U.S. patent application number 10/195101 was filed with the patent office on 2003-05-08 for human nim1 kinase.
This patent application is currently assigned to Incyte Genomics, Inc.. Invention is credited to Bandman, Olga, Bosotti, Roberta, Hodgson, David M., Isacchi, Antonella, Magnaghi, Paola, Molteni, Angela, Scacheri, Emanuela.
Application Number | 20030087317 10/195101 |
Document ID | / |
Family ID | 24086680 |
Filed Date | 2003-05-08 |
United States Patent
Application |
20030087317 |
Kind Code |
A1 |
Bandman, Olga ; et
al. |
May 8, 2003 |
Human NIM1 kinase
Abstract
The invention provides a human NIM1 kinase, an encoding nucleic
acid molecule, and an antibody that specifically binds the protein.
The invention also provides for the use of these compositions in
the characterization, diagnosis, and treatment of disorders
associated NIM1 kinase expression.
Inventors: |
Bandman, Olga; (Mountain
View, CA) ; Molteni, Angela; (Cantu, IT) ;
Magnaghi, Paola; (US) ; Bosotti, Roberta;
(Nerviano, IT) ; Scacheri, Emanuela; (US) ;
Isacchi, Antonella; (US) ; Hodgson, David M.;
(Ann Arbor, MI) |
Correspondence
Address: |
INCYTE GENOMICS, INC.
3160 PORTER DRIVE
PALO ALTO
CA
94304
US
|
Assignee: |
Incyte Genomics, Inc.
Palo Alto
CA
|
Family ID: |
24086680 |
Appl. No.: |
10/195101 |
Filed: |
July 11, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10195101 |
Jul 11, 2002 |
|
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09523849 |
Mar 13, 2000 |
|
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6458561 |
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Current U.S.
Class: |
435/7.23 ;
435/194; 435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/1205 20130101;
A61P 25/00 20180101; A61P 35/00 20180101 |
Class at
Publication: |
435/7.23 ;
435/69.1; 435/194; 435/320.1; 435/325; 536/23.2 |
International
Class: |
G01N 033/574; C07H
021/04; C12N 009/12; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. A purified nucleic acid molecule or a fragment thereof encoding
a protein comprising SEQ ID NO:2 or a portion thereof.
2. A purified protein comprising a polypeptide having an amino acid
sequence of SEQ ID NO:2.
3. A composition comprising the protein of claim 2 and a labeling
moiety.
4. A composition comprising the protein of claim 2 and a
pharmaceutical carrier.
5. A substrate upon which the protein of claim 2 is
immobilized.
6. An array element comprising the protein of claim 2.
7. An oligopeptide comprising residue M1 to residue A18 of the
protein of claim 2.
8. A method for detecting expression of a protein having the amino
acid sequence of SEQ ID NO:2 in a sample, the method comprising
performing an assay selected from two-dimensional polyacrylamide
electrophoresis, western analysis, mass spectrophotometry,
enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence
activated cell sorting, and array technology with the sample,
thereby detecting expression of the protein in the sample.
9. The method of claim 8 wherein the sample is from brain, breast,
cervix, colon, lung, ovary, and prostate.
10. The method of claim 8 wherein the expression when compared to
standards is diagnostic of brain disorders or cancers of the brain,
breast, cervix, colon, lung, ovary, and prostate.
11. A method for using a protein to screen a plurality of molecules
and compounds to identify at least one ligand, the method
comprising: a) combining the protein of claim 2 with a plurality of
molecules and compounds under conditions to allow specific binding;
and b) detecting specific binding, thereby identifying a ligand
that specifically binds the protein.
12. The method of claim 11 wherein the molecules and compounds are
selected from agonists, antagonists, bispecific molecules, DNA
molecules, small drug molecules, immunoglobulins, inhibitors,
mimetics, multispecific molecules, peptides, peptide nucleic acids,
pharmaceutical agent, proteins, and RNA molecules.
13. A method for using a protein to identify an antibody that
specifically binds the protein having the amino acid sequence of
SEQ ID NO:2 comprising: a) contacting a plurality of antibodies
with the protein of claim 2 under conditions to allow specific
binding, and b) detecting specific binding between an antibody and
the protein, thereby identifying an antibody that specifically
binds the protein.
14. The method of claim 13, wherein the antibodies are selected
from a polyclonal antibody, a monoclonal antibody, a chimeric
antibody, a recombinant antibody, a humanized antibody, a single
chain antibody, a Fab fragment, an F(ab').sub.2 fragment, an Fv
fragment; and an antibody-peptide fusion protein.
15. A method of using a protein to prepare and purify a polyclonal
antibody comprising: a) immunizing a animal with a 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; and e) dissociating the
antibodies from the protein, thereby obtaining purified polyclonal
antibodies.
16. 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 from culture monoclonal antibody
that specifically binds the protein.
17. A method for using a protein to diagnose a disorder comprising:
a) performing an assay to quantify the expression of the protein of
claim 2 in a sample; b) comparing the expression of the protein to
standards, thereby diagnosing a disorder.
18. The method of claim 17 wherein the sample is selected from
brain, breast, cervix, colon, lung, ovary, and prostate.
19. The method of claim 17 wherein expression is diagnostic of
brain disorders or cancers of the brain, breast, cervix, colon,
lung, ovary, and prostate.
20. A method for testing a molecule or compound for effectiveness
as an agonist comprising: a) exposing a sample comprising the
protein of claim 2 to the molecule or compound, and b) detecting
agonist activity in the sample.
21. A method for testing a molecule or compound for effectiveness
as an antagonist, the method comprising: a) exposing a sample
comprising the protein of claim 2 to a molecule or compound, and b)
detecting antagonist activity in the sample.
22. An isolated antibody that specifically binds a polypeptide
having an amino acid sequence of SEQ ID NO:2.
23. A polyclonal antibody produced by the method of claim 15.
24. A monoclonal antibody produced by the method of claim 16.
25. A method for using an antibody to detect expression of a
protein in a sample, the method comprising: a) combining the
antibody of claim 22 with a sample under conditions which allow the
formation of antibody:protein complexes; and b) detecting complex
formation, wherein complex formation indicates expression of the
protein in the sample.
26. The method of claim 25 wherein the sample is from brain,
breast, cervix, colon, lung, ovary, and prostate.
27. The method of claim 25 wherein complex formation is compared
with standards and is diagnostic of brain disorders and cancer.
28. A method for using an antibody to immunopurify a protein
comprising: a) attaching the antibody of claim 22 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.
29. A composition comprising an antibody of claim 22 and a labeling
moiety.
30. A kit comprising the composition of claim 29.
31. An array element comprising the antibody of claim 22.
32. A substrate upon which the antibody of claim 22 is
immobilized.
33. A composition comprising the monoclonal antibody of claim 24
and a pharmaceutical agent.
34. The composition of claim 33 wherein the composition is
lyophilized.
35. A method for using a composition to assess efficacy of a
molecule or compound, the method comprising: a) treating a sample
containing a protein having the amino acid sequence of SEQ ID NO:2
with a molecule or compound; b) contacting the sample with the
composition of claim 29 under conditions for complex formation; c)
determining the amount of complex formation; and d) comparing the
amount of complex formation in the treated sample with the amount
of complex formation in standards, wherein a difference in complex
formation indicates efficacy of the molecule or compound.
36. A method for using a composition to assess toxicity of a
molecule or compound, the method comprising: a) treating a sample
containing a protein having the amino acid sequence of SEQ ID NO:2
with a molecule or compound; b) contacting the protein in the
sample with the composition of claim 29 under conditions for
complex formation; c) determining the amount of complex formation;
and d) comparing the amount of complex formation in the treated
sample with the amount of complex formation in standards, wherein a
difference in complex formation indicates toxicity of the molecule
or compound.
37. A method for treating a cancer comprising administering to a
subject in need of therapeutic intervention the antibody of claim
22.
38. A method for treating a cancer comprising administering to a
subject in need of therapeutic intervention the composition of
claim 33.
39. A method for delivering a pharmaceutical agent to a cell
comprising: a) attaching the pharmaceutical agent to a bispecific
molecule identified by the method of claim 11; and b) administering
the bispecific molecule to a subject in need of therapeutic
intervention, wherein the bispecific molecule specifically binds
the protein having the amino acid sequence of SEQ ID NO:2 thereby
delivering the pharmaceutical agent to the cell.
40. An agonist that specifically binds the protein of claim 2.
41. A composition comprising an agonist of claim 40 and a
pharmaceutical carrier.
42. An antagonist that specifically binds the protein of claim
2.
43. A composition comprising the antagonist of claim 42 and a
pharmaceutical carrier.
44. A pharmaceutical agent that specifically binds the protein of
claim 2.
45. A composition comprising the pharmaceutical agent of claim 44
and a pharmaceutical carrier.
46. A small drug molecule that specifically binds the protein of
claim 2.
47. A composition comprising the small drug molecule of claim 46
and a pharmaceutical carrier.
48. An antisense molecule of 18 to 30 nucleotides in length that
specifically binds a portion of a polynucleotide of claim 1 or the
complement thereof wherein the antisense molecule inhibits
expression of the protein encoded by the polynucleotide.
49. The antisense molecule of claim 48 wherein the antisense
molecule comprises at least one modified internucleoside
linkage.
50. The antisense molecule of claim 49 wherein the modified
internucleoside linkage is a phosphorothioate linkage.
51. The antisense molecule of claim 48 wherein the antisense
molecule comprises at least one nucleotide analog.
52. The antisense molecule of claim 51 wherein the nucleotide
analog is a 5-methylcytosine.
Description
[0001] This application claims priority to U.S. Ser. No.
09/523,849, filed Mar. 13, 2000.
FIELD OF THE INVENTION
[0002] This invention relates to human NIM1 kinase, its encoding
nucleic acid and an antibody that specifically binds human NIM1
kinase and to the use of these compositions in the
characterization, diagnosis, and treatment of brain disorders and
cancers.
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 protein kinases of nematode, fly, rat, and man have common
chemical and structural features and modulate the same general
cellular activity. 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 disorders.
[0004] Protein kinases regulate many different cell proliferation,
differentiation, and signaling processes by adding phosphate groups
to proteins. Uncontrolled signaling has been implicated in a
variety of disease conditions including inflammation, cancer,
arteriosclerosis, and psoriasis. Reversible protein phosphorylation
is the main strategy for controlling activities of eukaryotic
cells. It is estimated that more than 1000 of the 10,000 proteins
active in a typical mammalian cell are phosphorylated. The high
energy phosphate which drives activation is generally transferred
from adenosine triphosphate (ATP) or guanosine triphosphate (GTP)
to a particular protein by protein kinases and removed from that
protein by protein phosphatases. Phosphorylation is roughly
analogous to turning on a molecular switch, and it occurs in
response to extracellular signals (mediated by such molecules as
hormones, neurotransmitters, and growth and differentiation
factors), cell cycle checkpoints, and environmental or nutritional
stresses. When the switch goes on, the appropriate protein kinase
activates a metabolic enzyme, regulatory protein, receptor,
cytoskeletal protein, ion channel or pump, or transcription
factor.
[0005] The protein kinases comprise the largest known protein
group, a superfamily of enzymes with widely varied functions and
specificities. They are usually named after their substrate, their
regulatory molecules, or some aspect of a mutant phenotype. With
regard to substrates, the protein kinases may be roughly divided
into two groups; those that phosphorylate tyrosine residues
(protein tyrosine kinases, PTK) and those that phosphorylate serine
or threonine residues (serine/threonine kinases, STK). A few
protein kinases have dual specificity and phosphorylate both
threonine and tyrosine residues.
[0006] Protein kinases may be categorized into families by the
different amino acid residues (generally between 5 and 100
residues) located on either side of, or inserted into loops of, the
catalytic domain. These residues allow the regulation of each
kinase as it recognizes and interacts with its target protein.
Almost all kinases contain a similar 250-300 amino acid catalytic
domain with 11 subdomains distributed across two lobes. The
N-terminal lobe, which contains subdomains I-IV, binds and orients
the ATP donor molecule. The larger C terminal lobe, which contains
subdomains VIA-XI, binds the protein substrate and carries out the
transfer of the gamma phosphate from ATP to the hydroxyl group of a
serine, threonine, or tyrosine residue. Subdomain V spans the N and
C terminal lobes.
[0007] Each of the 11 subdomains contain specific residues and
motifs or patterns of amino acids that are characteristic of that
subdomain and are highly conserved (Hardie and Hanks (1995) The
Protein Kinase Facts Books, Vol I, Academic Press, San Diego
Calif., pp. 7-20). In particular, two protein kinase signature
sequences have been identified in the kinase domain, the first
containing an active site lysine residue involved in ATP binding,
and the second containing an aspartate residue important for
catalytic activity. If a protein is found to contain the two
protein kinase signatures, the probability of that protein being a
protein kinase is close to 100% (MOTIFS search program, Accelrys,
San Diego Calif.; Bairoch et al. (1996) Nucleic Acids Res
24:189-196).
[0008] STK Families
[0009] The second messenger dependent protein kinases primarily
mediate the effects of second messengers such as cyclic AMP (cAMP),
cyclic GMP, inositol triphosphate, phosphatidylinositol,
3,4,5-triphosphate, cyclic ADP ribose, arachidonic acid,
diacylglycerol and calcium-calmodulin. The cyclic-AMP dependent
protein kinases (PKA) are important members of the STK family. cAMP
is an intracellular mediator of hormone action in all prokaryotic
and animal cells that have been studied. Such hormone-induced
cellular responses include thyroid hormone secretion, cortisol
secretion, progesterone secretion, glycogen breakdown, bone
resorption, and regulation of heart rate and force of heart muscle
contraction. PKA is found in all animal cells and is thought to
account for the effects of cAMP in most of these cells. Altered PKA
expression is implicated in a variety of disorders and diseases
including cancer, thyroid disorders, diabetes, atherosclerosis, and
cardiovascular disease (Isselbacher et al. (1994) Harrison's
Principles of Internal Medicine, McGraw-Hill, New York N.Y., pp.
416-431 and 1887).
[0010] Calcium-calmodulin (CaM) dependent protein kinases are also
members of STK family. Calmodulin is a calcium receptor that
mediates many calcium regulated processes by binding to target
proteins in response to the binding of calcium. The principle
target protein in these processes is CaM dependent protein kinases
(CaMK). CaMK are involved in regulation of smooth muscle
contraction, glycogen breakdown (phosphorylase kinase), and
neurotransmission (CaMK I and CaMK II). CaMK I phosphorylates a
variety of substrates including the neurotransmitter related
proteins synapsin I and II, the gene transcription regulator, CREB,
and the cystic fibrosis conductance regulator protein, CFTR
(Haribabu et al. (1995) EMBO J 14:3679-86). CaMK II also
phosphorylates synapsin at different sites and controls the
synthesis of catecholamines in the brain through phosphorylation
and activation of tyrosine hydroxylase. Many of the CaMK are
activated by phosphorylation in addition to binding to CaM. CaMK
may autophosphorylate or be phosphorylated by another kinase as
part of a "kinase cascade".
[0011] Another ligand-activated protein kinase is 5'-AMP-activated
protein kinase (AMPK; Dyck et al. (1996) J Biol Chem
271:17998-17803). Mammalian AMPK is a regulator of fatty acid and
sterol synthesis through phosphorylation of the enzymes acetyl-CoA
carboxylase and hydroxymethyl glutaryl-CoA reductase and mediates
responses of these pathways to cellular stresses such as heat shock
and depletion of glucose and ATP. AMPK is a heterotrimeric complex
comprised of a catalytic alpha subunit and two non-catalytic beta
and gamma subunits that are believed to regulate the activity of
the alpha subunit. Subunits of AMPK have a much wider distribution
in non-lipogenic tissues such as brain, heart, spleen, and lung
than expected. This distribution suggests that its role may extend
beyond regulation of lipid metabolism alone.
[0012] The mitogen-activated protein kinases (MAPK) are also
members of the STK family, and they regulate intracellular
signaling pathways. MAPK mediate signal transduction from the cell
surface to the nucleus via phosphorylation cascades. Several
subgroups have been identified, and each manifests different
substrate specificities and responds to distinct extracellular
stimuli (Egan and Weinberg (1993) Nature 365:781-783). MAP kinase
signaling pathways are present in mammalian cells as well as in
yeast. The extracellular stimuli which activate mammalian pathways
include epidermal growth factor, ultraviolet light, hyperosmolar
medium, heat shock, endotoxic lipopolysaccharide, and
pro-inflammatory cytokines such as tumor necrosis factor and
interleukin-1. Altered MAPK expression is implicated in a variety
of disease conditions including cancer, inflammation, immune
disorders, and disorders affecting growth and development.
[0013] Proliferation-related kinase (PRK) is a serum/cytokine
inducible STK that is involved in regulation of the cell cycle and
cell proliferation in human megakaroytic cells (Li et al. (1996) J
Biol Chem 271:19402-8). PRK is related to the polo family of STKs
implicated in cell division. PRK is downregulated in lung tumor
tissue and may be a proto-oncogene whose deregulated expression in
normal tissue leads to oncogenic transformation.
[0014] The cyclin-dependent protein kinases (CDKs) are another
group of STKs that control the progression of cells through the
cell cycle. Cyclins are small regulatory proteins that act by
binding to and activating CDKs which then trigger various phases of
the cell cycle by phosphorylating and activating selected proteins
involved in the mitotic process. CDKs are unique in that they
require multiple inputs to become activated. In addition to the
binding of cyclin, CDK activation requires the phosphorylation of a
specific threonine residue and the dephosphorylation of a specific
tyrosine residue.
[0015] The discovery of human NIM1 kinase, a nucleic acid molecule
encoding the protein and an antibody that specifically binds the
protein provides new compositions which are useful in the
characterization, diagnosis, and treatment of brain disorders and
cancers.
SUMMARY OF THE INVENTION
[0016] The invention is based on the discovery of human NIM1 kinase
(hNIM1), its encoding nucleic acid, and an antibody that
specifically binds the protein. The invention provides compositions
useful in the characterization, diagnosis, and treatment of brain
disorders and cancers of the brain, breast, cervix, colon, lung,
ovary, and prostate.
[0017] The invention provides a purified nucleic acid molecule
which encodes the human NIM1 kinase comprising SEQ ID NO:2. The
invention also provides a nucleic acid molecule comprising SEQ ID
NO:1 or the complement thereof. The invention also provides a
composition comprising the nucleic acid molecule having the nucleic
sequence of SEQ ID NO:1 or the complement thereof and a labeling
moiety. The invention further provides a mammalian variant of the
nucleic acid molecule selected from SEQ ID NOs:24-30. The invention
still further provides a fragment of at least 18 consecutive
nucleotides selected from about nucleotide 414 to about 1414 of SEQ
ID NO:1, SEQ ID NOs:3-30, or the complements thereof. In one
aspect, the invention provides a substrate upon which at least one
of these fragments or complements is immobilized. In a second
aspect, the invention provides a probe which can be used in methods
of detection, screening, and purification. In a further aspect, the
probe comprises a single stranded complementary RNA or DNA
molecule.
[0018] The invention provides a method for detecting a nucleic acid
molecule in a sample, the method comprising hybridizing a probe or
complementary nucleic acid molecule to at least one nucleic acid in
a sample, forming a hybridization complex; and detecting the
hybridization complex, wherein the presence of the hybridization
complex indicates the presence of the nucleic acid molecule in the
sample. In one aspect, the method further comprises amplifying the
nucleic acids of the sample prior to hybridization. The nucleic
acid molecule or a fragment or a complement thereof may comprise an
element on an array.
[0019] The invention provides a method for using a nucleic acid
molecule or a fragment or a complement thereof to screen a library
of molecules or compounds to identify at least one ligand which
specifically binds the nucleic acid molecule, the method comprising
combining the nucleic acid molecule with a library of molecules or
compounds under conditions allowing specific binding, and detecting
specific binding to the nucleic acid molecule, thereby identifying
a ligand which specifically binds the nucleic acid molecule. In one
aspect, the libraries of molecules and compounds include DNA
molecules, enhancers, peptide nucleic acids, peptides, proteins,
RNA molecules, and transcription factors.
[0020] The invention provides an expression vector comprising the
nucleic acid molecule and a host cell comprising the vector. The
invention also provides a method for producing a protein, the
method comprising culturing the host cell under conditions for the
expression of the protein and recovering the protein from the host
cell culture.
[0021] The invention provides a purified protein comprising a
polypeptide having an amino acid sequence of SEQ ID NO:2. The
invention also provides a composition comprising the protein and a
labeling moiety, a composition comprising the protein and a
pharmaceutical carrier, and an array element comprising the
protein. The invention further provides substrate upon which the
protein is immobilized.
[0022] The invention provides a method for detecting differential
expression of a protein having the amino acid sequence of SEQ ID
NO:1 in a sample, the method comprising performing an assay to
determine the amount of the protein in a sample; and comparing the
amount of protein to standards, thereby detecting differential
expression of the protein in the sample. In one aspect, the assay
is selected from two-dimensional polyacrylamide electrophoresis,
western analysis, mass spectrophotometry, enzyme-linked
immunosorbent assay , radioimmunoassay, fluorescence activated cell
sorting, and array technology. In a second aspect, the sample is
from brain, breast, cervix, colon, lung, ovary, and prostate. In a
third aspect, the protein is differentially expressed when compared
with standards and is diagnostic of brain disorders or cancer.
[0023] The invention provides a method for using a protein to
screen a plurality of molecules and compounds to identify at least
one ligand, the method comprising combining the protein with a
plurality of molecules and compounds under conditions to allow
specific binding; and detecting specific binding, thereby
identifying a ligand that specifically binds the protein. In one
aspect, the molecules and compounds are selected from agonists,
antagonists, bispecific molecules, DNA molecules, small drug
molecules, immunoglobulins, inhibitors, mimetics, multispecific
molecules, peptides, peptide nucleic acids, pharmaceutical agent,
proteins, and RNA molecules. The invention also provides a method
for using a protein to identify an antibody that specifically binds
the protein having the amino acid sequence of SEQ ID NO:2
comprising contacting a plurality of antibodies with the protein
under conditions to allow specific binding, and detecting specific
binding between an antibody and the protein, thereby identifying an
antibody that specifically binds the protein. In one aspect, the
plurality of antibodies are selected from a polyclonal antibody, a
monoclonal antibody, a chimeric antibody, a recombinant antibody, a
humanized antibody, a single chain antibody, a Fab fragment, an
F(ab').sub.2 fragment, an Fv fragment; and an antibody-peptide
fusion protein.
[0024] The invention provides a method of using a protein to
prepare and purify a polyclonal antibody comprising immunizing an
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; and
dissociating the antibodies from the protein, thereby obtaining
purified polyclonal antibodies. The invention also provides a
method of using a protein to prepare a monoclonal antibody
comprising immunizing an animal with a protein under conditions to
elicit an antibody response; isolating antibody-producing cells
from the animal; fusing the antibody-producing cells with
immortalized cells in culture to form monoclonal antibody producing
hybridoma cells; culturing the hybridoma cells; and isolating from
culture monoclonal antibody that specifically binds the
protein.
[0025] The invention provides a method for using a protein to
diagnose a cancer comprising performing an assay to quantify the
expression of the protein in a sample; and comparing the expression
of the protein to standards, thereby diagnosing cancer. In one
aspect, the sample is selected from brain, breast, cervix, colon,
lung, ovary, and prostate. In a second aspect, expression is
diagnostic of brain disorders or cancers. The invention also
provides a method for testing a molecule or compound for
effectiveness as an agonist comprising exposing a sample comprising
the protein to the molecule or compound, and detecting agonist
activity in the sample. The invention also provides a method for
testing a molecule or compound for effectiveness as an antagonist,
the method comprising exposing a sample comprising the protein of
to a molecule or compound, and detecting antagonist activity in the
sample.
[0026] The invention provides an isolated antibody that
specifically binds a polypeptide having an amino acid sequence of
SEQ ID NO:2. The invention also provides a polyclonal antibody and
a monoclonal antibody. The invention further provides a method for
using an antibody to detect expression of a protein in a sample,
the method comprising combining an antibody that specifically binds
the protein with a sample under conditions which allow the
formation of antibody:protein complexes; and detecting complex
formation, wherein complex formation indicates expression of the
protein in the sample. In one aspect, the sample is from brain,
breast, cervix, colon, lung, ovary, and prostate. In a second
aspect, complex formation is compared with standards and is
diagnostic of brain disorders and cancer. The invention further
provides a method for using an antibody to immunopurify a protein
comprising attaching an antibody that specifically binds the
protein to a substrate, exposing the antibody to a sample
containing protein under conditions to allow antibody:protein
complexes to form, dissociating the protein from the complex, and
collecting the purified protein.
[0027] The invention provides a composition comprising an antibody
that specifically binds the protein and a labeling moiety or a
pharmaceutical agent. In one aspect, the composition is
lyophilized. The invention also provides a kit comprising the
antibody, an array element comprising the antibody, and a substrate
upon which the antibody is immobilized.
[0028] The invention provides a method for using a composition to
assess efficacy of a molecule or compound, the method comprising
treating a sample containing the protein with a molecule or
compound; contacting the sample with the composition under
conditions for complex formation; determining the amount of complex
formation; and comparing the amount of complex formation in the
treated sample with the amount of complex formation in standards,
wherein a difference in complex formation indicates efficacy of the
molecule or compound. The invention provides a method for using a
composition to assess toxicity of a molecule or compound, the
method comprising treating a sample containing the protein with a
molecule or compound; contacting the sample with the composition
under conditions for complex formation; determining the amount of
complex formation; and comparing the amount of complex formation in
the treated sample with the amount of complex formation in
standards, wherein a difference in complex formation indicates
toxicity of the molecule or compound.
[0029] The invention provides a method for treating a cancer
comprising administering to a subject in need of therapeutic
intervention an antibody that specifically binds the protein. In
one aspect, the antibody is a monoclonal antibody. The invention
also provides a method for treating cancer comprising administering
to a subject in need of therapeutic intervention an antibody that
specifically binds the protein or a monoclonal antibody. The
invention further provides a method for delivering a therapeutic
agent to a cell comprising attaching the therapeutic agent to a
bispecific molecule identified; and administering the bispecific
molecule to a subject in need of therapeutic intervention, wherein
the bispecific molecule specifically binds the protein having the
amino acid sequence of SEQ ID NO:2 thereby delivering the
therapeutic agent to the cell.
[0030] The invention provides an agonist that specifically binds
the protein and a composition comprising an agonist and a
pharmaceutical carrier. The invention also provides an antagonist
that specifically binds the protein and a composition comprising
the antagonist and a pharmaceutical carrier. The invention further
provides a pharmaceutical agent that specifically binds the protein
and a composition comprising the pharmaceutical agent and a
pharmaceutical carrier. The invention still further provides a
small drug molecule that specifically binds the protein and a
composition comprising the small drug molecule and a pharmaceutical
carrier.
[0031] The invention provides an antisense molecule of 18 to 30
nucleotides in length that specifically binds a portion of a
polynucleotide encoding the human NIM1 kinase or the complement
thereof wherein the antisense molecule inhibits expression of the
protein encoded by the polynucleotide. In one aspect, the antisense
molecule comprises at least one modified internucleoside linkage
wherein the internucleoside linkage is a phosphorothioate linkage.
In a second aspect, the antisense molecule comprises at least one
nucleotide analog wherein the modified nucleotide analog is a
5-methylcytosine.
BRIEF DESCRIPTION OF THE FIGURES AND TABLE
[0032] FIGS. 1A, 1B, 1C, 1D, 1E, and 1F show the nucleic acid
molecule (SEQ ID NO: 1) encoding the human NIM1 kinase (SEQ ID
NO:2). The alignment was produced using MACDNASIS PRO software
(Hitachi Software Engineering, South San Francisco Calif.).
[0033] FIG. 2 shows electronic northern analysis of the highly
specific expression of human NIM1 kinase in brain tissue (74%) and
cancers of the breast (connective tissue) and prostate (genitalia,
male). The electronic northern was produced using the LIFESEQ Gold
database (Incyte Genomics, Palo Alto Calif.).
[0034] FIG. 3 shows a graph of transcript expression produced using
quantitative PCR in various cell lines and tissues. The x axis
shows fold expression; and the y axis, the cell lines (H460, A2780,
A375, HDF, HELA, DU145, MDA-MB231, U87-MG and BX-PC3) and tissues
(brain, colon, uterus and placenta).
[0035] FIGS. 4A, 4B, and 4C demonstrate the conserved chemical and
structural similarities among the catalytic domains of human NIM1
kinase (3317608CD1; SEQ ID NO:2), Caenorhabditis elegans STK
(g3877329; SEQ ID NO:31), Rattus norvegicus STK (g2052189; SEQ ID
NO:32), human C-TAK1 (g3089349; SEQ ID NO:33), Rattus norvegicus
salt-inducible kinase (g5672676; SEQ ID NO:34), Drosophila
melanogaster K78 protein kinase (g2564680; SEQ ID NO:35), and human
EMK1 (g1749794; SEQ ID NO:36). The alignment was produced using the
MEGALIGN program of LASERGENE software (DNASTAR, Madison Wis.).
[0036] FIG. 5 shows the human NIM1 (hNIM1) kinase assay. The
enzyme, hNIM1-GST; substrates: myelin basic protein (MBP) and
histone (HIST.1); and the positive control, ZAP70 (70 kd zeta-chain
(TCR) associated protein kinase). The designations + and - indicate
the presence or absence of the human NIM kinase and substrates in
the lane. Size markers in kilodaltons are shown along the right
side of the gel.
DESCRIPTION OF THE INVENTION
[0037] 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 only 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.
[0038] 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.
[0039] Definitions "Antibody" refers to intact immunoglobulin
molecule, a polyclonal antibody, a monoclonal antibody, a chimeric
antibody, a recombinant antibody, a humanized antibody, single
chain antibodies, a Fab fragment, an F(ab').sub.2 fragment, an Fv
fragment, and an antibody-peptide fusion protein.
[0040] "Antigenic determinant" refers to an antigenic or
immunogenic epitope, structural feature, or region of an
oligopeptide, peptide, or protein which is capable of inducing
formation of an antibody that specifically binds the protein.
Biological activity is not a prerequisite for immunogenicity.
[0041] "Array" refers to an ordered arrangement of at least two
nucleic acid molecules, proteins, or antibodies on a substrate. At
least one of the nucleic acid molecules, proteins, or antibodies
represents a control or standard, and the other nucleic acid
molecule, protein, or antibody is of diagnostic or therapeutic
interest. The arrangement of at least two and up to about 40,000
nucleic acid molecules, proteins, or antibodies on the substrate
assures that the size and signal intensity of each labeled complex,
formed between each nucleic acid molecule and at least one nucleic
acid, each protein and at least one ligand or antibody, or each
antibody and at least one protein to which the antibody
specifically binds, is individually distinguishable.
[0042] "Biologically active" refers to a protein having structural,
immunological, regulatory, or chemical functions of a naturally
occurring, recombinant or synthetic molecule.
[0043] A "bispecific molecule" has two different binding
specificities and can be bound to two different molecules or two
different sites on a molecule concurrently. Similarly, a
"multispecific molecule" can bind to multiple (more than two)
distinct targets, one of which may be a molecule on the surface of
an immune cell. Antibodies can perform as or be a part of
bispecific or multispecific molecules.
[0044] "Complementary" refer to the natural hydrogen bonding by
base pairing between purines and pyrimidines. For example, the
sequence A-C-G-T forms hydrogen bonds with its complements T-G-C-A
or U-G-C-A. Two single-stranded molecules may be considered
partially complementary, if only some of the nucleotides bond, or
completely complementary, if nearly all of the nucleotides bond.
The degree of complementarity between nucleic acid strands affects
the efficiency and strength of the hybridization and amplification
reactions.
[0045] A "composition" refers to the nucleic acid molecule and a
labeling moiety; a purified protein and a pharmaceutical carrier or
a heterologous, labeling or purification moiety; an antibody and a
labeling moiety or pharmaceutical agent; and the like.
[0046] "Derivative" refers to a nucleic acid molecule or a protein
that has been subjected to a chemical modification. Derivatization
of a nucleic acid molecule 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 nucleic acid molecule or a protein can also
involve the replacement of a hydrogen by an acetyl, acyl, alkyl,
amino, formyl, or morpholino group (for example, 5-methylcytosine).
Derivative molecules retain the biological activities of the
naturally occurring molecules but may confer longer lifespan or
enhanced activity.
[0047] "Differential expression" refers to an increased or
upregulated or a decreased or downregulated expression as detected
by absence, presence, or at least two-fold change in the amount of
transcribed messenger RNA or translated protein in a sample.
[0048] "Disorder" refers to conditions, diseases or syndromes in
which human NIM1 kinase or the mRNA encoding hNIM1 are
differentially expressed. These include disorders of the brain and
cancers of the brain, breast, cervix, colon, lung, ovary, and
prostate.
[0049] An "expression profile" is a representation of gene
expression in a sample. A nucleic acid expression profile is
produced using sequencing, hybridization, or amplification
technologies, transcript imaging as described in U.S. Pat. No.
5,840,484, incorporated herein by reference, and
guilt-by-association as described in U.S. Pat. No. 6,277,574, and
mRNAs or cDNAs from a sample. A protein expression profile,
although time delayed, mirrors the nucleic acid expression profile
and uses two-dimensional polyacrylamide electrophoresis (2D-PAGE),
mass spectrophotometry (MS), western analysis, enzyme-linked
immunosorbent assays (ELISAs), fluorescence activated cell sorting
(FACS), radioimmunoassays (RIAs), scintillation counters, and
arrays and antibodies or labeling moieties to detect protein
expression in a sample. The nucleic acids, proteins, or antibodies
may be used in solution or attached to a substrate, and their
detection is based on methods and labeling moieties well known in
the art.
[0050] "Fragment" refers to an Incyte clone or any part of a
nucleic acid molecule which retains a usable, functional
characteristic. Useful fragments are generally at least 18
consecutive nucleotides in length and include oligonucleotides
which may be used in hybridization, amplification or screening
technologies or in regulation of replication, transcription or
translation.
[0051] "Hybridization complex" refers to a complex between two
nucleic acid molecules by virtue of the formation of hydrogen bonds
between purines and pyrimidines.
[0052] "Identity" 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), CLUSTALW (Thompson et al. (1994)
Nucleic Acids Res 22:4673-4680), 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. "Similarity" uses the same
algorithms but takes conservative substitution of residues into
account. In proteins, similarity exceeds identity in that
substitution of a valine for a leucine or isoleucine, is counted in
calculating the reported percentage. Substitutions which are
considered to be conservative are well known in the art.
[0053] "Isolated" or "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.
[0054] "Labeling moiety" refers to any reporter molecule including
radionuclides, enzymes, fluorescent, chemiluminescent, or
chromogenic agents, substrates, cofactors, inhibitors, or magnetic
particles than can be attached to or incorporated into a
polynucleotide, protein, or antibody. Visible labels and dyes
include but are not limited to anthocyanins, .beta. glucuronidase,
biotin, BIODIPY, Coomassie blue, Cy3 and Cy5,
4,6-diamidino-2-phenylindole (DAPI), digoxigenin, fluorescein,
FITC, gold, green fluorescent protein (GFP), lissamine, luciferase,
phycoerythrin, rhodamine, spyro red, silver, streptavidin, and the
like. Radioactive markers include radioactive forms of hydrogen,
iodine, phosphorous, sulfur, and the like.
[0055] "Ligand" refers to any molecule, agent, or compound which
will bind specifically to a complementary site on a nucleic acid
molecule or protein. Such ligands stabilize or modulate the
activity of nucleic acid molecules or proteins of the invention and
may be composed of at least one of the following: inorganic and
organic substances including nucleic acids, proteins,
carbohydrates, fats, and lipids.
[0056] "hNIM1 kinase" refers to a purified enzyme obtained from any
mammalian species, including bovine, ovine, porcine, rodent,
canine, simian, and preferably the human species, and from any
source, whether natural, synthetic, semi-synthetic, or
recombinant.
[0057] "Nucleic acid molecule" refers to polynucleotide or cDNA, or
any fragment or complement thereof. It may be DNA or RNA of genomic
or synthetic origin, double-stranded or single-stranded, and
combined with carbohydrate, lipids, protein or other materials to
perform a particular activity such as transformation or form a
useful composition such as a peptide nucleic acid (PNA). It may
contain untranslated 5' or 3' regulatory regions and, rarely, an
intron.
[0058] "Oligonucleotide" is equivalent to the terms amplimer,
primer, or oligomer, is usually single stranded, extends about 18
to 60 nucleotides in length, and may be used in hybridization or
amplification technologies or in regulation of replication,
transcription, or translation.
[0059] "Oligopeptide" refers to a portion of a protein from about 5
to about 15 residues in length that is useful for making antibodies
or chimeric molecules.
[0060] A "pharmaceutical agent" may be an antibody, an antisense
molecule, a bispecific molecule, a multispecific molecule, a
peptide, a protein, a radionuclide, a small drug molecule, a
cytospecific or cytotoxic drug such as abrin, actinomyosin D,
cisplatin, crotin, doxorubicin, 5-fluorouracil, methotrexate,
ricin, vincristine, vinblastine, or any combination of these
elements.
[0061] The phrase "nucleic acid molecule encoding a protein" refers
to a nucleic acid molecule whose sequence closely aligns with
sequences that encode conserved regions, motifs or domains
identified by employing analyses well known in the art. These
analyses include BLAST (Altschul (1993) J Mol Evol 36:290-300;
Altschul et al. (1990) J Mol Biol 215:403-410) and BLAST2
(Altschul, supra) which provide identity within the conserved
region. Brenner et al. (1998; Proc Natl Acad Sci 95:6073-6078) who
analyzed BLAST for its ability to identify structural homologs by
sequence identity found 30% identity is a reliable threshold for
sequence alignments of at least 150 residues and 40% is a
reasonable threshold for alignments of at least 70 residues
(Brenner, page 6076, column 2).
[0062] "Protein" refers to an amino acid sequence, oligopeptide,
peptide, polypeptide or portions thereof whether naturally
occurring or synthetic.
[0063] "Portion" refers to any part of a protein used for any
purpose, but especially for the screening of a library of molecules
or compounds to identify a ligand.
[0064] "Sample" is used in its broadest sense as containing nucleic
acids, proteins, and antibodies. A sample may comprise a bodily
fluid such as ascites, blood, cerebrospinal fluid, lymph, semen,
sputum, urine and the like; 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 nucleic acid molecule in solution or
bound to a substrate; a cell; a tissue, a tissue biopsy, or a
tissue print; buccal cells, skin, hair, a hair follicle; and the
like.
[0065] "Specific binding" or "specifically binding" refers to the
interaction between two molecules. In the case of a polynucleotide,
specific binding may involve hydrogen bonding between sense and
antisense strands or between one strand and a protein which affects
its replication or transcription, intercalation of a molecule or
compound into the major or minor groove of the DNA molecule, or
interaction with at least one molecule which functions as a
transcription factor, enhancer, repressor, and the like. In the
case of a protein, specific binding may involve interactions with
polynucleotides, as described above or with molecules or compounds
such as agonists, antibodies, antagonists, and the like. Specific
binding is dependent upon the presence of structural features that
allow appropriate chemical or molecular interactions between
molecules.
[0066] "Substrate" refers to any rigid or semi-rigid support to
which polynucleotides, proteins, or antibodies are bound and
includes magnetic or nonmagnetic beads, capillaries or other
tubing, chips, fibers, filters, gels, membranes, plates, polymers,
slides, wafers, and microparticles with a variety of surface forms
including channels, columns, pins, pores, trenches, and wells.
[0067] The Invention
[0068] The invention is based on the discovery of a nucleic acid
molecule which encodes human NIM1 kinase and on the use of the
nucleic acid molecule, protein, and an antibody that specifically
binds the protein as compositions in the characterization,
diagnosis, and treatment of brain disorders and cancers.
[0069] The nucleic acid molecule encoding NIM1 kinase of the
present invention was first identified as a kinase by Block II
homology match between Incyte Clone 670279 from the cerebellum
library (CRBLNOT01) and a putative STK of C. elegans (g733122)
important for the polarity of zygote division and differentiation.
The full length nucleic acid molecule, Incyte Clone 3317608 (SEQ ID
NO:1) was sequenced and assembled from the Incyte LIFESEQ GOLD
database (Mar 99 release) template 200700.1, the assembly 670279CB1
and Incyte Clones (Library): 3317608H1 (PROSBPT03), 4313713H1
(BRAFNOT01), 4617082H1 (BRAYDIT01), 4711644H1 (BRAIHCT01),
2286324H1 (BRAINON01), 2286816H1 (BRAINON01), 2287217H1
(BRAINON01), 2286816R6 (BRAINON01), 2286816T6 (BRAINON01),
3317608T6 (PROSBPT03), 4201896T6 (BRAITUT29), 462481 1T6
(FIBRTXT02), 6559834H1 (BRAFNON02), 670279F1 (CRBLNOT01), 670279H1
(CRBLNOT01), 670279R1 (CRBLNOT01), 670279R6 (CRBLNOT01), 670279T6
(CRBLNOT01), and 4936446H1 (BRAXNOT03) and which are SEQ ID
NOs:3-23, respectively. Useful fragments of the nucleic acid
molecule that encodes SEQ ID NO:2 include a fragment of at least 18
consecutive nucleotides selected from about nucleotide 414 to about
nucleotide 1414 of SEQ ID NO:1 and SEQ ID NOs:3-23 or the
complements thereof. The Incyte clone 3317608CA has been designated
a verified reagent, and Incyte clones 2286816 and 4201896,
component sequences of SEQ ID NO:1 as listed above, were used as
array elements.
[0070] FIGS. 1A-1F show the sequence of the nucleic acid molecule
and its deduced translation into amino acids. Incyte Clone number
3317608 which contains the coding region for the human NIM1 kinase
has been deposited in the American Type Culture Collection (ATCC;
Manassas Va.) and has the Patent Deposit Designation: PT-1217.
[0071] As shown in FIG. 2, electronic northern analysis showed
highly differential expression of the transcript encoding NIM1
kinase in the nervous system. All of these libraries were from
brain; five were associated with cancer, three, with stroke, two,
with Huntington's disease and one, with epilepsy. The transcript
was found to be expressed only two other times in the LIFESEQ
database (Incyte Genomics) which contains 1039 libraries and over 5
million sequences, in cancerous breast fibroblasts, FIBRTXT02, and
in cancerous prostate, PROSBPT03.
[0072] As presented in FIG. 3, northern analysis was also performed
in the laboratory using quantitative PCR. Expression of human NIM1
kinase transcripts is shown in H460, A2780, A375, HDF, HELA, DU145,
MDA-MB231, U87-MG, and BX-PC3 cell lines and in brain, colon,
uterus and placenta tissues. Of particular note, human breast
carcinoma cell line, MDA-MB231, and brain tissue show approximately
100 and 155 fold expression of hNIM1 transcripts, respectively.
Greater than 5-fold expression was also seen in cell lines
representing carcinomas of the brain, cervix, colon, lung, ovary,
and prostate and in normal uterus and placenta.
[0073] NIM1 kinase comprising the amino acid sequence of SEQ ID
NO:2 is 436 amino acids in length and has a potential signal
sequence from M1 to A18, potential phosphorylation sites at
residues S56, T108, Ti14,S123,S169,S221,S282, Y288, T311, T332,
T349, S359,S420, and T429. The human NIM1 kinase, as shown in FIGS.
4A-4C, is used as the reference for numbering the conserved
residues, motifs, and subdomains of catalytic region of the kinases
(SEQ ID NOs:2 and 31-36) in the alignment. The residues, motifs and
subdomains are: subdomain 1 extends from G81 to V88; subdomain 2,
has conserved residues A101 and K103; subdomain 3 has the invariant
E121; subdomain extends from E151 to E 157 and shares the invariant
residues M150 and Y152; subdomain 6B which represents the catalytic
loop, extends from H194 to N201; subdomain 7, has the highly
conserved triplet, D214, F215, and G216; subdomain 8, extends from
T229 to F242 and contains the A238, P239, E240 motif; subdomain 9
has the invariant D253, subdomain 11 has the invariant R244 which
interacts with the APE motif of subdomain 8. The C terminal
boundary of the catalytic domain begins with H248, A249 and F250.
Both PFAM and PRINTS analyses confirm these kinase motifs and
domains. Useful portions of the polypeptide having the nucleic acid
sequence of SEQ ID NO:2 include a portion of at least 6 consecutive
amino acids selected from about residue 1 to about residue 295 of
SEQ ID NO:2.
[0074] FIG. 5 shows the human NIM1 kinase assay. hNIM1 kinase-GST
autophosphorylated as shown in lanes 1, 2, and 4 and phosphorylated
its substrates MBP, lane 2, and HIST. 1, lane 4. In the absence of
kinase, substrates MBP, lane 3, and HIST. 1, lane 5, show no
phosphorylation. Lane 6 shows the positive control ZAP70.
[0075] The results of a lung cancer study using the HumanGenome
GEM1 (HG1) microarray are shown in the table below. The first
column shows the log2(Cy3/Cy5); the second column, the description
of the Cy3 sample, normal tissue sample identified by donor number;
and the third column, the description of the Cy5 sample, cancer
tissue sample identified by donor number. Five of the cancer
samples are matched with cytologically normal tissue from the same
donor. Donor tissues are described in Example VII.
1 log2Cy3/Cy5 Description of Cy3 sample Description of Cy5 sample
1.04 Nrml, Dn7965 Adenocarcinoma, Dn7965 1.06 Nrml, Dn5795
Adenocarcinoma, Dn5795 1.06 Nrml, Dn7966 Adenocarcinoma, Dn7966
1.11 Nrml, Pool, Dn9007 Adenocarcinoma, Dn7976 1.14 Nrml, Dn7968
Squamous carcinoma, Dn7968 1.23 Nrml, Dn7188 Adenocarcinoma, Dn7188
1.39 Nrml, Pool, Dn9007 Adenocarcinoma, Dn7975
[0076] Differential expression of hNIM1 in non-small cell lung
cancers was considered to be significant if it exceeded two-fold or
log.sub.2.gtoreq.1.0. Expression was not seen in small cell lung
cancer or lung carcinoids.
[0077] Mammalian variants of the nucleic acid molecules encoding
the human NIM1 kinase were identified by using BLAST or BLAST2
(with default parameters), to identify clones in the LIFESEQ or
ZOOSEQ databases (Incyte Genomics) which aligned with SEQ ID NOs:1
and 3-23. The mammalian variants are ZOOSEQ database template (Dec
99 build) 216150.1 (SEQ ID NO:24) and Incyte clones: 701925441 HI
(RALITXS03), 701910632H 1 (RABYUNN02), 701905514H1 (RABYUNS09),
701293826H1 (RABXNOT04), and 700949543H1 (RASPNON02) from rat and
700706950H1 (MNBFNOT01) from monkey; SEQ ID NOs:25-30,
respectively.
[0078] The nucleic acid molecules and fragments thereof (SEQ ID
NOs:1 and 3-30) may be used in hybridization, amplification, and
screening technologies to identify and distinguish among SEQ ID
NO:1 and similar molecules in a sample. The human molecules and
their mammalian variants may be used to produce transgenic
organisms which model the human disorders and upon which potential
therapeutic treatments may be tested. Toxicology studies, clinical
trials, and subject/patient treatment profiles may be performed and
monitored using the nucleic acid molecules, proteins, antibodies
and molecules and compounds identified using the nucleic acid
molecules and proteins of the present invention.
[0079] Characterization and Use of the Invention
[0080] cDNA libraries
[0081] In a particular embodiment disclosed herein, mRNA was
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 clones listed above were isolated from
mammalian cDNA libraries. Three library preparations representative
of the invention are described in the EXAMPLES below. The consensus
sequences were chemically and/or electronically assembled from
fragments including Incyte clones and extension and/or shotgun
sequences using computer programs such as PHRAP (P Green,
University of Washington, Seattle Wash.), GELVIEW Fragment Assembly
system (Accelrys), and AUTOASSEMBLER application (Applied
Biosystems (ABI), Foster City Calif.). Clones, extension and/or
shotgun sequences are electronically assembled into clusters and/or
master clusters.
[0082] Sequencing
[0083] 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 Biosciences (APB), Piscataway N.J.), or
combinations of polymerases and proofreading exonucleases
(Invitrogen, Carlsbad Calif.). Preferably, sequence preparation is
automated with machines such as the DNA ENGINE thermal cycler
(PTC200; MJ Research, Watertown Mass.). Machines used for
sequencing include the PRISM 3700, 377 or 373 DNA sequencing
systems (ABI), the MEGABACE 1000 DNA sequencing system (APB), and
the like.
[0084] The sequences of the nucleic acid molecules presented in the
Sequence Listing were prepared by such automated methods and may
contain occasional sequencing errors and unidentified nucleotides,
designated with an N, that reflect state-of-the-art technology at
the time the cDNA was sequenced. In most sequences, vector, linker,
and polyA sequences have been masked using algorithms and programs
based on BLAST, dynamic programming, and dinucleotide nearest
neighbor analysis. Ns and single nucleotide polymorphisms (SNPs)
can be verified either by resequencing the cDNA or using algorithms
to compare multiple sequences that overlap the area in which the Ns
or SNPs occur. Both of these techniques are well known to and used
by those skilled in the art. A variety of algorithms useful in
comparing sequences are 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).
[0085] 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 nucleic acid molecules 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.
[0086] Extension of a Nucleic Acid Sequence
[0087] The sequences of the invention may be extended using various
PCR-based methods known in the art. For example, the XL-PCR kit
(ABI), 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 CO) 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 55C
to about 68C. When extending a sequence to recover regulatory
elements, it is preferable to use genomic, rather than cDNA
libraries.
[0088] Hybridization
[0089] The nucleic acid molecule 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 human NIM1 kinase, 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:3-30. 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 nucleic acid molecule 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 (APB).
[0090] 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. In solutions used for
some membrane based hybridizations, addition of an organic solvent
such as formamide allows the reaction to occur at a lower
temperature. Hybridization can be performed at low stringency with
buffers, such as 5.times.SSC with 1% sodium dodecyl sulfate (SDS)
at 60C, 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 45C (medium
stringency) or 68C (high stringency). At high stringency,
hybridization complexes will remain stable only where the nucleic
acid molecules 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 other 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.).
[0091] Arrays may be prepared and analyzed using methods known in
the art. cDNAs or oligonucleotides may be used as either probes or
elements on an array. The array can be used to monitor the
expression level of large numbers of genes simultaneously and to
identify genetic variants, mutations, and single nucleotide
polymorphisms. Arrays may be used to determine gene function; to
understand the genetic basis of a disorder; to diagnose a disorder;
and to develop and monitor the activities of therapeutic agents.
(See, e.g., 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; U.S. Pat. No. 5,605,662.)
[0092] 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 , yeast artificial chromosomes,
bacterial artificial chromosomes, bacterial P1 constructions, or
the cDNAs of libraries made from single chromosomes.
[0093] QPCR
[0094] QPCR is a method for quantifying a nucleic acid molecule
based on detection of a fluorescent signal produced during PCR
amplification (Gibson et al. (1996) Genome Res 6:995-1001; Heid et
al. (1996) Genome Res 6:986-994). Amplification is carried out
using machines such as the PRISM 7700 detection system (ABI) which
consists of a 96-well thermal cycler connected to a laser and
charge-coupled device (CCD) optics system. To perform QPCR, a PCR
reaction is carried out in the presence of a doubly labeled probe.
The probe, which is designed to anneal between the standard forward
and reverse PCR primers, is labeled at the 5' end by a flourogenic
reporter dye such as 6-carboxyfluorescein (6-FAM) and at the 3' end
by a quencher molecule such as 6-carboxy-tetramethyl-rhodamine
(TAMRA). As long as the probe is intact, the 3' quencher
extinguishes fluorescence by the 5' reporter. However, during each
primer extension cycle, the annealed probe is degraded as a result
of the intrinsic 5' to 3' nuclease activity of Taq polymerase
(Holland et al. (1991) Proc Natl Acad Sci 88:7276-7280). This
degradation separates the reporter from the quencher, and
fluorescence is detected every few seconds by the CCD. The higher
the starting copy number of the nucleic acid, the sooner an
increase in fluorescence is observed. A cycle threshold (C.sub.T)
value, representing the cycle number at which the PCR product
crosses a fixed threshold of detection is determined by the
instrument software. The C.sub.T is inversely proportional to the
copy number of the template and can therefore be used to calculate
either the relative or absolute initial concentration of the
nucleic acid molecule in the sample. The relative concentration of
two different molecules can be calculated by determining their
respective C.sub.T values (comparative C.sub.T method).
Alternatively, the absolute concentration of the nucleic acid
molecule can be calculated by constructing a standard curve using a
housekeeping molecule of known concentration. The process of
calculating C.sub.T values, preparing a standard curve, and
determining starting copy number is performed using SEQUENCE
DETECTOR 1.7 software (ABI).
[0095] Expression
[0096] A multitude of nucleic acid molecules encoding NIM1 kinase
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, nucleic acid molecule,
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).
[0097] 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.
[0098] Routine cloning, subcloning, and propagation of nucleic acid
sequences can be achieved using the multifunctional pBLUESCRIPT
vector (Stratagene, La Jolla Calif.) or pSPORT1 plasmid
(Invitrogen). Introduction of a nucleic acid sequence into the
multiple cloning site of these vectors disrupts the lacZ gene and
allows calorimetric 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.
[0099] 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, such as anthocyanins, GFP, .beta. glucuronidase,
luciferase and the like, may be propagated using culture
techniques. Visible markers are also used to quantify the amount of
protein expressed by the introduced genes. Verification that the
host cell contains the desired mammalian nucleic acid molecule is
based on DNA-DNA or DNA-RNA hybridizations or PCR amplification
techniques.
[0100] 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 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.
[0101] Recovery of Proteins from Cell Culture
[0102] Heterologous moieties engineered into a vector for ease of
purification include glutathione S-transferase (GST), calmodulin
binding peptide (CBP), 6-His, FLAG, MYC, and the like. GST, CBP,
and 6-His are purified using commercially available affinity
matrices such as immobilized glutathione, calmodulin, and
metal-chelate resins, respectively. FLAG and MYC are purified using
commercially available monoclonal and polyclonal antibodies. A
proteolytic cleavage site may be located between the desired
protein sequence and the heterologous moiety for ease of separation
following purification. Methods for recombinant protein expression
and purification are discussed in Ausubel (supra, unit 16) and are
commercially available.
[0103] Protein Identification
[0104] Several techniques have been developed which permit rapid
identification of proteins using high performance liquid
chromatography and mass spectrometry (MS). Beginning with a sample
containing proteins, the method is: 1) proteins are separated using
two-dimensional gel electrophoresis (2-DE), 2) selected proteins
are excised from the gel and digested with a protease to produce a
set of peptides; and 3) the peptides are subjected to mass spectral
analysis to derive peptide ion mass and spectral pattern
information. The MS information is used to identify the protein by
comparing it with information in a protein database (Shevenko et
al. (1996) Proc Natl Acad Sci 93:14440-14445).
[0105] Proteins are separated by 2-DE employing isoelectric
focusing (IEF) in the first dimension followed by SDS-PAGE in the
second dimension. For IEF, an immobilized pH gradient strip is
useful to increase reproducibility and resolution of the
separation. Alternative techniques may be used to improve
resolution of very basic, hydrophobic, or high molecular weight
proteins. The separated proteins are detected using a stain or dye
such as silver stain, Coomassie blue, or spyro red (Molecular
Probes, Eugene Oreg.) that is compatible with MS. Gels may be
blotted onto a PVDF membrane for western analysis and optically
scanned using a STORM scanner (APB) to produce a computer-readable
output which is analyzed by pattern recognition software such as
MELANIE (GeneBio, Geneva, Switzerland). The software annotates
individual spots by assigning a unique identifier and calculating
their respective x, y coordinates, molecular masses, isoelectric
points, and signal intensity. Individual spots of interest, such as
those representing differentially expressed proteins, are excised
and proteolytically digested with a site-specific protease such as
trypsin or chymotrypsin, singly or in combination, to generate a
set of small peptides, preferably in the range of 1-2 kDa. Prior to
digestion, samples may be treated with reducing and alkylating
agents, and following digestion, the peptides are then separated by
liquid chromatography or capillary electrophoresis and analyzed
using MS.
[0106] MS converts components of a sample into gaseous ions,
separates the ions based on their mass-to-charge ratio, and
determines relative abundance. For peptide mass fingerprinting
analysis, a MALDI-TOF (Matrix Assisted Laser
Desorption/Ionization-Time of Flight), ESI (Electrospray
Ionization), and TOF-TOF (Time of Flight/Time of Flight) machines
are used to determine a set of highly accurate peptide masses.
Using analytical programs, such as TURBOSEQUEST software (Finnigan,
San Jose Calif.), the MS data is compared against a database of
theoretical MS data derived from known or predicted proteins. A
minimum match of three peptide masses is used for reliable protein
identification. If additional information is needed for
identification, Tandem-MS may be used to derive information about
individual peptides. In tandem-MS, a first stage of MS is performed
to determine individual peptide masses. Then selected peptide ions
are subjected to fragmentation using a technique such as collision
induced dissociation (CID) to produce an ion series. The resulting
fragmentation ions are analyzed in a second round of MS, and their
spectral pattern may be used to determine a short stretch of amino
acid sequence (Dancik et al. (1999) J Comput Biol 6:327-342).
Assuming the protein is represented in the database, a combination
of peptide mass and fragmentation data, together with the
calculated MW and pI of the protein, will usually yield an
unambiguous identification. If no match is found, protein sequence
can be obtained using direct chemical sequencing procedures well
known in the art (cf. Creighton (1984) Proteins, Structures and
Molecular Properties, WH Freeman, New York N.Y.).
[0107] Chemical Synthesis of Peptides
[0108] 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 431A peptide synthesizer (ABI).
A protein or portion thereof may be 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, WH Freeman, New York
N.Y.).
[0109] Antibodies
[0110] Antibodies, or immunoglobulins (Ig), are components of
immune response expressed on the surface of or secreted into the
circulation by B cells. The prototypical antibody is a tetramer
composed of two identical heavy polypeptide chains (H-chains) and
two identical light polypeptide chains (L-chains) interlinked by
disulfide bonds which binds and neutralizes foreign antigens. Based
on their H-chain, antibodies are classified as IgA, IgD, IgE, IgG
or IgM. The most common class, IgG, is tetrameric while other
classes are variants or multimers of the basic structure.
[0111] Antibodies are described in terms of their two functional
domains. Antigen recognition is mediated by the Fab (antigen
binding fragment) region of the antibody, while effector functions
are mediated by the Fc (crystallizable fragment) region. The
binding of antibody to antigen triggers destruction of the antigen
by phagocytic white blood cells such as macrophages and
neutrophils. These cells express surface Fc receptors that
specifically bind to the Fc region of the antibody and allow the
phagocytic cells to destroy antibody-bound antigen. Fc receptors
are single-pass transmembrane glycoproteins containing about 350
amino acids whose extracellular portion typically contains two or
three Ig domains (Sears et al. (1990) J Immunol 144:371-378).
[0112] Preparation and Screening of Antibodies
[0113] Various hosts including mice, rats, rabbits, goats, llamas,
camels, and human cell lines may be immunized by injection with an
antigenic determinant. Adjuvants such as Freund's, mineral gels,
and surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemacyanin (KLH; Sigma-Aldrich), and dinitrophenol may be used to
increase immunological response. In humans, BCG (bacilli
Calmette-Guerin) and Cormebacterium parvum increase response. The
antigenic determinant may be an oligopeptide, peptide, or protein.
When the amount of antigenic determinant allows immunization to be
repeated, specific polyclonal antibody with high affinity can be
obtained (Klinman and Press (1975) Transplant Rev 24:41-83).
Oligopepetides which may contain between about five and about
fifteen amino acids identical to a portion of the endogenous
protein may be fused with proteins such as KLH in order to produce
antibodies to the chimeric molecule.
[0114] Monoclonal antibodies may be prepared using any technique
which provides for the production of antibodies by continuous cell
lines in culture. These include the hybridoma technique, the human
B-cell hybridoma technique, and the EBV-hybridoma technique (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).
[0115] Chimeric antibodies may be produced by techniques such as
splicing of mouse antibody genes to human antibody genes to obtain
a molecule with appropriate antigen specificity and biological
activity (Morrison et al. (1984) Proc Natl Acad Sci 81:6851-6855;
Neuberger et al. (1984) Nature 312:604-608; and Takeda et al.
(1985) Nature 314:452-454). Alternatively, techniques described for
antibody production may be adapted, using methods known in the art,
to produce specific, single chain antibodies. Antibodies with
related specificity, but of distinct idiotypic composition, may be
generated by chain shuffling from random combinatorial
immunoglobulin libraries (Burton (1991) Proc Natl Acad Sci
88:10134-10137). Antibody fragments which contain specific binding
sites for an antigenic determinant may also be produced. 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 (Huse et al. (1989)
Science 246:1275-1281).
[0116] Antibodies may also be produced by inducing production in
the lymphocyte population or by screening immunoglobulin libraries
or panels of highly specific binding reagents as disclosed in
Orlandi et al. (1989; Proc Natl Acad Sci 86:3833-3837) or Winter et
al. (1991; Nature 349:293-299). A protein may be used in screening
assays of phagemid or B-lymphocyte immunoglobulin libraries to
identify antibodies having a desired specificity. Numerous
protocols for competitive binding or immunoassays using either
polyclonal or monoclonal antibodies with established specificities
are well known in the art.
[0117] Antibody Specificity
[0118] Various methods such as Scatchard analysis combined with
radioimmunoassay techniques may be used to assess the affinity of
particular antibodies for a protein. Affinity is expressed as an
association constant, K.sub.a, which is defined as the molar
concentration of protein-antibody complex divided by the molar
concentrations of free antigen and free antibody under equilibrium
conditions. The K.sub.a determined for a preparation of polyclonal
antibodies, which are heterogeneous in their affinities for
multiple antigenic determinants, represents the average affinity,
or avidity, of the antibodies. The K.sub.a determined for a
preparation of monoclonal antibodies, which are specific for a
particular antigenic determinant, represents a true measure of
affinity. High-affinity antibody preparations with K.sub.a ranging
from about 10.sup.9 to 10.sup.12 L/mole are commonly used in
immunoassays in which the protein-antibody complex must withstand
rigorous manipulations. Low-affinity antibody preparations with
K.sub.a ranging from about 10.sup.6 to 10.sup.7 L/mole are
preferred for use in immunopurification and similar procedures
which ultimately require dissociation of the protein, preferably in
active form, from the antibody (Catty (1988) Antibodies, Volume I:
A Practical Approach, IRL Press, Washington D.C.; Liddell and Cryer
(1991) A Practical Guide to Monoclonal Antibodies, John Wiley &
Sons, New York N.Y.).
[0119] The titer and avidity of polyclonal antibody preparations
may be further evaluated to determine the quality and suitability
of such preparations for certain downstream applications. For
example, a polyclonal antibody preparation containing about 5-10 mg
specific antibody/ml, is generally employed in procedures requiring
precipitation of protein-antibody complexes. Procedures for making
antibodies, evaluating antibody specificity, titer, and avidity,
and guidelines for antibody quality and usage in various
applications, are discussed in Catty (supra) and Ausubel (supra)
pp. 11.1-11.31.
[0120] Diagnostics
[0121] Differential expression of hNIM1 is associated with brain
disorders such as such as Alzheimer's disease, amyotrophic lateral
sclerosis, cerebral palsy, dementia, Down's syndrome, multiple
sclerosis, muscular dystrophy, cerebral neoplasms, Parkinson's
disease, schizophrenia, and in particular, epilepsy, Huntington's
chorea and stroke; and such as adenocarcinoma, leukemia, lymphoma,
melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular,
cancers of the brain, breast, cervix, colon, lung, ovary, and
prostate. Differential expression can be detected using the
protein, the nucleic acid molecule encoding hNIM1, or an antibody
that specifically binds hNIM1 and at least one of the assays
below.
[0122] Nucleic Acid Assays
[0123] The nucleic acid molecules, fragments, oligonucleotides,
complementary RNA and DNA molecules, and PNAs may be used in assays
to detect and quantify differential expression. Differential
expression has been associated with brains disorders and cancers.
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 and described in the EXAMPLES.
[0124] Protein Assays
[0125] Two-dimensional polyacrylamide electrophoresis (2D-PAGE) may
be used in combination with Bradford analysis, scintillation
counting, mass spectrophotometry (MS) and immunological methods
well known in the art to quantitate hNIM1 expression in samples.
These assays are described in Ausubel (supra), units 10.1-10.7.
[0126] Immunological methods for detecting and measuring complex
formation as a measure of protein expression using either specific
polyclonal or monoclonal antibodies are known in the art. Examples
of such techniques include western analysis, enzyme-linked
immunosorbent assays (ELISAs), radioimmunoassays (RIAs),
fluorescence-activated cell sorting (FACS) and protein and antibody
arrays. Such immunoassays typically involve the measurement of
complex formation between the protein and an antibody that
specifically binds the protein. These assays and their comparison
with standards are well known in the art (Ausubel, supra, unit
10.1-10.6). A two-site, monoclonal-based immunoassay utilizing
antibodies reactive to two non-interfering epitopes is preferred,
but a competitive binding assay may be employed (Pound (1998)
Immunochemical Protocols, Humana Press, Totowa N.J.).
[0127] Protein assays are useful for diagnosing diseases that show
differential protein expression. Normal or standard values for
protein expression are established by combining samples taken from
a normal mammalian or human subject with antibodies that
specifically bind the protein under conditions for complex
formation. Standard values for complex formation in normal and
diseased tissues are established by various methods, often
photometric means. Then complex formation as it is expressed in a
subject sample is compared with the standard values. Deviation from
the normal standard and toward the diseased standard provides
parameters for disease diagnosis or prognosis while deviation away
from the diseased and toward the normal standard may be used to
evaluate treatment efficacy.
[0128] Recently, antibody arrays have allowed the development of
techniques for high-throughput screening of recombinant antibodies.
Such methods use robots to pick and grid bacteria containing
antibody genes, and a filter-based ELISA to screen and identify
clones that express antibody fragments. Because liquid handling is
eliminated, and the clones are arrayed from master stocks; the same
antibodies can be spotted multiple times and screened against
multiple antigens simultaneously. Antibody arrays are highly useful
in the identification of differentially expressed proteins and,
because of ease in handling and time saved, will become the
standard for diagnosis and prognosis (de Wildt et al. (2000) Nature
Biotechnol 18:989-94).
[0129] Expression Profiles
[0130] A gene expression profile comprises the expression of a
plurality of nucleic acid molecules or proteins as measured using
the assays above with a sample. The nucleic acid molecules or
antibodies that specifically bind hNIM1 may be used as elements on
a array to produce a expression profile. Once the differences in
gene expression between healthy and diseased tissues or cells are
assessed, the array is used to diagnose, to stage, or to monitor
the progression or treatment of a disease.
[0131] For example, a nucleic acid molecule 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.
[0132] By analyzing changes in patterns of gene expression, a
disorder can be diagnosed at earlier stages before the patient is
symptomatic. The invention can be used to formulate a prognosis and
to design a treatment regimen. The invention can also be used to
monitor the efficacy of treatment. For treatments with known side
effects, the array is employed to improve the treatment regimen. A
dosage that results in a more normal genetic expression pattern is
indicative of successful treatment. Expression patterns associated
with the onset of undesirable side effects are avoided. This
approach may be more sensitive and rapid than waiting for the
patient to show inadequate improvement, or to manifest side
effects, before altering the course of treatment.
[0133] In one embodiment, animal models which mimic a human disease
can be used to characterize expression profiles associated with a
particular disorder or treatment of that disorder. Novel treatment
regimens may be tested in these animal models using arrays to
establish and then follow expression over time. In addition, arrays
may be used with cell cultures or tissues removed from animal
models to rapidly screen large numbers of candidate drug molecules,
looking for ones that produce an expression profile similar to
those of known therapeutic drugs, with the expectation that
molecules with the same expression profile will likely have similar
therapeutic effects. Thus, the invention provides the means to
rapidly determine the molecular mode of action of a drug.
[0134] Assays may also be used to evaluate the efficacy of a
particular therapeutic treatment regimen in animal studies, in
clinical trials or in treating an individual patient. Once the
presence of a disorder 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 years.
[0135] Labeling of Molecules for Assay
[0136] A wide variety of labeling moieties 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 kits such as those supplied
by Promega (Madison Wis.) or APB for incorporation of a labeled
nucleotide such as .sup.32P-dCTP (APB), Cy3-dCTP or Cy5-dCTP
(Qiagen-Operon, Alameda Calif.), or an 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).
[0137] Therapeutics
[0138] Chemical and structural similarity, in the context of the
kinase catalytic domain, exists between regions of human NIM1
kinase (SEQ ID NO:2) and the kinases of C. elegans STK (g3877329;
SEQ ID NO:31), R. norvegicus STK (g2052189; SEQ ID NO:32), human
C-TAK1 (g3089349; SEQ ID NO:33), R. norvegicus salt-inducible
kinase (g5672676; SEQ ID NO:34), D. melanogaster K78 protein kinase
(g2564680; SEQ ID NO:35), and human EMK1 (g1749794; SEQ ID NO:36)
shown in FIGS. 4A-4C. In addition, gene expression is highly
associated with brain disorders and cancers of the brain, breast,
cervix, colon, lung, ovary, and prostate as shown in FIGS. 2 and 3
and the microarray data. Disorders associated with differential
expression of hNIM1 include Alzheimer's disease, amyotrophic
lateral sclerosis, cerebral palsy, dementia, Down's syndrome,
epilepsy, Huntington's disease, multiple sclerosis, muscular
dystrophy, cerebral neoplasms, Parkinson's disease, schizophrenia,
stroke, and cancers such as adenocarcinoma, leukemia, lymphoma,
melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular,
cancers of the brain, breast, cervix, colon, lung, ovary, and
prostate.
[0139] In one embodiment, when decreased expression or activity of
hNIM1 is desired, an antibody, antagonist, inhibitor, a
pharmaceutical agent or a composition containing one or more of
these molecules may be delivered to a subject in need of such
treatment. Such delivery may be effected by methods well known in
the art and may include delivery by an antibody that specifically
binds the protein. For therapeutic use, monoclonal antibodies are
used to block an active site, inhibit dimer formation, trigger
apoptosis, and the like.
[0140] In another embodiment, when increased expression or activity
of hNIM1 is desired, the protein, an agonist, an enhancer, a
pharmaceutical agent or a composition containing one or more of
these molecules may be delivered to a subject in need of such
treatment. Such delivery may be effected by methods well known in
the art and may include delivery of a pharmaceutical agent by an
antibody or other bispecific molecule specifically targeted to the
protein.
[0141] In an additional embodiment, a vector expressing the nucleic
acid molecule, or the complement thereof, may be administered to a
subject to modulate expression of hNIM1.
[0142] Any of the nucleic acid molecules, proteins, agonists,
antagonists, antibodies, bispecific molecules, vectors, or
pharmaceutical agents may be administered in combination with other
therapeutics. Selection of 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 effect treatment of a
particular disorder at a lower dosage of each agent.
[0143] Modification of Gene Expression Using Nucleic Acids
[0144] 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 NIM1 kinase.
Oligonucleotides designed with reference to the transcription
initiation site 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 of nucleic acid molecules or fragments thereof may be
screened to identify those which specifically bind a regulatory,
nontranslated sequence.
[0145] Natural ribozymes or synthetic 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.
[0146] Sense or antisense nucleic acids 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, or the modification of adenine, cytidine, guanine,
thymine, and uridine with acetyl-, methyl-, thio-groups renders the
molecule less available to endogenous endonucleases.
[0147] Nucleic Acid Therapeutics
[0148] The nucleic acid molecules of the invention can be used in
gene therapy. The nucleic acid molecules in expression vectors can
be delivered ex vivo to target cells, such as cells of bone marrow.
Once stable integration and transcription are confirmed, the bone
marrow may be reintroduced into the subject. These techniques are
known in the art and have been used to treat breast cancer.
Expression of the protein encoded by the nucleic acid molecule may
correct a disorder associated with mutation of a normal sequence,
reduction or loss of an endogenous target protein, or overepression
of an endogenous or mutant protein. Alternatively, nucleic acid
molecules may be delivered in vivo using vectors such as
retrovirus, adenovirus, adeno-associated virus, herpes simplex
virus, and bacterial plasmids. Non-viral methods of gene delivery
include cationic liposomes, polylysine conjugates, artificial viral
envelopes, and direct injection of DNA (Anderson (1998) Nature
392:25-30; Dachs et al. (1997) Oncol Res 9:313-325; Chu et al.
(1998) J Mol Med 76(3-4):184-192; Weiss et al. (1999) Cell Mol Life
Sci 55(3):334-358; Agrawal (1996) Antisense Therapeutics, Humana
Press, Totowa N.J.; and August et al. (1997) Gene Therapy (Advances
in Pharmacology, Vol. 40), Academic Press, San Diego Calif.).
[0149] Monoclonal Antibody Therapeutics
[0150] Antibodies, and in particular monoclonal antibodies, that
specifically bind a particular protein, enzyme, or receptor and
block its expression or activity in the disease process are now
being used therapeutically. The first widely accepted therapeutic
antibodies were HERCEPTIN (Trastuzumab, Genentech, S. San Francisco
Calif.) and GLEEVEC (imatinib mesylate, Norvartis Pharmaceuticals,
East Hanover N.J.). HERCEPTIN is a humanized antibody approved for
the treatment of HER2 positive metastatic breast cancer. It is
designed to bind and block the function of overexpressed HER2
protein. GLEEVEC is indicated for the treatment of patients with
Philadelphia chromosome positive (Ph+) chronic myeloid leukemia
(CML) in blast crisis, accelerated phase, or in chronic phase after
failure of interferon-alpha therapy. A second indication for
GLEEVEC is treatment of patients with KIT (CD117) positive
unresectable and/or metastatic malignant gastrointestinal stromal
tumors. Other monoclonal antibodies are in various stages of
clinical trials for indications such as prostate cancer, lymphoma,
melanoma, pneumococcal infections, rheumatoid arthritis, psoriasis,
systemic lupus erythematosus, and the like.
[0151] Screening and Purification Assays
[0152] The nucleic acid molecule encoding hNIM1 may be used to
screen a library of molecules or compounds for specific binding
affinity. The libraries may be aptamers, DNA molecules, PNAs,
peptides, proteins such as transcription factors, enhancers, and
repressors, RNA molecules, and other ligands which regulate the
activity, replication, transcription, or translation of the nucleic
acid molecule in the biological system. The assay involves
combining the nucleic acid molecule or a fragment thereof with the
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
nucleic acid molecule.
[0153] In one embodiment, the nucleic acid molecule of the
invention may be incubated with a library of isolated and 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 polynucleotide may be incubated with
nuclear extracts from biopsied and/or cultured cells and tissues.
Specific binding between the polynucleotide and a molecule or
compound in the nuclear extract is initially determined by gel
shift assay and may be later confirmed by raising antibodies
against that molecule or compound. When these antibodies are added
into the assay, they cause a supershift in the gel-retardation
assay.
[0154] In another embodiment, the nucleic acid molecule may be used
to purify a molecule or compound using affinity chromatography
methods well known in the art. In one embodiment, the
polynucleotide 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 polynucleotide. The molecule or
compound which is bound to the polynucleotide may be released from
the polynucleotide by increasing the salt concentration of the
flow-through medium and collected.
[0155] In a further embodiment, hNIM1 or a portion thereof may be
used to purify a ligand from a sample. A method for using a
mammalian protein or a portion thereof to purify a ligand would
involve combining the protein 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 thereby
releasing purified ligand.
[0156] In a preferred embodiment, hNIM1 kinase may be used to
screen libraries 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 polypeptide on
their cell surface can be used in screening assays. The cells are
screened against libraries or a plurality of ligands and the
specificity of binding or formation of complexes between the
expressed polypeptide and the ligand may be measured. Depending on
the kind of library being screened, the assay may be used to
identify agonists, antagonists, antibodies, DNA molecules, small
drug molecules, immunoglobulins, inhibitors, mimetics, peptides,
peptide nucleic acids, proteins, and RNA molecules or any other
ligand, which specifically binds the protein.
[0157] 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 polypeptide or oligopeptide or
portion thereof. Molecules or compounds identified by screening may
be used in a mammalian model system to evaluate their toxicity or
efficacy.
[0158] Pharmaceutical Compositions
[0159] Pharmaceutical compositions may be formulated and
administered, to a subject in need of such treatment, to attain a
therapeutic effect. Such compositions contain the instant protein,
agonists, antibodies specifically binding the protein, antagonists,
inhibitors, or mimetics of the protein. Compositions may be
manufactured by conventional means such as mixing, dissolving,
granulating, dragee-making, levigating, emulsifying, encapsulating,
entrapping, or lyophilizing. The composition may be provided as a
salt, formed with acids such as hydrochloric, sulfuric, acetic,
lactic, tartaric, malic, and succinic, or as a lyophilized powder
which may be combined with a sterile buffer such as saline,
dextrose, or water. These compositions may include auxiliaries or
excipients which facilitate processing of the active compounds.
[0160] Auxiliaries and excipients may include coatings, fillers or
binders including sugars such as lactose, sucrose, mannitol,
glycerol, or sorbitol; starches from corn, wheat, rice, or potato;
proteins such as albumin, gelatin and collagen; cellulose in the
form of hydroxypropylmethyl-cellulose, methyl cellulose, or sodium
carboxymethylcellulose; gums including arabic and tragacanth;
lubricants such as magnesium stearate or talc; disintegrating or
solubilizing agents such as the, agar, alginic acid, sodium
alginate or cross-linked polyvinyl pyrrolidone; stabilizers such as
carbopol gel, polyethylene glycol, or titanium dioxide; and
dyestuffs or pigments added for identify the product or to
characterize the quantity of active compound or dosage.
[0161] These compositions may be administered by any number of
routes including oral, intravenous, intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal,
subcutaneous, intraperitoneal, intranasal, enteral, topical,
sublingual, or rectal.
[0162] The route of administration and dosage will determine
formulation; for example, oral administration may be accomplished
using tablets, pills, dragees, capsules, liquids, gels, syrups,
slurries, or suspensions; parenteral administration may be
formulated in aqueous, physiologically compatible buffers such as
Hanks' solution, Ringer's solution, or physiologically buffered
saline. Suspensions for injection may be aqueous, containing
viscous additives such as sodium carboxymethyl cellulose or dextran
to increase the viscosity, or oily, containing lipophilic solvents
such as sesame oil or synthetic fatty acid esters such as ethyl
oleate or triglycerides, or liposomes. Penetrants well known in the
art are used for topical or nasal administration.
[0163] Toxicity and Therapeutic Efficacy
[0164] A therapeutically effective dose refers to the amount of
active ingredient which ameliorates symptoms or condition. For any
compound, a therapeutically effective dose can be estimated from
cell culture assays using normal and neoplastic cells or in animal
models. Therapeutic efficacy, toxicity, concentration range, and
route of administration may be determined by standard
pharmaceutical procedures using experimental animals.
[0165] The therapeutic index is the dose ratio between therapeutic
and toxic effects--LD50 (the dose lethal to 50% of the
population)/ED50 (the dose therapeutically effective in 50% of the
population)--and large therapeutic indices are preferred. Dosage is
within a range of circulating concentrations, includes an ED50 with
little or no toxicity, and varies depending upon the composition,
method of delivery, sensitivity of the patient, and route of
administration. Exact dosage will be determined by the practitioner
in light of factors related to the subject in need of the
treatment.
[0166] Dosage and administration are adjusted to provide active
moiety that maintains therapeutic effect. Factors for adjustment
include the severity of the disease state, general health of the
subject, age, weight, and gender of the subject, diet, time and
frequency of administration, drug combination(s), reaction
sensitivities, and tolerance/response to therapy. Long-acting
pharmaceutical compositions may be administered every 3 to 4 days,
every week, or once every two weeks depending on half-life and
clearance rate of the particular composition.
[0167] Normal dosage amounts may vary from 0.1 .mu.g, up to a total
dose of about 1 g, depending upon the route of administration. The
dosage of a particular composition may be lower when administered
to a patient in combination with other agents, drugs, or hormones.
Guidance as to particular dosages and methods of delivery is
provided in the pharmaceutical literature. Further details on
techniques for formulation and administration may be found in the
latest edition of Remington's Pharmaceutical Sciences (Mack
Publishing, Easton Pa.).
[0168] Model Systems
[0169] 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.
[0170] Toxicology
[0171] 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.
[0172] 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.
[0173] 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.
[0174] 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.
[0175] 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.
[0176] Transgenic Models
[0177] 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.
[0178] Embryonic Stem Cells
[0179] 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.
[0180] 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.
[0181] Knockout Analysis
[0182] 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.
[0183] Knockin Analysis
[0184] 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.
[0185] Non-Human Primate Model
[0186] 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.
[0187] In additional embodiments, the nucleic acid molecules 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 nucleic acid molecules that are currently known,
including, but not limited to, such properties as the triplet
genetic code and specific base pair interactions.
EXAMPLES
[0188] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention. For purposes of example, preparation of the human
cerebellum (CRBLNOT01), prostate (PROSBPT03), and normalized brain
(BRAINON01) libraries will be described.
[0189] I Tissues
[0190] Cerebellum
[0191] The tissue used for cerebellum library construction was
obtained from a 69 year-old, Caucasian male (RT95-05-0301;
International Institute for Advanced Medicine, Exton PA). The
frozen tissue was homogenized and lysed using a POLYTRON
homogenizer (PT-3000; Brinkmann Instruments, Westbury N.J.). The
reagents and extraction procedures were used as supplied in the RNA
Isolation kit (Stratagene). 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 hr at 25,000 rpm at
ambient temperature. The RNA was extracted twice with phenol
chloroform, pH 8.0, and once with acid phenol, pH 4.0; precipitated
using 0.3 M sodium acetate and 2.5 volumes of ethanol; resuspended
in water; and treated with DNAse for 15 min at 37C. The RNA was
isolated with the OLIGOTEX kit (Qiagen, Chatsworth Calif.) and used
to construct the cDNA library.
[0192] Prostate
[0193] Diseased prostate tissue removed from a 59-year-old
Caucasian male during a radical prostatectomy was used to construct
the PROSBPT03 library. Pathology for the tumor indicated
adenocarcinoma, Gleason grade 3+3, with microscopic foci involving
the right and left sides peripherally. The tumor was confined and
did not involve the capsule. High-grade prostatic intraepithelial
neoplasia was identified on the right side peripherally. The
patient presented with elevated prostate specific antigen. Family
history included cerebrovascular disease in both parents and
prostate cancer in a sibling.
[0194] II cDNA Library Construction
[0195] To construct the cDNA library, polyA RNA was used according
to the recommended protocols in the SUPERSCRIPT Plasmid system
(Invitrogen). First strand cDNA synthesis was accomplished using
oligo d(T) priming, and second strand synthesis was performed using
a combination of DNA polymerase I, E. coli ligase, and RNAse H. The
cDNA was blunted with T4 polymerase, and a Sal I linker was added
to the blunt end of the cDNA. The Sal I adapted, double stranded
cDNAs were the digested with Not I and fractionated on a SEPHAROSE
CL4B column (APB).
[0196] Those cerebellum cDNAs exceeding 400 bp were ligated into
pSPORT1 plasmid (Invitrogen) which was subsequently transformed
into DH5.alpha. competent cells (Invitrogen). Those prostate cDNAs
exceeding 400 bp were ligated into the NotI and EcoRI sites of the
pINCY plasmid (Incyte Genomics) which was subsequently transformed
into competent DH5.alpha. or ELECTROMAX DH10B competent cells (Life
Technologies).
[0197] Normalization
[0198] For purposes of example, the normalization of the human
brain library (BRAINON01) is described. About 4.9.times.10.sup.6
independent clones of the BRAINOT03 plasmid library in E. coli
strain DH12S competent cells (Invitrogen) were grown in liquid
culture under carbenicillin (25 mg/l) and methicillin (1 mg/ml)
selection following transformation by electroporation. To reduce
the number of excess cDNA copies according to their abundance
levels in the library, the cDNA library was normalized in a single
round according to the procedure of Soares et al. (1994, Proc Natl
Acad Sci 91:9228-9232), with the following modifications. The
primer to template ratio in the primer extension reaction was
increased from 2:1 to 10:1. The dNTP concentration in the reaction
was reduced to 150 .mu.M for each dNTP to allow the generation of
longer (400 to 1000 nt) primer extension products. The reannealing
hybridization was extended from 13 to 48 hr. The single stranded
DNA circles of the normalized library were purified by
hydroxyapatite chromatography, converted to partially
double-stranded by random priming, and electroporated into E. coli
strain DH10B competent cells (Invitrogen).
[0199] III Isolation and Sequencing of cDNA Clones
[0200] 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). This 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
inoculated into 1 ml of sterile TERRIFIC BROTH (BD Biosciences,
Sparks Md.) with carbenicillin at 25 mg/l and glycerol at 0.4%; 2)
after being cultured for 19 hours, the cells were lysed with 0.3 ml
of lysis buffer; and 3) following isopropanol precipitation of the
lysate, the plasmid DNA pellet was resuspended in 0.1 ml of
distilled water. Following the last step in the protocol, samples
were transferred to a 96-well block for storage at 4C.
[0201] The cDNAs were prepared for sequencing using the MICROLAB
2200 system (Hamilton, Reno Nev.) in combination with 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
PRISM 377 sequencing system (ABI) or the MEGABASE 1000 DNA
sequencing system (APB). Most of the cDNAs were sequenced according
to standard ABI protocols and kits (with solution volumes of
0.25.times.-1.0.times. concentrations). In the alternative, cDNAs
were sequenced using APB solutions and dyes.
[0202] IV Extension of a Polynucleotide Sequence
[0203] The nucleic acid molecules were extended using a 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 OLIGO software (Molecular Biology Insights), or another
appropriate program, 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 68C to about 72C. Any stretch of
nucleotides that would result in hairpin structures and
primer-primer dimerizations was avoided.
[0204] Selected cDNA libraries were used as templates to extend the
sequence. If more than one extension is desired, additional or
nested sets of primers are designed and used with libraries that
have been size-selected to include larger cDNAs and contain more
sequences with the 5' and upstream regions of genes. A randomly
primed library is used if an oligo d(T) library does not yield a
full length cDNA. Genomic libraries are used for extension 5' of
the promoter binding region in order to obtain regulatory
elements.
[0205] 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 (Amersham Pharmacia Biotech), ELONGASE enzyme (Life
Technologies), and Pfu DNA polymerase (Stratagene), with the
following parameters for primer pair PCI A and PCI B (Incyte
Genomics): 1: 94C, three min; 2: 94C, 15 sec; 3: 60C, one min; 4:
68C, two min; 5: 2, 3, and 4 repeated 20 times; 6: 68C, five min;
and 7: storage at 4C. In the alternative, the parameters for primer
pair T7 and SK+ (Stratagene) were as follows: 1: 94C, three min; 2:
94C, 15 sec; 3: 57C, one min; 4: 68C, two min; 5: 2, 3, and 4
repeated 20 times; 6: 68C, five min; and 7: storage at 4C.
[0206] 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
Life Sciences, Acton MA) 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 mini-gel to
determine which reactions were successful in extending the
sequence.
[0207] The extended nucleotide sequences 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 37C in
384-well plates in LB/2.times. carbenicillin liquid media.
[0208] The cells were lysed, and DNA was amplified using primers,
Taq DNA polymerase (APB) and Pfu DNA polymerase (Stratagene) with
the following parameters: 1: 94C, three min; 2: 94C, 15 sec; 3:
60C, one min; 4: 72C, two min; 5: 2, 3, and 4 repeated 29 times; 6:
72C, five min; and 7: storage at 4C. DNA was quantified using
PICOGREEN quantitative 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 PRISM BIGDYE terminator cycle
sequencing kit (ABI).
[0209] V Homology Searching of Nucleic Acid Molecules and Their
Deduced Proteins
[0210] The nucleic acid molecules 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 BLAST 2 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).
[0211] 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 electronic stringency for an exact match was set at 70, and the
conservative lower limit for an exact match was set at
approximately 40 (with 1-2% error due to uncalled bases).
[0212] The BLAST software suite, freely available sequence
comparison algorithms (NCBI, Bethesda Md.) includes various
sequence analysis programs including "blastn", that is used to
align known nucleic acid molecules, BLAST 2 that is used for direct
pairwise comparison of either nucleic or amino acid molecules.
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 x drop-off: 50; Expect: 10; Word Size: 11; and
Filter: on. Identity or similarity may be measured over the entire
length of a sequence or some smaller portion thereof. Brenner
(supra) 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.
[0213] The mammalian nucleic acid molecules 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.
[0214] 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.
[0215] 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 analyze 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.
[0216] 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.).
[0217] The nucleic acid molecule 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.
[0218] VI Northern Analysis
[0219] Northern blots are used to detect the presence of a
transcript of a gene and involves the hybridization of a labeled
nucleotide sequence to a membrane on which RNAs from a particular
cell type or tissue have been bound. This technology is well known
in the art, and the protocol has been clearly set forth in the
manuals of Sambrook (supra, ch 7) and Ausubel (supra, unit 4),
incorporated by reference herein.
[0220] Electronic Northern Analysis
[0221] Computer techniques applying BLAST were used to search for
identical or related molecules in nucleotide databases such as
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.
[0222] The results of the electronic northern performed at a
product score of 70 are shown in FIG. 2. Analysis involved the
categorization of cDNA libraries by system, organ/tissue and cell
type. The categories included cardiovascular system, connective
tissue (specifically cancerous breast fibroblasts), digestive
system, embryonic structures, endocrine system, exocrine glands,
female and male genitalia (specifically cancerous prostate), germ
cells, hemic and 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 expressing the sequence
was counted and shown over the total number of libraries in that
category. Electronic northern analysis can be used to support data
from other methodologies such as hybridization,
guilt-by-association and array technologies.
[0223] VII Hybridization Technologies and Analyses
[0224] Sample Preparation
[0225] Normal and cancerous lung tissue samples were obtained from
the Roy Castle International Centre for Lung Cancer Research
(Liverpool UK). All samples are described by donor identification
number in the table below. The first column shows the donor ID; the
second, donor age/sex; and the third column, a description of these
non-small cell lung cancers.
2 Donor ID Age/Sex* Sample and Description 5795 71/F Adenocarcinoma
7188 54/M Adenocarcinoma, stage III 7965 54/F Adenocarcinoma, stage
III 7966 39/F Adenocarcinoma, stage III 7968 58/M Squamous
carcinoma, stage III 7975 62/M Adenocarcarcinoma, stage III 7976
U/U Non small cell cancer, stage III 9007 U/U Normal lung, pool *U
= unknown
[0226] Immobilization of Nucleic Acid Molecules on a Substrate
[0227] Nucleic acid molecules are applied to a substrate by one of
the following methods. A mixture of nucleic acid molecules is
fractionated by gel electrophoresis and transferred to a nylon
membrane by capillary transfer. Alternatively, the nucleic acid
molecules are individually ligated to a vector and inserted into
bacterial host cells to form a library. The nucleic acid molecules
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 37C 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).
[0228] In the second method, nucleic acid molecules 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 (Amersham Pharmacia Biotech). 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 MA) 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
110C 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 60C; then the arrays are
washed in 0.2% SDS and rinsed in distilled water as before.
[0229] The HUMAN GENOME GEM series 1 microarray (Incite Genomic)
used in the lung cancer study contains 9,766 array elements which
represent 7,612 annotated clusters and 1,382 unannotated
clusters.
[0230] Probe Preparation for Membrane Hybridization
[0231] Hybridization probes derived from the nucleic acid molecules
of the Sequence Listing are employed for screening cDNAs, mRNAs, or
genomic DNA in membrane-based hybridizations. Probes are prepared
by diluting the nucleic acid molecules to a concentration of 40-50
ng in 45 .mu.l TE buffer, denaturing by heating to 100C for five
min, and briefly centrifuging. The denatured nucleic acid molecule
is then added to a REDIPRIME tube (Amersham Pharmacia Biotech),
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 37C 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
(Amersham Pharmacia Biotech). The purified probe is heated to 100C
for five min, snap cooled for two min on ice, and used in
membrane-based hybridizations as described below.
[0232] Probe Preparation for Polymer Coated Slide Hybridization
[0233] Hybridization probes derived from mRNA isolated from samples
are employed for screening nucleic acid molecules 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 37C for two hr. The reaction
mixture is then incubated for 20 min at 85C, 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 65C 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.
[0234] Membrane-based Hybridization
[0235] Membranes are pre-hybridized in hybridization solution
containing 1% Sarkosyl and lx high phosphate buffer (0.5 M NaCl,
0.1 M Na.sub.2HPO.sub.4, 5 mM EDTA, pH 7) at 55C 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 55C
for 16 hr. Following hybridization, the membrane is washed for 15
min at 25C in 1 mM Tris (pH 8.0), 1% Sarkosyl, and four times for
15 min each at 25C 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 -70C, developed, and examined
visually.
[0236] Polymer Coated Slide-based Hybridization
[0237] Probe is heated to 65C for five min, centrifuged five min at
9400 rpm in a 5415C 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 60C. The arrays are washed for 10 min at 45C in
1.times.SSC, 0.1% SDS, and three times for 10 min each at 45C in
0.1.times.SSC, and dried.
[0238] 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 equal
numbers of probes derived from both biological samples give a
distinct combined fluorescence (Shalon WO95/35505).
[0239] 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 20X
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.
[0240] The output of the photomultiplier tube is digitized using a
12-bit RTI-835H analog-to-digital (AID) 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).
[0241] VIII QPCR
[0242] QPCR was used to examine expression of human NIM1 kinase in
various cell lines and tissues. The cell lines, obtained from
sources such as ATCC, were H460 human non-small cell lung
carcinoma, A2780 human ovarian carcinoma line, A375 human melanoma
cell line, HDF human dermal fibroblasts, HELA human cervix
carcinoma, DU145 androgen-independent prostate carcinoma cell line,
MDA-MB231 human mammary tumor cells, U87 glioblastoma tumor cells,
and BX-PC3 pancreatic cancer cells. The cell lines were plated in
culture dishes and grown in RPM1 supplemented with 10% fetal bovine
serum, 2 mM glutamine at 37C in 5% CO.sub.2 until they were 90%
confluent. The tissues, brain, colon, uterus and placenta, were
obtained as MTN blots (Human I, II, III, and IV) from Clontech.
[0243] QPCR was performed using the PRISM 7700 Detection system and
TAQMAN assay reagents (TAQMAN Universal PCR Master mix) according
to manufacturer instructions (all ABI). All reactions were
performed in triplicate.
[0244] The primers used in the reaction included SEQ ID NOs:37-39.
Relative quantification was done using 18s RNA as the standard. The
linearity of this standardization procedure was described in Spiess
and Ivell (1999; Biotechniques 26:46-50, incorporated herein by
reference).
[0245] IX Complementary Nucleic Acid Molecules
[0246] Molecules complementary to the nucleic acid molecule, or a
fragment thereof, are used to detect, decrease, or inhibit gene
expression. Although use of oligonucleotides comprising from about
15 to about 30 base pairs is described, the same procedure is used
with larger or smaller fragments or their derivatives (PNAs).
Appropriate oligonucleotides are selected using OLIGO software
(Molecular Biology Insights). To inhibit transcription by
preventing promoter binding, a complementary oligonucleotide is
designed to bind to the most unique 5' sequence, most preferably
about 10 nucleotides before the initiation codon of the open
reading frame. To inhibit translation, a complementary
oligonucleotide is designed to prevent ribosomal binding to the
mRNA encoding the protein.
[0247] In addition to using antisense molecules constructed to
interrupt transcription or translation, modifications of gene
expression can be obtained by designing antisense molecules to
genomic sequences (such as enhancers or introns) or even to
trans-acting regulatory genes. Similarly, antisense inhibition can
be achieved using Hogeboom base-pairing methodology, also known as
"triple helix" base pairing. Antisense molecules involved in triple
helix pairing compromise the ability of the double helix to open
sufficiently for the binding of polymerases, transcription factors,
or regulatory molecules.
[0248] Such antisense molecules are placed in expression vectors
and used to transform preferred cells or tissues. This may include
introduction of the expression vector into 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 or
other reproducing lineage for long term or stable gene therapy.
Transient expression may last 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.
[0249] Stable transformation of appropriate dividing cells with a
vector encoding the antisense molecule can produce 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 antisense
molecules to compromise or entirely eliminate activity of the
nucleic acid molecule encoding the mammalian protein.
[0250] X Expression of NIM1 Kinase
[0251] Expression of the human NIM1 kinase was achieved using
monkey and insect cell-based expression systems. The cDNA was
purified using QIAPREP spin miniprep kit and PLASMID MAXI kit (both
Qiagen) accordingly to the manufacturer's instructions.
[0252] For transient expression, the cDNA was cloned into the
pcDNA3.1(-)/myc-His B vector (Invitrogen) and the vector
pcDNA3-Nim1 was transformed into COS1-1 cells using CaPO.sub.4
transfection kit 2-463335 (Eppendorf--5 Prime, Boulder Colo.).
4.times.10.sup.6 cells were seeded in 10 cm tissue culture dishes
24 hr before transformation. On the day of transformation,
CaPO.sub.4-DNA precipitate was obtained using 500 ml 2.times. DNA
precipitation buffer, 62 ml M CaC.sub.2, 10 mg (10 ml) pcDNA3-NIM1,
and 428 ml water. After the mixture was incubated at room
temperature for 20 min, it was slowly added to 9 ml of culture
medium (DMEM, 10% fetal calf serum, 2 mM glutamine, 10 mg/ml
penicillin, and 10 mg/ml streptomycin). Cells were incubated at 37C
at 5% CO.sub.2 for 4 h. The medium was replaced with fresh medium,
and cells were incubated for 48 additional hours.
[0253] Sf21 insect cells were cotransformed according to
instructions supplied with the BACULOGOLD transfection kit (BD
Pharmingen, San Diego Calif.). 2.times.10.sup.6 cells were seeded
in one 6 cm tissue culture dish and incubated at 27C for 15 min. 4
mg (4 ml) of pVL1392/GST-NIM1 expression vector were combined with
0.5 mg (0.5 ml) of BACULOGOLD DNA and incubated at room temperature
for 5 min. The culture medium (TNM-FH) was removed and replaced
with 1 ml of Buffer A. The DNA mixture was diluted in 1 ml of
Buffer B and added drop by drop to the cotransfection plate. After
the plate was incubated at 27C for 4 hours, the medium was
replaced, and the cells were incubated for 5 days.
[0254] The recombinant viruses were subjected to three cycles of
amplification to obtain a 10.sup.7 pfu/ml viral stock.
1.2.times.10.sup.7 Sf21 insect cells were infected with 10 ml of
viral stock and incubated at 27C. After 3 days, the cells were
lysed, and the protein was purified.
[0255] XI Protein Purification
[0256] His-Purification
[0257] COS-1 cells were centrifuged at 1000 rpm, resuspended in 4
ml of lysis buffer (5 mM imidazole+0.5 mM NaCl+20 mM
Na.sub.2HPO.sub.4+Complete protease inhibitor cocktail tablets
(Roche Applied Science, Indianapolis Ind.) and sonicated. The
soluble fraction was recovered by centrifugation at 10000 rpm for
10 min at 4C. The recombinant Nim1 kinase was purified by
immobilized metal ion affinity chromatography (Invitrogen)
according to manufacturer instructions. Purity was determined by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie
blue staining. Protein concentration was determined using the
Bradford method.
[0258] GST-Purification
[0259] Sf21 cells were centrifuged at 800 rpm, resuspended in 10 ml
of lysis buffer (PBS+1 mM orthovanadate+20 mM DTT+Complete protease
inhibitor cocktail tablets (Roche Applied Science), and sonicated.
The soluble fraction was recovered by centrifugation at 10000 rpm
for 10 min at 4C. The recombinant Nim1 kinase was purified by
affinity chromatography on glutathione-SEPHAROSE resin (APB)
following the manufacturer's instructions. Purity was determined
using SDS-PAGE followed by Coomassie blue staining. Protein
concentration was determined using the Bradford method.
[0260] For Western blot analysis, proteins were separated by
SDS-PAGE and analyzed by immunoblotting (with either GST-HRP
conjugated antibody or His-HRP conjugated antibody, Santa Cruz
Biotechnology, Heidelberg Germany) using standard materials and
techniques (ECL apparatus, APB).
[0261] XII Characterization of the Protein
[0262] In vitro translation of Incyte Clone number 3317608 was
accomplished using the TNT T7 system (Promega) according to the
manufacturer's instructions. SDS-PAGE analysis revealed the
presence of a 48 kd protein. The size of the protein was confirmed
by expressing pcDNA3-Nim1 as a template. As previously described,
Nim1 kinase was expressed in both mammalian and insect cells. The
purified protein was subjected to SDS-PAGE and revealed a 50 kd
protein (including myc-His tag) and an 80 kd protein (including GST
tag) respectively.
[0263] XIII Human NIM1 Kinase Assay
[0264] An in vitro kinase assay was performed by incubating 100 ng
of recombinant Nim1-GST with 2 .mu.g of myelin basic protein (MBP)
or histone (HIST) in 20 .mu.l of kinase buffer (50 mM Hepes, pH
7.5, 3 mM MgCl.sub.2 and MnCl.sub.2, 10 mM DTT and 6 .mu.M NaOVa)
containing 10 .mu.Ci of [.gamma.-.sup.32P]-ATP (3000 Ci/mmol; APB)
for 30 min at 37C. The reaction was stopped by adding sample buffer
and heating to 100C for 5 min. Samples were analyzed by SDS-PAGE,
the gels were dried and subjected to autoradiographic analysis.
[0265] XIV Production of NIM1 Kinase Specific Antibodies
[0266] NIM1 kinase 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 NIM1 kinase is analyzed using LASERGENE software
(DNASTAR) to determine regions of high immunogenicity. An
immunogenic 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 431A peptide synthesizer (ABI) using
Fmoc-chemistry and coupled to KLH (Sigma-Aldrich) by reaction with
N-maleimidobenzoyl-N-hydr- oxysuccinimide ester to increase
immunogenicity.
[0267] 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 twelve weeks,
antisera are drawn and tested for specific recognition of hNIM1.
Antisera that reacts positively with hNIM1 are affinity purified on
a column containing beaded agarose resin to which the epitope has
been conjugated. The column is equilibrated using 12 mL IMMUNOPURE
Gentle Binding buffer (Pierce Chemical, Rockford Ill.). Three ml of
rabbit antisera are combined with one ml of binding buffer and
added to the top of the column. The antisera are allowed to bind to
the epitope with gentle shaking at room temperature for 30 min. The
column is allowed to settle for 30 min, drained by gravity flow,
and washed with 16 ml binding buffer (4.times.4 ml additions of
buffer). The antibody is eluted in one ml fractions with IMMUNOPURE
Gentle Elution buffer (Pierce), and absorbance at 280 nm is
determined. Peak fractions are pooled and dialyzed against 50 mM
Tris, pH 7.4, 100 mM NaCl, and 10% glycerol. After dialysis, the
concentration of the purified antibody is determined using the BCA
assay (Pierce), aliquoted, and frozen.
[0268] XV Purification of Naturally Occurring Protein Using
Specific Antibodies
[0269] Naturally occurring or recombinant mammalian protein is
purified by immunoaffinity chromatography using antibodies that
specifically bind the protein. An immunoaffinity column is
constructed by covalently coupling the anti-NIM1 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.
[0270] XVI Screening Molecules for Specific Binding with the
Nucleic Acid Molecule or Protein
[0271] The nucleic acid molecule, 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), respectively. Libraries of candidate molecules or
compounds previously arranged on a substrate are incubated in the
presence of labeled nucleic acid molecule 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.
[0272] XVII Two-Hybrid Screen
[0273] A yeast two-hybrid system, MATCHMAKER LexA Two-Hybrid system
(Clontech Laboratories, Palo Alto Calif.), is used to screen for
peptides that bind the mammalian protein of the invention. A
nucleic acid molecule 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 30C until the colonies have grown up and can be
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 P-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.
[0274] 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 30C. A sample of the culture is plated on SD/-Trp/-Ura
media and incubated at 30C 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 nucleic acid molecule encoding a protein that
physically interacts with the mammalian protein, can be isolated
from the yeast cells and characterized.
[0275] All patents and publications mentioned in the specification
are herein incorporated by reference. Various modifications and
variations of the described method and system of the invention will
be apparent to those skilled in the art without departing from the
scope and spirit of the invention. Although the invention has been
described in connection with specific preferred embodiments, it
should be understood that the invention as claimed should not be
unduly limited to such specific embodiments. Indeed, various
modifications of the 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
39 1 2060 DNA Homo sapiens misc_feature Incyte Clone No 3317608CB1
1 cagagatgag atcccgcagc agggacgtgg gggcctccca ggggcattta cgcaccagag
60 tgcaagattc tctggccatc aagggaaata gcaaacagaa gcctttgtcc
tggggcacag 120 ccacctacca caaagcatca gactccacgt ctggccagaa
agttcctgga gtcccatcag 180 gccagtgggt atgtaacatg tgcctaattg
tacagctaga gcctgcaagt tcaacgtgag 240 ggaaggtggg aaatgtcttg
agtgaggcga gcagctcctg gctgggctgg gcagactcag 300 ctaccacgtt
cactgccttc ctctcactaa agccgagagg gaggctgctc agctctcagg 360
aaaactcttt tgaaccctgg gcacctgctg tcctcagttg gcatctccca ccctctgagc
420 ctcttctgct cctgcacaac ctgcctcttc gctgagatgg agacgtgagc
ccccgtggac 480 gatgactgca gtgtatatga atggaggtgg cctggtgaac
ccccactatg cccggtggga 540 tcggcgcgac agtgtagaaa gtggctgtca
gaccgagagt agcaaggtgg gtgaggaggg 600 acagccccgc cagctgacgc
ccttcgagaa actgacacag gacatgtccc aggatgagaa 660 ggtggtgagg
gagatcacgc tggggaaacg gataggcttc taccgaattc gaggggaaat 720
cggaagtgga aacttctccc aagtgaagct tgggattcac tccctaacca aagaaaaggt
780 ggccattaag atcctggaca agaccaagtt agaccagaaa acccagaggc
tactatcccg 840 agaaatctcc agcatggaaa agctgcacca tcccaacatc
atccgccttt acgaagtggt 900 ggagacccta tccaagctgc acttggtgat
ggagtatgca gggggtgggg agctcttcgg 960 aaaaattagc actgagggga
agctctctga accagaaagc aagctcatct tctcccagat 1020 tgtgtctgcc
gtgaagcaca tgcatgaaaa ccaaattatt catagagatc tgaaagcaga 1080
aaatgtattc tataccagta atacttgtgt gaaggtgggc gattttggat tcagcacagt
1140 aagcaaaaaa ggtgaaatgc tgaacacttt ctgtgggtct cctccctacg
ctgcgcctga 1200 actcttccgg gacgagcact acatcggcat ttacgtggat
atctgggcct tgggggtgct 1260 tttgtacttc atggtgactg gcaccatgcc
atttcgggca gaaaccgtgg ccaaactaaa 1320 aaagagcatc ctcgagggca
catacagtgt accgccgcac gtgtcagagc cctgccaccg 1380 actcatccga
ggagtccttc agcagatccc cacggagagg tacggaatcg actgcatcat 1440
gaatgatgaa tggatgcaag gggtgccata ccctacacct ttggaacctt tccaactgga
1500 tcccaaacat ttgtcggaaa ccagcactct caaggaagaa gaaaatgagg
tcaaaagcac 1560 tttagaacat ttgggcatta cagaagagca tattcgaaat
aaccaaggga gagatgctcg 1620 cagctcaatc acaggggtct atagaattat
tttacataga gtccaaagga agaaggcttt 1680 ggaaagtgtc ccagtcatga
tgctaccaga ccctaaagaa agagacctca aaaaagggtc 1740 ccgtgtctac
agagggataa gacacacatc caaattttgc tcgattttat aaattgcact 1800
agactgcttg taactaacca agatgattgt tgctgcttct aaattttttt caaggacaac
1860 ttgagtggag acatttttgt aatttttaaa taaacttaaa tttgagatat
gcaaaaaaaa 1920 aaaaaaaaag ggcggccgcc gactagtgag ctcgtcgacc
cgggaattaa ttccggaccg 1980 gtacctgcag gcgtaccagc tttccctata
gtggagtccg tattaaactt ggccgtaatc 2040 atggcataac ttgttccctg 2060 2
436 PRT Homo sapiens misc_feature Incyte ID No 3317608CD1 2 Met Thr
Ala Val Tyr Met Asn Gly Gly Gly Leu Val Asn Pro His 1 5 10 15 Tyr
Ala Arg Trp Asp Arg Arg Asp Ser Val Glu Ser Gly Cys Gln 20 25 30
Thr Glu Ser Ser Lys Val Gly Glu Glu Gly Gln Pro Arg Gln Leu 35 40
45 Thr Pro Phe Glu Lys Leu Thr Gln Asp Met Ser Gln Asp Glu Lys 50
55 60 Val Val Arg Glu Ile Thr Leu Gly Lys Arg Ile Gly Phe Tyr Arg
65 70 75 Ile Arg Gly Glu Ile Gly Ser Gly Asn Phe Ser Gln Val Lys
Leu 80 85 90 Gly Ile His Ser Leu Thr Lys Glu Lys Val Ala Ile Lys
Ile Leu 95 100 105 Asp Lys Thr Lys Leu Asp Gln Lys Thr Gln Arg Leu
Leu Ser Arg 110 115 120 Glu Ile Ser Ser Met Glu Lys Leu His His Pro
Asn Ile Ile Arg 125 130 135 Leu Tyr Glu Val Val Glu Thr Leu Ser Lys
Leu His Leu Val Met 140 145 150 Glu Tyr Ala Gly Gly Gly Glu Leu Phe
Gly Lys Ile Ser Thr Glu 155 160 165 Gly Lys Leu Ser Glu Pro Glu Ser
Lys Leu Ile Phe Ser Gln Ile 170 175 180 Val Ser Ala Val Lys His Met
His Glu Asn Gln Ile Ile His Arg 185 190 195 Asp Leu Lys Ala Glu Asn
Val Phe Tyr Thr Ser Asn Thr Cys Val 200 205 210 Lys Val Gly Asp Phe
Gly Phe Ser Thr Val Ser Lys Lys Gly Glu 215 220 225 Met Leu Asn Thr
Phe Cys Gly Ser Pro Pro Tyr Ala Ala Pro Glu 230 235 240 Leu Phe Arg
Asp Glu His Tyr Ile Gly Ile Tyr Val Asp Ile Trp 245 250 255 Ala Leu
Gly Val Leu Leu Tyr Phe Met Val Thr Gly Thr Met Pro 260 265 270 Phe
Arg Ala Glu Thr Val Ala Lys Leu Lys Lys Ser Ile Leu Glu 275 280 285
Gly Thr Tyr Ser Val Pro Pro His Val Ser Glu Pro Cys His Arg 290 295
300 Leu Ile Arg Gly Val Leu Gln Gln Ile Pro Thr Glu Arg Tyr Gly 305
310 315 Ile Asp Cys Ile Met Asn Asp Glu Trp Met Gln Gly Val Pro Tyr
320 325 330 Pro Thr Pro Leu Glu Pro Phe Gln Leu Asp Pro Lys His Leu
Ser 335 340 345 Glu Thr Ser Thr Leu Lys Glu Glu Glu Asn Glu Val Lys
Ser Thr 350 355 360 Leu Glu His Leu Gly Ile Thr Glu Glu His Ile Arg
Asn Asn Gln 365 370 375 Gly Arg Asp Ala Arg Ser Ser Ile Thr Gly Val
Tyr Arg Ile Ile 380 385 390 Leu His Arg Val Gln Arg Lys Lys Ala Leu
Glu Ser Val Pro Val 395 400 405 Met Met Leu Pro Asp Pro Lys Glu Arg
Asp Leu Lys Lys Gly Ser 410 415 420 Arg Val Tyr Arg Gly Ile Arg His
Thr Ser Lys Phe Cys Ser Ile 425 430 435 Leu 3 1051 DNA Homo sapiens
misc_feature Incyte Template No 200700.1 3 gctgcaccat cccaacatca
tccgccttta cgaagtggtg gagaccctat ccaagctgca 60 cttggtgatg
gagtatgcag ggggtgggga gctcttcgga aaaattagca ctgaggggaa 120
gctctctgaa ccagaaagca agctcatctt ctcccagatt gtgtctgccg tgaagcacat
180 gcatgaaaac caaattattc atagagatct gaaagcagaa aatgtattct
ataccagtaa 240 tacttgtgtg aaggtgggcg attttggatt cagcacagta
agcaaaaaag gtgaaatgct 300 gaacactttc tgtgggtctc ctccctacgc
tgcgcctgaa ctcttccggg acgagcacta 360 catcggcatt tacgtggata
tctgggcctt gggggtgctt ttgtacttca tggtgactgg 420 caccatgcca
tttcgggcag aaaccgtggc caaactaaaa aagagcatcc tcgagggcac 480
atacagtgta ccgccgcacg tgtcagagcc ctgccaccga ctcatccgag gagtccttca
540 gcagatcccc acggagaggt acggaatcga ctgcatcatg aatgatgaat
ggatgcaagg 600 ggtgccatac cctacacctt tggaaccttt ccaactggat
cccaaacatt tgtcggaaac 660 cagcactctc aaggaagaag aaaatgaggt
caaaagcact ttagaacatt tgggcattac 720 agaagagcat attcgaaata
accaagggag agatgctcgc agctcaatca caggggtcta 780 tagaattatt
ttacatagag tccaaaggaa gaaggctttg gaaagtgtcc cagtcatgat 840
gctaccagac cctaaagaaa gagacctcaa aaaagggtcc cgtgtctaca gagggataag
900 acacacatcc aaattttgct cgattttata aattgcacta gactgcttgt
aactaaccaa 960 gatgattgtt gctgcttcta aatttttttc aaggacaact
tgagtggaga catttttgta 1020 atttttaaat aaacttaaat ttgagatatg c 1051
4 1507 DNA Homo sapiens misc_feature Incyte Clone No 670279CB1 4
gagcctcttc tgctcctgca caacctgcct cttcgctgag atggagacgt gagcccccgt
60 ggacgatgac tgcagtgtat atgaatggag gtggcctggt gaacccccca
ctatgcccgg 120 tgggatcggc gcgacagtgt agaaagtggc tgtcagaccg
agagtagcaa ggtgggtgag 180 gagggacagc cccgccagct gacgcccttc
gagaaactga cacaggacat gtcccaggat 240 gagaaggtgg tgagggagat
cacgctgggg aaacggatag gcttctaccg aattcgaggg 300 gaaatcggaa
gtggaaactt ctcccaagtg aagcttggga ttcactccct aaccaaagaa 360
aaggtggcca ttaagatcct ggacaagacc aagttagacc agaaaaccca gaggctacta
420 tcccgagaaa tctccagcat ggaaaagctg caccatccca acatcatccg
cctttacgaa 480 gtggtggaga ccctatccaa gctgcacttg gtgatggagt
atgcaggggg tggggagctc 540 ttcggaaaaa ttagcactga ggggaagctc
tctgaaccag aaagcaagct catcttctcc 600 cagattgtgt ctgccgtgaa
gcacatgcat gaaaaccaaa ttattcatag agatctgaaa 660 gcagaaaatg
tattctatac cagtaatact tgtgtgaagg tgggcgattt tggattcagc 720
acagtaagca aaaaaggtga aatgctgaac actttctgtg ggtctcctcc ctacgctgcg
780 cctgaactct tccgggacga gcactacatc ggcatttacg tggatatctg
ggccttgggg 840 gtgcttttgt acttcatggt gactggcacc atgccatttc
gggcagaaac cgtggccaaa 900 ctaaaaaaga gcatcctcga gggcacatac
agtgtaccgc cgcacgtgtc agagccctgc 960 caccgactca tccgaggagt
ccttcagcag atccccacgg agaggtacgg aatcgactgc 1020 atcatgaatg
atgaatggat gcaaggggtg ccatacccta cacctttgga acctttccaa 1080
ctggatccca aacatttgtc ggaaaccagc actctcaagg aagaagaaaa tgaggtcaaa
1140 agcactttag aacatttggg cattacagaa gagcatattc gaaataacca
agggagagat 1200 gctcgcagct caatcacagg ggtctataga attattttac
atagagtcca aaggaagaag 1260 gctttggaaa gtgtcccagt catgatgcta
ccagacccta aagaaagaga cctcaaaaaa 1320 gggtcccgtg tctacagagg
gataagacac acatccaaat tttgctcgat tttataaatt 1380 gcactagact
gcttgtaact aaccaagatg attgttgctg cttctaaatt tttttcaagg 1440
acaacttgag tggagacatt tttgtaattt ttaaataaac ttaaatttga gatatgcaaa
1500 aaaaaaa 1507 5 258 DNA Homo sapiens misc_feature Incyte Clone
No 3317608H1 5 cagagatgag atcccgcagc agggacgtgg gggcctccca
ggggcattta cgcaccagag 60 tgcaagattc tctggccatc aagggaaata
gcaaacagaa gcctttgtcc tggggcacag 120 ccacctacca caaagcatca
gactccacgt ctggccagaa agttcctgga gtcccatcag 180 gccagtgggt
atgtaacatg tgcctaattg tacagctaga gcctgcaagt tcaacgtgag 240
ggaaggtggg aaatgtct 258 6 290 DNA Homo sapiens misc_feature Incyte
Clone No 4313713H1 6 ggagtatgca gggggtgggg agctcttcgg aaaaattagt
actgagggga agctctctga 60 accagaaagc aagctcatct tctcccagat
tgtgtctgcc gtgaagcaca tgcatgaaaa 120 ccaaattatt catagagatc
tgaaagcaga aaatgtttct ataccagtaa tacttgtgtg 180 aaggtgggcg
attttggatt cagcacagta agcaaaaaag gtgaaatgct gaacatttct 240
gtgggtctcc tccctacgct gcgctgaact cttccgggga cgagcattac 290 7 256
DNA Homo sapiens misc_feature Incyte Clone No 4617082H1 7
agcaaaaaag gtgaaatgct gaacactttc tgtgggtctc ctccctacgc tgcgcctgaa
60 ctcttccggg acgagcacta catcggcatt tacgtggata tctgggcctt
gggggtgctt 120 ttgtacttca tggtgactgg caccatgcca tttcgggcag
aaaccgtggc caaactaaaa 180 aagagcatcc tcgngggcac atacagtgta
ccgccgcacg tgtcagagcc ctgccaccga 240 ntcatccgag gagtct 256 8 250
DNA Homo sapiens misc_feature Incyte Clone No 4711644H1 8
ctcactaaag ccgagaggga ggctgctcag ctctcaggaa aactcttttg aaccctgggc
60 acctgctgtc ctcagttggc atctcccacc ctctgagcct cttctgctcc
tgcacaacct 120 gcctcttcgc tgagatggag acgtgagccc ccgtggacga
tgactgcagt gtatatgaat 180 ggaggtggcc tggtgaaccc ccactatgcc
cggtgggatc ggcgcganag tgtagaaagt 240 ggctgtcaga 250 9 243 DNA Homo
sapiens misc_feature Incyte Clone No 2286324H1 9 gaattatttt
acatagagtc caaaggaaga aggctttgga aagtgtccca gtcatgatgc 60
taccagaccc taaagaaaga gacctcaaaa aagggtcccg tgtctacaga gggataagac
120 acacatccaa attttgctcg attttataaa ttgcactaga ctgcttgtaa
ctaaccaaga 180 tgattgttgc tgcttctaaa tttttttcaa ggacaacttg
agtggagaca tttttgtaat 240 ttt 243 10 232 DNA Homo sapiens
misc_feature Incyte Clone No 2286816H1 10 gaattatttt acatagagtc
caaaggaaga aggctttgga aagtgtccca gtcatgatgc 60 taccagaccc
taaagaaaga gacctcaaaa aagggtcccg tgtctacaga gggataagac 120
acacatccaa attttgctcg attttataaa ttgcactaga ctgcttgtaa ctaaccaaga
180 tgattgttgc tgcttctaaa tttttttcaa ggacaacttg agtggagaca tt 232
11 88 DNA Homo sapiens misc_feature Incyte Clone No 2287217H1 11
caanagaaag aaacctcaaa aaagggtccc gtgtctacag agggataaga cacacatcca
60 aattttgctc gattttataa attgcact 88 12 270 DNA Homo sapiens
misc_feature Incyte Clone No 2286816R6 12 gaattatttt acatagagtc
caaaggaaga aggctttgga aagtgtccca gtcatgatgc 60 taccagaccc
taaagnaaga gacctcaaaa aagggtcccg tgtctacaga gggataagac 120
acacatccaa attttgctcg attttataaa ttgcactaga ctgcttgtaa ctaaccaaga
180 tgattgttgc tgcttctaaa tttttttcaa ggacaacttg agtggagaca
tttttgtaat 240 tttttaaata aacttaaatt tgagatatgc 270 13 230 DNA Homo
sapiens misc_feature Incyte Clone No 2286816T6 13 tgtctccact
caagttgtcc ttgaaaaaaa tttagaagca gcaacaatca tcttggttag 60
ttacaagcag tctagtgcaa tttataaaat cgagcaaaat ttggatgtgt gtcttatccc
120 tctgtagaca cgggaccctt ttttgaggtc tctttcttta gggtctggta
gcatcatgac 180 tgggacactt tccaaagcct tcttcctttg gactctatgt
aaaataattc 230 14 468 DNA Homo sapiens misc_feature Incyte Clone No
3317608T6 14 tctccactca agttgtcctt gaaaaaaatt tagaagcagc aacaatcatc
ttggttagtt 60 acaagcagtc tagtgcaatt tataaaatcg agcaaaattt
ggatgtgtgt cttatccctc 120 tgtagacacg ggaccctttt ttgaggtctc
tttctttagg gtctggtagc atcatgactg 180 ggacactttc caaagccttc
ttcctttgga ctctatgtaa aataattcta tagacccctg 240 tgattgagct
gcgagcatct ctcccttggt tatttcgaat atgctcttct gtaatgccca 300
aatgttctaa agtgcttttg acctcatttt cttcttcctt gagagtgctg gtttccgaca
360 aatgtttggg atccagttgg aaagggtcca aangtgtaag gtatggcacc
ccttgcatcc 420 attcatcatt catgatgcag tcgattccgt aactctccgt gggggatc
468 15 485 DNA Homo sapiens misc_feature Incyte Clone No 4201896T6
15 ttcactaatt acaaaaatgt ctccactcaa gttgtccttg acaaaaattt
agaagcagca 60 acaatcatct tggttagtta caagcagtct agtgcaattt
ataaaatcga gcaaaatttg 120 gatgtgtgtc ttatccctct gtagacacgg
gacccttttt tgaggtctct ttctttaggg 180 tctggtagca tcatgactgg
gacactttcc aaagccttct tcctttggac tctatgtaaa 240 ataattctat
agacccctgt gattgagctg cgagcatctc tcccttggtt atttcgaata 300
tgctcttctg taatgcccaa atgttctaaa gtgcttttga cctcattttc ttcttccttg
360 agagtgctgg tttccgacca atgtttggga tccagttgga aaggttccaa
aggtgtaggg 420 tatggcaccc cttgcctcca ttcatcattc ctgatgccgt
cgattccgta cctctccgtg 480 gggat 485 16 423 DNA Homo sapiens
misc_feature Incyte Clone No 4624811T6 16 aaatgtctcc actcaagttg
tccttgaaaa aaatttagaa gcagcaacaa tcatcttggt 60 tagttacaag
cagtctagtg caatttataa aatcgagcaa aatttggatg tgtgtcttat 120
ccctctgtag acacgggacc cttttttgag gtctctttct ttagggtctg gtagcatcat
180 gactgggaca ctttccaaag ccttcttcct ttggactcta tgtaaaataa
ttctatagac 240 ccctgtgatt gagctgcgag catctctccc ttggttattt
cgaatatgct cttctgtaat 300 gcccaaatgt tctaaagtgc ttttgacctc
attttcttct tccttgagag tgctggtttc 360 cgacaaatgt ttgggatcca
gttggaaagg ttccaaaggt gtagggtatg gcaccccttg 420 cat 423 17 564 DNA
Homo sapiens misc_feature Incyte Clone No 6559834H1 17 attgaggccg
cgggaatcca aaagggaaac aaacgaaacc agatgaaaaa aagtgattcc 60
aagtcaagat ttcaaatgga aatctaagaa aaggtggcca ttaagatcct ggacaagacc
120 aagttagacc agaaaaccca gaggctacta tcccgagaaa tctccagcat
ggaaaagctg 180 caccatccca acatcatccg cctttacgaa gtggtggaga
ccctatccaa gctgcacttg 240 gtgatggagt atgcaggggg tggggagctc
ttcggaaaaa ttagcactga ggggaagctc 300 tctgaaccag aaagcaagct
catcttctcc cagattgtgt ctgccgtgaa gcacatgcat 360 gaaaaccaaa
ttattcatag agatctgaaa gcagaaaatg tattctatac cagtaatact 420
tgtgtgaagg tgggcgattt tggattcagc acagtaagca aaaaaggtga aatgctgaac
480 actttctgtg ggtctcctcc ctacgctgcg cctgaactct tccgggacga
gcactacatc 540 ggcatttacg tggatatctg ggcc 564 18 598 DNA Homo
sapiens misc_feature Incyte Clone No 670279F1 18 gcatatctca
aatttaagtt tatttaaaaa ttacaaaaat gtctccactc aagttgtcct 60
tgaaaaaaat ttagaagcag caacaatcat cttggttagt tacaagcagt ctagtgcaat
120 ttataaaatc gagcaaaatt tggatgtgtg tcttatccct ctgtagacac
gggacccttt 180 tttgaggtct ctttctttag ggtctggtag catcatgact
gggacanttt ccaaagcctt 240 cttcctttgg actctatgta aaataattct
atagacccct gtgattgagc tgcgagcatc 300 tctcccttgg ttatttcgaa
tatgctcttc tgtaatgccc aaatgttcta aagtgctttt 360 gacctcattt
tcttcttcct tgagagtgct ggtttccgac aaatgtttgg gatccagttg 420
gaaaggttcc aaaggtgtag ggtatggcac cccttgcatc cattcatcat tcatgatgca
480 gtcgattccg tacctctccg tggggatctg ctgaaggact cctcggatga
gtcggtggca 540 gggctctgac acgtgcggcg gtacactgta tgtgccctcg
aggatgctct tttttagt 598 19 288 DNA Homo sapiens misc_feature Incyte
Clone No 670279H1 19 gctgcaccat cccaacatna tccgccttta cgaagtggtg
gagaccctat ccaagctgca 60 cttggtgatg gagtatgcag ggggtgggga
gctcttcgga aaaattagca ctgaggggaa 120 gctctctgaa ccagaaagca
agctcatctt ctnccagatt gtgtctgccg tgaagcacat 180 gcatgaaaac
caaattattc atagagatct gaaagcagaa aatgtattct ataccagtaa 240
tacttgtgtg aaggtgggcg attttggntt cagcacagta agcaaaaa 288 20 546 DNA
Homo sapiens misc_feature Incyte Clone No 670279R1 20 gctgcaccat
cccaacatca tccgccttta cgagtggtgg agaccctatc caagctgcac 60
ttggtgatgg agtatgcagg gggtggggag ctcttcggaa aaattagcac tgaggggaag
120 ctctctgaac cagaaagcaa gctcatcttc tcccagattg tgtctgccgt
gaagcacatg 180 catgaaaacc aaattattca tagagatctg aaagcagaaa
atgtattcta taccagtaat 240 acttgtgtga aggtgggcga ttttggattc
agcacagtaa gcaaaaaagg tgaaatgctg 300 aacactttct gtgggtctcc
tccctacgct gcgcctgaac tcttccggga cgagcactac 360 atcggcattt
acgtggatat ctgggccttg ggggtgcttt tgtacttcat ggtgactggc 420
accatgccat ttcgggcaga aaccgtggcc aaactaaaaa agagcatcct cgagggcaca
480 tacagtgtac cgccgcacgt gtcagagccc tgccaccgac tcatccgagg
agtncttcag 540 cagatc 546 21 383 DNA Homo sapiens misc_feature
Incyte Clone No 670279R6 21 gctgcaccat cccaacatca tccgccttta
cgaatggtgg agaccctatc caagctgcac 60 ttggtgatgg agtatgcagg
nggtggggag ctcttcggaa aaattagcac tgaggggaag 120 ctctctgaac
cagaaagcaa gctcatnttc tcccagattg tgtctgccgt gaagcacatg 180
catgaaaacc aaattattca tagagatctg aaagcagaaa
atgtattcta taccagtaat 240 acttgtgtga aggtgggcga ttttggattc
agcacagtaa gcaaaaaagg tgaaatgctg 300 aacactttct gtgggtctcc
tccctacgct gcgcctgaac tcttccggga cgagcactac 360 atcgggcatt
tacgtgggat atc 383 22 537 DNA Homo sapiens misc_feature Incyte
Clone No 670279T6 22 aatgtctcca ctcaagttgt ccttgaaaaa aatttagaag
cagcaacaat catcttggtt 60 agttacaagc agtctagtgc aatttataaa
atcgagcaaa atttggatgt gtgtcttatc 120 cctctgtaga cacgggaccc
ttttttgagg tctctttctt tagggtctgg tagcatcatg 180 actgggacac
tttccaaagc cttcttcctt tggactctat gtaaaataat tctatagacc 240
cctgtgattg agctgcgagc atctctccct tggttatttc gaatatgctc ttctgtaatg
300 cccaaatgtt ctaaagtgct tttgacctca ttttcttctt ccttgagagt
gctggtttcc 360 gacaaatgtt tgggatccag ttggaaaggt tccaaaggtg
tagggtatgg caccccttgc 420 atccattcat cattcatgat gcagtcgatt
ccgtacctct ccgtggggat ctgctgaagg 480 actcctcgga tgagtcggtg
gcanggcctg acacgtgcgg cggtacactg tatgtgc 537 23 279 DNA Homo
sapiens misc_feature Incyte Clone No 4936446H1 23 gcatcatgaa
tgatgaatgg atgcaagggg tgccataccc tacacctttg gaacctttcc 60
aactggatcc caaacatttg tcgnaaacca gcactctcaa ggaagaagaa aatgaggtca
120 aaagcacttt agaacatttg ggcattacag aagagcatat tcgaaataac
caagggagag 180 atgctcgcag ctcaatcaca ggggtctata gaattatttt
acatagagtc caaaggaaga 240 aggctttgga aagtgtccca gtcatgatgc
taccagacc 279 24 488 DNA Rattus norvegicus misc_feature Incyte
Template No 216150.1 24 aaaagttgca ccatcccaac attgtccgtc tttatgaagt
cgtggagacc ctgtccaagc 60 tccacctagt gatggagtat gcaggaggtg
gggagctctt tgggaaaatt agcaccgagg 120 ggaaactttc tgaaccggaa
agcaagctca tcttctccca gatcgtgtct gccgtgaagc 180 aacggcatga
gaaccaaatt atccacagag atctgaaagc agaaaacgtc ttctatacca 240
gtagcacttg tgtgaaggtg ggggattttg gattcagcac cgtaagtaag aaaggtgaga
300 tgctgaacac cttctgtggg tctccgccct acgctgcacc ggaactcttc
cgtgacgagc 360 actatgttgg cgtttatgtg gatatctggg ccttgggtgt
ccttttgtac ttcatggtga 420 ctggtacgat gccatttcga gcagaaaccg
tggccaaact gaaaaagagc atcctcgatg 480 gtgcctac 488 25 272 DNA Rattus
norvegicus misc_feature Incyte Clone No 701925441H1 25 agccgcgcca
gctgacacct ttcgagaaac tgactcagga catgtgccaa gatgagaagg 60
tggtgaggga gatcacgctg gggaaacgca taggcttcta tcgaattcga ggggagatcg
120 gaagcggaaa cttttcccag gtcaagctgg gaattcactc cctaaccaaa
gaaaaggtgg 180 ccattaagat tctggacaaa accaagttag accagaaaac
ccaaaggctg ttatccagag 240 aaatttccag cagccggaaa tgcaccatcc ca 272
26 303 DNA Rattus norvegicus misc_feature Incyte Clone No
701910632H1 26 aagttgcacc atcccaacat tgtccgtctt tatgaagtcg
tggagaccct gtccaagctc 60 cacctagtga tggagtatgc aggaggtggg
gagctctttg ggaaaattag caccgagggg 120 aaactttctg aaccggaaag
caagctcatc ttctcccaga tcgtgtctgc cgtgaagcaa 180 atnccatgan
aaccaaatta tccacagaga tctgaaagca gaaaacgtct tctataccag 240
tagcacttgt gtgaaggtgg gggattttgg attcagcacc gtaagtnaga naggtgagat
300 gct 303 27 299 DNA Rattus norvegicus misc_feature Incyte Clone
No 701905514H1 27 ganaaccaaa ttatccacag agatctgana gcagaaaacg
tcttctatac cagtagcact 60 tgtgtgaagg tgggggattt tggattcagc
accgtaagta agaaaggtga gatgctgaac 120 accttctgtg ggtctccgcc
ctacgctgca ccggaactct tccgtgacga gcactatgtt 180 ggcgtttatg
tggatatctg ggcctgggtg tccttttgta cttcatggtg actggtacga 240
tgccattcga gcagaaaccg tggccaaact gaaaaagagc atcctcgagg gtgcctaca
299 28 258 DNA Rattus norvegicus misc_feature Incyte Clone No
701293826H1 28 aatggatgcg aggggtgccg tacccctccc ctctggaacc
tttccaactg gatcctaaac 60 atttgtcgga aactagcacc ctcaaagaag
aagaaaacga ggtgaaaagc actttagagc 120 acttggggat cacagacgaa
catatccgga ataaccaagg gagagacgct cgaagctcta 180 tcacgggggt
ctatagaatc attttacatc gagtgcaaag aagaaaagcc tggaaagtgt 240
gccaatggcg acactacc 258 29 113 DNA Rattus norvegicus misc_feature
Incyte Clone No 700949543H1 29 tacctggcag gcgtaccagc tttccctata
agtggagtcg tattaggagc ttgggcgtan 60 atccatgggt ccataggctg
ttttcctgtg tggaaattgt tnatcncgct cca 113 30 300 DNA Macaca
fascicularis misc_feature Incyte Clone No 700706950H1 30 gaagtcgtgg
agaccctatc taagctgcac ttggtgatgg aatatgcagg gggtggggag 60
ctgttcggaa aaattagcac tgaggggaag ctctctgaac cagaaagcaa gctcatcttc
120 tcccagattg tgtctgccgt gaagcactgc atgaaaccaa ttattcacga
gtctgaagca 180 gaaatgtatc natacagtat actgtgtgaa gtggcgattt
ggatcancag taattaaaaa 240 gtgaatnnaa cnttnngtgg ctctccatnt
gacgaccttc ggcgncntan cgnatncgtg 300 31 334 PRT Caenorhabditis
elegans misc_feature GenBank Accession No g3877329 31 Met Ser Glu
Lys Thr Gln Tyr Glu Arg Ala Ile Leu Gln Leu Asn 1 5 10 15 Asn Asp
Pro Val Val His Lys Glu Val Trp Ala Cys Ile Val Ser 20 25 30 Tyr
Gly Lys Arg Lys Leu Trp Phe Gln Val Ala Leu Gly Arg Arg 35 40 45
Ile Gly Phe Tyr Arg Leu Gly Lys Glu Leu Gly Ala Gly Asn Phe 50 55
60 Ser Lys Val Lys Leu Gly Val His Gln Leu Thr Lys Glu Lys Val 65
70 75 Ala Val Lys Ile Met Asp Lys Ala Lys Met Asp Ala Lys Ala Gln
80 85 90 Lys Leu Leu Ser Arg Glu Ile Gln Ala Met Glu Glu Met Asn
His 95 100 105 Pro Asn Ile Val Lys Leu Phe Glu Val Val Glu Thr Leu
Thr Arg 110 115 120 Val His Leu Val Ile Glu Tyr Ala Ser Gly Gly Glu
Leu Tyr Thr 125 130 135 Tyr Val His Glu Arg Gly Lys Leu Thr Glu Gly
Asp Ala Lys Pro 140 145 150 Leu Phe Ala Gln Ile Val Ser Ala Val Ser
His Met His Ser Arg 155 160 165 Asn Ile Val His Arg Asp Ile Lys Ala
Glu Asn Val Met Phe Ser 170 175 180 Ser Pro Asn Thr Val Lys Leu Val
Asp Phe Gly Phe Ser Cys Leu 185 190 195 Val Asp Arg Glu Gln Met Leu
Arg Thr Phe Cys Gly Ser Pro Pro 200 205 210 Tyr Ala Ala Pro Glu Leu
Phe Gln Asp Thr Ser Tyr Ala Gly Glu 215 220 225 Leu Val Asp Val Trp
Ala Leu Gly Val Leu Leu Phe Phe Met Leu 230 235 240 Ile Gly Val Thr
Pro Phe Lys Ala Glu Thr Val Pro Asp Met Lys 245 250 255 Val Leu Ile
Thr Ala Gly Lys Tyr Gln Ile Pro Asp Tyr Val Ser 260 265 270 Leu Leu
Ala Thr Glu Leu Ile Lys Ser Met Leu Lys Thr Asp Thr 275 280 285 Gly
Gln Arg Ala Asp Ile Asp Ser Val Lys Lys His Phe Trp Met 290 295 300
Arg Asp Cys Arg Phe Thr Lys Ser Tyr Leu Ser Ile Lys Ala Thr 305 310
315 Ala Lys Ile Asp Asn Glu Glu Glu Lys Lys Ala Ile Asp Asp Lys 320
325 330 Val Ile Phe Val 32 793 PRT Rattus norvegicus misc_feature
GenBank Accession No g2052189 32 Met Ser Ala Arg Thr Pro Leu Pro
Thr Val Asn Glu Arg Asp Thr 1 5 10 15 Glu Asn His Thr Ser Val Asp
Gly Tyr Thr Glu Thr His Ile Pro 20 25 30 Pro Thr Lys Ser Ser Ser
Arg Gln Asn Ile Pro Arg Cys Arg Asn 35 40 45 Ser Ile Thr Ser Ala
Thr Asp Glu Gln Pro His Ile Gly Asn Tyr 50 55 60 Arg Leu Gln Lys
Thr Ile Gly Lys Gly Asn Phe Ala Lys Val Lys 65 70 75 Leu Ala Arg
His Val Leu Thr Gly Arg Glu Val Ala Val Lys Ile 80 85 90 Ile Asp
Lys Thr Gln Leu Asn Pro Thr Ser Leu Gln Lys Leu Phe 95 100 105 Arg
Glu Val Arg Ile Met Lys Ile Leu Asn His Pro Asn Ile Val 110 115 120
Lys Leu Phe Glu Val Ile Glu Thr Glu Lys Thr Leu Tyr Leu Val 125 130
135 Met Glu Tyr Ala Ser Gly Gly Glu Val Phe Asp Tyr Leu Val Ala 140
145 150 His Gly Arg Met Lys Glu Lys Glu Ala Arg Ala Lys Phe Arg Gln
155 160 165 Ile Val Ser Ala Val Gln Tyr Cys His Gln Lys Cys Ile Val
His 170 175 180 Arg Asp Leu Lys Ala Glu Asn Leu Leu Leu Asp Ala Asp
Met Asn 185 190 195 Ile Lys Ile Ala Asp Phe Gly Phe Ser Asn Glu Phe
Thr Val Gly 200 205 210 Asn Lys Leu Asp Thr Phe Cys Gly Ser Pro Pro
Tyr Ala Ala Pro 215 220 225 Glu Leu Phe Gln Gly Lys Lys Tyr Asp Gly
Pro Glu Val Asp Val 230 235 240 Trp Ser Leu Gly Val Ile Leu Tyr Thr
Leu Val Ser Gly Ser Leu 245 250 255 Pro Phe Asp Gly Gln Asn Leu Lys
Glu Leu Arg Glu Arg Val Leu 260 265 270 Arg Gly Lys Tyr Arg Val Pro
Phe Tyr Met Ser Thr Asp Cys Glu 275 280 285 Asn Leu Leu Lys Lys Leu
Leu Val Leu Asn Pro Ile Lys Arg Gly 290 295 300 Ser Leu Glu Gln Ile
Met Lys Asp Arg Trp Met Asn Val Gly His 305 310 315 Glu Glu Glu Glu
Leu Lys Pro Tyr Ser Glu Pro Glu Leu Asp Leu 320 325 330 Asn Asp Ala
Lys Arg Ile Asp Ile Met Val Thr Met Gly Phe Ala 335 340 345 Arg Asp
Glu Ile Asn Asp Ala Leu Val Ser Gln Lys Tyr Asp Glu 350 355 360 Val
Met Ala Thr Tyr Ile Leu Leu Gly Arg Lys Pro Pro Glu Phe 365 370 375
Glu Gly Gly Glu Ser Leu Ser Ser Gly Asn Leu Cys Gln Arg Ser 380 385
390 Arg Pro Ser Ser Asp Leu Asn Asn Ser Thr Leu Gln Ser Pro Ala 395
400 405 His Leu Lys Val Gln Arg Ser Ile Ser Ala Asn Gln Lys Gln Arg
410 415 420 Arg Phe Ser Asp His Ala Gly Pro Ser Ile Pro Pro Ala Val
Ser 425 430 435 Tyr Thr Lys Arg Pro Gln Ala Asn Ser Val Glu Ser Glu
Gln Lys 440 445 450 Glu Glu Trp Asp Lys Asp Thr Ala Arg Arg Leu Gly
Ser Thr Thr 455 460 465 Val Gly Ser Lys Ser Glu Val Thr Ala Ser Pro
Leu Val Gly Pro 470 475 480 Asp Arg Lys Lys Ser Ser Ala Gly Pro Ser
Asn Asn Val Tyr Ser 485 490 495 Gly Gly Ser Met Thr Arg Arg Asn Thr
Tyr Val Cys Glu Arg Ser 500 505 510 Thr Asp Arg Tyr Ala Ala Leu Gln
Asn Gly Arg Asp Ser Ser Leu 515 520 525 Thr Glu Met Ser Ala Ser Ser
Met Ser Ser Thr Gly Ser Thr Val 530 535 540 Ala Ser Ala Gly Pro Ser
Ala Arg Pro Arg His Gln Lys Ser Met 545 550 555 Ser Thr Ser Gly His
Pro Ile Lys Val Thr Leu Pro Thr Ile Lys 560 565 570 Asp Gly Ser Glu
Ala Tyr Arg Pro Gly Thr Ala Gln Arg Val Pro 575 580 585 Ala Ala Ser
Pro Ser Ala His Ser Ile Ser Ala Ser Thr Pro Asp 590 595 600 Arg Thr
Arg Phe Pro Arg Gly Ser Ser Ser Arg Ser Thr Phe His 605 610 615 Gly
Glu Gln Leu Arg Glu Arg Arg Ser Ala Ala Tyr Ser Gly Pro 620 625 630
Pro Ala Ser Pro Ser His Asp Thr Ala Ala Leu Ala His Ala Arg 635 640
645 Arg Gly Thr Ser Thr Gly Ile Ile Ser Lys Ile Thr Ser Lys Phe 650
655 660 Val Arg Arg Asp Pro Ser Glu Gly Glu Ala Ser Gly Arg Thr Asp
665 670 675 Thr Ala Arg Gly Ser Ser Gly Glu Pro Lys Asp Lys Glu Glu
Gly 680 685 690 Lys Glu Ala Lys Pro Arg Ser Leu Arg Phe Thr Trp Ser
Met Lys 695 700 705 Thr Thr Ser Ser Met Asp Pro Asn Asp Met Val Arg
Glu Ile Arg 710 715 720 Lys Val Leu Asp Ala Asn Thr Cys Asp Tyr Glu
Gln Arg Glu Arg 725 730 735 Phe Leu Leu Phe Cys Val His Gly Asp Ala
Arg Gln Asp Ser Leu 740 745 750 Val Gln Trp Glu Met Glu Val Cys Lys
Leu Pro Arg Leu Ser Leu 755 760 765 Asn Gly Val Arg Phe Lys Arg Ile
Ser Gly Thr Ser Ile Ala Phe 770 775 780 Lys Asn Ile Ala Ser Lys Ile
Ala Asn Glu Leu Lys Leu 785 790 33 729 PRT Homo sapiens
misc_feature GenBank Accession No g3089349 33 Met Ser Thr Arg Thr
Pro Leu Pro Thr Val Asn Glu Arg Asp Thr 1 5 10 15 Glu Asn His Thr
Ser His Gly Asp Gly Arg Gln Glu Val Thr Ser 20 25 30 Arg Thr Ser
Arg Ser Gly Ala Arg Cys Arg Asn Ser Ile Ala Ser 35 40 45 Cys Ala
Asp Glu Gln Pro His Ile Gly Asn Tyr Arg Leu Leu Lys 50 55 60 Thr
Ile Gly Lys Gly Asn Phe Ala Lys Val Lys Leu Ala Arg His 65 70 75
Ile Leu Thr Gly Arg Glu Val Ala Ile Lys Ile Ile Asp Lys Thr 80 85
90 Gln Leu Asn Pro Thr Ser Leu Gln Lys Leu Phe Arg Glu Val Arg 95
100 105 Ile Met Lys Ile Leu Asn His Pro Asn Ile Val Lys Leu Phe Glu
110 115 120 Val Ile Glu Thr Glu Lys Thr Leu Tyr Leu Ile Met Glu Tyr
Ala 125 130 135 Ser Gly Gly Glu Val Phe Asp Tyr Leu Val Ala His Gly
Arg Met 140 145 150 Lys Glu Lys Glu Ala Arg Ser Lys Phe Arg Gln Ile
Val Ser Ala 155 160 165 Val Gln Tyr Cys His Gln Lys Arg Ile Val His
Arg Asp Leu Lys 170 175 180 Ala Glu Asn Leu Leu Leu Asp Ala Asp Met
Asn Ile Lys Ile Ala 185 190 195 Asp Phe Gly Phe Ser Asn Glu Phe Thr
Val Gly Gly Lys Leu Asp 200 205 210 Thr Phe Cys Gly Ser Pro Pro Tyr
Ala Ala Pro Glu Leu Phe Gln 215 220 225 Gly Lys Lys Tyr Asp Gly Pro
Glu Val Asp Val Trp Ser Leu Gly 230 235 240 Val Ile Leu Tyr Thr Leu
Val Ser Gly Ser Leu Pro Phe Asp Gly 245 250 255 Gln Asn Leu Lys Glu
Leu Arg Glu Arg Val Leu Arg Gly Lys Tyr 260 265 270 Arg Ile Pro Phe
Tyr Met Ser Thr Asp Cys Glu Asn Leu Leu Lys 275 280 285 Arg Phe Leu
Val Leu Asn Pro Ile Lys Arg Gly Thr Leu Glu Gln 290 295 300 Ile Met
Lys Asp Arg Trp Ile Asn Ala Gly His Glu Glu Asp Glu 305 310 315 Leu
Lys Pro Phe Val Glu Pro Glu Leu Asp Ile Ser Asp Gln Lys 320 325 330
Arg Ile Asp Ile Met Val Gly Met Gly Tyr Ser Gln Glu Glu Ile 335 340
345 Gln Glu Ser Leu Ser Lys Met Lys Tyr Asp Glu Ile Thr Ala Thr 350
355 360 Tyr Leu Leu Leu Gly Arg Lys Ser Ser Glu Leu Asp Ala Ser Asp
365 370 375 Ser Ser Ser Ser Ser Asn Leu Ser Leu Ala Lys Val Arg Pro
Ser 380 385 390 Ser Asp Leu Asn Asn Ser Thr Gly Gln Ser Pro His His
Lys Val 395 400 405 Gln Arg Ser Val Ser Ser Ser Gln Lys Gln Arg Arg
Tyr Ser Asp 410 415 420 His Ala Gly Pro Ala Ile Pro Ser Val Val Ala
Tyr Pro Lys Arg 425 430 435 Ser Gln Thr Ser Thr Ala Asp Gly Asp Leu
Lys Glu Asp Gly Ile 440 445 450 Ser Ser Arg Lys Ser Ser Gly Ser Ala
Val Gly Gly Lys Gly Ile 455 460 465 Ala Pro Ala Ser Pro Met Leu Gly
Asn Ala Ser Asn Pro Asn Lys 470 475 480 Ala Asp Ile Pro Glu Arg Lys
Lys Ser Ser Thr Val Pro Ser Ser 485 490 495 Asn Thr Ala Ser Gly Gly
Met Thr Arg Arg Asn Thr Tyr Val Cys 500 505 510 Ser Glu Arg Thr Thr
Ala Asp Arg His Ser Val Ile Gln Asn Gly 515 520 525 Lys Glu Asn Ser
Thr Ile Pro Asp Gln Arg Thr Pro Val Ala Ser 530 535 540 Thr His Ser
Ile Ser Ser Ala Ala Thr Pro Asp Arg Ile Arg Phe 545 550 555 Pro Arg
Gly Thr Ala Ser Arg Ser Thr Phe His Gly
Gln Pro Arg 560 565 570 Glu Arg Arg Thr Ala Thr Tyr Asn Gly Pro Pro
Ala Ser Pro Ser 575 580 585 Leu Ser His Glu Ala Thr Pro Leu Ser Gln
Thr Arg Ser Arg Gly 590 595 600 Ser Thr Asn Leu Phe Ser Lys Leu Thr
Ser Lys Leu Thr Arg Ser 605 610 615 Arg Asn Val Ser Ala Glu Gln Lys
Asp Glu Asn Lys Glu Ala Lys 620 625 630 Pro Arg Ser Leu Arg Phe Thr
Trp Ser Met Lys Thr Thr Ser Ser 635 640 645 Met Asp Pro Gly Asp Met
Met Arg Glu Ile Arg Lys Val Leu Asp 650 655 660 Ala Asn Asn Cys Asp
Tyr Glu Gln Arg Glu Arg Phe Leu Leu Phe 665 670 675 Cys Val His Gly
Asp Gly His Ala Glu Asn Leu Val Gln Trp Glu 680 685 690 Met Glu Val
Cys Lys Leu Pro Arg Leu Ser Leu Asn Gly Val Arg 695 700 705 Phe Lys
Arg Ile Ser Gly Thr Ser Ile Ala Phe Lys Asn Ile Ala 710 715 720 Ser
Lys Ile Ala Asn Glu Leu Lys Leu 725 34 776 PRT Rattus norvegicus
misc_feature GenBank Accession No g5672676 34 Met Val Ile Met Ser
Glu Phe Ser Ala Val Pro Thr Gly Thr Gly 1 5 10 15 Gln Gly Gln Gln
Lys Pro Leu Arg Val Gly Phe Tyr Asp Val Glu 20 25 30 Arg Thr Leu
Gly Lys Gly Asn Phe Ala Val Val Lys Leu Ala Arg 35 40 45 His Arg
Val Thr Lys Thr Gln Val Ala Ile Lys Ile Ile Asp Lys 50 55 60 Thr
Arg Leu Asp Ser Ser Asn Leu Glu Lys Ile Tyr Arg Glu Val 65 70 75
Gln Leu Met Lys Leu Leu Asn His Pro Asn Ile Ile Lys Leu Tyr 80 85
90 Gln Val Met Glu Thr Lys Asp Met Leu Tyr Ile Val Thr Glu Phe 95
100 105 Ala Lys Asn Gly Glu Met Phe Asp Tyr Leu Thr Ser Asn Gly His
110 115 120 Leu Ser Glu Asn Glu Ala Arg Lys Lys Phe Trp Gln Ile Leu
Ser 125 130 135 Ala Val Glu Tyr Cys His Asn His His Ile Val His Arg
Asp Leu 140 145 150 Lys Thr Glu Asn Leu Leu Leu Asp Gly Asn Met Asp
Ile Lys Leu 155 160 165 Ala Asp Phe Gly Phe Gly Asn Phe Tyr Lys Pro
Gly Glu Pro Leu 170 175 180 Ser Thr Trp Cys Gly Ser Pro Pro Tyr Ala
Ala Pro Glu Val Phe 185 190 195 Glu Gly Lys Glu Tyr Glu Gly Pro Gln
Leu Asp Ile Trp Ser Leu 200 205 210 Gly Val Val Leu Tyr Val Leu Val
Cys Gly Ser Leu Pro Phe Asp 215 220 225 Gly Pro Asn Leu Pro Thr Leu
Arg Gln Arg Val Leu Glu Gly Arg 230 235 240 Phe Arg Ile Pro Phe Phe
Met Ser Gln Asp Cys Glu Thr Leu Ile 245 250 255 Arg Arg Met Leu Val
Val Asp Pro Ala Lys Arg Ile Thr Ile Ala 260 265 270 Gln Ile Arg Gln
His Arg Trp Met Gln Ala Asp Pro Thr Leu Leu 275 280 285 Gln Gln Asp
Asp Pro Ala Phe Ser Met Gln Gly Tyr Thr Ser Asn 290 295 300 Leu Gly
Asp Tyr Asn Glu Gln Val Leu Gly Ile Met Gln Ala Leu 305 310 315 Gly
Ile Asp Arg Gln Arg Thr Val Glu Ser Leu Gln Asn Ser Ser 320 325 330
Tyr Asn His Phe Ala Ala Ile Tyr Tyr Leu Leu Leu Glu Arg Leu 335 340
345 Arg Glu His Arg Ser Thr Gln Pro Ser Ser Arg Ala Thr Pro Ala 350
355 360 Pro Ala Arg Gln Pro Gln Leu Arg Asn Ser Asp Leu Ser Ser Leu
365 370 375 Glu Val Pro Gln Glu Ile Leu Pro Cys Asp Pro Phe Arg Pro
Ser 380 385 390 Leu Leu Cys Pro Gln Pro Gln Ala Leu Ala Gln Ser Val
Leu Gln 395 400 405 Ala Glu Ile Asp Cys Asp Leu His Ser Ser Leu Gln
Pro Leu Phe 410 415 420 Phe Pro Leu Asp Thr Asn Cys Ser Gly Val Phe
Arg His Arg Ser 425 430 435 Ile Ser Pro Ser Ser Leu Leu Asp Thr Ala
Ile Ser Glu Glu Ala 440 445 450 Arg Gln Gly Pro Ser Leu Glu Glu Glu
Gln Glu Val Gln Glu Pro 455 460 465 Leu Pro Gly Ser Thr Gly Arg Arg
His Thr Leu Ala Glu Val Ser 470 475 480 Thr His Phe Ser Pro Leu Asn
Pro Pro Cys Ile Ile Val Ser Ser 485 490 495 Ser Ala Ala Val Ser Pro
Ser Glu Gly Thr Ser Ser Asp Ser Cys 500 505 510 Leu Pro Phe Ser Ala
Ser Glu Gly Pro Ala Gly Leu Gly Gly Gly 515 520 525 Leu Ala Thr Pro
Gly Leu Leu Gly Thr Ser Ser Pro Val Arg Leu 530 535 540 Ala Ser Pro
Phe Leu Gly Ser Gln Ser Ala Thr Pro Val Leu Gln 545 550 555 Ser Gln
Ala Gly Leu Gly Ala Thr Val Leu Pro Pro Val Ser Phe 560 565 570 Gln
Glu Gly Arg Arg Ala Ser Asp Thr Ser Leu Thr Gln Gly Leu 575 580 585
Lys Ala Phe Arg Gln Gln Leu Arg Lys Asn Ala Arg Thr Lys Gly 590 595
600 Phe Leu Gly Leu Asn Lys Ile Lys Gly Leu Ala Arg Gln Val Cys 605
610 615 Gln Ser Ser Ile Arg Gly Ser Arg Gly Gly Met Ser Thr Phe His
620 625 630 Thr Pro Ala Pro Ser Ser Gly Leu Gln Gly Cys Thr Ala Ser
Ser 635 640 645 Arg Glu Gly Arg Ser Leu Leu Glu Glu Val Leu His Gln
Gln Arg 650 655 660 Leu Leu Gln Leu Gln His His Ser Ala Val Ser Ser
Asp Tyr Gln 665 670 675 Gln Ala Pro Gln Leu Ser Pro Val Pro Tyr Val
Leu Thr Pro Cys 680 685 690 Asp Gly Leu Leu Val Ser Gly Ile Pro Leu
Leu Pro Thr Pro Leu 695 700 705 Leu Gln Pro Gly Met Ser Pro Val Ala
Ser Ala Ala Gln Leu Leu 710 715 720 Asp Ala His Leu His Ile Ser Ala
Gly Pro Val Ala Leu Pro Thr 725 730 735 Gly Pro Leu Pro Gln Cys Leu
Thr Arg Leu Ser Pro Ser Cys Asp 740 745 750 Pro Ala Gly Leu Pro Gln
Gly Asp Cys Glu Met Glu Asp Leu Thr 755 760 765 Ser Gly Gln Arg Gly
Thr Phe Val Leu Val Gln 770 775 35 604 PRT Drosophila melanogaster
misc_feature GenBank Accession No g2564680 35 Met Thr Ala Ala Asn
Thr Asn Lys Thr Thr Asp Lys Glu Asn Asp 1 5 10 15 Pro Gly Pro Asn
Thr Ser Ile Ser Thr Thr Ala Thr Pro Pro Ser 20 25 30 Ala Ala Ala
Gln Asn Val Gly Gly Cys Val Gly Ser Ser Gly Gly 35 40 45 Arg Ser
Ser Pro Lys Phe Xaa Ser Tyr Val Asn Gly Asn Gly Tyr 50 55 60 Gly
Val Tyr Lys Ile Ile Lys Thr Leu Gly Lys Gly Asn Phe Ala 65 70 75
Lys Val Lys Leu Ala Ile His Leu Pro Thr Gly Arg Glu Val Ala 80 85
90 Ile Lys Leu Ile Asp Lys Thr Ala Leu Asn Thr Ile Ala Arg Gln 95
100 105 Lys Leu Tyr Arg Glu Val Asn Ile Met Lys Lys Leu Asn His Pro
110 115 120 Asn Ile Val Arg Leu Leu Gln Val Ile Glu Ser Glu Arg Thr
Leu 125 130 135 Tyr Leu Val Met Glu Tyr Val Ser Gly Gly Glu Leu Phe
Asn Tyr 140 145 150 Leu Val Lys Asn Gly Arg Met Arg Glu Arg Asp Ala
Arg Val Leu 155 160 165 Phe Arg Gln Leu Val Ser Ala Ile Glu Tyr Cys
His Ser Lys Ser 170 175 180 Ile Val His Arg Asp Leu Lys Ala Glu Asn
Leu Leu Leu Asp Gln 185 190 195 Gln Met Lys Leu Lys Ile Ala Asp Phe
Gly Phe Ser Thr Thr Phe 200 205 210 Glu Pro Lys Ala Pro Leu Glu Thr
Phe Cys Gly Ser Pro Pro Tyr 215 220 225 Ala Ala Pro Glu Leu Phe Lys
Gly Lys Lys Tyr Ser Gly Pro Glu 230 235 240 Val Asp Ser Trp Ser Leu
Gly Val Val Leu Tyr Thr Leu Val Ser 245 250 255 Gly Ser Leu Pro Phe
Asp Gly Thr Asn Leu Lys Glu Leu Arg Asp 260 265 270 Arg Val Leu Arg
Gly Lys Tyr Arg Val Pro Tyr Tyr Val Ser Ile 275 280 285 Glu Cys Glu
Ser Leu Xaa Arg Lys Phe Leu Val Leu Asn Pro Thr 290 295 300 Gln Arg
Thr Ser Leu Ser Ala Val Met Ala Asp Arg Trp Ile Asn 305 310 315 Met
Gly Tyr Glu Gln Gly Asn Gly Leu Arg Pro Phe Gln Glu Lys 320 325 330
Pro Met Asp Leu His Asp Val Asn Arg Leu Ser Leu Leu Ser Asn 335 340
345 Met Gly His Lys Pro Arg Asp Val Xaa Gln Ser Leu Lys Asn Gln 350
355 360 Lys Phe Asp Asp Ile Tyr Cys Ala Tyr Met Leu Leu Asp Val Ala
365 370 375 Lys Pro Arg Ser Thr Ala Cys Ser Glu Lys Ser Gly Ser Ser
Phe 380 385 390 Arg Glu Thr Pro Thr Ala Met Pro Gly Ser Ser Arg Ile
Pro Val 395 400 405 Pro Ile Ala Ala Pro Asn Val Thr Ile Ser Gln Val
Thr Phe Ala 410 415 420 Leu Asp Lys Ser Thr Pro Asn Arg Pro Gly Ala
Thr Ser Ile Arg 425 430 435 Pro Met Ala Pro Arg Leu Ala Asn Ala Leu
Thr Pro Leu Pro Leu 440 445 450 Thr Pro Pro Pro Lys Lys Tyr Ile Cys
Cys Ser Ala Ser Lys Ala 455 460 465 Ala Asn Pro Arg Arg Ser Glu Pro
Ser Ser Ile Pro Gln Ser Ala 470 475 480 Met Pro Lys Lys Gly Val Gly
Ser Pro Val Asp Val Lys Thr Thr 485 490 495 Leu Leu Ser Ala Gln Arg
Lys Leu Ala Val Asn His Lys Leu Thr 500 505 510 Ser Ala Ser His Gln
Ile Arg Ser Pro Ile Thr Gln Ser Ser Ser 515 520 525 Gln Ala Ser Glu
Cys Thr Arg Thr Pro Pro Thr His Phe Glu Met 530 535 540 Leu Asp Ser
Thr Ser Thr Pro Leu Lys Val Leu Lys Leu Val Ala 545 550 555 Ser Asn
Ser Gln Thr Pro Pro Ser Thr Glu Asn Ile Asn Arg Pro 560 565 570 Thr
Arg Val Gly Phe Phe Ser Lys Leu Ser Ala Arg Phe Val Arg 575 580 585
Arg Ser Leu His Lys Gly Glu Lys Asp Ile Ser Glu Gln Gly Arg 590 595
600 Asn Leu Thr Lys 36 745 PRT Homo sapiens misc_feature GenBank
Accession No g1749794 36 Met Ile Arg Gly Arg Asn Ser Ala Thr Ser
Ala Asp Glu Gln Pro 1 5 10 15 His Ile Gly Asn Tyr Arg Leu Leu Lys
Thr Ile Gly Lys Gly Asn 20 25 30 Phe Ala Lys Val Lys Leu Ala Arg
His Ile Leu Thr Gly Lys Glu 35 40 45 Val Ala Val Lys Ile Ile Asp
Lys Thr Gln Leu Asn Ser Ser Ser 50 55 60 Leu Gln Lys Leu Phe Arg
Glu Val Arg Ile Met Lys Val Leu Asn 65 70 75 His Pro Asn Ile Val
Lys Leu Phe Glu Val Ile Glu Thr Glu Lys 80 85 90 Thr Leu Tyr Leu
Val Met Glu Tyr Ala Ser Gly Gly Glu Val Phe 95 100 105 Asp Tyr Leu
Val Ala His Gly Arg Met Lys Glu Lys Glu Ala Arg 110 115 120 Ala Lys
Phe Arg Gln Ile Val Ser Ala Val Gln Tyr Cys His Gln 125 130 135 Lys
Phe Ile Val His Arg Asp Leu Lys Ala Glu Asn Leu Leu Leu 140 145 150
Asp Ala Asp Met Asn Ile Lys Ile Ala Asp Phe Gly Phe Ser Asn 155 160
165 Glu Phe Thr Phe Gly Asn Lys Leu Asp Thr Phe Cys Gly Ser Pro 170
175 180 Pro Tyr Ala Ala Pro Glu Leu Phe Gln Gly Lys Lys Tyr Asp Gly
185 190 195 Pro Glu Val Asp Val Trp Ser Leu Gly Val Ile Leu Tyr Thr
Leu 200 205 210 Val Ser Gly Ser Leu Pro Phe Asp Gly Gln Asn Leu Lys
Glu Leu 215 220 225 Arg Glu Arg Val Leu Arg Gly Lys Tyr Arg Ile Pro
Phe Tyr Met 230 235 240 Ser Thr Asp Cys Glu Asn Leu Leu Lys Lys Phe
Leu Ile Leu Asn 245 250 255 Pro Ser Lys Arg Gly Thr Leu Glu Gln Ile
Met Lys Asp Arg Trp 260 265 270 Met Asn Val Gly His Glu Asp Asp Glu
Leu Lys Pro Tyr Val Glu 275 280 285 Pro Leu Pro Asp Tyr Lys Asp Pro
Arg Arg Thr Glu Leu Met Val 290 295 300 Ser Met Gly Tyr Thr Arg Glu
Glu Ile Gln Asp Ser Leu Val Gly 305 310 315 Gln Arg Tyr Asn Glu Val
Met Ala Thr Tyr Leu Leu Leu Gly Tyr 320 325 330 Lys Ser Ser Glu Leu
Glu Gly Asp Thr Ile Thr Leu Lys Pro Arg 335 340 345 Pro Ser Ala Asp
Leu Thr Asn Ser Ser Ala Gln Phe Pro Ser His 350 355 360 Lys Val Gln
Arg Ser Val Ser Ala Asn Pro Lys Gln Arg Arg Phe 365 370 375 Ser Asp
Gln Ala Gly Pro Ala Ile Pro Thr Ser Asn Ser Tyr Ser 380 385 390 Lys
Lys Thr Gln Ser Asn Asn Ala Glu Asn Lys Arg Pro Glu Glu 395 400 405
Asp Arg Glu Ser Gly Arg Lys Ala Ser Ser Thr Ala Lys Val Pro 410 415
420 Ala Ser Pro Leu Pro Gly Leu Glu Arg Lys Lys Thr Thr Pro Thr 425
430 435 Pro Ser Thr Asn Ser Val Leu Ser Thr Ser Thr Asn Arg Ser Arg
440 445 450 Asn Ser Pro Leu Leu Glu Arg Ala Ser Leu Gly Gln Ala Ser
Ile 455 460 465 Gln Asn Gly Lys Asp Ser Leu Thr Met Pro Gly Ser Arg
Ala Ser 470 475 480 Thr Ala Ser Ala Ser Ala Ala Val Ser Ala Ala Arg
Pro Arg Gln 485 490 495 His Gln Lys Ser Met Ser Ala Ser Val His Pro
Asn Lys Ala Ser 500 505 510 Gly Leu Pro Pro Thr Glu Ser Asn Cys Glu
Val Pro Arg Pro Ser 515 520 525 Thr Ala Pro Gln Arg Val Pro Val Ala
Ser Pro Ser Ala His Asn 530 535 540 Ile Ser Ser Ser Gly Gly Ala Pro
Asp Arg Thr Asn Phe Pro Arg 545 550 555 Gly Val Ser Ser Arg Ser Thr
Phe His Ala Gly Gln Leu Arg Gln 560 565 570 Val Arg Asp Gln Gln Asn
Leu Pro Tyr Gly Val Thr Pro Ala Ser 575 580 585 Pro Ser Gly His Ser
Gln Gly Arg Arg Gly Ala Ser Gly Ser Ile 590 595 600 Phe Ser Lys Phe
Thr Ser Lys Phe Val Arg Arg Asn Leu Asn Glu 605 610 615 Pro Glu Ser
Lys Asp Arg Val Glu Thr Leu Arg Pro His Val Val 620 625 630 Gly Ser
Gly Gly Asn Asp Lys Glu Lys Glu Glu Phe Arg Glu Ala 635 640 645 Lys
Pro Arg Ser Leu Arg Phe Thr Trp Ser Met Lys Thr Thr Ser 650 655 660
Ser Met Glu Pro Asn Glu Met Met Arg Glu Ile Arg Lys Val Leu 665 670
675 Asp Ala Asn Ser Cys Gln Ser Glu Leu His Glu Lys Tyr Met Leu 680
685 690 Leu Cys Met His Gly Thr Pro Gly His Glu Asp Phe Val Gln Trp
695 700 705 Glu Met Glu Val Cys Lys Leu Pro Arg Leu Ser Leu Asn Gly
Val 710 715 720 Arg Phe Lys Arg Ile Ser Gly Thr Ser Met Ala Phe Lys
Asn
Ile 725 730 735 Ala Ser Lys Ile Ala Asn Glu Leu Lys Leu 740 745 37
28 DNA Homo sapiens misc_feature primer forward 37 ggttagttac
aagcagtcta gtgcaatt 28 38 25 DNA Homo sapiens misc_feature primer
reverse 38 tgctaccaga ccctaaagaa agaga 25 39 28 DNA Homo sapiens
misc_feature Primer taqman 39 ccctctgtag acacgggacc cttttttg 28
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