the temperature at which hybridization is performed, and background
signals can be reduced by the use of detergents such as Sarkosyl or
TRITON X-100 (Sigma-Aldrich, St. Louis Mo.) and a blocking agent
such as denatured salmon sperm DNA. Selection of components and
conditions for hybridization are well known to those skilled in the
art and are reviewed in Ausubel (supra) and Sambrook et al. (1989)
Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,
Plainview N.Y.
[0067] Arrays may be prepared and analyzed using methods well known
in the art. Oligonucleotides or cDNAs may be used as hybridization
probes or targets to monitor the expression level of large numbers
of genes simultaneously or to identify genetic variants, mutations,
and single nucleotide polymorphisms. Arrays may be used to
determine gene function; to understand the genetic basis of a
condition, disease, or disorder; to diagnose a condition, disease,
or disorder; and to develop and monitor the activities of
therapeutic agents. (See, e.g., Brennan et al. (1995) U.S. Pat. No.
5,474,796; Schena et al. (1996) Proc Natl Acad Sci 93:10614-10619;
Heller et al. (1997) Proc Natl Acad Sci 94:2150-2155; and Heller et
al. (1997) U.S. Pat. No. 5,605,662.)
[0068] Hybridization probes are also useful in mapping the
naturally occurring genomic sequence. The probes may be hybridized
to a particular chromosome, a specific region of a chromosome, or
an artificial chromosome construction. Such constructions include
human artificial chromosomes (HAC), yeast artificial chromosomes
(YAC), bacterial artificial chromosomes (BAC), bacterial PI
constructions, or the cDNAs of libraries made from single
chromosomes.
[0069] Expression
[0070] Any one of a multitude of cDNAs encoding NRP may be cloned
into a vector and used to express the protein, or portions thereof,
in host cells. The nucleic acid sequence can be engineered by such
methods as DNA shuffling (U.S. Pat. No. 5,830,721) and
site-directed mutagenesis to create new restriction sites, alter
glycosylation patterns, change codon preference to increase
expression in a particular host, produce splice variants, extend
half-life, and the like. The expression vector may contain
transcriptional and translational control elements (promoters,
enhancers, specific initiation signals, and polyadenylated 3'
sequence) from various sources which have been selected for their
efficiency in a particular host. The vector, cDNA, and regulatory
elements are combined using in vitro recombinant DNA techniques,
synthetic techniques, and/or in vivo genetic recombination
techniques well known in the art and described in Sambrook (supra,
ch. 4, 8, 16 and 17).
[0071] A variety of host systems may be transformed with an
expression vector. These include, but are not limited to, bacteria
transformed with recombinant bacteriophage, plasmid, or cosmid DNA
expression vectors; yeast transformed with yeast expression
vectors; insect cell systems transformed with baculovirus
expression vectors; plant cell systems transformed with expression
vectors containing viral and/or bacterial elements, or animal cell
systems (Ausubel supra, unit 16). For example, an adenovirus
transcription/translation complex may be utilized in mammalian
cells. After sequences are ligated into the E1 or E3 region of the
viral genome, the infective virus is used to transform and express
the protein in host cells. The Rous sarcoma virus enhancer or SV40
or EBV-based vectors may also be used for high-level protein
expression.
[0072] Routine cloning, subcloning, and propagation of nucleic acid
sequences can be achieved using the multifunctional PBLUESCRIPT
vector (Stratagene, La Jolla Calif.) or PSPORT1 plasmid (Life
Technologies). Introduction of a nucleic acid sequence into the
multiple cloning site of these vectors disrupts the lacZ gene and
allows colorimetric screening for transformed bacteria. In
addition, these vectors may be useful for in vitro transcription,
dideoxy sequencing, single strand rescue with helper phage, and
creation of nested deletions in the cloned sequence.
[0073] For long term production of recombinant proteins, the vector
can be stably transformed into cell lines along with a selectable
or visible marker gene on the same or on a separate vector. After
transformation, cells are allowed to grow for about 1 to 2 days in
enriched media and then are transferred to selective media.
Selectable markers, antimetabolite, antibiotic, or herbicide
resistance genes, confer resistance to the relevant selective agent
and allow growth and recovery of cells which successfully express
the introduced sequences. Resistant clones identified either by
survival on selective media or by the expression of visible markers
may be propagated using culture techniques. Visible markers are
also used to estimate the amount of protein expressed by the
introduced genes. Verification that the host cell contains the
desired cDNA is based on DNA-DNA or DNA-RNA hybridizations or PCR
amplification techniques.
[0074] The host cell may be chosen for its ability to modify a
recombinant protein in a desired fashion. Such modifications
include acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, acylation and the like. Post-translational processing
which cleaves a "prepro" form may also be used to specify protein
targeting, folding, and/or activity. Different host cells available
from the ATCC (Manassas Va.) which have specific cellular machinery
and characteristic mechanisms for post-translational activities may
be chosen to ensure the correct modification and processing of the
recombinant protein.
[0075] Recovery of Proteins from Cell Culture
[0076] Heterologous moieties engineered into a vector for ease of
purification include glutathione S-transferase (GST), 6.times.His,
FLAG, MYC, and the like. GST and 6-His are purified using
commercially available affinity matrices such as immobilized
glutathione and metal-chelate resins, respectively. FLAG and MYC
are purified using commercially available monoclonal and polyclonal
antibodies. For ease of separation following purification, a
sequence encoding a proteolytic cleavage site may be part of the
vector located between the protein and the heterologous moiety.
Methods for recombinant protein expression and purification are
discussed in Ausubel (supra, unit 16) and are commercially
available.
[0077] Chemical Synthesis of Peptides
[0078] Proteins or portions thereof may be produced not only by
recombinant methods, but also by using chemical methods well known
in the art. Solid phase peptide synthesis may be carried out in a
batchwise or continuous flow process which sequentially adds
.alpha.-amino- and side chain-protected amino acid residues to an
insoluble polymeric support via a linker group. A linker group such
as methylamine-derivatized polyethylene glycol is attached to
poly(styrene-co-divinylbenzene) to form the support resin. The
amino acid residues are N-.alpha.-protected by acid labile Boc
(t-butyloxycarbonyl) or base-labile Fmoc
(9-fluorenylmethoxycarbonyl). The carboxyl group of the protected
amino acid is coupled to the amine of the linker group to anchor
the residue to the solid phase support resin. Trifluoroacetic acid
or piperidine are used to remove the protecting group in the case
of Boc or Fmoc, respectively. Each additional amino acid is added
to the anchored residue using a coupling agent or pre-activated
amino acid derivative, and the resin is washed. The full length
peptide is synthesized by sequential deprotection, coupling of
derivitized amino acids, and washing with dichloromethane and/or
N,N-dimethylformamide. The peptide is cleaved between the peptide
carboxy terminus and the linker group to yield a peptide acid or
amide. (Novabiochem 1997/98 Catalog and Peptide Synthesis Handbook,
San Diego Calif. pp. S1-S20). Automated synthesis may also be
carried out on machines such as the ABI 431A peptide synthesizer
(Applied Biosystems). A protein or portion thereof may be
substantially purified by preparative high performance liquid
chromatography and its composition confirmed by amino acid analysis
or by sequencing (Creighton (1984) Proteins, Structures and
Molecular Properties, W H Freeman, New York N.Y.).
[0079] Preparation and Screening of Antibodies
[0080] Various hosts including goats, rabbits, rats, mice, humans,
and others may be immunized by injection with NRP or any portion
thereof. Adjuvants such as Freund's, mineral gels, and surface
active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemacyanin
(KLH), and dinitrophenol may be used to increase immunological
response. The oligopeptide, peptide, or portion of protein used to
induce antibodies should consist of at least about five amino
acids, more preferably ten amino acids, which are identical to a
portion of the natural protein. Oligopeptides may be fused with
proteins such as KLH in order to produce antibodies to the chimeric
molecule.
[0081] Monoclonal antibodies may be prepared using any technique
which provides for the production of antibodies by continuous cell
lines in culture. These include, but are not limited to, the
hybridoma technique, the human B-cell hybridoma technique, and the
EBV-hybridoma technique. (See, e.g., Kohler et al. (1975) Nature
256:495-497; Kozbor et al. (1985) J. Immunol Methods 81:31-42; Cote
et al. (1983) Proc Natl Acad Sci 80:2026-2030; and Cole et al.
(1984) Mol Cell Biol 62:109-120.)
[0082] Alternatively, techniques described for antibody production
may be adapted, using methods known in the art, to produce
epitope-specific, single chain antibodies. Antibody fragments which
contain specific binding sites for epitopes of the protein may also
be generated. For example, such fragments include, but are not
limited to, F(ab')2 fragments produced by pepsin digestion of the
antibody molecule and Fab fragments generated by reducing the
disulfide bridges of the F(ab)2 fragments. Alternatively, Fab
expression libraries may be constructed to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity. (See, e.g., Huse et al. (1989) Science
246:1275-1281.)
[0083] The NRP or a portion thereof may be used in screening assays
of phagemid or B-lymphocyte immunoglobulin libraries to identify
antibodies having the desired specificity. Numerous protocols for
competitive binding or immunoassays using either polyclonal or
monoclonal antibodies with established specificities are well known
in the art. Such immunoassays typically involve the measurement of
complex formation between the protein and its specific antibody. A
two-site, monoclonal-based immunoassay utilizing monoclonal
antibodies reactive to two non-interfering epitopes is preferred,
but a competitive binding assay may also be employed (Pound (1998)
Immunochemical Protocols, Humana Press, Totowa N.J.).
[0084] Labeling of Molecules for Assay
[0085] A wide variety of reporter molecules and conjugation
techniques are known by those skilled in the art and may be used in
various nucleic acid, amino acid, and antibody assays. Synthesis of
labeled molecules may be achieved using commercially available kits
(Promega, Madison Wis.) for incorporation of a labeled nucleotide
such as .sup.32P-dCTP (APB), Cy3-dCTP or Cy5-dCTP (Operon
Technologies, Alameda Calif.), or amino acid such as
.sup.35S-methionine (APB). Nucleotides and amino acids may be
directly labeled with a variety of substances including
fluorescent, chemiluminescent, or chromogenic agents, and the like,
by chemical conjugation to amines, thiols and other groups present
in the molecules using reagents such as BIODIPY or FITC (Molecular
Probes, Eugene Oreg.).
DIAGNOSTICS
[0086] The cDNAs, fragments, oligonucleotides, complementary RNA
and DNA molecules, and PNAs and may be used to detect and quantify
differential gene expression for diagnosis of a disorder. Similarly
antibodies which specifically bind NRP may be used to quantitate
the protein. Disorders associated with differential expression
include intestine cancer, breast cancer, uterine cancer, liver
cancer, brain cancer, and kidney cancer. The diagnostic assay may
use hybridization or amplification technology to compare gene
expression in a biological sample from a patient to standard
samples in order to detect differential gene expression.
Qualitative or quantitative methods for this comparison are well
known in the art.
[0087] For example, the cDNA or probe may be labeled by standard
methods and added to a biological sample from a patient under
conditions for the formation of hybridization complexes. After an
incubation period, the sample is washed and the amount of label (or
signal) associated with hybridization complexes, is quantified and
compared with a standard value. If complex formation in the patient
sample is significantly altered (higher or lower) in comparison to
either a normal or disease standard, then differential expression
indicates the presence of a disorder.
[0088] In order to provide standards for establishing differential
expression, normal and disease expression profiles are established.
This is accomplished by combining a sample taken from normal
subjects, either animal or human, with a cDNA under conditions for
hybridization to occur. Standard hybridization complexes may be
quantified by comparing the values obtained using normal subjects
with values from an experiment in which a known amount of a
purified sequence is used. Standard values obtained in this manner
may be compared with values obtained from samples from patients who
were diagnosed with a particular condition, disease, or disorder.
Deviation from standard values toward those associated with a
particular disorder is used to diagnose that disorder.
[0089] Such assays may also be used to evaluate the efficacy of a
particular therapeutic treatment regimen in animal studies or in
clinical trials or to monitor the treatment of an individual
patient. Once the presence of a condition is established and a
treatment protocol is initiated, diagnostic assays may be repeated
on a regular basis to determine if the level of expression in the
patient begins to approximate that which is observed in a normal
subject. The results obtained from successive assays may be used to
show the efficacy of treatment over a period ranging from several
days to months.
[0090] Immunological Methods
[0091] Detection and quantification of a protein using either
specific polyclonal or monoclonal antibodies are known in the art.
Examples of such techniques include enzyme-linked immunosorbent
assays (ELISAs), radioimmunoassays (RIAs), and fluorescence
activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay utilizing monoclonal antibodies reactive to two
non-interfering epitopes is preferred, but a competitive binding
assay may be employed. (See, e.g., Coligan et al. (1997) Current
Protocols in Immunology, Wiley-Interscience, New York N.Y.; and
Pound, supra.)
THERAPEUTICS
[0092] Chemical and structural similarity, in particular the
alpha-beta hydrolase fold and the androgen receptor signatures,
exists between regions of NRP1 (2227688; SEQ ID NO:1), NRP2
(3507515; SEQ ID NO:2), mouse Ndr2 (g6141566; SEQ ID NO:32), and
human RTP (g1596167; SEQ ID NO:33) as shown in FIGS. 3A, 3B, and
3C. In addition, differential expression is associated with cancer,
particularly cancers of the intestine, breast, uterus, liver,
brain, and kidney as shown in Tables 1 and 2. NRP clearly plays a
role in cancer, particularly cancers of the intestine, breast,
uterus, liver, brain, and kidney.
[0093] In the treatment of conditions associated with increased
expression of NRP, it is desirable to decrease expression or
protein activity. In one embodiment, the an inhibitor, antagonist
or antibody of the protein may be administered to a subject to
treat a condition associated with increased expression or activity.
In another embodiment, a pharmaceutical composition comprising an
inhibitor, antagonist or antibody in conjunction with a
pharmaceutical carrier may be administered to a subject to treat a
condition associated with the increased expression or activity of
the endogenous protein. In an additional embodiment, a vector
expressing the complement of the cDNA or fragments thereof may be
administered to a subject to treat the disorder.
[0094] In the treatment of conditions associated with decreased
expression of NRP, it is desirable to increase expression or
protein activity. In one embodiment, the protein, an agonist or
enhancer may be administered to a subject to treat a condition
associated with decreased expression or activity. In another
embodiment, a pharmaceutical composition comprising the protein, an
agonist or enhancer in conjunction with a pharmaceutical carrier
may be administered to a subject to treat a condition associated
with the decreased expression or activity of the endogenous
protein. In an additional embodiment, a vector expressing cDNA may
be administered to a subject to treat the disorder.
[0095] Any of the cDNAs, complementary molecules, or fragments
thereof, proteins or portions thereof, vectors delivering these
nucleic acid molecules or expressing the proteins, and their
ligands may be administered in combination with other therapeutic
agents. Selection of the agents for use in combination therapy may
be made by one of ordinary skill in the art according to
conventional pharmaceutical principles. A combination of
therapeutic agents may act synergistically to affect treatment of a
particular disorder at a lower dosage of each agent.
[0096] Modification of Gene Expression Using Nucleic Acids
[0097] Gene expression may be modified by designing complementary
or antisense molecules (DNA, RNA, or PNA) to the control, 5', 3',
or other regulatory regions of the gene encoding NRP.
Oligonucleotides designed to inhibit transcription initiation are
preferred. Similarly, inhibition can be achieved using triple helix
base-pairing which inhibits the binding of polymerases,
transcription factors, or regulatory molecules (Gee et al. In:
Huber and Carr (1994) Molecular and Immunologic Approaches, Futura
Publishing, Mt. Kisco N.Y., pp. 163-177). A complementary molecule
may also be designed to block translation by preventing binding
between ribosomes and mRNA. In one alternative, a library or
plurality of cDNAs may be screened to identify those which
specifically bind a regulatory, nontranslated sequence.
[0098] Ribozymes, enzymatic RNA molecules, may also be used to
catalyze the specific cleavage of RNA. The mechanism of ribozyme
action involves sequence-specific hybridization of the ribozyme
molecule to complementary target RNA followed by endonucleolytic
cleavage at sites such as GUA, GUU, and GUC. Once such sites are
identified, an oligonucleotide with the same sequence may be
evaluated for secondary structural features which would render the
oligonucleotide inoperable. The suitability of candidate targets
may also be evaluated by testing their hybridization with
complementary oligonucleotides using ribonuclease protection
assays.
[0099] Complementary nucleic acids and ribozymes of the invention
may be prepared via recombinant expression, in vitro or in vivo, or
using solid phase phosphoramidite chemical synthesis. In addition,
RNA molecules may be modified to increase intracellular stability
and half-life by addition of flanking sequences at the 5' and/or 3'
ends of the molecule or by the use of phosphorothioate or 2'
O-methyl rather than phosphodiesterase linkages within the backbone
of the molecule. Modification is inherent in the production of PNAs
and can be extended to other nucleic acid molecules. Either the
inclusion of nontraditional bases such as inosine, queosine, and
wybutosine, and or the modification of adenine, cytidine, guanine,
thymine, and uridine with acetyl-, methyl-, thio-groups renders the
molecule less available to endogenous endonucleases.
[0100] Screening and Purification Assays
[0101] The cDNA encoding NRP may be used to screen a library of
molecules or compounds for specific binding affinity. The libraries
may be aptamers, DNA molecules, RNA molecules, PNAs, peptides,
proteins such as transcription factors, enhancers, repressors, and
other ligands which regulate the activity, replication,
transcription, or translation of the endogenous gene. The assay
involves combining a polynucleotide with a library of molecules
under conditions allowing specific binding, and detecting specific
binding to identify at least one molecule which specifically binds
the single-stranded or double-stranded molecule.
[0102] In one embodiment, the cDNA of the invention may be
incubated with a plurality of purified molecules or compounds and
binding activity determined by methods well known in the art, e.g.,
a gel-retardation assay (U.S. Pat. No. 6,010,849) or a reticulocyte
lysate transcriptional assay. In another embodiment, the cDNA may
be incubated with nuclear extracts from biopsied and/or cultured
cells and tissues. Specific binding between the cDNA and a molecule
or compound in the nuclear extract is initially determined by gel
shift assay and may be later confirmed by recovering and raising
antibodies against that molecule or compound. When these antibodies
are added into the assay, they cause a supershift in the
gel-retardation assay.
[0103] In another embodiment, the cDNA may be used to purify a
molecule or compound using affinity chromatography methods well
known in the art. In one embodiment, the cDNA is chemically reacted
with cyanogen bromide groups on a polymeric resin or gel. Then a
sample is passed over and reacts with or binds to the cDNA. The
molecule or compound which is bound to the cDNA may be released
from the cDNA by increasing the salt concentration of the
flow-through medium and collected.
[0104] In a further embodiment, the protein or a portion thereof
may be used to purify a ligand from a sample. A method for using a
protein or a portion thereof to purify a ligand would involve
combining the protein or a portion thereof with a sample under
conditions to allow specific binding, detecting specific binding
between the protein and ligand, recovering the bound protein, and
using an appropriate chaotropic agent to separate the protein from
the purified ligand.
[0105] In a preferred embodiment, NRP may be used to screen a
plurality of molecules or compounds in any of a variety of
screening assays. The portion of the protein employed in such
screening may be free in solution, affixed to an abiotic or biotic
substrate (e.g. borne on a cell surface), or located
intracellularly. For example, in one method, viable or fixed
prokaryotic host cells that are stably transformed with recombinant
nucleic acids that have expressed and positioned a peptide on their
cell surface can be used in screening assays. The cells are
screened against a plurality or libraries of ligands, and the
specificity of binding or formation of complexes between the
expressed protein and the ligand may be measured. Specific binding
between the protein and molecule may be measured. Depending on the
particular kind of library being screened, the assay may be used to
identify DNA molecules, RNA molecules, peptide nucleic acids,
peptides, proteins, mimetics, agonists, antagonists, antibodies,
immunoglobulins, inhibitors, and drugs or any other ligand, which
specifically binds the protein.
[0106] In one aspect, this invention comtemplates a method for high
throughput screening using very small assay volumes and very small
amounts of test compound as described in U.S. Pat. No. 5,876,946,
incorporated herein by reference. This method is used to screen
large numbers of molecules and compounds via specific binding. In
another aspect, this invention also contemplates the use of
competitive drug screening assays in which neutralizing antibodies
capable of binding the protein specifically compete with a test
compound capable of binding to the protein. Molecules or compounds
identified by screening may be used in a mammalian model system to
evaluate their toxicity, diagnostic, or therapeutic potential.
[0107] Pharmacology
[0108] Pharmaceutical compositions are those substances wherein the
active ingredients are contained in an effective amount to achieve
a desired and intended purpose. The determination of an effective
dose is well within the capability of those skilled in the art. For
any compound, the therapeutically effective dose may be estimated
initially either in cell culture assays or in animal models. The
animal model is also used to achieve a desirable concentration
range and route of administration. Such information may then be
used to determine useful doses and routes for administration in
humans.
[0109] A therapeutically effective dose refers to that amount of
protein or inhibitor which ameliorates the symptoms or condition.
Therapeutic efficacy and toxicity of such agents may be determined
by standard pharmaceutical procedures in cell cultures or
experimental animals, e.g., ED.sub.50 (the dose therapeutically
effective in 50% of the population) and LD.sub.50 (the dose lethal
to 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index, and it may be
expressed as the ratio, LD.sub.50/ED.sub.50. Pharmaceutical
compositions which exhibit large therapeutic indexes are preferred.
The data obtained from cell culture assays and animal studies are
used in formulating a range of dosage for human use.
[0110] Model Systems
[0111] Animal models may be used as bioassays where they exhibit a
phenotypic response similar to that of humans and where exposure
conditions are relevant to human exposures. Mammals are the most
common models, and most infectious agent, cancer, drug, and
toxicity studies are performed on rodents such as rats or mice
because of low cost, availability, lifespan, reproductive
potential, and abundant reference literature. Inbred and outbred
rodent strains provide a convenient model for investigation of the
physiological consequences of under- or over-expression of genes of
interest and for the development of methods for diagnosis and
treatment of diseases. A mammal inbred to over-express a particular
gene (for example, secreted in milk) may also serve as a convenient
source of the protein expressed by that gene.
[0112] Toxicology
[0113] Toxicology is the study of the effects of agents on living
systems. The majority of toxicity studies are performed on rats or
mice. Observation of qualitative and quantitative changes in
physiology, behavior, homeostatic processes, and lethality in the
rats or mice are used to generate a toxicity profile and to assess
potential consequences on human health following exposure to the
agent.
[0114] Genetic toxicology identifies and analyzes the effect of an
agent on the rate of endogenous, spontaneous, and induced genetic
mutations. Genotoxic agents usually have common chemical or
physical properties that facilitate interaction with nucleic acids
and are most harmful when chromosomal aberrations are transmitted
to progeny. Toxicological studies may identify agents that increase
the frequency of structural or functional abnormalities in the
tissues of the progeny if administered to either parent before
conception, to the mother during pregnancy, or to the developing
organism. Mice and rats are most frequently used in these tests
because their short reproductive cycle allows the production of the
numbers of organisms needed to satisfy statistical
requirements.
[0115] Acute toxicity tests are based on a single administration of
an agent to the subject to determine the symptomology or lethality
of the agent. Three experiments are conducted: 1) an initial
dose-range-finding experiment, 2) an experiment to narrow the range
of effective doses, and 3) a final experiment for establishing the
dose-response curve.
[0116] Subchronic toxicity tests are based on the repeated
administration of an agent. Rat and dog are commonly used in these
studies to provide data from species in different families. With
the exception of carcinogenesis, there is considerable evidence
that daily administration of an agent at high-dose concentrations
for periods of three to four months will reveal most forms of
toxicity in adult animals.
[0117] Chronic toxicity tests, with a duration of a year or more,
are used to demonstrate either the absence of toxicity or the
carcinogenic potential of an agent. When studies are conducted on
rats, a minimum of three test groups plus one control group are
used, and animals are examined and monitored at the outset and at
intervals throughout the experiment.
[0118] Transgenic Animal Models
[0119] Transgenic rodents that over-express or under-express a gene
of interest may be inbred and used to model human diseases or to
test therapeutic or toxic agents. (See, e.g., U.S. Pat. No.
5,175,383 and U.S. Pat. No. 5,767,337.) In some cases, the
introduced gene may be activated at a specific time in a specific
tissue type during fetal or postnatal development. Expression of
the transgene is monitored by analysis of phenotype, of
tissue-specific mRNA expression, or of serum and tissue protein
levels in transgenic animals before, during, and after challenge
with experimental drug therapies.
[0120] Embryonic Stem Cells
[0121] Embryonic (ES) stem cells isolated from rodent embryos
retain the potential to form embryonic tissues. When ES cells are
placed inside a carrier embryo, they resume normal development and
contribute to tissues of the live-born animal. ES cells are the
preferred cells used in the creation of experimental knockout and
knockin rodent strains. Mouse ES cells, such as the mouse 129/SvJ
cell line, are derived from the early mouse embryo and are grown
under culture conditions well known in the art. Vectors used to
produce a transgenic strain contain a disease gene candidate and a
marker gen, the latter serves to identify the presence of the
introduced disease gene. The vector is transformed into ES cells by
methods well known in the art, and transformed ES cells are
identified and microinjected into mouse cell blastocysts such as
those from the C57BL/6 mouse strain. The blastocysts are surgically
transferred to pseudopregnant dams, and the resulting chimeric
progeny are genotyped and bred to produce heterozygous or
homozygous strains.
[0122] ES cells derived from human blastocysts may be manipulated
in vitro to differentiate into at least eight separate cell
lineages. These lineages are used to study the differentiation of
various cell types and tissues in vitro, and they include endoderm,
mesoderm, and ectodermal cell types which differentiate into, for
example, neural cells, hematopoietic lineages, and
cardiomyocytes.
[0123] Knockout Analysis
[0124] In gene knockout analysis, a region of a mammalian gene is
enzymatically modified to include a non-mammalian gene such as the
neomycin phosphotransferase gene (neo; Capecchi (1989) Science
244:1288-1292). The modified gene is transformed into cultured ES
cells and integrates into the endogenous genome by homologous
recombination. The inserted sequence disrupts transcription and
translation of the endogenous gene. Transformed cells are injected
into rodent blastulae, and the blastulae are implanted into
pseudopregnant dams. Transgenic progeny are crossbred to obtain
homozygous inbred lines which lack a functional copy of the
mammalian gene. In one example, the mammalian gene is a human
gene.
[0125] Knockin Analysis
[0126] ES cells can be used to create knockin humanized animals
(pigs) or transgenic animal models (mice or rats) of human
diseases. With knockin technology, a region of a human gene is
injected into animal ES cells, and the human sequence integrates
into the animal cell genome. Transformed cells are injected into
blastulae and the blastulae are implanted as described above.
Transgenic progeny or inbred lines are studied and treated with
potential pharmaceutical agents to obtain information on treatment
of the analogous human condition. These methods have been used to
model several human diseases.
[0127] Non-Human Primate Model
[0128] The field of animal testing deals with data and methodology
from basic sciences such as physiology, genetics, chemistry,
pharmacology and statistics. These data are paramount in evaluating
the effects of therapeutic agents on non-human primates as they can
be related to human health. Monkeys are used as human surrogates in
vaccine and drug evaluations, and their responses are relevant to
human exposures under similar conditions. Cynomolgus and Rhesus
monkeys (Macaca fascicularis and Macaca mulatta, respectively) and
Common Marmosets (Callithrix jacchus) are the most common non-human
primates (NHPs) used in these investigations. Since great cost is
associated with developing and maintaining a colony of NHPs, early
research and toxicological studies are usually carried out in
rodent models. In studies using behavioral measures such as drug
addiction, NHPs are the first choice test animal. In addition, NHPs
and individual humans exhibit differential sensitivities to many
drugs and toxins and can be classified as a range of phenotypes
from "extensive metabolizers" to "poor metabolizers" of these
agents.
[0129] In additional embodiments, the cDNAs which encode the
protein may be used in any molecular biology techniques that have
yet to be developed, provided the new techniques rely on properties
of cDNAs that are currently known, including, but not limited to,
such properties as the triplet genetic code and specific base pair
interactions.
EXAMPLES
[0130] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention. The preparation of the human SEMVNOT01 seminal vesicle
library will be described.
[0131] I cDNA Library Construction
[0132] The SEMVNOT01 cDNA library was constructed from
microscopically normal seminal vesicles removed from a 58-year-old
Caucasian male (specimen #0759B). Pathology for the associated
tumor tissue indicated adenocarcinoma of the prostate, Gleason
grade 3+2. The frozen tissue was homogenized and lysed using a
POLYTRON homogenizer (Brinkmann Instruments, Westbury N.J.) in
guanidinium isothiocyanate solution. The lysate was centrifuged
over a 5.7 M CsCl cushion using an SW28 rotor in an L8-70M
ultracentrifuge (Beckman Coulter, Fullerton Calif.) for 18 hours at
25,000 rpm at ambient temperature. The RNA was extracted with acid
phenol, pH 4.7, precipitated using 0.3 M sodium acetate and 2.5
volumes of ethanol, resuspended in RNAse-free water, and treated
with DNAse at 37 C. The RNA was reextracted and precipitated as
before. The mRNA was isolated with the OLIGOTEX kit (Qiagen,
Chatsworth Calif.) and used to construct the cDNA library.
[0133] The mRNA was handled according to the recommended protocols
in the SUPERSCRIPT plasmid system (Life Technologies) which
contains a NotI primer-adaptor designed to prime the first strand
cDNA synthesis at the poly(A) tail of mRNAs. Double stranded cDNA
was blunted, ligated to EcoRI adaptors and digested with NotI (New
England Biolabs, Beverly Mass.). The cDNAs were fractionated on a
SEPHAROSE CL4B column (APB), and those cDNAs exceeding 400 bp were
ligated into pINCY plasmid (Incyte Genomics). The plasmid pINCY was
subsequently transformed into DH5.alpha. competent cells (Life
Technologies).
[0134] II Construction of pINCY Plasmid
[0135] The plasmid was constructed by digesting the PSPORT1 plasmid
(Life Technologies) with EcoRI restriction enzyme (New England
Biolabs, Beverly Mass.) and filling the overhanging ends using
Klenow enzyme (New England Biolabs) and 2'-deoxynucleotide
5'-triphosphates (dNTPs). The plasmid was self-ligated and
transformed into the bacterial host, E. coli strain JM109.
[0136] An intermediate plasmid, pSPORT 1-.DELTA.RI, which showed no
digestion with EcoRI, was digested with Hind III (New England
Biolabs); and the overhanging ends were filled in with Klenow and
dNTPs. A linker sequence was phosphorylated, ligated onto the 5'
blunt end, digested with EcoRI, and self-ligated. Following
transformation into JM109 host cells, plasmids were isolated and
tested for preferential digestibility with EcoRI, but not with Hind
III. A single colony that met this criteria was designated pINCY
plasmid.
[0137] After testing the plasmid for its ability to incorporate
cDNAs from a library prepared using NotI and EcoRI restriction
enzymes, several clones were sequenced; and a single clone
containing an insert of approximately 0.8 kb was selected from
which to prepare a large quantity of the plasmid. After digestion
with NotI and EcoRI, the plasmid was isolated on an agarose gel and
purified using a QIAQUICK column (Qiagen) for use in library
construction.
[0138] III Isolation and Sequencing of cDNA Clones
[0139] Plasmid DNA was released from the cells and purified using
either the MINIPREP kit (Edge Biosystems, Gaithersburg Md.) or the
REAL PREP 96 plasmid kit (Qiagen). A kit consists of a 96-well
block with reagents for 960 purifications. The recommended protocol
was employed except for the following changes: 1) the bacteria were
cultured in 1 ml of sterile TERRIFIC BROTH (APB) with carbenicillin
at 25 mg/l and glycerol at 0.4%; 2) after inoculation, the cells
were cultured for 19 hours and then lysed with 0.3 ml of lysis
buffer; and 3) following isopropanol precipitation, the plasmid DNA
pellet was resuspended in 0.1 ml of distilled water. After the last
step in the protocol, samples were transferred to a 96-well block
for storage at 4 C.
[0140] The cDNAs were prepared for sequencing using the MICROLAB
2200 system (Hamilton) in combination with the DNA ENGINE thermal
cyclers (MJ Research). The cDNAs were sequenced by the method of
Sanger and Coulson (1975; J Mol Biol 94:441-448) using an ABI PRISM
377 sequencing system (Applied Biosystems) or the MEGABACE 1000 DNA
sequencing system (APB). Most of the isolates were sequenced
according to standard ABI protocols and kits (Applied Biosystems)
with solution volumes of 0.25.times.-1.0.times. concentrations. In
the alternative, cDNAs were sequenced using solutions and dyes from
APB.
[0141] IV Extension of cDNA Sequences
[0142] The cDNAs were extended using the cDNA clone and
oligonucleotide primers. One primer was synthesized to initiate 5'
extension of the known fragment, and the other, to initiate 3'
extension of the known fragment. The initial primers were designed
using commercially available primer analysis software to be about
22 to 30 nucleotides in length, to have a GC content of about 50%
or more, and to anneal to the target sequence at temperatures of
about 68 C. to about 72 C. Any stretch of nucleotides that would
result in hairpin structures and primer-primer dimerizations was
avoided.
[0143] Selected cDNA libraries were used as templates to extend the
sequence. If more than one extension was necessary, additional or
nested sets of primers were designed. Preferred libraries have been
size-selected to include larger cDNAs and random primed to contain
more sequences with 5' or upstream regions of genes. Genomic
libraries are used to obtain regulatory elements, especially
extension into the 5' promoter binding region.
[0144] High fidelity amplification was obtained by PCR using
methods such as that taught in U.S. Pat. No. 5,932,451. PCR was
performed in 96-well plates using the DNA ENGINE thermal cycler (MJ
Research). The reaction mix contained DNA template, 200 nmol of
each primer, reaction buffer containing Mg.sup.2+,
(NH.sub.4).sub.2SO.sub.4, and .beta.-mercaptoethanol, Taq DNA
polymerase (APB), ELONGASE enzyme (Life Technologies), and Pfu DNA
polymerase (Stratagene), with the following parameters for primer
pair PCI A and PCI B (Incyte Genomics): Step 1: 94 C., three min;
Step 2: 94 C., 15 sec; Step 3: 60 C., one min; Step 4: 68 C., two
min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 C.,
five min; Step 7: storage at 4 C. In the alternative, the
parameters for primer pair T7 and SK+ (Stratagene) were as follows:
Step 1: 94 C., three min; Step 2: 94 C., 15 sec; Step 3: 57 C., one
min; Step 4: 68 C., two min; Step 5: Steps 2, 3, and 4 repeated 20
times; Step 6: 68 C., five min; Step 7: storage at 4 C.
[0145] The concentration of DNA in each well was determined by
dispensing 100 .mu.l PICOGREEN quantitation reagent (0.25% reagent
in 1.times. TE, v/v; Molecular Probes) and 0.5 .mu.l of undiluted
PCR product into each well of an opaque fluorimeter plate (Corning,
Acton Mass.) and allowing the DNA to bind to the reagent. The plate
was scanned in a Fluoroskan II (Labsystems Oy) to measure the
fluorescence of the sample and to quantify the concentration of
DNA. A 5 .mu.l to 10 .mu.l aliquot of the reaction mixture was
analyzed by electrophoresis on a 1% agarose minigel to determine
which reactions were successful in extending the sequence.
[0146] The extended clones were desalted, concentrated, transferred
to 384-well plates, digested with CviJI cholera virus endonuclease
(Molecular Biology Research, Madison Wis.), and sonicated or
sheared prior to religation into pUC18 vector (APB). For shotgun
sequences, the digested nucleotide sequences were separated on low
concentration (0.6 to 0.8%) agarose gels, fragments were excised,
and the agar was digested with AGARACE enzyme (Promega). Extended
clones were religated using T4 DNA ligase (New England Biolabs)
into pUC18 vector (APB), treated with Pfu DNA polymerase
(Stratagene) to fill-in restriction site overhangs, and transfected
into E. coli competent cells. Transformed cells were selected on
antibiotic-containing media, and individual colonies were picked
and cultured overnight at 37 C. in 384-well plates in LB/2.times.
carbenicillin liquid media.
[0147] The cells were lysed, and DNA was amplified using primers,
Taq DNA polymerase (APB) and Pfu DNA polymerase (Stratagene) with
the following parameters: Step 1: 94 C., three min; Step 2: 94 C.,
15 sec; Step 3: 60 C., one min; Step 4: 72 C., two min; Step 5:
steps 2, 3, and 4 repeated 29 times; Step 6: 72 C., five min; Step
7: storage at 4 C. DNA was quantified using PICOGREEN quantitation
reagent (Molecular Probes) as described above. Samples with low DNA
recoveries were reamplified using the conditions described above.
Samples were diluted with 20% dimethylsulfoxide (DMSO; 1:2, v/v),
and sequenced using DYENAMIC energy transfer sequencing primers and
the DYENAMIC DIRECT cycle sequencing kit (APB) or the ABI PRISM
BIGDYE terminator cycle sequencing kit (Applied Biosystems).
[0148] V Homology Searching of cDNA Clones and Their Deduced
Proteins
[0149] The cDNAs of the Sequence Listing or their deduced amino
acid sequences were used to query databases such as GenBank,
SwissProt, BLOCKS, and the like. These databases that contain
previously identified and annotated sequences or domains were
searched using BLAST or BLAST2 to produce alignments and to
determine which sequences were exact matches or homologs. The
alignments were to sequences of prokaryotic (bacterial) or
eukaryotic (animal, fungal, or plant) origin. Alternatively,
algorithms such as the one described in Smith and Smith (1992,
Protein Engineering 5:35-51) could have been used to deal with
primary sequence patterns and secondary structure gap penalties.
All of the sequences disclosed in this application have lengths of
at least 49 nucleotides, and no more than 12% uncalled bases (where
N is recorded rather than A, C, G, or T).
[0150] As detailed in Karlin (supra), BLAST matches between a query
sequence and a database sequence were evaluated statistically and
only reported when they satisfied the threshold of 10.sup.-25 for
nucleotides and 10.sup.-14 for peptides. Homology was also
evaluated by product score calculated as follows: the % nucleotide
or amino acid identity [between the query and reference sequences]
in BLAST is multiplied by the % maximum possible BLAST score [based
on the lengths of query and reference sequences] and then divided
by 100. In comparison with hybridization procedures used in the
laboratory, the stringency for an exact match was set from a lower
limit of about 40 (with 1-2% error due to uncalled bases) to a 100%
match of about 70.
[0151] The BLAST software suite (NCBI, Bethesda Md.;
http://www.ncbi.nlm.nih.gov/gorf/b12.html), includes various
sequence analysis programs including "blastn" that is used to align
nucleotide sequences and BLAST2 that is used for direct pairwise
comparison of either nucleotide or amino acid sequences. BLAST
programs are commonly used with gap and other parameters set to
default settings, e.g.: Matrix: BLOSUM62; Reward for match: 1;
Penalty for mismatch: -2; Open Gap: 5 and Extension Gap: 2
penalties; Gap.times.drop-off: 50; Expect: 10; Word Size: 11; and
Filter: on. Identity is measured over the entire length of a
sequence. Brenner et al. (1998; Proc Natl Acad Sci 95:6073-6078,
incorporated herein by reference) analyzed BLAST for its ability to
identify structural homologs by sequence identity and found 30%
identity is a reliable threshold for sequence alignments of at
least 150 residues and 40%, for alignments of at least 70
residues.
[0152] The cDNAs of this application were compared with assembled
consensus sequences or templates found in the LIFESEQ GOLD database
(Incyte Genomics). Component sequences from cDNA, extension, full
length, and shotgun sequencing projects were subjected to PHRED
analysis and assigned a quality score. All sequences with an
acceptable quality score were subjected to various pre-processing
and editing pathways to remove low quality 3' ends, vector and
linker sequences, polyA tails, Alu repeats, mitochondrial and
ribosomal sequences, and bacterial contamination sequences. Edited
sequences had to be at least 50 bp in length, and low-information
sequences and repetitive elements such as dinucleotide repeats, Alu
repeats, and the like, were replaced by "Ns" or masked.
[0153] Edited sequences were subjected to assembly procedures in
which the sequences were assigned to gene bins. Each sequence could
only belong to one bin, and sequences in each bin were assembled to
produce a template. Newly sequenced components were added to
existing bins using BLAST and CROSSMATCH. To be added to a bin, the
component sequences had to have a BLAST quality score greater than
or equal to 150 and an alignment of at least 82% local identity.
The sequences in each bin were assembled using PHRAP. Bins with
several overlapping component sequences were assembled using DEEP
PHRAP. The orientation of each template was determined based on the
number and orientation of its component sequences.
[0154] Bins were compared to one another, and those having local
similarity of at least 82% were combined and reassembled. Bins
having templates with less than 95% local identity were split.
Templates were subjected to analysis by STITCHER/EXON MAPPER
algorithms that determine the probabilities of the presence of
splice variants, alternatively spliced exons, splice junctions,
differential expression of alternative spliced genes across tissue
types or disease states, and the like. Assembly procedures were
repeated periodically, and templates were annotated using BLAST
against GenBank databases such as GBpri. An exact match was defined
as having from 95% local identity over 200 base pairs through 100%
local identity over 100 base pairs and a homolog match as having an
E-value (or probability score) of .ltoreq.1.times.10.sup.-8. The
templates were also subjected to frameshift FASTx against GENPEPT,
and homolog match was defined as having an E-value of
.ltoreq.1.times.10.sup.-8. Template analysis and assembly was
described in U.S. Ser. No. 09/276,534, filed Mar. 25, 1999.
[0155] Following assembly, templates were subjected to BLAST,
motif, and other functional analyses and categorized in protein
hierarchies using methods described in U.S. Ser. No. 08/812,290 and
U.S. Ser. No. 08/811,758, both filed Mar. 6, 1997; in U.S. Ser. No.
08/947,845, filed Oct. 9, 1997; and in U.S. Ser. No. 09/034,807,
filed Mar. 4, 1998. Then templates were analyzed by translating
each template in all three forward reading frames and searching
each translation against the PFAM database of hidden Markov
model-based protein families and domains using the HMMER software
package (Washington University School of Medicine, St. Louis Mo.;
http://pfam.wustl.edu/). The cDNA was further analyzed using
MACDNASIS PRO software (Hitachi Software Engineering), and
LASERGENE software (DNASTAR) and queried against public databases
such as the GenBank rodent, mammalian, vertebrate, prokaryote, and
eukaryote databases, SwissProt, BLOCKS, PRINTS, PFAM, and
Prosite.
[0156] VI Chromosome Mapping
[0157] Radiation hybrid and genetic mapping data available from
public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for Genome Research (WIGR), and Gnthon are used
to determine if any of the cDNAs presented in the Sequence Listing
have been mapped. Any of the fragments of the cDNA encoding NRP
that have been mapped result in the assignment of all related
regulatory and coding sequences mapping to the same location. The
genetic map locations are described as ranges, or intervals, of
human chromosomes. The map position of an interval, in cM (which is
roughly equivalent to 1 megabase of human DNA), is measured
relative to the terminus of the chromosomal p-arm.
[0158] VII Hybridization Technologies and Analyses
[0159] Immobilization of cDNAs on a Substrate
[0160] The cDNAs are applied to a substrate by one of the following
methods. A mixture of cDNAs is fractionated by gel electrophoresis
and transferred to a nylon membrane by capillary transfer.
Alternatively, the cDNAs are individually ligated to a vector and
inserted into bacterial host cells to form a library. The cDNAs are
then arranged on a substrate by one of the following methods. In
the first method, bacterial cells containing individual clones are
robotically picked and arranged on a nylon membrane. The membrane
is placed on LB agar containing selective agent (carbenicillin,
kanamycin, ampicillin, or chloramphenicol depending on the vector
used) and incubated at 37 C. for 16 hr. The membrane is removed
from the agar and consecutively placed colony side up in 10% SDS,
denaturing solution (1.5 M NaCl, 0.5 M NaOH), neutralizing solution
(1.5 M NaCl, 1 M Tris, pH 8.0), and twice in 2.times.SSC for 10 min
each. The membrane is then UV irradiated in a STRATALINKER
UV-crosslinker (Stratagene).
[0161] In the second method, cDNAs are amplified from bacterial
vectors by thirty cycles of PCR using primers complementary to
vector sequences flanking the insert. PCR amplification increases a
starting concentration of 1-2 ng nucleic acid to a final quantity
greater than 5 .mu.g. Amplified nucleic acids from about 400 bp to
about 5000 bp in length are purified using SEPHACRYL-400 beads
(APB). Purified nucleic acids are arranged on a nylon membrane
manually or using a dot/slot blotting manifold and suction device
and are immobilized by denaturation, neutralization, and UV
irradiation as described above. Purified nucleic acids are
robotically arranged and immobilized on polymer-coated glass slides
using the procedure described in U.S. Pat. No. 5,807,522.
Polymer-coated slides are prepared by cleaning glass microscope
slides (Corning, Acton Mass.) by ultrasound in 0.1% SDS and
acetone, etching in 4% hydrofluoric acid (VWR Scientific Products,
West Chester Pa.), coating with 0.05% aminopropyl silane (Sigma
Aldrich) in 95% ethanol, and curing in a 110 C. oven. The slides
are washed extensively with distilled water between and after
treatments. The nucleic acids are arranged on the slide and then
immobilized by exposing the array to UV irradiation using a
STRATALINKER UV-crosslinker (Stratagene). Arrays are then washed at
room temperature in 0.2% SDS and rinsed three times in distilled
water. Non-specific binding sites are blocked by incubation of
arrays in 0.2% casein in phosphate buffered saline (PBS; Tropix,
Bedford Mass.) for 30 min at 60 C.; then the arrays are washed in
0.2% SDS and rinsed in distilled water as before.
[0162] Probe Preparation for Membrane Hybridization
[0163] Hybridization probes derived from the cDNAs of the Sequence
Listing are employed for screening cDNAs, mRNAs, or genomic DNA in
membrane-based hybridizations. Probes are prepared by diluting the
cDNAs to a concentration of 40-50 ng in 45 .mu.l TE buffer,
denaturing by heating to 100 C. for five min, and briefly
centrifuging. The denatured cDNA is then added to a REDIPRIME tube
(APB), gently mixed until blue color is evenly distributed, and
briefly centrifuged. Five .mu.l of [.sup.32P]dCTP is added to the
tube, and the contents are incubated at 37 C. for 10 min. The
labeling reaction is stopped by adding 5 .mu.l of 0.2M EDTA, and
probe is purified from unincorporated nucleotides using a
PROBEQUANT G-50 microcolumn (APB). The purified probe is heated to
100 C. for five min, snap cooled for two min on ice, and used in
membrane-based hybridizations as described below.
[0164] Probe Preparation for Polymer Coated Slide Hybridization
[0165] Hybridization probes derived from mRNA isolated from samples
are employed for screening cDNAs of the Sequence Listing in
array-based hybridizations. Probe is prepared using the GEMbright
kit (Incyte Genomics) by diluting mRNA to a concentration of 200 ng
in 9 .mu.l TE buffer and adding 5 .mu.l 5.times. buffer, 1 .mu.l
0.1 M DTT, 3 .mu.l Cy3 or Cy5 labeling mix, 1 .mu.l RNase
inhibitor, 1 .mu.l reverse transcriptase, and 5 .mu.l 1.times.
yeast control mRNAs. Yeast control mRNAs are synthesized by in
vitro transcription from noncoding yeast genomic DNA (W. Lei,
unpublished). As quantitative controls, one set of control mRNAs at
0.002 ng, 0.02 ng, 0.2 ng, and 2 ng are diluted into reverse
transcription reaction mixture at ratios of 1:100,000, 1:10,000,
1:1000, and 1:100 (w/w) to sample mRNA respectively. To examine
mRNA differential expression patterns, a second set of control
mRNAs are diluted into reverse transcription reaction mixture at
ratios of 1:3, 3:1, 1:10, 10:1, 1:25, and 25:1 (w/w). The reaction
mixture is mixed and incubated at 37 C. for two hr. The reaction
mixture is then incubated for 20 min at 85 C., and probes are
purified using two successive CHROMA SPIN+TE 30 columns (Clontech,
Palo Alto Calif.). Purified probe is ethanol precipitated by
diluting probe to 90 .mu.l in DEPC-treated water, adding 2 .mu.l 1
mg/ml glycogen, 60 .mu.l 5 M sodium acetate, and 300 .mu.l 100%
ethanol. The probe is centrifuged for 20 min at 20,800.times.g, and
the pellet is resuspended in 12 .mu.l resuspension buffer, heated
to 65 C. for five min, and mixed thoroughly. The probe is heated
and mixed as before and then stored on ice. Probe is used in high
density array-based hybridizations as described below.
[0166] Membrane-based Hybridization
[0167] Membranes are pre-hybridized in hybridization solution
containing 1% Sarkosyl and 1.times. high phosphate buffer (0.5 M
NaCl, 0.1 M Na.sub.2HPO.sub.4, 5 mM EDTA, pH 7) at 55 C. for two
hr. The probe, diluted in 15 ml fresh hybridization solution, is
then added to the membrane. The membrane is hybridized with the
probe at 55 C. for 16 hr. Following hybridization, the membrane is
washed for 15 min at 25 C. in 1 mM Tris (pH 8.0), 1% Sarkosyl, and
four times for 15 min each at 25 C. in 1 mM Tris (pH 8.0). To
detect hybridization complexes, XOMAT-AR film (Eastman Kodak,
Rochester N.Y.) is exposed to the membrane overnight at -70 C.,
developed, and examined visually.
[0168] Polymer Coated Slide-based Hybridization
[0169] Probe is heated to 65 C. for five min, centrifuged five min
at 9400 rpm in a 5415 C. microcentrifuge (Eppendorf Scientific,
Westbury N.Y.), and then 18 .mu.l is aliquoted onto the array
surface and covered with a coverslip. The arrays are transferred to
a waterproof chamber having a cavity just slightly larger than a
microscope slide. The chamber is kept at 100% humidity internally
by the addition of 140 .mu.l of 5.times.SSC in a corner of the
chamber. The chamber containing the arrays is incubated for about
6.5 hr at 60 C. The arrays are washed for 10 min at 45 C. in
1.times.SSC, 0.1% SDS, and three times for 10 min each at 45 C. in
0.1.times.SSC, and dried.
[0170] Hybridization reactions are performed in absolute or
differential hybridization formats. In the absolute hybridization
format, probe from one sample is hybridized to array elements, and
signals are detected after hybridization complexes form. Signal
strength correlates with probe mRNA levels in the sample. In the
differential hybridization format, differential expression of a set
of genes in two biological samples is analyzed. Probes from the two
samples are prepared and labeled with different labeling moieties.
A mixture of the two labeled probes is hybridized to the array
elements, and signals are examined under conditions in which the
emissions from the two different labels are individually
detectable. Elements on the array that are hybridized to
substantially equal numbers of probes derived from both biological
samples give a distinct combined fluorescence (Shalon
WO95/35505).
[0171] Hybridization complexes are detected with a microscope
equipped with an Innova 70 mixed gas 10 W laser (Coherent, Santa
Clara Calif.) capable of generating spectral lines at 488 nm for
excitation of Cy3 and at 632 nm for excitation of Cy5. The
excitation laser light is focused on the array using a 20.times.
microscope objective (Nikon, Melville N.Y.). The slide containing
the array is placed on a computer-controlled X-Y stage on the
microscope and raster-scanned past the objective with a resolution
of 20 micrometers. In the differential hybridization format, the
two fluorophores are sequentially excited by the laser. Emitted
light is split, based on wavelength, into two photomultiplier tube
detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater
N.J.) corresponding to the two fluorophores. Appropriate filters
positioned between the array and the photomultiplier tubes are used
to filter the signals. The emission maxima of the fluorophores used
are 565 nm for Cy3 and 650 nm for Cy5. The sensitivity of the scans
is calibrated using the signal intensity generated by the yeast
control mRNAs added to the probe mix. A specific location on the
array contains a complementary DNA sequence, allowing the intensity
of the signal at that location to be correlated with a weight ratio
of hybridizing species of 1:100,000.
[0172] The output of the photomultiplier tube is digitized using a
12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog
Devices, Norwood Mass.) installed in an IBM-compatible PC computer.
The digitized data are displayed as an image where the signal
intensity is mapped using a linear 20-color transformation to a
pseudocolor scale ranging from blue (low signal) to red (high
signal). The data is also analyzed quantitatively. Where two
different fluorophores are excited and measured simultaneously, the
data are first corrected for optical crosstalk (due to overlapping
emission spectra) between the fluorophores using the emission
spectrum for each fluorophore. A grid is superimposed over the
fluorescence signal image such that the signal from each spot is
centered in each element of the grid. The fluorescence signal
within each element is then integrated to obtain a numerical value
corresponding to the average intensity of the signal. The software
used for signal analysis is the GEMTOOLS program (Incyte
Genomics).
[0173] VIII Electronic Analysis
[0174] BLAST was used to search for identical or related molecules
in the GenBank or LIFESEQ databases (Incyte Genomics). The product
score for human and rat sequences was calculated as follows: the
BLAST score is multiplied by the % nucleotide identity and the
product is divided by (5 times the length of the shorter of the two
sequences), such that a 100% alignment over the length of the
shorter sequence gives a product score of 100. The product score
takes into account both the degree of similarity between two
sequences and the length of the sequence match. For example, with a
product score of 40, the match will be exact within a 1% to 2%
error, and with a product score of at least 70, the match will be
exact. Similar or related molecules are usually identified by
selecting those which show product scores between 8 and 40.
[0175] Electronic northern analysis was performed at a product
score of 70 and is shown in Tables 1 and 2. All sequences and cDNA
libraries in the LIFESEQ database were categorized by system,
organ/tissue and cell type. The categories included cardiovascular
system, connective tissue, digestive system, embryonic structures,
endocrine system, exocrine glands, female and male genitalia, germ
cells, hemic/immune system, liver, musculoskeletal system, nervous
system, pancreas, respiratory system, sense organs, skin,
stomatognathic system, unclassified/mixed, and the urinary tract.
For each category, the number of libraries in which the sequence
was expressed were counted and shown over the total number of
libraries in that category. In a non-normalized library, expression
levels of two or more are significant.
[0176] IX Complementary Molecules
[0177] Molecules complementary to the cDNA, from about 5 (PNA) to
about 5000 bp (complement of a cDNA insert), are used to detect or
inhibit gene expression. Detection is described in Example VII. To
inhibit transcription by preventing promoter binding, the
complementary molecule is designed to bind to the most unique 5'
sequence and includes nucleotides of the 5' UTR upstream of the
initiation codon of the open reading frame. Complementary molecules
include genomic sequences (such as enhancers or introns) and are
used in "triple helix" base pairing to compromise the ability of
the double helix to open sufficiently for the binding of
polymerases, transcription factors, or regulatory molecules. To
inhibit translation, a complementary molecule is designed to
prevent ribosomal binding to the mRNA encoding the protein.
[0178] Complementary molecules are placed in expression vectors and
used to transform a cell line to test efficacy; into an organ,
tumor, synovial cavity, or the vascular system for transient or
short term therapy; or into a stem cell, zygote, or other
reproducing lineage for long term or stable gene therapy. Transient
expression lasts for a month or more with a non-replicating vector
and for three months or more if appropriate elements for inducing
vector replication are used in the transformation/expression
system.
[0179] Stable transformation of appropriate dividing cells with a
vector encoding the complementary molecule produces a transgenic
cell line, tissue, or organism (U.S. Pat. No. 4,736,866). Those
cells that assimilate-and replicate sufficient quantities of the
vector to allow stable integration also produce enough
complementary molecules to compromise or entirely eliminate
activity of the cDNA encoding the protein.
[0180] X Expression of NRP
[0181] Expression and purification of the protein are achieved
using either a mammalian cell expression system or an insect cell
expression system. The pUB6/V5-His vector system (Invitrogen,
Carlsbad Calif.) is used to express NRP in CHO cells. The vector
contains the selectable bsd gene, multiple cloning sites, the
promoter/enhancer sequence from the human ubiquitin C gene, a
C-terminal V5 epitope for antibody detection with anti-V5
antibodies, and a C-terminal polyhistidine (6.times.His) sequence
for rapid purification on PROBOND resin (Invitrogen). Transformed
cells are selected on media containing blasticidin.
[0182] Spodoptera frugiperda (Sf9) insect cells are infected with
recombinant Autographica californica nuclear polyhedrosis virus
(baculovirus). The polyhedrin gene is replaced with the cDNA by
homologous recombination and the polyhedrin promoter drives cDNA
transcription. The protein is synthesized as a fusion protein with
6.times.his which enables purification as described above. Purified
protein is used in the following activity and to make
antibodies
[0183] XI Production of Antibodies
[0184] NRP is purified using polyacrylamide gel electrophoresis and
used to immunize mice or rabbits. Antibodies are produced using the
protocols below. Alternatively, the amino acid sequence of NRP is
analyzed using LASERGENE software (DNASTAR) to determine regions of
high antigenicity. An antigenic epitope, usually found near the
C-terminus or in a hydrophilic region is selected, synthesized, and
used to raise antibodies. Typically, epitopes of about 15 residues
in length are produced using an ABI 431A peptide synthesizer
(Applied Biosystems) using Fmoc-chemistry and coupled to KLH
(Sigma-Aldrich) by reaction with
N-maleimidobenzoyl-N-hydroxysuccinimide ester to increase
antigenicity.
[0185] Rabbits are immunized with the epitope-KLH complex in
complete Freund's adjuvant. Immunizations are repeated at intervals
thereafter in incomplete Freund's adjuvant. After a minimum of
seven weeks for mouse or twelve weeks for rabbit, antisera are
drawn and tested for antipeptide activity. Testing involves binding
the peptide to plastic, blocking with 1% bovine serum albumin,
reacting with rabbit antisera, washing, and reacting with
radio-iodinated goat anti-rabbit IgG. Methods well known in the art
are used to determine antibody titer and the amount of complex
formation.
[0186] XII Purification of Naturally Occurring Protein Using
Specific Antibodies
[0187] Naturally occurring or recombinant protein is purified by
immunoaffinity chromatography using antibodies which specifically
bind the protein. An immunoaffinity column is constructed by
covalently coupling the antibody to CNBr-activated SEPHAROSE resin
(APB). Media containing the protein is passed over the
immunoaffinity column, and the column is washed using high ionic
strength buffers in the presence of detergent to allow preferential
absorbance of the protein. After coupling, the protein is eluted
from the column using a buffer of pH 2-3 or a high concentration of
urea or thiocyanate ion to disrupt antibody/protein binding, and
the protein is collected.
[0188] XIII Screening Molecules for Specific Binding with the cDNA
or Protein
[0189] The cDNA, or fragments thereof, or the protein, or portions
thereof, are labeled with .sup.32P-dCTP, Cy3-dCTP, or Cy5-dCTP
(APB), or with BIODIPY or FITC (Molecular Probes, Eugene Oreg.),
respectively. Libraries of candidate molecules or compounds
previously arranged on a substrate are incubated in the presence of
labeled cDNA or protein. After incubation under conditions for
either a nucleic acid or amino acid sequence, the substrate is
washed, and any position on the substrate retaining label, which
indicates specific binding or complex formation, is assayed, and
the ligand is identified. Data obtained using different
concentrations of the nucleic acid or protein are used to calculate
affinity between the labeled nucleic acid or protein and the bound
molecule.
[0190] XIV Two-Hybrid Screen
[0191] A yeast two-hybrid system, MATCHMAKER LexA Two-Hybrid system
(Clontech Laboratories, Palo Alto Calif.), is used to screen for
peptides that bind the protein of the invention. A cDNA encoding
the protein is inserted into the multiple cloning site of a pLexA
vector, ligated, and transformed into E. coli. cDNA, prepared from
mRNA, is inserted into the multiple cloning site of a pB42AD
vector, ligated, and transformed into E. coli to construct a cDNA
library. The pLexA plasmid and pB42AD-cDNA library constructs are
isolated from E. coli and used in a 2:1 ratio to co-transform
competent yeast EGY48[p8op-lacZ] cells using a polyethylene
glycol/lithium acetate protocol. Transformed yeast cells are plated
on synthetic dropout (SD) media lacking histidine (-His),
tryptophan (-Trp), and uracil (-Ura), and incubated at 30 C. until
the colonies have grown up and are counted. The colonies are pooled
in a minimal volume of 1.times. TE (pH 7.5), replated on
SD/-His/-Leu/-Trp/-Ura media supplemented with 2% galactose (Gal),
1% raffinose (Raf), and 80 mg/ml 5-bromo-4-chloro-3-indolyl
.beta.-d-galactopyranoside (X-Gal), and subsequently examined for
growth of blue colonies. Interaction between expressed protein and
cDNA fusion proteins activates expression of a LEU2 reporter gene
in EGY48 and produces colony growth on media lacking leucine
(-Leu). Interaction also activates expression of
.beta.-galactosidase from the p8op-lacZ reporter construct that
produces blue color in colonies grown on X-Gal.
[0192] Positive interactions between expressed protein and cDNA
fusion proteins are verified by isolating individual positive
colonies and growing them in SD/-Trp/-Ura liquid medium for 1 to 2
days at 30 C. A sample of the culture is plated on SD/-Trp/-Ura
media and incubated at 30 C. until colonies appear. The sample is
replica-plated on SD/-Trp/-Ura and SD/-His/-Trp/-Ura plates.
Colonies that grow on SD containing histidine but not on media
lacking histidine have lost the pLexA plasmid. Histidine-requiring
colonies are grown on SD/Gal/Raf/X-Gal/-Trp/-Ura, and white
colonies are isolated and propagated. The pB42AD-cDNA plasmid,
which contains a cDNA encoding a protein that physically interacts
with the protein, is isolated from the yeast cells and
characterized.
[0193] XV NRP Assay
[0194] Assays of transcriptional regulation of NRP are performed in
human carcinoma cell lines as described by Ulrix et al. (supra).
NRP expression is monitored in the presence and absence of
androgens, retinoic acid, homocysteine, or nickel compounds. Total
RNA is determined by Northern blot analysis.
[0195] All patents and publications mentioned in the specification
are incorporated by reference herein. 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 described modes for carrying out the invention
that 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
33 1 371 PRT Homo sapiens misc_feature Incyte ID No 2227688CD1 1
Met Ala Glu Leu Gln Glu Val Gln Ile Thr Glu Glu Lys Pro Leu 1 5 10
15 Leu Pro Gly Gln Thr Pro Glu Ala Ala Lys Glu Ala Glu Leu Ala 20
25 30 Ala Arg Ile Leu Leu Asp Gln Gly Gln Thr His Ser Val Glu Thr
35 40 45 Pro Tyr Gly Ser Val Thr Phe Thr Val Tyr Gly Thr Pro Lys
Pro 50 55 60 Lys Arg Pro Ala Ile Leu Thr Tyr His Asp Val Gly Leu
Asn Tyr 65 70 75 Lys Ser Cys Phe Gln Pro Leu Phe Gln Phe Glu Asp
Met Gln Glu 80 85 90 Ile Ile Gln Asn Phe Val Arg Val His Val Asp
Ala Pro Gly Met 95 100 105 Glu Glu Gly Ala Pro Val Phe Pro Leu Gly
Tyr Gln Tyr Pro Ser 110 115 120 Leu Asp Gln Leu Ala Asp Met Ile Pro
Cys Val Leu Gln Tyr Leu 125 130 135 Asn Phe Ser Thr Ile Ile Gly Val
Gly Val Gly Ala Gly Ala Tyr 140 145 150 Ile Leu Ala Arg Tyr Ala Leu
Asn His Pro Asp Thr Val Glu Gly 155 160 165 Leu Val Leu Ile Asn Ile
Asp Pro Asn Ala Lys Gly Trp Met Asp 170 175 180 Trp Ala Ala His Lys
Leu Thr Gly Leu Thr Ser Ser Ile Pro Glu 185 190 195 Met Ile Leu Gly
His Leu Phe Ser Gln Glu Glu Leu Ser Gly Asn 200 205 210 Ser Glu Leu
Ile Gln Lys Tyr Arg Asn Ile Ile Thr His Ala Pro 215 220 225 Asn Leu
Asp Asn Ile Glu Leu Tyr Trp Asn Ser Tyr Asn Asn Arg 230 235 240 Arg
Asp Leu Asn Phe Glu Arg Gly Gly Asp Ile Thr Leu Arg Cys 245 250 255
Pro Val Met Leu Val Val Gly Asp Gln Ala Pro His Glu Asp Ala 260 265
270 Val Val Glu Cys Asn Ser Lys Leu Asp Pro Thr Gln Thr Ser Phe 275
280 285 Leu Lys Met Ala Asp Ser Gly Gly Gln Pro Gln Leu Thr Gln Pro
290 295 300 Gly Lys Leu Thr Glu Ala Phe Lys Tyr Phe Leu Gln Gly Met
Gly 305 310 315 Tyr Met Ala Ser Ser Cys Met Thr Arg Leu Ser Arg Ser
Arg Thr 320 325 330 Ala Ser Leu Thr Ser Ala Ala Ser Val Asp Gly Asn
Arg Ser Arg 335 340 345 Ser Arg Thr Leu Ser Gln Ser Ser Glu Ser Gly
Thr Leu Ser Ser 350 355 360 Gly Pro Pro Gly His Thr Met Glu Val Ser
Cys 365 370 2 402 PRT Homo sapiens misc_feature Incyte ID No
3507515CD1 2 Met Ala Glu Leu Gln Glu Val Gln Ile Thr Glu Glu Lys
Pro Leu 1 5 10 15 Leu Pro Gly Gln Thr Pro Glu Ala Ala Lys Thr His
Ser Val Glu 20 25 30 Thr Pro Tyr Gly Ser Val Thr Phe Thr Val Tyr
Gly Thr Pro Lys 35 40 45 Pro Lys Arg Pro Ala Ile Leu Thr Tyr His
Asp Val Gly Leu Asn 50 55 60 Tyr Lys Ser Cys Phe Gln Pro Leu Phe
Gln Phe Glu Asp Met Gln 65 70 75 Glu Ile Ile Gln Asn Phe Val Arg
Val His Val Asp Ala Pro Gly 80 85 90 Met Glu Glu Gly Ala Pro Val
Phe Pro Leu Gly Tyr Gln Tyr Pro 95 100 105 Ser Leu Asp Gln Leu Ala
Asp Met Ile Pro Cys Val Leu Gln Tyr 110 115 120 Leu Asn Phe Ser Thr
Ile Ile Gly Val Gly Val Gly Ala Gly Ala 125 130 135 Tyr Ile Leu Ala
Arg Tyr Ala Leu Asn His Pro Asp Thr Val Glu 140 145 150 Gly Leu Val
Leu Ile Asn Ile Asp Pro Asn Ala Lys Gly Trp Met 155 160 165 Asp Trp
Ala Ala His Lys Leu Thr Gly Leu Thr Ser Ser Ile Pro 170 175 180 Glu
Met Ile Leu Gly His Leu Phe Ser Gln Glu Glu Leu Ser Gly 185 190 195
Asn Ser Glu Leu Ile Gln Lys Tyr Arg Asn Ile Ile Thr His Ala 200 205
210 Pro Asn Leu Asp Asn Ile Glu Leu Tyr Trp Asn Ser Tyr Asn Asn 215
220 225 Arg Arg Asp Leu Asn Phe Glu Arg Gly Gly Asp Ile Thr Leu Arg
230 235 240 Cys Pro Val Met Leu Val Val Gly Asp Gln Ala Pro His Glu
Asp 245 250 255 Ala Val Val Glu Cys Asn Ser Lys Leu Asp Pro Thr Gln
Thr Ser 260 265 270 Phe Leu Lys Met Ala Asp Ser Gly Gly Gln Pro Gln
Leu Thr Gln 275 280 285 Pro Gly Lys Leu Thr Glu Ala Phe Lys Tyr Phe
Leu Gln Gly Met 290 295 300 Gly Tyr Met Ala Ser Ser Cys Met Thr Arg
Leu Ser Arg Ser Arg 305 310 315 Thr Ala Ser Leu Thr Ser Ala Ala Ser
Val Asp Gly Xaa Arg Ser 320 325 330 Arg Ser Arg Thr Leu Ser Gln Ser
Ser Glu Ser Gly Thr Leu Phe 335 340 345 Phe Gly Gly Pro Arg Gly His
Thr Met Gly Gly Leu Leu Leu Asn 350 355 360 Gly Pro Cys Cys Pro Arg
Val Gly Pro Ser Pro Gln Leu Xaa Gln 365 370 375 Ser Asn Leu Xaa Gly
Ala Glu Arg Gly His Trp Gly His Arg Lys 380 385 390 Gln Arg Gly Lys
Arg Ala Asp Ser Trp Arg Gly Arg 395 400 3 2055 DNA Homo sapiens
misc_feature Incyte ID No 2227688CB1 3 agagcaggcg tcgggacgca
gcaaagagag gagagacccc agagtcagaa ggagtgagaa 60 ccctgacccc
taatcccact gcatccagcc aataggagcc cagccaccat ggcggagctg 120
caggaggtgc agatcacaga ggagaagcca ctgttgccag gacagacgcc tgaggcggcc
180 aaggaggctg agttagctgc ccgaatcctc ctggaccagg gacagactca
ctctgtggag 240 acaccatacg gctctgtcac tttcactgtc tatggcaccc
ccaaacccaa acgcccagcg 300 atccttacct accacgatgt gggactcaac
tataaatctt gcttccagcc actgtttcag 360 ttcgaggaca tgcaggaaat
cattcagaac tttgtgcggg ttcatgtgga tgcccctgga 420 atggaagagg
gagcccctgt gttccctttg ggatatcagt acccatctct ggaccagctt 480
gcagacatga tcccttgcgt cctgcagtac ctaaatttct ctacaataat tggagttggt
540 gttggagctg gagcctacat cctggcgaga tatgctctta accacccgga
cactgttgaa 600 ggtcttgtcc tcatcaacat tgatcccaat gccaagggtt
ggatggattg ggcagcccac 660 aagctaacag gcctcacctc ttccattccg
gagatgatcc ttggacatct tttcagccag 720 gaagagctct ctggaaattc
tgagttgata caaaagtaca gaaatatcat tacacatgca 780 cccaacctgg
ataacattga attgtactgg aacagctaca acaaccgccg agacctgaac 840
tttgagcgtg gaggtgatat caccctcagg tgtcctgtga tgctggtggt aggagaccaa
900 gcacctcatg aagatgcagt ggtggaatgt aactcaaaac tggaccccac
ccagacctcg 960 ttcctcaaga tggctgactc cggaggtcag ccccagctga
ctcagccagg caagctgacc 1020 gaggccttca agtacttcct gcaaggcatg
ggctacatgg cctcatcctg catgactcgc 1080 ctgtcccggt ctcgtacagc
ctctctgacc agtgcagcat ccgttgatgg caaccggtcc 1140 cgctctcgca
ccctgtccca gagcagcgag tctggaactc tttcttcggg gcccccgggg 1200
cacaccatgg aggtctcctg ttgaatggcc cttgttgccc tagagtggga cccagccctc
1260 acctccccca gagctaacct gggaggtgct gaaggggcat tgggccaccg
taagcaaggg 1320 aaaaagggca gatcatgcgg ggagatgacc ttgatctttg
attgctaccc taaccttgac 1380 ctttaacccg tgattccccc cagctcctgg
aagagatgtc ctaatatctc ttagggaccc 1440 agacccctaa attctcctcc
tcccccattt tgatgttaag gtggagaggg catatgcatc 1500 ctctgtcctg
atctaggtgt ctatagctga ggggtaagag gttgttgtag ttgtcctggt 1560
gcctccatca gactctccct acttgtccca tatttgcaag gggaggggat ttggggctgg
1620 ggctccattc accaaagctg aggtggcttc tcattaaccc tttaggactc
tgaagggtat 1680 ggacctacgt gaatgtgtgt cagggggaga cttgctggtg
ggttagtggt cctcaggatg 1740 tgatagaaac atccagtgta aaaaggaagt
tggaatggga gttggcgggc agtgaacgag 1800 tgtggggaag gattggtgct
ggggcaacag gaaggggcct ggggccgttt ggctgcacta 1860 actttggtag
ctcagtgtgc atctagagtg ggactgggga gggagctaag cttgggctgg 1920
gctgcttggg gcttggcata gggtggaaag ggctaccctg gggctctgac cacactgtag
1980 tatgtgtgga gggtgccctc ccgtctccca caacttctgc tataacaata
aactgtagag 2040 gaatctgaaa aaaaa 2055 4 228 DNA Homo sapiens
misc_feature Incyte ID No 2227688H1 4 gggttggatg gattgggcag
cccacaagct aacaggcctc acctcttcca ttccggagan 60 gatccttgga
catcttttca gccaggaaga gctctctgga aattctgagt tgatacaaaa 120
gtacagaaat atcattacac atgcacccaa cctggataac attgaattgt actggaacag
180 ctacaacaac cgccgagacc tgaactttga gcgtggaggt gatatcac 228 5 285
DNA Homo sapiens misc_feature Incyte ID No 3507515H1 5 ggcgaggggc
cgtaggcctg gnaaggcgcc agcgnngncc ggcgggcggt ngtgattgat 60
ccgcgtcccc tggagctgga ggctcggggg aaagggccag aacggagcgg gcgctcggtt
120 gctgcgcaca aaggctgagg ctccaagagc tgcagggcgt gtttgggacc
ccagagtcag 180 aaggagtgag aaccctgacc cctaatccca ctgcatccag
ccaataggag cccagccacc 240 atggcggagc tgcaggaggt gcagatcaca
gaggagaagc cactg 285 6 844 DNA Homo sapiens misc_feature Incyte ID
No 027805X7 6 cgccccccct ccccgccttg ttgtcctact tctcccggag
cagccggaga gcaggcgtcg 60 ggacgcagca aagagaggag aggccaccat
ggcggagctg caggaggtgc agatcncaga 120 ggagaagcca ctgttgccag
gacagacgcc tgaggcggcc aagactcact ctgtggagac 180 accatacggc
tctgtcactt tcactgtcta tggcaccccc aaacccaaac gcccagcgat 240
ccttacctac cacgatgtgg gactcaacta taaatcttgc ttccagccac tgtttcnntt
300 cgaggacatg caggaaatca ttcagaactt tgtgcgggtt catgtggatg
cccctggaat 360 ggaagaggga gcccctgtgt tccctttggg atatcagtac
ccatctctgg accagcttgc 420 agacatgatc ccttgcgtcc tgcagtacct
aaatttctct acaataattg gagttggtgt 480 tggagctgga gcctacatcc
tggcgagata tgctcttaac cacccggaca ctgttgaagg 540 tcttgtcctc
ntcnacattg atcccaatgc caagggttgg atggattggg cagcccacaa 600
gctaacaggc ctcacctctt ccattccnga gatgatcctt ggacatcttt tcagccagga
660 aaaactctct ggaaattctg aattgataca aaatacagaa atatcnttac
ncatgcacca 720 acctggataa cattgaattg tactggaaca ctacnacaac
cgccaanact gaactttgan 780 cgtggaagta tatcaccncn ggtttctgtg
atctggtggt aggaaacaac ccctcttgaa 840 gagc 844 7 528 DNA Homo
sapiens misc_feature Incyte ID No 1300824F1 7 gctgcccccg gacactgttn
taaggtcttg tcctcatcaa cattgatccc aatgccaagg 60 gttggatgga
ttgggcagcc cacaagctaa caggcctcac ctcttccatt ccggagatga 120
tccttggaca tcttttcagc caggaagagc tctctggaaa ttctgagttg atacaaaagt
180 acagaaatat cattacacat gcacccaacc tggataacat tgaattgtac
tggaacagct 240 acaacaaccg ccgagacctg aactttgagc gtggaggtga
tatcaccctc aggtgtcctg 300 tgatgctggt ggtaggagac caagcacctc
atgaagatgc agtggtggaa tgtaactcaa 360 aaactggacc ccacccagac
ctcgttcctc aagatngctg actccggagg tcagccccag 420 ctgactcagc
caggaagctg accganggct tcaagtactt ctgcaaggca tgggctanat 480
tggctcatcc tgcatgactc gctgtccggg tctcgtaaaa gctctctt 528 8 593 DNA
Homo sapiens misc_feature Incyte ID No 1384447F1 8 gcaaggcatg
ggctacatgg cctcatcctg catgactcgc ctgtcccggt ctcgtacagc 60
ctctctgacc agtgcagcat ccgttgatgg caaccggtcc cgctctcgca ccctgtccca
120 gagcagcgag tctggaactc tttcttcggg gcccccgggg cacaccatgg
aggtctcctg 180 ttgaatggcc cttgttgccc tagagtggga cccagccctc
acctccccca gagctaacct 240 gggaggtgct gaaggggcat tgggccaccg
taagcaaggg aaaaagggca gatcatgcgg 300 ggagatgacc ttgatctttg
attgctaccc taaccttgac ctttaanccg tgattccccc 360 cagtcctgga
agagatgtcc taatatctct tagggaccag acccctaaat tctcctcctc 420
ccccattttg atgttaaggt ggagaaggca tatgatctct gtcctgatct agtgtctaat
480 aagcttaagg ggtaagaggt tgttgtagtt gtcctggtgc tccatcagat
tctcccantt 540 ggcccatatt gcaaagggaa gggatttggg gtnggggtcc
atttaaccaa agt 593 9 785 DNA Homo sapiens misc_feature Incyte ID No
1291929T1 9 tacagtttat tgttatanca gaagttgtgg gagangggag ggcacctcca
cacatactac 60 agtgtggtca gagccccagg gtagcccttt ccaccctatg
ccaagcccca agcagcccag 120 cccaagctta gctccctccc cagtcccact
ctagatgcac actgagctac caaagttagt 180 gcagccaaac ggccccaggn
cccttcctgt tgccccagca ccaatccttc cccacactcg 240 ttcactgccc
gccaactccc attccaactt cctttttaca ctggatgttt ctatcacatc 300
ctgaggacca ctaacccacc agcaagtctc cccctgacac acattcacgt aggtccatac
360 ccttcagagt cctaaagggt taatgagaag cacctcagct ttggtgaatg
gagcccagcc 420 caaatccctc cccttgcaaa tatggcaagt agggagagtt
gatggaggac caggacaact 480 acaacaactt tttaccccna agtatagaca
cctagattag gncagaggnt natatgcctc 540 ttcaacttaa cacccaaatg
ggggaggagg ngaatttagg ggtctggggc ctaagnggtn 600 ttagggnatt
ctcttccang agcttggggg ggatcaccgg ggttnaaagg tcaaagggtt 660
aagggtggca antcaaagnt tcaagggtat nttcccccgg atggatttgg ccctttttnc
720 ccttggntta nngggngggc cnaattgncc cctttaggga acttncccag
ggtttagtct 780 tgggg 785 10 225 DNA Homo sapiens misc_feature
Incyte ID No 1367779H1 10 gaaggggcct ggggccgttt ggctgcacta
actttggtag ctcagtgtgc atctagagtg 60 ggactgggga gggagctaag
cttgggctgg gctgcttggg gcttggcata gggtggaaag 120 ggctaccctg
gggctctgac cacactgtag tatgtgtgga gggtgccctc ccgtctccca 180
caacttctgc tataacaata aactgtagag gaatctgaaa aaaaa 225 11 1726 DNA
Homo sapiens misc_feature Incyte ID No 3507515CT1 11 aggcgccgta
ggctggaagc gccagcgctg ccggcgggcg gtgtgattga tccgcgtccc 60
ctggagctgg aggctcgggg gaaagggcca gcacggagcg ggcgctcggt tgctgcgcac
120 aaaggctgag gctccaagag ctgcagggcg tgtttgggac cccagagtca
gaaggagtga 180 gaaccctgac ccctaatccc actgcatcca gccaatagga
gcccagccac catggcggag 240 ctgcaggagg tgcagatcac agaggagaag
ccactgttgc caggacagac gcctgaggcg 300 gccaagactc actctgtgga
gacaccatac ggctctgtca ctttcactgt ctatggcacc 360 cccaaaccca
aacgcccagc gatccttacc taccacgatg tgggactcaa ctataaatct 420
tgcttccagc cactgtttca gttcgaggac atgcaggaaa tcattcagaa ctttgtgcgg
480 gttcatgtgg atgcccctgg aatggaagag ggagcccctg tgttcccttt
gggatatcag 540 tacccatctc tggaccagct tgcagacatg atcccttgcg
tcctgcagta cctaaatttc 600 tctacaataa ttggagttgg tgttggagct
ggagcctaca tcctggcgag atatgctctt 660 aaccacccgg acactgttga
aggtcttgtc ctcatcaaca ttgatcccaa tgccaagggt 720 tggatggatt
gggcagccca caagctaaca ggcctcacct cttccattcc ggagatgatc 780
cttggacatc ttttcagcca ggaagagctc tctggaaatt ctgagttgat acaaaagtac
840 agaaatatca ttacacatgc acccaacctg gataacattg aattgtactg
gaacagctac 900 aacaaccgcc gagacctgaa ctttgagcgt ggaggtgata
tcaccctcag gtgtcctgtg 960 atgctggtgg taggagacca agcacctcat
gaagatgcag tggtggaatg taactcaaaa 1020 ctggacccca cccagacctc
gttcctcaag atggctgact ccggaggtca gccccagctg 1080 actcagccag
gcaagctgac cgaggccttc aagtacttcc tgcaaggcat gggctacatg 1140
gcctcatcct gcatgactcg cctgtcccgg tctcgtacag cctctctgac cagtgcagca
1200 tccgttgatg gcnaccggtc ccgctctcgc accctgtccc agagcagcga
gtctggaact 1260 cttttcttcg ggggcccccg ggggcacacc atgggaggtc
tcctgttgaa tggcccttgt 1320 tgccctagag tgggacccag ccctcagctc
ccncagagta acctgngagg tgctgaaagg 1380 gggcattggg gccaccgtaa
gcaaagggga aaaagggcag attcatggcg ggggagatga 1440 ccttgattct
ttgaattgnn aancctaanc ttgaacttta anccgtgatt cccccccagc 1500
tcctgggaag angaggtcct aatnatctct taagggaccc cagaacccct aaaattnctc
1560 cgtcttnccc cattttgaag gtnaaagggg aaaaggggna tatggaatcc
tctgttccng 1620 gatttaaggg gtccaaangt tgagggggna aaaggttgtg
gnaattggtc cctggtggct 1680 ccatcaagaa tttccnaaat tgtcccanat
tttgnaaggg gggggt 1726 12 584 DNA Homo sapiens misc_feature Incyte
ID No 3507515F6 12 ggcgaggngc cgtaggcctg gaaggcgcca gcgnngccgg
cgggcggtgt gattgatccg 60 cgtcccctgg agctggaggc tcgggggaaa
gggccagcac ggagcgggcg ctcggttgct 120 gcgcacaaag gctgaggctc
caagagctgc agggcgtgtt tgggacccca gagtcagaag 180 gagtgagaac
cctgacccct aatcccactg catccagcca ataggagccc agccaccatg 240
gcggantgca ggaggtgcag atcacagagg agaagccact gttgccagga cagacgcctg
300 aggcggccaa gactcactct gtggagacac catacggctc tgtcactttc
actgtctatg 360 gcacccccaa acccaaacgc ccagcgatcc ttacctacca
cgatgtggga ctcaactata 420 aatcttgctt ccagccactg tttcagttcg
aggacatgca ggaaatcatt cagaactttg 480 tgcgggttca tgtggatgcc
cctggaatgg aagagggagc ccctgtgttc cctttgggat 540 atcagtaccc
atctctggac cagcttgcag acatgatccc tttg 584 13 578 DNA Homo sapiens
misc_feature Incyte ID No 1214191R1 13 gcttgcttcc agccactgtt
tcagttcgag gacatgcagg aaatcattca gaactttgtg 60 cgggttcatg
tggatgcccc tggaatggaa gnncggagcc cctgtgttcc ctttgggata 120
tcagtaccca tctctggacc agcttgcaga catgatccct tgcgtcctgc agtacctaaa
180 tttctctaca ataattggag ttggtgttgg agctggagcc tacatcctgg
cgagatatgc 240 tcttaaccaa ccggacactg ttgaaggtct tgtcctcatc
aacattgatt ccaatgccaa 300 gggttggatg gattgggcag cccacaagct
aacaggctca nctcttccat tccggagatg 360 atccttggac atcttttcag
ccaggaagag tctctggaaa ttctgagttg atacaaagta 420 cagaaatatc
attacacatg caaccaacct ggataacatt gaattgtact ggaacagcta 480
caacaaccgg ccgagacctg aactttgagc gtggaggtga tatcaccctc aggtgtcctg
540 tgatgctggt ggtagcagac caagcanctc atgaagat 578 14 569 DNA Homo
sapiens misc_feature Incyte ID No 3364735F6 14 gttggagctg
gagcctacat cctggcgaga tatgctctta accacccgga cactgttgaa 60
ggtcttgtcc tcatcaacat tgatcccaat gccaagggtt ggatggattg ggcagcccac
120 aagctaacag gcctcacctc ttccattccg gagatgatcn ttggacatct
tttcagccag 180 gaagagctct ctggaaattc tgagttgata caaaagtaca
gaaatatcat tacacatgca 240 cccaacctgg ataacattga attgtactgg
aacagctaca acaaccgccg agacctgaac 300 tttgagcgtg gaggtgatat
caccctcagg tgtcctgtga tgctggtggt aggagaccaa 360 gcacctcatg
aagatgcagt ggtggaatgt aactcaaaat tggaccccca cccagacctc 420
gttcctcaag atggctgact ccggaggtca gccccagctg actcagccag gcaagctgac
480 cganggcctt caagtacttt cctgcaggca
tgggctanat ggcctcatct gatgactngc 540 ttgtccgggt tcgtanagcc
tnttgacca 569 15 913 DNA Homo sapiens misc_feature Incyte ID No
1289617F1 15 ggaaattctg agttgataca aaagtacaga aatatcatta cacatgcacc
caacctggat 60 aacattgaat tgtactggaa cagctacaac aaccgccgag
acctgaactt tgagcgtgga 120 ggtgatatca ccctcaggtg tcctgtgatg
ctggtggtag gagaccaagc acctcatgaa 180 gatgcagtgg tggaatgtaa
ctcaaaactg gaccccaccc agacctcgtt cctcaagatg 240 gctgactccg
gaggtcagcc ccagctgact cagccaggca agctgaccga ggccttcaag 300
tacttcctgc aaggcatggg ctacatggcc tcatcctgca tgactcgcct gtcccggtct
360 cgtacagcct ctctgaccag tgcagcatcc gttgatggcn accggtcccg
ctctcgcacc 420 ctgtcccaga gcagcgagtc tggaactctt ttcttcgggg
gcccccgggg gcacaccatg 480 ggaggtctcc tgttgaatgg cccttgttgc
cctagagtgg gacccagccc tcagctcccn 540 cagagtaacc tgngaggtgc
tgaaaggggg cattggggcc accgtaagca aaggggaaaa 600 agggcagatt
catggcgggg gagatgacct tgattctttg aattgnnaan cctaancttg 660
aactttaanc cgtgattccc ccccagctcc tgggaagang aggtcctaat natctcttaa
720 gggaccccag aacccctaaa attnctccgt cttnccccat tttgaaggtn
aaaggggaaa 780 aggggnatat ggaatcctct gttccnggat ttaaggggtc
caaangttga gggggnaaaa 840 ggttgtggna attggtccct ggtggctcca
tcaagaattt ccnaaattgt cccanatttt 900 gnaagggggg ggt 913 16 265 DNA
Macaca fascicularis misc_feature Incyte ID No 700718124H1 16
ccaaacccaa acgcccagcg atccttacct accacgatgt gggactcaac tataaatctt
60 gcttccagcc actgtttcaa ttcggggaca tgcaggaaat cattcagaac
ttcgtgcggg 120 ttcatgtgga tgcccctgga atggaagagg gagcccctgt
gttccctttg ggatatcagt 180 acccatctct ggaccagctt gcagacatga
tcccttgcgt cctacagtac ctaaattttt 240 ctacagtaat tggagttggt gttgg
265 17 264 DNA Macaca fascicularis misc_feature Incyte ID No
700710954H1 17 acttccgcag ccaggcaagc tgactgaggc cttcaagtac
ttcctgcaag gcatgggcta 60 catggcctca tcctgcatga ctcgcctgtc
ccggtctcgt acggcctctc taaccagtgc 120 agcatccatt gatggcaacc
ggtcccgctc tcgcaccctg tctcagagca gcgagtctgg 180 aactctttct
tcggggcccc cggtgcacac catggaggtc tcctgttgaa tgacccttgt 240
tgccctagtg tgggacccag ccct 264 18 240 DNA Macaca fascicularis
misc_feature Incyte ID No 700707576H1 18 ctgtcccggt ctcgtacggc
ctctctaacc agtgcagcat ccattgatgg caaccggtcc 60 cgctctcgca
ccctgtctca gagcagcgag tctggaactc tttcttcggg gcccccgggg 120
cacaccagga ggtctcctgt tgaatgaccc ttgttgccct agtgtgggac ccagccctca
180 cctcccccag aactaacctg ggaggtgctg aaggggcatt gggccagagt
aagcaaggga 240 19 264 DNA Macaca fascicularis misc_feature Incyte
ID No 700715158H1 19 ggcagcggct gcagcaggca tcatggcgga ctgcaggagg
tgcagatcac agaggagaag 60 ccactgctgc caggacagac gcctgaggcg
gccaagattc actctgtgga gacaccgtat 120 ggctctgtca ctttcactgt
ctatggcacc cccaaaccca aacgcccagc gatccttacc 180 taccacgatg
tgggactcaa ctataaatct tgcttccagc cactgtttca attcggggac 240
atgcaggaaa tcattcagaa cttc 264 20 329 DNA Macaca fascicularis
misc_feature Incyte ID No 700705541H1 20 tgcactccaa actggacccg
accaccacga ccttcctgaa gatggcagac tccggagggc 60 tgccccaggt
cacacagcca gggaagctga ctgaagcctt caaatacttc ctgcaaggca 120
tgggctacat gccctcagcc agcatgaccc gcctggcacg ctcacgcact gcatccctca
180 ccagtgccag ctcggtggat ggcagccgcc cacaggcctg cacccactcg
gagagcagcg 240 aggggctggg ccaggtcaac cacaccatgg aggtgtcctg
ttgaagccct cgatcccgct 300 gacgacgccc acctgtccgc ccacgtcga 329 21
131 DNA Macaca fascicularis misc_feature Incyte ID No 700719804H1
21 agcggacggc tgcagcagac cccagagtca ggagtgagaa cgctgacccc
taatcccact 60 gcatccagcc aataggagcc cagtaagtga ccccacctcg
caggctgcag gctccttcct 120 gtgcaggcat c 131 22 642 DNA Canis
familiaris misc_feature Incyte ID No 702769047H1 22 ggcgggaccg
gttgccatca atggacgccg cactggtcag cgaggccgtg cgcgatcgcg 60
acaggcgagt catgcaggac gaggccatgt agcccatgcc ttgcaggaag tacttgaagg
120 cctcggtcag cttgcctggc tgcgtcagct ggggctgacc tccagagtcg
gccatcttga 180 gaaaagaggt ctgggtgggg tccagctttg agttacactc
caccactgca tcttcatggg 240 gtgcttggtc tcccaccacc agcatcacag
ggcacctgag ggtgacggca ccgccacgct 300 ccaggttcag gtctcggcga
ttgttgtagc tgttccagta cagttcaatg ttctccaggt 360 tgggcgcatg
tgtgatgatg tttctgtact tctgtatcag ctccgagttt ccagacagct 420
cctcctggct gaaaagatgt ccgaggatca tctccggaat ggaagaggtg agacctgtta
480 gcttgtgggc cgcccagtcc atccaaccct tggcattggg atcaatgttg
atgaggacaa 540 gcccctcgac tgtatccggg tgggtcagag catatcgtga
caggatgtag gctcagctcc 600 aacaccaact ccaattattg tggagaaatt
caggtactgc ag 642 23 651 DNA Canis familiaris misc_feature Incyte
ID No 702763624H1 23 ctcggtcagc ttgcctggct gcgtcagctg gggctgacct
ccagagtcgg ccatcttgag 60 aaaagaggtc tgggtggggt ccagctttga
gttacactcc accactgcat cttcatgggg 120 tgcttggtct cccaccacca
gcatcacagg gcacctgagg gtgacggcac cgccacgctc 180 caggttcagg
tctcggcgat tgttgtagct gttccagtac agttcaatgt tctccaggtt 240
gggcgcatgt gtgatgatgt ttctgtactt ctgtatcagc tccgagtttc cagacagctc
300 ctcctggctg aaaagatgtc cgaggatcat ctccggaatg gaagaggtga
gacctgttag 360 cttgtgggcc gcccagtcca tccaaccctt ggcattggga
tcaatgttga tgaggacaag 420 cccctcgact gtatccgggt gggtcagagc
atatcgtgac aggatgtagg ctccagctcc 480 aacaccaact ccaattattg
tggagaaatt caggtactgc agaatgcaag ggatcatgtc 540 cgcgagctgg
tccagagacg ggtactgata ccccaaaggg aacacgggag cccctcttcc 600
attccaggcg catccacatg aaccgcacga agtctgaatg atttctgcat g 651 24 692
DNA Canis familiaris misc_feature Incyte ID No 702776664H1 24
ctgtactgga acagctacaa caatcgccga gacctgaacc tggagcgtgg cggtgccgtc
60 accctcaggt gccctgtgat gctggtggtg ggagaccaag caccccatga
agatgcagtg 120 gtggagtgta actcaaagct ggaccccacc cagacctctt
ttctcaagat ggccgactct 180 ggaggtcagc cccagctgac gcagccaggc
aagctgaccg aggccttcaa gtacttcctg 240 caaggcatgg gctacatggc
ctcgtcctgc atgactcgcc tgtcgcgatc gcgcacggcc 300 tcgctgacca
gtgcggcgtc cattgatggc aaccggtccc gctcccgcac cctgtcgcag 360
ggcagcgagt ctgggactct cccttcaggg ccgccagggc ataccatgga ggtctcctgc
420 tgaatggcct cggttgccct gctcaccgga cccagccctc acctccgcct
gcactaacct 480 gggaggtcct agggcgctgg gccagagtaa gggaggacgg
gtggatcatg tggggagatg 540 accttgatct ttgattgcta ccctaaactt
gactctaacc tgtgattccc ctcagctcct 600 gagagatgcc ctaatatcta
gatattagga cagatgtcta aatatctctt agggacccag 660 accctaaatt
atcctctctt cagatctctg aa 692 25 633 DNA Canis familiaris
misc_feature Incyte ID No 702778279H1 25 gcagatcaca gaggagaagc
cgctgttgcc aggcagacgc ccgagacggc caaggttatg 60 agaccccttg
accgactccc aggcccgagt tccagacctg caggcatccc ctcgcctcct 120
gccgccccaa atccataatc tcattctaat ccgcacccat gttttctcca cctggaccca
180 cgtctctctc cccgccgccg ccacgggagt ccaggcctgt gccccgtcac
tgcactaacc 240 ttcatcctct tcatgtctct ttgtcatctc tttctgtctc
ttgtcttggt ctttttgccc 300 acctgtcatt gtctgtgtct gtctctccat
ctgtgactac acggctgtgt gtctctgtgc 360 atannnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnccagga ggctgagtta 420 gctgcccgaa
tcctcctgga ccggggacag actcactctg tggagacacc gtatggctct 480
gtcactttta ctgtctatgg gacccccaag cccaaacgcc cagcgatact cacctaccat
540 gatgtaggac tcaactataa gtcttgcttc cagccgctct ttcagttcgg
ggacatgcag 600 gaaatcattc agaacttcgt gcggttcatg tgg 633 26 461 DNA
Canis familiaris misc_feature Incyte ID No 702249907H1 26
gagggcacgt gtcccctctc cctaatctag gtgtccatag ataagggata agaggttgtg
60 gtagtcgtcc catggtgccc ccaccagact ctccctgctt accccactgc
aaggggaggg 120 gacttggggc tgggactcca ttcaccaaag ctgatatggc
ttctcattaa cccttcagat 180 ctctgaagag tatgggccta aatgaatgtg
tgtcagagaa ggcttgctgg tgggttagtg 240 gtcctcaggg tggggtagag
acatccagtg tgtgaaacgg aagtcggaat ggtgggctgt 300 gaacaagtct
gagggaagga tcagagctgg ggcaatagga aggggcctgg ggccattggc 360
tgcactaact ttggtagctg tgtagacagt gtgtgtctac agtgggaggg ggagggagct
420 cagctgggac agggctgctt ggggcttggc gtaggggtgg g 461 27 610 DNA
Rattus norvegicus misc_feature Incyte ID No 702025443H1 27
tgtcacgata cgctctgaac cacccggaca ccgttgaagg tcttgttctc atcaacattg
60 atcccaacgc caagggctgg atggattggg cagcccacaa gttaaccggc
cttacgtctt 120 ccattccgga gatgattctt gggcaccttt tcagccagga
agagctttct ggaaattctg 180 aattgataca gaagtataga agtctcatca
cacacgcgcc caacctggag aacatcgaac 240 tgtattggaa cagttacaac
aaccgccgag acctgaactt tgagcgaggt ggtgagatga 300 ccctcaagtg
ccccgtgatg ctggtggtag gagaccaagc acctcatgaa gatgccgtgg 360
tggaatgtaa ttcaaaactg gaccccacac agacctcatt cctcaagatg gcggactctg
420 gaggtcagcc gcagctgact cagccaggca agctgacaga agctttcaag
tacttcgtgc 480 aaggcatggg ctacatggcc tcatcctgta tgactcgcct
gtctcggtct cgcacagcat 540 ctctgaccag tgcggatcca tcgatggcag
tcggtcccga tcccgcaccc tgtcgcagag 600 tagcgagtct 610 28 533 DNA
Rattus norvegicus misc_feature Incyte ID No 702028546H2 28
atgatgtagg actcaactat aaatcttgct tccagccact gtttcagttc ggggatatgc
60 aagagatcat acagaacttc gtgcgggtcc atgtggatgc ccctggaatg
gaagaggggg 120 cacctgtgtt tcctctgggg taccagtacc catctctgga
ccagcttgca gacatgattc 180 cttgcatcct gcagtactta aatttctcta
cgataattgg agttggcgtt ggagctggag 240 catacattct gtcacgatac
gctctgaacc acccggacac cgttgaaggt cttgttctca 300 tcaacattga
tcccaacgcc aagggctgga tggattgggc agcccacaag ttaaccggcc 360
ttacgtcttc cattccggag atgattcttg ggcacctttt cagccaggaa gagctttctg
420 gaaattctga attgatacag aagtatagaa gtctcatcac acacgcgccc
aacctggaga 480 acatcgaact gtattggaac agttacaaca accgccgaga
cctgaacttt gag 533 29 540 DNA Rattus norvegicus misc_feature Incyte
ID No 702160576H1 29 gccactgttg ccaggacaga cgcctgaggc tgccaagact
cattctgtgg agacacctta 60 tggctccgtc acttttaccg tgtatggcac
ccccaaaccc aaacgtccag cgatattcac 120 ctaccatgat gtaggactca
actataaatc ttgcttccag ccactgtttc agttcgggga 180 tatgcaagag
atcatacaga acttcgtgcg ggtccatgtg gatgcccctg gaatggaaga 240
gggggcacct gtgtttcctc tggggtacca gtacccatct ctggaccagc ttgcagacat
300 gattccttgc atcctgcagt acttaaattt ctctacgata attggagttg
gcgttggagc 360 tggagcatac attctgtcac gatacgctct gaaccacccg
gacaccgttg aaggtcttgt 420 tctcatcaac attgatccca acgccaaggg
ctggatggat tgggcagccc acaagttaac 480 cggccttacg tcttccattc
cggagatgat tcttgggcac cttttcagcc aggaagagct 540 30 489 DNA Rattus
norvegicus misc_feature Incyte ID No 701938896H1 30 cccgaatcct
cctggaccag gacagactca ttctgtggag acaccttatg gctccgtcac 60
ttttaccgtg tatggcaccc cctaacccaa acgtccagcg atattcaact aacaaggatg
120 taggactcaa ctataaatct tgcttccagc cactgtttca gttcggggat
atgcaagaga 180 tcatacagaa cttcgtgcgg gtccatgtgg atgcccctgg
aatggaagag ggggcacctg 240 tgtttcctct ggggtaccag tacccatctc
tggaccagct tgcagacatg attccttgca 300 tcctgcagta cttaaatttc
tctacgataa ttggagttgg cgttggagct ggagcataca 360 ttctgtcacg
atacgctctg aaccacccgg acaccgttga aggtcttgtt ctcatcaaca 420
ttgatcccaa cgccaagggc tggatggatt gggcagccca caagttaacc gggcttacgt
480 cttccattc 489 31 317 DNA Rattus norvegicus misc_feature Incyte
ID No 701646914H1 31 gcggctgcag caggccacca tggcagagct tcaggaggtg
cagatcactg aggagaagcc 60 actgttgcca ggacagacgc ctgaggctgc
caaggaggct gagttagctg cccgaatcct 120 cctggaccag ggacagactc
attctgtgga gacaccttat ggctccgtca cttttaccgt 180 gtatggcacc
cccaaaccca tacgtccagc gatattcacc taccatgatg taggactcaa 240
ctataaatct tgcttccagc cactgtttca gttcggggat atgcaagaga tcatacagaa
300 cttcgtgcgg gtccatg 317 32 371 PRT Mus musculus misc_feature
GenBank ID No g6141566 32 Met Ala Glu Leu Gln Glu Val Gln Ile Thr
Glu Glu Lys Pro Leu 1 5 10 15 Leu Pro Gly Gln Thr Pro Glu Thr Ala
Lys Glu Ala Glu Leu Ala 20 25 30 Ala Arg Ile Leu Leu Asp Gln Gly
Gln Thr His Ser Val Glu Thr 35 40 45 Pro Tyr Gly Ser Val Thr Phe
Thr Val Tyr Gly Thr Pro Lys Pro 50 55 60 Lys Arg Pro Ala Ile Phe
Thr Tyr His Asp Val Gly Leu Asn Tyr 65 70 75 Lys Ser Cys Phe Gln
Pro Leu Phe Arg Phe Gly Asp Met Gln Glu 80 85 90 Ile Ile Gln Asn
Phe Val Arg Val His Val Asp Ala Pro Gly Met 95 100 105 Glu Glu Gly
Ala Pro Val Phe Pro Leu Gly Tyr Gln Tyr Pro Ser 110 115 120 Leu Asp
Gln Leu Ala Asp Met Ile Pro Cys Ile Leu Gln Tyr Leu 125 130 135 Asn
Phe Ser Thr Ile Ile Gly Val Gly Val Gly Ala Gly Ala Tyr 140 145 150
Ile Leu Ser Arg Tyr Ala Leu Asn His Pro Asp Thr Val Glu Gly 155 160
165 Leu Val Leu Ile Asn Ile Asp Pro Asn Ala Lys Gly Trp Met Asp 170
175 180 Trp Ala Ala His Lys Leu Thr Gly Leu Thr Ser Ser Ile Pro Asp
185 190 195 Met Ile Leu Gly His Leu Phe Ser Gln Glu Glu Leu Ser Gly
Asn 200 205 210 Ser Glu Leu Ile Gln Lys Tyr Arg Gly Ile Ile Gln His
Ala Pro 215 220 225 Asn Leu Glu Asn Ile Glu Leu Tyr Trp Asn Ser Tyr
Asn Asn Arg 230 235 240 Arg Asp Leu Asn Phe Glu Arg Gly Gly Glu Thr
Thr Leu Lys Cys 245 250 255 Pro Val Met Leu Val Val Gly Asp Gln Ala
Pro His Glu Asp Ala 260 265 270 Val Val Glu Cys Asn Ser Lys Leu Asp
Pro Thr Gln Thr Ser Phe 275 280 285 Leu Lys Met Ala Asp Ser Gly Gly
Gln Pro Gln Leu Thr Gln Pro 290 295 300 Gly Lys Leu Thr Glu Ala Phe
Lys Tyr Phe Leu Gln Gly Met Gly 305 310 315 Tyr Met Ala Ser Ser Cys
Met Thr Arg Leu Ser Arg Ser Arg Thr 320 325 330 Ala Ser Leu Thr Ser
Ala Ala Ser Ile Asp Gly Ser Arg Ser Arg 335 340 345 Ser Arg Thr Leu
Ser Gln Ser Ser Glu Ser Gly Thr Leu Pro Ser 350 355 360 Gly Pro Pro
Gly His Thr Met Glu Val Ser Cys 365 370 33 394 PRT Homo sapiens
misc_feature GenBank ID No g1596167 33 Met Ser Arg Glu Met Gln Asp
Val Asp Leu Ala Glu Val Lys Pro 1 5 10 15 Leu Val Glu Lys Gly Glu
Thr Ile Thr Gly Leu Leu Gln Glu Phe 20 25 30 Asp Val Gln Glu Gln
Asp Ile Glu Thr Leu His Gly Ser Val His 35 40 45 Val Thr Leu Cys
Gly Thr Pro Lys Gly Asn Arg Pro Val Ile Leu 50 55 60 Thr Tyr His
Asp Ile Gly Met Asn His Lys Thr Cys Tyr Asn Pro 65 70 75 Leu Phe
Asn Tyr Glu Asp Met Gln Glu Ile Thr Gln His Phe Ala 80 85 90 Val
Cys His Val Asp Ala Pro Gly Gln Gln Asp Gly Ala Ala Ser 95 100 105
Phe Pro Ala Gly Tyr Met Tyr Pro Ser Met Asp Gln Leu Ala Glu 110 115
120 Met Leu Pro Gly Val Leu Gln Gln Phe Gly Leu Lys Ser Ile Ile 125
130 135 Gly Met Gly Thr Gly Ala Gly Ala Tyr Ile Leu Thr Arg Phe Ala
140 145 150 Leu Asn Asn Pro Glu Met Val Glu Gly Leu Val Leu Ile Asn
Val 155 160 165 Asn Pro Cys Ala Glu Gly Trp Met Asp Trp Ala Ala Ser
Lys Ile 170 175 180 Ser Gly Trp Thr Gln Ala Leu Pro Asp Met Val Val
Ser His Leu 185 190 195 Phe Gly Lys Glu Glu Met Gln Ser Asn Val Glu
Val Val His Thr 200 205 210 Tyr Arg Gln His Ile Val Asn Asp Met Asn
Pro Gly Asn Leu His 215 220 225 Leu Phe Ile Asn Ala Tyr Asn Ser Arg
Arg Asp Leu Glu Ile Glu 230 235 240 Arg Pro Met Pro Gly Thr His Thr
Val Thr Leu Gln Cys Pro Ala 245 250 255 Leu Leu Val Val Gly Asp Ser
Ser Pro Ala Val Asp Ala Val Val 260 265 270 Glu Cys Asn Ser Lys Leu
Asp Pro Thr Lys Thr Thr Leu Leu Lys 275 280 285 Met Ala Asp Cys Gly
Gly Leu Pro Gln Ile Ser Gln Pro Ala Lys 290 295 300 Leu Ala Glu Ala
Phe Lys Tyr Phe Val Gln Gly Met Gly Tyr Met 305 310 315 Pro Ser Ala
Ser Met Thr Arg Leu Met Arg Ser Arg Thr Ala Ser 320 325 330 Gly Ser
Ser Val Thr Ser Leu Asp Gly Thr Arg Ser Arg Ser His 335 340 345 Thr
Ser Glu Gly Thr Arg Ser Arg Ser His Thr Ser Glu Gly Thr 350 355 360
Arg Ser Arg Ser His Thr Ser Glu Gly Ala His Leu Asp Ile Thr 365 370
375 Pro Asn Ser Gly Ala Ala Gly Asn Ser Ala Gly Pro Lys Ser Met 380
385 390 Glu Val Ser Cys
* * * * *
References