JP2003532419A - Cytoskeletal binding protein - Google Patents

Abstract

  (57) [Summary] The present invention provides polynucleotides that identify and encode human cytoskeletal binding protein (CYSKP) and CYSKP. The present invention also provides expression vectors, host cells, antibodies, agonists and antagonists. The present invention also provides a method for diagnosing, treating or preventing a disease associated with abnormal expression of CYSKP.

Description

Detailed Description of the Invention

[0001]

【Technical field】

The present invention relates to nucleic acid and amino acid sequences of cytoskeletal binding proteins. The present invention also relates to diagnosis / treatment / prevention of cell proliferation abnormality, autoimmune / inflammatory disease, vesicle transporting property, neurogenicity, cell motility, reproductive system disease and muscular disease using these sequences. The invention further relates to the evaluation of the effect of exogenous compounds on the expression of nucleic acid and amino acid sequences of cytoskeletal binding proteins.

[0002]

BACKGROUND OF THE INVENTION

Cytoskeleton, a cytoplasmic protein fiber, is involved in cell shape, structure, and movement
. The cytoskeleton supports the cell membrane, forms tracks, and organelles and other elements
Move in the cytosol along the tracks of. The cytoskeleton has a dynamic structure and
The cells have various shapes and are capable of directional movement. Furthermore, the molecule is
It is sequestered at specific cell locations by interaction with the vesicle skeletal binding protein. main
Cytoskeletal fibers required are microfilaments, microtubules and intermediate filaments
. Motor proteins such as myosin, dynein and kinesin drive fiber movement
Do or move along the fibers. Cytoskeletal membrane anchors attach fibers to cell membranes
However, accessory or binding proteins modify fiber structure or activity
. Other proteins that bind to the cytoskeleton are processes such as secretion and intracellular signaling
Play a role in. (For the cytoskeleton, Lodish, H. et al. (1995)Mole cular Cell Biology  See Scientific American Books, New York NY. )Microtubules and binding proteins Tubulin Microtubules, which are 24 nm diameter cytoskeletal fibers, play multiple roles within the cell. bundle
The formed microtubules form cilia and flagella, both of which are flagellated extensions of the cell membrane.
Are required to clear substances on the epithelium and flagella are required for sperm swimming. Red blood cell
The peripheral regions of platelet and microtubules are important for maintaining the flexibility of these cells. cell
Organelles, membrane vesicles and proteins are transported intracellularly along microtubule tracks
. For example, microtubules run through nerve cell axons, between cell bodies and nerve endings.
It enables bidirectional transport of substances and membrane vesicles. Provide these vesicles to the nerve endings.
Failure to supply, interferes with nerve signaling. Spindle-loaded microtubules also
It is important for chromosome movement during cell division. Between stable and short-lived microtubule populations
Both are intracellular. Microtubules are macromolecules composed of GTP-bound tubulin protein subunits.
Each subunit is a heterodimer of α-tubulin and β-tubulin
So, each has an isoform. α-tubulin is acetylated, polyglutamic
Many of the glycation, cleavage of two amino acids (Δ2 tubulin formation), tyrosine, etc.
Post-translational modification is performed. In some cases, these modifications affect microtubule stability.
To do. Due to hydrolysis of GTP, tubulin subunits are attached to the ends of microtubules.
Add and combine. This subunit interacts with the head and tail to produce protophyllamen.
The protofilaments interact with each other to form microtubules.
Is forming. Microtubules are polar, with one end containing α-tubulin and the other end.
The ends are surrounded by β-tubulin and the two ends are
Is different. Each microtubule is usually composed of 13 protofilaments,
In some cases, 11 or 15 protofilament microtubules. Cilia and flagella are two
Contains microtubules. Microtubules are known as centrosomes or microtubule formation centers (MTOCs)
It grows from a special structure. The center of microtubule formation is one or two centrioles
Included, it has a set of three microtubule windmills. At the bottom of cilia or flagella
The basal body, which is the center of formation, contains one centriole (central grain). For center formation
The γ-tubulin present is the nuclear form of polymerization of α- and β-tubulin heterodimers.
Important for growth, it does not polymerize in microtubules. The area around the protein center can be seen in MTOC.
And is involved in the assembly of microtubules.Microtubule-binding protein Microtubule-associated protein (MAP) is involved in microtubule assembly and stabilization. Assembly
One major family of microtubule-binding proteins, re-MAP, is
It is also identified in non-neuronal cells. Assembly MAP is a small
It is also responsible for bridging small tubes. These MAPs contain basic microtubule-binding domains and acidic probes.
It forms two domains, the projection domain. Projection
The main is the binding site for membranes, intermediate filaments or other microtubules. Sequence analysis
The assembly MAP can be divided into two types, Type I and Type II, based on
I can. Type IMAP includes MAP1A and MAP1B, which are large filamentous components that are co-purified in microtubules.
It is a child and is often expressed in the brain and testis. These are positively charged amino acid sequences
Contains several repeats of erves to neutralize negatively charged tubulin and
This leads to tube stabilization. Are MAP1A and MAP1B each a single precursor polypeptide?
, Which is then processed proteolytically to yield one heavy chain and one light chain.
Form. Another light chain, LC3, is 16.4 kDa (kilodalton) and contains MAP1A, MAP1B, and small
Combine with the tube. LC3 is synthesized from sources other than MAP1A or MAP1B transcripts
 Expression is important for the regulation of MAP1A or MAP1B microtubule-binding activity during cell proliferation
(Mann, S. S. et al. (1994) J. Biol. Chem. 269: 11492-11.
497). Type II MAPs, including MAP2a, MAP2b, MAP2c, MAP4 and Tau, are microtubule-bound
It is characterized by having three to four copies of the main 18-residue sequence. MAP2a
, MAP2b and MAP2c are found only in dendrites, and MAP4 is found in non-neuronal cells.
Therefore, Tau is found in axons and dendrites of nerve cells. Another Tau mRNA
Pricing causes the presence of multiple forms of the Tau protein. Al
Zheimer's disease, Pick's disease, progressive supranuclear palsy, corticobasal degeneration and home
Frontal temporal dementia and paternity associated with chromosome 17
Phosphorylation of Tau is altered in neurodegenerative diseases such as Kinson's disease. Tau phosphoric acid
The change in denaturation disrupts the microtubule network, leading to the formation of Tau aggregates in neurons.
Occurs (Spillantini, M.G. and Goedert, M. (1998) Trends Neurosci. 21: 4
28-433). Microtubule stability is also regulated by cutting microtubule length. AAA
Adenosine triphosphate degrading enzyme (ATPase) protein belonging to superfamily
Katanin uses ATP hydrolysis energy to cleave and degrade stable microtubules.
Katanine is responsible for microtubule release from centrosomes, regulation of spindle assembly, and cell perimeter.
It is responsible for promoting microtubule turnover during the transitional phase (Hartman, J.J. and Va.
le, R.D. (1999) Science 286: 782-785). Microtubule aggregation is associated with several diseases. Extraskeletal mucoid cartilage obtained from humans
Tumors contained parallel arrays of microtubules within the rough endoplasmic reticulum (Suzuki, T. et al. (19
88) J. Pathol. 156: 51-57). Microtubule aggregates are Chin infected with hepatitis C virus
It was also found in hepatocytes collected from pansies. Monoc made into these aggregates
A protein called p44 (or microtubule aggregation protein) by the monoclonal antibody
Quality has been detected (Maeda, T. et al. (1989) J. Gen. Virol. 70: 1401-1407). p
44 human homologues induced by interferon-α and interferon-β
But not by interferon-γ. P44 is an interfero
It is believed to be a mediator in the antiviral action of arsenin (Kitamura, A. et al. (1994
) Eur. J. Biochem. 224: 877-883).Dynein-related motor protein Dynein is a (-) end-directed motor protein that acts on microtubules. Cytoplasmic
There are two types of dynein, which are axonal and. Cytoplasmic dynein is a substance along the cytoplasmic microtubule
Are involved in the migration of cells from the nerve endings to the cell body and endocytic vesicles.
Is involved in the transport of erythrocytes to lysosomes. Intracytoplasmic dynein is also involved in mitosis
It is reported to be involved. Axon dynein also moves flagella and cilia
Involved. Dynein on one microtubule doublet (two sequences) is adjacent to the microtubule doublet.
It moves like "walking" along the let. This slip creates bending force that causes flagella
Or beat the cilia. Dynein has a natural mass of between 1000 and 2000 kilodaltons
And has two or three heads that produce force, which are due to hydrolysis of ATP.
Driven. This head is linked to the base domain via a stalk,
The basal domain is composed of a very different number of attached intermediate and light chains.
ItKinesin-related motor protein Kinesins are (+) end-directed motor proteins that act on microtubules. Protota
The ips kinesin molecule is involved in the transport of membrane-bound vesicles and organelles. This feature is God
It is of particular importance for axonal transport in transcellular cells. Also for all cell types
Kinesin is important for vesicular transport from the Golgi complex to the endoplasmic reticulum. This role
Are extremely important for maintaining the uniqueness and functionality of these secretory organelles.
It Kinesin is a ubiquitous, conserved protein family of over 50 proteins.
Lee, which are the primary amino acid sequence, domain structure, motility and cell
Grouped into at least eight subfamilies based on function. (Moore, J.D. and
And S.A. Endow (1996) Bioessays 18: 207-219, Hoyt, A.M. (1994) Curr. Opin.
 See Cell Biol. 6: 63-68. ) The prototype kinesin molecule consists of two polypeptides.
It is a heterotetramer consisting of heavy chain (KHCs) and two polypeptide light chains (KLC). KHC
The subunit is usually called "kinesin". KHC is about 1000 amino acids in length
Yes, KLC is about 550 amino acids in length. Two KHCs are dimerized into three
Form rod-shaped molecules with secondary structure in different parts. Add ATP to one end of the molecule.
There are spherical motor domains that function in water splitting and microtubule binding. Kinesin exercise
Maine is highly conserved and has 70% or more identity. Motor domain
There is an α-helical coiled-coil region that mediates dimerization. To the other end of the molecule
Has a fan-shaped tail attached to the molecular cargo. This tail has a KHC C-terminus and two KLC
Formed by interaction. Kinesin-related proteins belong to a further branched subfamily of kinesins
Called (KRPs), many of these act during eukaryotic mitosis (Hoyt,Previous Out ). Some are required by KRP for spindle assembly.in vivo andin v itro  In analysis, these KRPs exert their force on the microtubules that make up the spindle,
Have been suggested to cause segregation of. This activity requires KRP phosphorylation.
Required Failure to build a spindle leads to unsuccessful mitosis or chromosome
An aneuploidy occurs, the latter characteristic of cancer cells. In addition, centromere protein E
Intrinsic KRP, termed the mitotic chromosome centromere, and the opposite spindle
Involved in separation into poles.Microfilaments and related proteins Actin Microfilaments, which are cytoskeletal filaments with a diameter of 7-9 nm, are migrating cells
, Is important for cell shape, cell adhesion, cell division and muscle contraction. micro
The assembly and disassembly of filaments allows cells to change their morphology.
Microfilaments are the most abundant intracellular proteins in eukaryotic cells
It is a polymer of actin. Human cells contain six isoforms of actin
ing. There are three α-actins in different muscle types, non-muscle β-actin and
And non-muscle γ-actin are in non-muscle cells, and other γ-actins are intestinal smooth muscle cells.
It is in. G-actin, a monomer of actin, is a polar spiral F-actin molecule.
It polymerizes into Lament and the hydrolysis of ATP to ADP occurs simultaneously. Actin filament
Bind together to form bundles and networks that provide a framework for supporting the plasma membrane,
Shape a cell. These bundles and networks connect to the cell membrane. In muscle cells
, A thin filament containing actin during contraction contains the motor protein myosin
Slide through a thick filament. Other actin-related filaments are actin
It is not part of the cytoskeleton, but rather binds to microtubules and dynein.Actin-binding protein Actin-binding proteins crosslink, cleave and stabilize actin filaments, or
It is involved in the sequestration of actin monomers. Some of the actin-binding proteins
It has multiple functions. Actin filament bundles and networks are both
It is retained by the tin-crosslinked protein. These proteins are
It has a total of two actin binding sites, one for each actin. Short cross-linked proteins
Cross-linking proteins that promote bundle formation, while longer and more flexible networks
Promote formation. Calmodulin-like calcium-binding domain of actin cross-linking protein
The main allows calcium regulation of the crosslinks. Group I cross-linking proteins
It has a unique actin-binding domain, 30 Kd protein, EF-1a, fasc
in) and scruin. Group II cross-linked proteins consist of 7,000-MW
It has a cutin-binding domain and includes villin and dematin.
. Group III cross-linking proteins have a pair of 26,000-MW actin binding domains and
Actinin, fimbrin, spectrin, dystrophin, ABP120 and
Includes filamin. By cleaving the protein, cut it into shorter pieces or block its ends.
The length of the actin filament is regulated by this. Protein cut
Examples include gCAP39, severin (fragmin), gelsolin and villin.
Protein capping forms a cap at the end of the actin filament
, You cannot cut the filament. CapZ for protein capping
Includes lopomodulin and tensin. Tensins found in focal adhesions also crosslink actin filaments. Extracellular mat
Integrin activity by lix causes tyrosine, serine and threonine residues
Phosphorylation of tensin on the base occurs, which phosphorylation is transformed by an oncogene.
It also occurs in damaged cells. Tensins have an SH2 domain, and other tyrosine
In some cases, it may bind to phosphorylated proteins (Lo, S.H. et al. (1997) J. Cell Biol. 1
36: 1349-1361). At the N-terminus of tensinyeast(Saccharomyces cerevisiae)Estimation
Contains a region of homology to the catalytic domain of tyrosine phosphatase (PTP) on
There is. This PTP domain of tensin mediates binding to phosphorylated polypeptides
(Haynie, D.T. and Ponting, C.P. (1996) Protein Sci. 5: 2643-2646)
. Mice that do not have the tensin gene have abnormal kidneys and loss of tensin
Shows weakening of adhesion spots in (Lo,Above). The proteins thymosin and profilin sequester actin monomers within the cell matrix,
Allows the presence of unpolymerized actin. Profilin also has an actin monomer.
Stimulates the formation of F-actin by effectively lowering the critical concentration required for addition (Gertler, F.
B. et al. (1996) Cell 87: 227-239). The actin-binding proteins tropomyosin, troponin, and caldesmon are muscle
Regulates meat contraction in response to calcium. This tropomyosin protein is muscle
Found on cells and non-muscle cells, is α-helical and forms a coiled-coil dimer
is doing. Tropomyosin in striated muscle interacts with the troponin complex and actin
To regulate muscle contraction (PROSITE PDOC00290 Tropomyosin Signature
). Is the tropomyosin complex troponin-T, troponin-I and troponin-C?
It is composed of Troponin-T is tropomyosin, bound troponin-I and troponin-T.
Binds Ponin-C to tropomyosin. The actin-binding proteins tropomyosin, troponin, and caldesmon are muscle
Regulates meat contraction in response to calcium. This tropomyosin protein is muscle
Found on cells and non-muscle cells, is α-helical and forms a coiled-coil dimer
is doing. Striated muscle tropomyosin interacts with the troponin complex and actin
Mediates and regulates muscle contraction (PROSITE PDOC00290 tropomyosin signature)
. Is the tropomyosin complex troponin-T, troponin-I and troponin-C?
It is composed of Troponin-T is tropomyosin, bound troponin-I and troponin-T.
Binds Ponin-C to tropomyosin. Many proteins involved in the regulation of actin assembly are, for example, alpha activators.
Actin racks such as nin, spectrin and fimbrin
Between proteins, such as the calponin homology (CH) domain found in bridge proteins
It has the characteristics of an interaction domain. Between extracellular matrix receptors and cytoskeleton
Other proteins that are mainly localized in focal adhesions, the macromolecular complex that mediates contact, are:
It has a protein-protein interaction motif known as the LIM domain. For example,
Zyxin is involved in the spatial regulation of actin assembly and has three tandem types.
Contains the LIM domain. Dixin is also α due to its proline-rich N-terminus
It interacts with actinin (Beckerle, M. C. (1997) BioEssays 19: 949-957).
Cytoskeletal proteins are associated with several diseases. Muscular dystrophy, Neff
Pathological conditions such as Rose syndrome and dilated cardiomyopathy are related to the differential expression of α-actinin-3.
(Vainzof, M. et al. (1997) Neuropediatrics 28: 223-228; Smoyer,
 W.E. and Mundel, P. (1998) J. Mol. Med. 76: 172-183; Sussman, M.A. et al.
 (1998) J. Clin. Invest. 101: 51-61). α-actinin and some MAPs are flat
Elderly people and patients with neurodegenerative diseases such as Alzheimer's disease present in the field body
(Maciver, S.K. and Harrington, C.R. (1995) Ne.
uroreport. 6: 1985-1988). With unusual actin bundling proteins
Some actinin-4 appears to be involved in cell motility of metastatic cancer cells. Other diseases
Immature staining often associated with division of cells from tumor tissue
Body condensation (Murnane, J.P. (1995) Cancer Metastasis Rev. 14: 17-29) and
There is an important role for axon and assembly MAP in illness pathogenesis (Sodeik, B.
 (1997) J. Cell Biol. 136: 1007-1021).Intermediate filaments and related proteins Intermediate filament (IF) is a cytoskeletal fiber with a diameter of 10 nm
And the middle diameter of the microtubule diameter. These play a structural role in the cell
To strengthen and organize cells. IF is particularly abundant in epithelial cells and neurons
. IF is extremely stable, and unlike microfilaments and microtubules
Does not get involved. IF proteins include acidic keratin, basic keratin, desmin, GF
A protein (glial fibrillary acidic protein), peripherin, nerve fibrils,
Contains Sutin and lamins. IF has a central α-helical rod region interrupted by a short non-helical linker segment
. This rod-shaped region is often defined by a non-spiral head-and-tail domain.
Are classified. The rod-shaped regions of the intermediate filament protein bind to form a coil
Form a decoil dimer. The IF is produced from the dimer through a highly ordered construction process.
Jijiru Unlike the construction of microfilaments and microtubules, ATP or GTP for IF construction
Neither is required. IF-related proteins (IFAPs) mediate interactions between IFs and with other cellular structures.
Through. IFAP crosslinks IF in bundles or networks, or at the plasma membrane
Crosslink. It may also crosslink IF into microfilaments and microtubule cytoskeletons
. Microtubules and IF are particularly closely related. IFAP includes BPAG1, Placoglobin, and
Smoplakin I, Desmoplakin II, plectin, ankyri
n), filaggrin and lamin B receptor. The N-terminal portion of ankyrin is involved in specific protein-protein interactions.
It consists of repeats of 33 amino acid motifs that are repeats. Different ankyrin repeats
Tubulin, anion exchange protein, voltage-gated sodium channel, Na⁺/
K⁺-To be involved in binding with ATPase and neurofascin,
The variable region within the motif is responsible for specific protein binding. Ankyrin Mochi
F also NF-κ-B and in yeast the cell cycle proteins CDC10, SW14 and SW
It is in a transcription factor such as 16.Drosophila Notch, LIN-12 and GLP-1C. eleg ans  Proteins associated with tissue differentiation, such as, also contain ankyrin-like repeats
It Lux et al. (1990; Nature 344: 36-42) described ankyrin-like repeats as "integrated".
Acts as ankyrin and integrates integral membrane proteins, tubulin and other proteins
It has been shown to form binding sites for proteins.Cytoskeletal membrane anchor Cytoskeletal fibers are attached to the plasma membrane by specific proteins. These connections
Adhesion is important for cell shape maintenance and muscle contraction. In red blood cells, spect
The phosphorus-actin cytoskeleton consists of three proteins, band 4.1, ankyrin and adene.
It is attached to the cell membrane by adducing. If this bond is defective, it will be abnormal.
Cells of different shapes, which are broken down more rapidly by the spleen, leading to anemia.
Wake up. In platelets, the spectrin-actin cytoskeleton also
It is bound to the membrane by a giraffe and the second actin network
Attached to the membrane by In muscle cells, the protein dystrophin
Binds actin microfilaments to the plasma membrane.
Mutations in the gene result in Dusenne muscular dystrophy. Adhesive bond
And contact plaques, actin fibrils are associated with the peripheral membrane protein α-actin.
It is bound to the cell membrane by cutinin and vinculin. IF is also attached to the membrane by a cytoskeletal membrane anchor. Nuclear lamina accepts lamin B
It is attached to the inner surface of the nuclear envelope by the body. Vimentin IF is pre-treated with Ankyrin
It is attached to the plasma membrane by cutin. Desmosomes and hemidesmososo
The epithelial cells of the organ and the skin are connected at the membrane junction. With these membrane junctions
Shear deformation forces are distributed throughout the epithelial cell layer, thus providing strength and rigidity to the epithelial cells.
Is given. The IF of epithelial cells is desmoked by procoglobin and desmoplakin.
It adheres to the some (sticky spot). The protein that binds IF to the hemidesmosome is
I don't understand. Desmin IF surrounds the sarcomere in muscle, paranemin,
It is bound to the plasma membrane by synemin and ankyrin.Protein of erythrocyte membrane skeleton Oxygen distribution in vertebrate bodies is affected by red blood cells. Oxygen is the surrounding water or
From the atmosphere, it diffuses through gill epithelium or lung epithelial type I cells. Then oxygen
Enter the blood circulation directly through the blood capillary endothelium, pass through the membrane of red blood cells,
Stored in soluble oxyhemoglobin of high quality. Oxygen from hemoglobin in the organ
It is released at each site and sent from red blood cells to other target cells. Red blood cell membrane and other red
The structure of cell membranes other than blood cells is designed to enable efficient diffusion of oxygen to the intracellular part.
Needs to be carried. Erythrocyte membranes are i) cholesterol rich with many bilayer transmembrane proteins embedded in them.
Rich phospholipid bilayer, ii) external glycosylphosphatidylinositol anchors
-Protein (GPI protein), and iii) Laminate inner surface of bilayer
It is composed of erythrocyte skeleton or membrane skeleton. For bilayer-penetrating proteins
Anion exchangers, glycophorins, sugar transporters and various cation transporters and
Pump is included. Erythrocyte GPI protein contains acetylcholinesterase and
Includes complement response regulator (CD55). Skeletal proteins are the lining of the plasma membrane,
Or it is organized on the surface inside the cytoplasm. Protein 4.1 for these proteins
, Proteins p 55, α and β spectrin, actin and dematin, tro
Actin-binding proteins such as pomyosin and tropomodulin are included.
α and β spectrin arein vivoTo form a heterotetramer. Spect
Trin heterotetramer is a two-dimensional network of lining with a hexagonal mesh.
Be organized into. This network consists of β-spectrin, ankyrin and anion
By a protein complex containing the recombinant and protein 4.2
, By the “triangular” interaction between glycophorin C and protein p 55,
Bound to the bilayer transmembrane protein. Structural function of erythrocyte membrane proteins
Variants are found in various tissues. Variants include, for example, anion exchangers,
From multigene families such as nkyrin or spectrin, or
Transcribed from a single gene family such as protein 4.1 or protein 4.2
It mRNA transcripts undergo tissue-specific alternative splicing. Many congenital hemolytic
Anemia results from mutations in the above-mentioned genes that encode erythrocyte membrane proteins
It For example, hereditary elliptocytosis is a head-to-head self of spectrin tetramer.
Resulting from mutations in the sequence of the spectrin gene at or near the binding region
Or a bump in the protein 4.1 gene that decreases the level of protein 4.1.
Naturally caused by mutation. In another example, hereditary spherocytosis is an ankyrin gene, negative
On-exchange gene, protein 4.2 gene, or α and β spectrin inheritance
Associated with mutations in offspring. (Delaunay J. (1995) Transfus. Clin. Biol.
 2: 207-216.) Protein 4.1 is an 80 kilodalton erythrocyte membrane protein with four functional domains.
It is a pak quality. These domains include i) actin-binding proteins and transmembrane
Homologous to the ERM (Ezrin / Radixin / Moesin) family of cross-protein binding
A 30 kDa basic N-terminal domain (Tsukita, S. et al. (1997) Trends Biochem.
Sci. 22: 53-58), ii) a 16 kDa hydrophilic molecule containing a protein kinase C phosphorylation site.
Sex domain, iii) cAMP dependence important for interaction with spectrin and actin
Highly charged domain of 10 kDa that contains the phosphorylation site of the active protein kinase, and iv)
 It has a 22/24 kDa acidic domain. Protein 4.1 is structurally and functionally related
Is a member of the protein 4.1 family. The protein 4.1 family has many
One of evolutionarily related protein superfamily including rosin phosphatase
(Baklouti, F. et al. (1997) Genomics 39: 289-302.). Contrary to the precise localization of protein 4.1 lining in mature enucleated erythrocytes,
The protein 4.1 epitope (antigenic determinant) is responsible for the cytoplasmic part of the nucleated cell and the nuclear skeleton.
Seen there. In particular, protein 4.1 was found in the nuclear skeleton during the interphase
To the spindle during rupture, around the chromosomes at the end of mitosis, and to the midbody during cytokinesis
Exists. (Krauss, S.W. et al. (1997) J. Cell Biol. 137: 275-289.) The differential expression of the protein 4.1 gene, which results in multiple mRNA splice variants, is variable in different genes.
It has been observed in the tissues of pigs and rodents. Gene structure and mRNA splice variants
Comparison reveals conservation of extreme genomic sequences of protein 4.1 across species
became. Successful identification and sequencing of the 5'UTR of human and rodent mRNA species
Not, but this is probably not technical during the nucleotide sequencing reaction.
It seems to be due to the GC rich areas that cause complications. (Baklouti, before
Out; Conboy, J.G. (1988) Proc. Natl. Acad. Sci. 85: 9062-9065) Analysis of proteins within the ERM family of proteins revealed that the N-terminal domain was CD4
It interacts with transmembrane proteins of intracellular domains such as 4 and the C-terminal domain
It was shown to bind to cutin. Both interactions result in the Rho-GTP protein complex
Is involved in the interaction of polyphosphoinositides with serine / tyrosine kinases.
, Also related to the activity of tyrosine kinases. Many phosphorylated parts of ERM protein
The rank is preserved.in vivo ERM protein expression is restricted to tissues such as endothelium
However, the blockage of ERM protein gene expression is released under cell culture conditions.
Be done. (Tsukita,Above.) The lining actin cytoskeleton is involved in various membrane-based processes, in which
Require large amounts of functional plasticity of related molecular components. Homology to band 4.1
A family of proteins that reconstitute the actin cytoskeleton in response to various stimuli.
It is involved in weaving and is probably involved in transmembrane signaling. This
Of the family include tyrosine phosphatases, tyrosine kinase substrates and tumors
Candidate suppressor genes are included (Arpin M, et al. (1994) Curr. Opin. Cell Biol.
 6: 136-141). Disruptions in cytoskeletal protein interactions have been identified in many conditions and diseases
ing. Neurofibromatosis type 2 is an autosomal dominant disorder of the nervous system. Neurofibroma
Schwann cells isolated from symptomatic type 2 patients differ from control Schwann cells,
It has a characteristic morphology and growth parameters. Remnants associated with neurofibromatosis type 2
The gene has been identified and is called NF2. schwannomin or with Merlin
Known NF2 gene product is a member of the protein 4.1 superfamily
It has been shown that mutations in the NF2 gene are associated with this disease. (Ro
senbaum, C. et al. (1998) Neurobiol. Dis. 5: 55-64.) Furthermore, the formation of psoriasis is red blood.
Due to mutational expression or distribution in the epidermal epithelium of analogues of globular protein 4.1
Is. (Shimizu, T. (1996) Histol. Histopathol. 11: 495-501.)
Erythrocytes with mutations in protein and protein 4.1Pl asmodium falciparum) It was shown that the susceptibility to invasion by P. (Face
r, C.A. (1995) Parasitol Res. 81: 52-57.)
By the antibody produced by the
And most of the neurofibrillary tangles in the hippocampus were stained. 68kDa protein
Was probably identified as a brain analog of erythrocyte protein 4.1. (Sihag, R.K
. Et al. (1994) Brain Res. 656: 14-26.) Development of novel cytoskeletal binding proteins and polynucleotides encoding them
At first glance, it is possible to meet the demands in the art by providing new compositions.  This novel composition is useful for cell proliferative disorders, autoimmune / inflammatory diseases, impaired vesicle transport.
, Diagnosis, prevention and treatment of neurological diseases, cell motility, reproductive diseases and muscle diseases
And for the expression of nucleic acid and amino acid sequences of cytoskeletal binding proteins.
It is also useful for assessing the effects of foreign compounds.

[0003]

SUMMARY OF THE INVENTION

The present invention is generically referred to as "CYSKP", individually as "CYSKP-1", "CYSKP-2",
"CYSKP-3", "CYSKP-4", "CYSKP-5", "CYSKP-6", "CYSKP-7", "CYSKP"
-8 "," CYSKP-9 "," CYSKP-10 "," CYSKP-11 "," CYSKP-12 "," CYSKP-13 "
, "CYSKP-14", "CYSKP-15", "CYSKP-16", "CYSKP-17", "CYSKP-18"
, "CYSKP-19", "CYSKP-20", "CYSKP-21", "CYSKP-22", "CYSKP-23",
"CYSKP-24", "CYSKP-25", "CYSKP-26", "CYSKP-27", "CYSKP-28", "
CYSKP-29 "," CYSKP-30 "," CYSKP-31 "," CYSKP-32 "," CYSKP-33 "," CY "
There is provided a purified polypeptide which is a cytoskeletal binding protein designated SKP-34 ". In one embodiment, the invention provides (a) a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34, (b) an amino acid sequence selected from the group having SEQ ID NO: 1-34 A naturally occurring polypeptide having an amino acid sequence that is at least 90% identical to (c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34, or (d) Provided is a substantially isolated polypeptide selected from the group comprising an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34. In one embodiment, SEQ I
Provided is a substantially isolated polypeptide comprising the amino acid sequence of D NO: 1-34. The present invention also provides (a) a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34, (b) at least 90% amino acid sequence selected from the group having SEQ ID NO: 1-34 A natural polypeptide containing an amino acid sequence having homology of (c
) A biologically active fragment of a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34, or (d) a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34 And a substantially isolated polynucleotide encoding a polypeptide comprising an immunogenic fragment of. In one embodiment, the polynucleotide encodes a polypeptide selected from the group having SEQ ID NO: 1-34. In another embodiment, the polynucleotide is selected from the group having SEQ ID NO: 35-68. The invention further comprises (a) a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34, (b) at least 90% amino acid sequence selected from the group having SEQ ID NO: 1-34 A natural polypeptide having an amino acid sequence that is identical to (c
) A biologically active fragment of a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34, or (d) a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34 Recombinant polynucleotides having a promoter sequence operably linked to a polynucleotide encoding a polypeptide comprising an immunogenic fragment of. In one embodiment, the invention provides cells transformed with the recombinant polynucleotide. In another embodiment, the present invention provides a genetic transformant containing the recombinant polynucleotide. Furthermore, the present invention relates to (a) a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34, and (b) an amino acid sequence selected from the group having SEQ ID NO: 1-34, and 90% A natural polypeptide having the same amino acid sequence as above,
(C) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34, and (d) an amino acid sequence selected from the group having SEQ ID NO: 1-34 And a method for producing a polypeptide selected from the group consisting of an immunogenic fragment of the polypeptide having: The production method comprises (a) a step of culturing cells transformed with the recombinant polynucleotide under conditions suitable for expression of the polypeptide, and (b) a step of receiving the polypeptide thus expressed. And the recombinant polynucleotide has a promoter sequence operably linked to the polynucleotide encoding the polypeptide. The invention further comprises at least 90 amino acid sequences selected from the group having: (a) an amino acid sequence selected from the group having SEQ ID NO: 1-34; and (b) an amino acid sequence selected from the group having SEQ ID NO: 1-34. A native polypeptide having an amino acid sequence that is% identical, (c) a biologically active fragment of the polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34, (d) SEQ ID There is provided a substantially isolated antibody which specifically binds to a polypeptide selected from the group consisting of an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group having NO: 1-34. The invention further comprises (a) a polynucleotide having a polynucleotide sequence selected from the group having SEQ ID NO: 35-68 and (b) a polynucleotide sequence selected from the group having SEQ ID NO: 35-68. A natural polynucleotide having a polynucleotide sequence that is at least 90% identical, a polynucleotide complementary to the polynucleotide of (c) (a), and a polynucleotide complementary to the polynucleotide of (d) (b). And a substantially isolated polynucleotide selected from the group consisting of (e) (a) to (d) RNA equivalents. In one embodiment the polynucleotide has at least 60 contiguous nucleotides. The invention further provides a method of detecting a target polynucleotide in a sample.
Here, the target polynucleotide is (a) a polynucleotide having a polynucleotide sequence selected from the group having SEQ ID NO: 35-68, and (b) SEQ ID NO: 35-68.
A naturally occurring polynucleotide having a polynucleotide sequence that is at least 90% identical to a polynucleotide sequence selected from the group having: (c) a polynucleotide complementary to the polynucleotide of (a), and (d) (b ), And a polynucleotide sequence selected from the group comprising a polynucleotide complementary to the polynucleotide of (a) or an RNA equivalent of (e) (a) to (d). The detection method comprises the steps of: (a) hybridizing the sample with a probe containing at least 20 consecutive nucleotides consisting of a sequence complementary to the target polynucleotide in the sample;
(B) The process of detecting the presence / absence of a hybridization complex, and optionally detecting the amount of the complex, if any, forms a hybridization complex between the probe and the target polynucleotide. Under conditions like
The probe specifically hybridizes to the target polynucleotide. In one embodiment, the probe comprises at least 60 contiguous nucleotides. The invention also provides a method of detecting a target polynucleotide in a sample.
Here, the target polynucleotide is (a) a polynucleotide having a polynucleotide sequence selected from the group having SEQ ID NO: 35-68, and (b) SEQ ID NO: 35-68.
A naturally occurring polynucleotide having a polynucleotide sequence that is at least 90% identical to a polynucleotide sequence selected from the group having: (c) a polynucleotide complementary to the polynucleotide of (a), and (d) (b A) A polynucleotide complementary to the polynucleotide and a sequence of a polynucleotide selected from the group comprising (e) (a) to (d) RNA equivalents. The detection method includes (a) a step of amplifying a target polynucleotide or a fragment thereof using polymerase chain reaction amplification, and (b) detecting the presence / absence of the target polynucleotide or a fragment thereof, and detecting the target polynucleotide or the fragment thereof. If a fragment is present, it optionally includes the step of detecting its amount. The invention further provides a component comprising an effective amount of the polypeptide and a pharmaceutically acceptable excipient, wherein the effective amount of the polypeptide is (a) an amino acid selected from the group having SEQ ID NO: 1-34. A polypeptide having a sequence, (b) a native polypeptide having an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group having SEQ ID NO: 1-34, and (c) SEQ ID NO: A biologically active fragment of a polypeptide having an amino acid sequence selected from the group having 1-34, or (d) SEQ ID NO: 1-34
Selected from the group comprising immunogenic fragments of polypeptides having an amino acid sequence selected from the group having In one example, a composition comprising an amino acid sequence selected from the group having SEQ ID NO: 1-34 is provided. Further, the present invention provides a method for treating a disease associated with decreased expression of functional CYSKP or a symptom thereof, which comprises administering the composition to a patient. The invention also relates to (a) a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34, (b) at least 90% amino acid sequence selected from the group having SEQ ID NO: 1-34 A naturally occurring polypeptide comprising an amino acid sequence that is identical to (c
) A biologically active fragment of a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34, or (d) a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34 The present invention provides a method for screening a compound for confirming the effectiveness as a agonist of a polypeptide comprising the immunogenic fragment of. The screening method includes (a) exposing a sample having a polypeptide to a compound, and (b) detecting an agonist activity in the sample. Alternatively, the invention provides a composition comprising an agonist compound identified by this method and a suitable pharmaceutical excipient. In a further alternative, the present invention provides a method of treating a disease or condition associated with reduced expression of functional CYSKP, comprising administering the composition to a patient. The invention further comprises (a) a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34, (b) at least 90% amino acid sequence selected from the group having SEQ ID NO: 1-34 A naturally occurring polypeptide having an amino acid sequence that is
c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34, or (d) a polymorph having an amino acid sequence selected from the group having SEQ ID NO: 1-34 Methods of screening compounds to confirm their effectiveness as antagonists of polypeptides, including immunogenic fragments of peptides, are provided. The screening method includes (a) exposing a sample containing the polypeptide to a compound, and (b) detecting the agonist activity in the sample. In one embodiment, the invention provides a component comprising an antagonist compound identified by this method and a pharmaceutically acceptable excipient. In another embodiment, the functional CYSKP
A method of treating a disease or medical condition associated with the overexpression of, comprising the step of administering a component to a patient in need of such treatment. Furthermore, the present invention provides (a) a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34, (b) an amino acid sequence selected from having SEQ ID NO: 1-34, and 90% or more of A naturally occurring polypeptide having an identical amino acid sequence, (c)
A biologically active fragment of a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34, or (d) a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34 Methods of screening for compounds that modulate the activity of a polypeptide, including immunogenic fragments, are provided. This screening method
(A) binding the polypeptide to at least one compound under suitable conditions, and (b) detecting binding of the polypeptide to the test compound, thereby specifically binding to the polypeptide. Identifying compounds that bind. The invention further comprises at least 90 amino acid sequences selected from the group having: (a) an amino acid sequence selected from the group having SEQ ID NO: 1-34; and (b) an amino acid sequence selected from the group having SEQ ID NO: 1-34. A native polypeptide having an amino acid sequence that is% identical, (c) a biologically active fragment of the polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34, (d) SEQ ID Provided is a method for screening a compound that regulates the activity of a polypeptide selected from the group comprising an immunogenic fragment of the polypeptide having an amino acid sequence selected from the group having NO: 1-34. The screening method includes (a) a step of mixing the polypeptide with at least one test compound under conditions where the activity of the polypeptide is allowed, and (b) a step of calculating the activity of the polypeptide in the presence of the test compound. And (c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, the change in the activity of the polypeptide in the presence of the test compound. Means a compound that modulates the activity of the polypeptide. The invention further provides methods of screening compounds to confirm the efficacy of mutated expression of a target polynucleotide. The target polynucleotide has SEQ ID NO:
Includes sequences selected from the group having 35-68. The screening method includes (a) exposing a sample containing the target polynucleotide to a compound, and (b) detecting mutated expression of the target polynucleotide. The invention further comprises (a) treating a biological sample containing nucleic acid with a test compound, and (b) (i) a polynucleotide comprising a polynucleotide sequence selected from the group having SEQ ID NO: 35-68. , (Ii) a natural polynucleotide comprising a polynucleotide sequence that is at least 90% identical to a polynucleotide sequence selected from the group having SEQ ID NO: 35-68, (iii) a sequence complementary to (i) Having a polynucleotide, (iv) a polynucleotide complementary to the polynucleotide of (ii), and (v
) Hybridizing nucleic acids of the treated biological sample with a probe composed of at least 20 contiguous nucleotides of a polynucleotide selected from the group comprising RNA equivalents of (i) to (iv); A method for calculating the toxicity of a test compound including is provided. Hybridization occurs under conditions such that a specific hybridization complex is formed between the probe and the target polynucleotide in the biological sample, the target polynucleotide being (i) SEQ ID NO: 35-
A polynucleotide comprising a polynucleotide sequence selected from the group having 68, (ii
) A natural polynucleotide comprising a polynucleotide sequence that is at least 90% identical to a polynucleotide sequence selected from the group having SEQ ID NO: 35-68, (ii)
i) a polynucleotide having a sequence complementary to the polynucleotide of (i), (iv)
A polynucleotide complementary to the polynucleotide of (ii), of (v) (i)-(iv)
Select from the group containing RNA equivalents. Alternatively, the target polynucleotide is (i) above.
~ (V) a fragment of the polynucleotide sequence selected from the group comprising: (c) quantifying the amount of hybridization complex; Differences in the amount of hybridization complex in the treated biological sample, including the step of comparing with the amount of hybridization complex in the untreated biological sample, are indicative of toxicity of the test compound.

[Description of the present invention]

Although the proteins, nucleotide sequences and methods of the invention are described, it is to be understood that the invention is not limited to the particular apparatus, materials and methods described, but may be modified. Further, the technical terms used herein are merely used for the purpose of describing specific examples, and are not intended to limit the scope of the present invention which is limited only by the claims. Please also understand. It should be noted that in the present specification and claims, "a", "the (s)" and the like which refer to the singular include the plural unless the context clearly dictates otherwise. Therefore,
For example, “a host cell” includes a plurality of host cells, “antibody” includes a plurality of antibodies, and equivalents well known to those skilled in the art are also included. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, unless otherwise defined. Although any apparatus, material, or method similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable apparatus, materials, or methods are described herein. All publications mentioned in this invention are cited for the purpose of describing and disclosing the cells, protocols, reagents and vectors which were reported in the publications and which may be relevant to the invention. . Nothing in this specification is admitted that the invention is not entitled to antedate such disclosure by virtue of prior art. Definitions The term "CYSKP" is substantially purified obtained from all species including natural, synthetic, semi-synthetic or recombinant (especially mammals including bovine, ovine, porcine, murine, equine and human). Refers to the amino acid sequence of CYSKP. The term "agonist" refers to a molecule that enhances or mimics the biological activity of CYSKP. This agonist interacts directly with CYSKP or acts with components of the biological pathway involving PKIN to regulate the activity of CYSKP, including proteins, nucleic acids, carbohydrates,
It may include a small molecule, any other compound or composition. The term "allelic variant" refers to another form of the gene encoding CYSKP. Allelic variants may be created from at least one mutation in the nucleic acid sequence. It can also be made from mutant RNA or polypeptides. The structure or function of the polypeptide may or may not be mutated. A gene may have no natural allelic variants, or one or several natural allelic variants. Common mutagenic changes, which generally give rise to allelic variants, are ascribed to spontaneous deletions, additions or substitutions of nucleotides. Each of these changes, alone or in combination with other changes, can occur once or several times within a given sequence. A "mutant" nucleic acid sequence encoding CYSKP refers to a polypeptide that has the same polypeptide as CYSKP or at least one of the functional properties of CYSKP, even though various nucleotide deletions, insertions, or substitutions have occurred. This definition includes inappropriate or unexpected hybridization of a CYSKP-encoding polynucleotide sequence with an allelic variant at a position other than the normal chromosomal locus, as well as specific oligonucleotide probes of the CYSKP-encoding polynucleotide. Includes polymorphisms that are easily or difficult to detect using. The encoded protein can also be "altered", resulting in silent changes and deletion of amino acid residues that are functionally equivalent to CYSKP,
It may include insertions or substitutions. Intentional amino acid substitutions are similar in terms of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of residues within the range where biologically or immunologically CYSKP activity is retained. Can be based on. For example,
Negatively charged amino acids include aspartic acid and glutamic acid, and positively charged amino acids include lysine and arginine. Amino acids having uncharged polar side chains with similar hydrophilicity values include asparagine and glutamine, and serine and threonine. Amino acids having uncharged side chains with similar hydrophilicity values include leucine, isoleucine and valine, glycine and alanine, phenylalanine and tyrosine. The terms "amino acid" and "amino acid sequence" refer to oligopeptides, peptides, polypeptides, protein sequences, or any fragment thereof, including naturally occurring and synthetic molecules. When "amino acid sequence" refers to the sequence of a naturally occurring protein molecule, "
"Amino acid sequence" and like terms are not intended to limit the amino acid sequence to the complete and intact amino acid sequence associated with the protein molecule described. The term "amplification" relates to making copies of nucleic acid sequences. Amplification is typically performed using the polymerase chain reaction (PCR) technique well known to those skilled in the art. The term "antagonist" is a molecule that inhibits or attenuates the biological activity of CYSKP. Antagonists are antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition that directly interacts with CYSKP or interacts with components of biological pathways involving PKIN to regulate CYSKP activity. May include proteins such as things. The term “antibody” refers to Fab and F (ab ′) ₂ capable of binding an antigenic determinant, and fragments thereof,
Refers to intact molecules such as Fv fragments. Antibodies that bind CYSKP polypeptides can be made using intact molecules or fragments thereof that contain small peptides that immunize the antibody.
The polypeptide or oligopeptide used to immunize an animal (mouse, rat, rabbit, etc.) can be derived from translated or chemically synthesized RNA, and can be conjugated to a carrier protein if desired. is there. Commonly used carriers that chemically bind to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The binding peptide is used to immunize the animal. The term "antigenic determinant" refers to the region (ie, epitope) of a molecule that makes contact with a particular antibody. When a host animal is immunized with a protein or protein fragment, multiple regions of the protein can induce the production of antibodies that specifically bind to an antigenic determinant (a specific region or three-dimensional structure of the protein). An antigenic determinant can compete with an intact antigen for binding to an antibody (ie, an immunogen used to induce an immune response). As used herein, "antisense" refers to any composition capable of base pairing with the sense (coding) strand of a particular nucleic acid sequence. Antisense components include DNA, RNA, peptide nucleic acid (PNA), oligonucleotides having a modified backbone chain such as phosphorothioic acid, methylphosphonic acid or benzylphosphonic acid, and 2'-methoxyethyl sugar or 2 '. There are oligonucleotides having modified saccharides such as -methoxyethoxy sugar and oligonucleotides having modified bases such as 5-methylcytosine, 2-deoxyuracil or 7-deaza-2'-deoxyguanosine. Antisense molecules can be produced by any method including chemical synthesis or transcription. Complementary antisense molecules, once introduced into cells,
It forms base pairs with the natural nucleic acid sequences formed by cells and forms duplexes that interfere with transcription or translation. The expression "negative" or "minus" is the antisense strand, and the expression "positive" or "plus" is the sense strand. The term "biologically active" refers to a protein having the structural, regulatory, or biochemical function of a naturally occurring molecule. Similarly, the term “immunologically active” or “immunogenicity” means that a natural or recombinant CYSKP, synthetic CYSKP or any oligopeptide thereof is capable of producing a specific immune response in a suitable animal or cell. Refers to the ability to induce and bind to a particular antibody. The term "complementary" refers to the relationship between two single-stranded nucleic acid sequences that anneal by base pairing. For example, the sequence "5'AG-T3 '" binds to the complementary sequence "3'TC-A5'". "Composition comprising a given polynucleotide sequence" or "composition comprising a given amino acid sequence" refers broadly to any composition that comprises a given polynucleotide or amino acid sequence. This component may include a dry formulation or an aqueous solution.
A composition comprising a polynucleotide sequence encoding CYSKP or a fragment of CYSKP can be used as a hybridization probe. This probe can be stored in a freeze-dried state and can be bound to a stabilizer such as sugar. For hybridization, the probe is dispersed in an aqueous solution containing salts (eg NaCl), detergents (eg sodium dodecyl sulfate; SDS) and other constituent elements (eg Denhardt's solution, skim milk powder, salmon sperm DNA etc.). Can be made. The “consensus sequence” is obtained by repeating analysis of a DNA sequence to separate unnecessary bases and extending in 5 ′ and / or 3 ′ direction using an XL-PCR kit (PE Biosystems, Foster City CA). Re-sequenced nucleic acid sequence, or GELVI
EW fragment construction system (GCG, Madison, WI) or Phrap (University of Washin
gton, Seattle WA) etc. refers to a nucleic acid sequence constructed from one or more overlapping cDNAs or ESTs or genomic DNA fragments using a computer program for constructing fragments. Some sequences are both extended and assembled to determine the consensus sequence.
The term "conservative amino acid substitution" refers to a substitution that does not significantly alter the properties of the original protein. That is, the substitution does not significantly change the structure or function of the protein,
The structure of the protein, in particular its function, is preserved. The table below shows the amino acids that can be substituted for the original amino acids in the protein and the amino acids that are recognized as conservative amino acid substitutions.

[0004] Conservative amino acid substitutions typically involve (a) a polypeptide background in the substitution region.
Structure, eg β-sheet or α-helical structure, (b) charge of the molecule at the substitution site or
Are hydrophobic and / or retain most of the (c) side chains. The term "deletion" is a change in an amino acid sequence that lacks one or more amino acid residues, or
Refers to changes in the nucleic acid sequence that lack one or more nucleotides. The term "derivative" refers to a chemically modified polynucleotide or polypeptide
. For example, hydrogen of an alkyl group, an acyl group, a hydroxyl group or an amino group
Substitutions can be included in the chemical modification of the polynucleotide sequence. Polynucleotide induction
The body retains at least one biological or immunological function of the natural molecule
It encodes a polypeptide. Polypeptide derivatives may be glycosylated, polyethylene
PEGylation, or any similar process of inducible origin
Retaining at least one biological or immunological function from
Is a polypeptide modified by the process. A "detectable label" is a polynucleotide or polynucleotide capable of producing a measurable signal.
A reporter molecule or enzyme that is covalently or non-covalently bound to a peptide. "Differential expression" is determined by comparing at least two different samples
Upregulated, increased or unregulated, or decreased, downregulated, or defective genes or
Refers to protein expression. Such comparisons can be made, for example, with post-treatment samples and untreated samples.
Sample or pathological sample and a normal sample. The term "fragment" refers to a unique portion of CYSKP or a polynucleotide encoding CYSKP.
Min, which is identical to the parent sequence but longer than that sequence
Means a short one. Fragments are 1 nucleotide / amino from the full length of the defined sequence.
It may have a length less than the length minus the acid residues. For example, some fragments are 5-1
It may have 000 consecutive nucleotides or amino acid residues. Probe, plastic
Fragments used as immers, antigens, therapeutic molecules, or for other purposes
Is at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 75,
100, 150, 250 or 500 consecutive nucleotides or amino acids
It can be residue length. Fragments can be preferentially selected from specific regions of the molecule. For example
, A polypeptide fragment is the first 250 or 500 amino acids as shown in the given sequence.
A length selected from noic acids (or the first 25% or 50% of the polypeptide)
Can have consecutive amino acids. These lengths are clearly given as examples
Examples of the present invention are supported by the specification including sequence listings, tables and drawings.
It may be of any desired length. The fragment of SEQ ID NO: 35-68 differs from other sequences in the genome from which it was obtained, for example.
Region of unique polynucleotide sequence that unambiguously identifies SEQ ID NO: 35-68.
Including. Certain fragments of SEQ ID NO: 35-68 are, for example, hybridized or amplified.
Technique, or similar analogs that distinguish SEQ ID NO: 35-68 from related polynucleotide sequences.
Useful for methods. The exact fragment length of SEQ ID NO: 35-68 that matches a fragment
Areas are routinely measured by techniques common in the art based on the purpose of the fragment.
it can. A fragment with SEQ ID NO: 1-34 is encoded by a fragment with SEQ ID NO: 35-68.
To be done. A fragment of SEQ ID NO: 1-34 specifically identifies SEQ ID NO: 1-34
Contains a region of unique amino acid sequence. For example, one fragment of SEQ ID NO: 1-34 is SE
As an immunogenic peptide in the development of antibodies that specifically recognize Q ID NO: 1-34
It is useful. The exact length and region of the fragment of SEQ ID NO: 1-34 that matches a fragment is
, Can be routinely determined by techniques common in the art based on the purpose of the fragment
. A "full length" polynucleotide sequence is at least one translation initiation codon (eg,
Methionine), an open reading frame and a translation stop codon.
It is a column. A "full length" polynucleotide sequence is a "full length" polypeptide sequence.
To code. The term "homology" refers to sequence similarity, that is, two or more polynucleotide sequences or two
The sequence identities are compatible between the sequences of the above polypeptide sequences. The term "percent identity" or "% identity" for polynucleotide sequences
"Sex" means two or more that are aligned using a standardized algorithm.
Is the percentage of matching residues between the polynucleotide sequences. Like this
The algorithm is the sequence to compare to optimize the alignment between the two sequences.
And insert the gap in a standardized and reproducible way,
A more significant comparison is possible. The percent match between polynucleotide sequences is determined by MEGALIGN version 3.12e sequence alignment.
Default path for the CLUSTAL V algorithm, such as that built into the
It can be determined using a parameter. This program is based on the LASERGENE software package.
Part of a cage (a suite of molecular biological analysis programs) (DNASTAR, Madison WI)
It This CLUSTAL V is from Higgins, D.G. and P.M.Sharp (1989) CABIOS 5: 151-1.
53, Higgins, D.G. et al. (1992) CABIOS 8: 189-191. Polynucle
For paired alignments of octido sequences, the default parameters are Ktuple = 2, ga
p penalty = 5, window = 4, “diagonals saved” = 4. "Weighted
The residue weight table was selected as the default. The percent identity is
CL as the "percentage of similarity" between the aligned polynucleotide sequences
Reported by USTAL V. Alternatively, a set of commonly used and freely available sequence comparison algorithms is
National Center for Biotechnology (NCBI) Basic Local Alignment Search T
ool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 2
15: 403-410), which is NCBI and Internet (Bethesda, MD).
available from several sources, including http://www.ncbi.nlm.nih.gov/BLAST/)
It This suite of BLAST software includes known polynucleotide sequences and various data
Database used for alignment with another polynucleotide sequence in the database.
Various sequence analysis programs are included, including "tn". "BLAST 2 Sequences"
Tools called are available that directly compare pairs of two nucleotide sequences
Used for. "BLAST 2 Sequences" is available at http://www.ncbi.nlm.nih.gov/g
You can access orf / b12.html and use it interactively. `` BLAST 2 Sequences
The tool can be used for both blastn and blastp (described below).
BLAST programs are generally configured with gaps and other default settings.
It is used with the parameter of. For example, when comparing two nucleotide sequences,
Some people found that the "BLAST 2 Sequences" tool Ver set with default parameters
Will use blastn with sion 2.0.12 (April-21-2000). Default parameter
An example of setting the data is shown below. 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 Filter: on The match rate is a measure of the fully defined sequence length (eg, defined by a particular sequence number)
It can be compared and measured. Alternatively, a shorter length, for example a larger defined
Fragments obtained from the row (eg at least 20, 30, 40, 50, 70, 100
Or the percentage of contiguous nucleotides) compared to the length of
May be. The lengths listed here are for example only, and should not be considered as tables, figures and sequence lists.
Fragments of any sequence length supported by the sequences described herein, including
It should be understood that can be used to describe the length over which the rate of agreement can be measured. Nucleic acid sequences that do not show a high degree of homology nevertheless show degeneracy in the genetic code.
The cause may encode similar amino acid sequences. Nucleic acid distribution using this degeneracy
All nucleic acid sequences encode proteins that are substantially the same, causing in-sequence variation.
It is to be understood that a large number of nucleic acid sequences such as The term “percent identity” or “% identity” as used in polypeptide sequences
”Means that there are two or more ports aligned using a standardized algorithm.
Refers to the percentage of matching residues between the polypeptide sequences. Polypeptide sequence
The method of instrumentation is known. Alignment considering conservative amino acid substitutions
There is also a method. Such conservative substitutions, which have already been detailed, usually involve the acidity of the substitution site and
Because it preserves hydrophobicity, it also preserves the structure (and thus function) of the polypeptide. The percent match between polypeptide sequences is determined by the MEGALIGN version 3.12e sequence alignment
Default parameters of the CLUSTAL V algorithm, as built into the program.
Can be determined using the data (see above for a description of it already). CLUSTAL V
Is used to align the polypeptide sequences in pairs and the default pattern
The parameters are set as Ktuple = 1, gap penalty = 3, window = 5, “diagonals saved” = 5.
Set. Select the PAM250 matrix as the default residue weight table. Poly
Aligned polypeptide sequences as well as nucleotide alignments
The percent identity of a pair of is calculated by CLUSTAL V as the "percentage of similarity".
Will be reported. Alternatively, the NCBI BLAST software suite may be used. For example, two polypep
When making pairwise comparisons of tide sequences, some were set with default parameters.
Use blastp with the BLAST 2 Sequences tool Version 2.0.12 (Apr-21-2000)
Will An example of setting default parameters is shown below. Matrix: BLOSUM62 Open Gap: 11 and Extension Gap: 1 penalties Gap x drop-off: 50 Expect: 10 Word Size: 3 Filter: on The match rate is a fully defined (eg defined by a particular SEQ ID NO) polypeptide.
It can be measured by comparison with the length of the sequence. The concordance rate is a sequence or shorter length,
Eg fragments obtained from larger defined polypeptide sequences (eg less
And a fragment of 15, 20, 30, 40, 50, 70 or 150 consecutive residues)
The match rate may be measured by comparing with the length of the. The lengths listed here are just exemplary
No more than supporting the sequences described herein, including tables, figures and sequence listings.
Using a fragment of any sequence length that has been truncated, it is of a certain length and
It should be understood that it may explain the length with which the fatality can be measured. "Human Artificial Chromosome (HAC)" is a DNA sequence of approximately 6 kb (kilobase) to 10 Mb in size.
All elements necessary for segregation and maintenance of stable chromosomal replication, which may include sequences
It is a linear small chromosome containing. The term "humanized antibody" mimics a more human antibody while retaining its original binding ability
Thus, it refers to an antibody molecule in which the amino acid sequence of the non-antigen binding region has been altered. “Hybridization” means that under the specified hybridization conditions,
Annelin forms a base pair with a complementary single strand with a single-stranded polynucleotide
It is the process of Specific hybridization is high for two nucleic acid sequences
It is an indication that they share homology. Under conditions that allow annealing,
A specific hybridization complex is formed, which allows hybridization to continue after the washing process.
I'm still playing. The washing step is a step in the hybridization process.
It is particularly important in determining linguisticity, and in more stringent conditions
Reduces non-specific binding (ie, pair binding between nucleic acid strands that are not perfectly matched). Nuclear
Acceptable conditions for acid sequence annealing are routine for one of ordinary skill in the art.
To decide. Permissive conditions may be constant during hybridization experiments, but may be
The purification conditions are chosen to obtain the desired stringency and
It can be modified during the experiment to also obtain specificity. Annealing allowed
The conditions are, for example, at a temperature of 68 ° C., about 6 × SSC, about 1% (w / v) SDS, and
Approximately 100 μg / ml shear denatured salmon sperm DNA is included. In general, hybridization stringency is to some extent a wash step.
Can be expressed in terms of the temperature at which it is run. Such cleaning temperatures are usually
Approximately 5 to 20 ° C lower than the melting point (Tm) of the specific sequence at a given ionic strength and pH
To choose. This Tm is perfectly consistent under a given ionic strength and pH
The temperature at which 50% of the target sequence hybridizes to the probe. Formula to calculate Tm
And nucleic acid hybridization conditions are well known, see Sambrook et al.
9)Molecular Cloning: A Laboratory Manual, Second Edition, Volume 1-3, Cold Spring Har
bor Press, Plainview NY, see especially Chapter 2 of Volume 2. High stringency hybridization between polynucleotides of the invention
At about 68 ° C in the presence of about 0.2 x SSC and about 0.1% SDS.
Including the degree. Alternatively, it is carried out at temperatures of 65 ° C, 60 ° C, 55 ° C, 42 ° C. SSC concentration
Can vary from about 0.1 to 2 x SSC in the presence of about 0.1% SDS. Normally,
Blocking agents are used to block non-specific hybridization. Such blocking reagents
For example, about 100 to 200 μg / ml of sheared and denatured salmon sperm DNA
Is included. Specified under certain conditions, such as RNA and DNA hybridization
Organic solvents such as formamide at a concentration of about 35-50% v / v can also be used.
. Useful variations of wash conditions will be apparent to those of skill in the art. Hybrida
Ization is an evolutionary process between nucleotides, especially under conditions of high stringency.
May indicate similarities. Such similarities in nucleotides and nucleotides
It strongly suggests a similar role for the encoded polypeptide. The term "hybridization complex" refers to hydrogen bonds between complementary bases.
, Refers to the complex of two nucleic acid sequences formed. The hybridization complex
, Can be formed in the dissolved state (C₀t or R₀analysis etc.). Alternatively, one of the nucleic acid sequences
The other nucleic acid sequence is present in solution and the other nucleic acid sequence is a solid support (eg paper, membrane, filter
, Chips, pins or glass slides, or other suitable substrate
Or between the two nucleic acid sequences that are immobilized on the substrate on which the nucleic acid is immobilized.
Can be made. The term "insertion" or "addition" refers to one or more amino acid residues or nucleotides.
Refers to changes in the amino acid sequence or nucleic acid sequence that are respectively added. "Immune response" is associated with inflammation, trauma, immune disorders, contagious or inherited disorders
It can refer to the symptoms. These conditions are species that can act on the cellular and systemic defense system.
Expression of various factors such as cytokines, chemokines, and other signaling molecules
Can be characterized by: The term “immunogenic fragment” when introduced into a living organism such as a mammal
, CYSKP polypeptide or oligopeptide fragments that induce an immune response
Point to. The term "immunogenic fragment" is also disclosed herein or well known in the art.
Polypeptide fragments or oligopeptides of CYSKP useful for any antibody production method
Including fragments. The term "microarray" refers to a plurality of polynucleotides, polypeptides or substrates on a substrate.
Or refers to the composition of other compounds. The term "element" or "array element" is unique to a microarray
A polynucleotide, polypeptide or other compound having a specified position
Refers to something. The term "modulation" refers to a change in the activity of CYSKP. For example, by adjusting the CYSKP
Protein activity, binding properties, or other biological or functional properties or
Changes in immunological properties occur. The terms "nucleic acid" and "nucleic acid sequence" refer to nucleotides, oligonucleotides, polynucleotides
Refers to cleotide or fragments thereof. The terms "nucleic acid" and "nucleic acid sequence" refer to geno
DNA or RNA of murine or synthetic origin, single-stranded or double-stranded
Is DNA or RNA that may represent the sense or antisense strand, or the peptide core
It may also refer to acid (PNA) or any DNA-like or RNA-like substance. "Functionally linked" means that the first and second nucleic acid sequences have a functional relationship.
Refers to a certain state. For example, a promoter may affect the transcription or expression of a coding sequence.
If so, the promoter is operably linked to the coding sequence.
. Need to join two protein coding regions in the same reading frame
, The functionally linked DNA sequences are very close together, or
Can be continuous. "Peptide nucleic acid (PNA)" is a peptide of amino acid residues ending in lysine.
Oligonucleosides bound to the backbone of at least about 5 nucleotides in length
Refers to antisense molecules or anti-gene agents, including tide. The terminal lysine is a component
Give solubility to. PNA preferentially binds and transcribes complementary single-stranded DNA or RNA
It stops the elongation of PNA in cells
Life can be extended. "Post-translational modifications" of CYSKP include lipidation, glycosylation, phosphorylation, acetylation,
Racemization, proteolytic cleavage and other modifications known in the art can be included. This
These processes can occur synthetically or biochemically. Biochemical modification of CYSKP
It depends on the enzymatic environment and may vary from cell type to cell type. A "probe" is a test for identical or allelic nucleic acid sequences or related nucleic acid sequences.
Nucleic acid encoding CYSKP, their complementary sequences, or their fragments used for export
It is an array. The probe may be an isolated oligonucleotide or polynucleotide.
Rheotide attached to a detectable label or reporter molecule
. Typical labels include radioactive isotopes, ligands, chemiluminescent reagents and enzymes.
is there. A "primer" is a target polynucleotide that forms complementary base pairs.
Is a short nucleic acid, usually a DNA oligonucleotide, that can be annealed to.
The primer can then be extended to the target DNA strand by a DNA polymerase enzyme. Plastic
Immer pairs are used to amplify nucleic acid sequences (and by, for example, polymerase chain reaction (PCR) (and
Identification). Probes and primers, such as those used in the present invention, typically include at least a known sequence.
It contains 15 consecutive nucleotides. Longer probe to increase specificity
And primers, such as at least 20, 25, 30, 4 of the disclosed nucleic acid sequences.
0, 50, 60, 70, 80, 90, 100 or 150 consecutive nucleoti
Probes and primers may be used. Much longer than this
Some probes and primers are also available. Included in the specification, including tables, drawings and sequence listings
It was understood that any length of supported nucleotides could be used
Yes. For the preparation and use of the probe and the primer, see, for example, Sambrook, J.
 Et al (1989)Molecular Cloning: A Laboratory Manual, Second Edition, Volume 1-3, Cold Sp
ring Harbor Press, Plainview NY, Ausubel, F.M. et al. (1987)Current Protoc ols in Molecular Biology , Greene Pubi. Assoc. & Wiley-Intersciences, New
 York NY, Innis et al. (1990)PCR Protocols, A Guide to Methods and Applicati ons  Please refer to Academic Press, San Diego CA, etc. PCR primer pairs
Computer programs for the purposes of, for example Primer (Version 0.5, 1991, W
hitehead Institute for Biomedical Research, Cambridge MA)
Can be obtained from known sequences. The choice of oligonucleotides to use as primers is for such purposes
This is done using software well known in the art. For example OLIGO 4.06
Software is useful for selecting PCR primer pairs of up to 100 nucleotides each
With oligonucleotides and large polynucleotides up to 5,000
Available from input polynucleotide sequences of up to 32 kilobases
It is also useful for analysis. Similar primer selection programs have extended capacity.
Built-in additional features. For example, the PrimOU primer selection program (test
One from the Genome Center at the University of Texas Southwestern Medical Center in Dallas, Texas
Available to the public) to select specific primers from the megabase sequence.
It is possible and therefore useful for designing primers throughout the genome.
Primer3 primer selection program (Whitehead Institute / MIT Center for Geno
(available from me Research, Cambridge MA1)
A "non-priming library (mispri
ming libaray) ”can be entered. Primer3 is especially useful for microarrays.
Useful for selecting gonucleotides. (Solutions for the latter two primer selection programs
Change the source code from your own source to meet your specific needs
You may. ) PrimerGen Program (British Human Genome Manager in Cambridge, UK)
Papping Project-Publicly available from the Resource Center)
Design primers based on sequence alignment, and
Hybridized with either the maximum conserved region or the minimum conserved region of the imported nucleic acid sequence.
It is possible to select a primer that can be removed. Therefore, this program is unique
To identify conserved oligonucleotide and polynucleotide fragments.
It is for. Oligonucleotides and polynucleotides identified by any of the above selection methods
Fragments of the oligonucleotide may be used in hybridization techniques such as PCR or.
Or as a sequencing primer, as a microarray element, or
Or a fully or partially complementary polynucleotide in a sample of nucleic acids
It is useful as a specific probe. For the method of selecting oligonucleotides, see above
It is not limited to law. As used herein, a "recombinant nucleic acid" is not a naturally occurring sequence and is the separation of two or more sequences.
It is an array that artificially combines different segments. This artificial combination is often
Achieved by chemical synthesis, but more commonly by artificial manipulation of isolated segments of nucleic acids.
Depending on the gene, such as those described in Sambrook et al.
Achieve by engineering method. The term "recombinant nucleic acid" simply means adding or replacing a part of nucleic acid.
It also includes a deleted mutant nucleic acid. Often recombinant nucleic acids contain promoter sequences
Included are operably linked nucleic acid sequences. Such recombinant nucleic acids are
An essential element, such as used to transform a cell
Can be anything. Alternatively, such recombinant nucleic acid is an essential element of the viral vector.
For example, it can be based on vaccinia virus. Vaccinia virus
It is used for vaccination of mammals expressing recombinant nucleic acid and protects mammals.
Induces an immune response. A "regulatory element" is a nucleic acid sequence usually derived from the untranslated region of a gene,
Enhancers, promoters, introns and 5'and 3'untranslated regions (UTR)
including. A regulatory element is a host that regulates transcription, translation or RNA stability.
Interacts with viral proteins. A "reporter molecule" is a chemical or chemical used to label nucleic acids, amino acids or antibodies.
Or biochemical part. Reporter molecules include radionuclides, enzymes, fluorescent agents,
Chemiluminescent agents, color formers, substrates, cofactors, inhibitors, magnetic particles and others in the field
There are known ingredients in. As used herein, the term “RNA equivalent” to a DNA sequence refers to a reference DNA sequence.
Consists of the same linear nucleic acid sequence, but with the nitrogenous base thymine replaced by uracil.
The backbone of the sugar chain consists of ribose rather than deoxyribose. The term "sample" is used in its broadest sense. CYSKP, CYSKP
Sample that is presumed to contain the nucleic acid
Chromosomes and subcellular organelles, membranes, cells and solutions isolated from
Genomic DNA, RNA, and cDNA that are present or immobilized on a substrate, tissues, and tissue purines
And the like. The terms "specific binding" and "specifically bind" refer to a protein or peptide
With an agonist, antibody, antagonist, small molecule, or any natural or
Refers to the interaction with a synthetic binding composition. This interaction is protein specific
The structure of (eg, an antigenic determinant or epitope) that the binding molecule recognizes
It means that it depends on whether or not it exists. For example, the antibody is an epitope
When specific for "A", it includes unbound labeled "A" and antibody.
In the reaction solution, an unlabeled “polypeptide containing epitope A or unbound” is added.
The presence of "A" reduces the amount of labeled A that binds to the antibody. The term "substantially purified" means that after being removed from its natural environment, isolated or
The isolated nucleic acid sequence or amino acid sequence, which naturally binds
At least about 60% removed, preferably about 75% or more removed,
It is also preferable that 90% or more is removed. "Substitution" is one or more amino acids or nucleotides that are different amino acids.
Or to replace with nucleotides. The term "substrate" refers to any suitable solid or semi-solid support, including film and film.
Targets, chips, slides, wafers, fibers, magnetic or non-magnetic beads, gels
, Tubes, plates, polymers, microparticles, capillaries. The substrate is a wall,
It can have various surface morphologies such as grooves, pins, channels, holes, etc.
Polynucleotide or polypeptide binds. A "transcribed image" is a specific cell type or
Refers to the pattern of collective gene expression by tissue. "Transformation" is the process by which exogenous DNA is introduced into recipient cells. form
Quality conversion can be achieved by natural or artificial conditions according to various methods known in the art.
Inserting an exogenous nucleic acid sequence into a prokaryotic or eukaryotic host cell
It can be based on any known method of The method of transformation is the host cell to be transformed.
Select by type. Transformation methods include, but are not limited to:
Infection, electroporation (electroporation), heat shock, lipofection
There is a method of using a gun and a particle gun. In "transformed" cells were introduced
Can replicate as a plasmid in which DNA replicates autonomously or as part of the host chromosome
Included are stably transformed cells that are capable. Furthermore, once in a limited time
Also included are cells that occasionally express the introduced DNA or RNA. As used herein, "genetic transformant" is any organism and is not meant to be limiting.
One or several cells of an organism, including porcine animals and plants,
For example, a heterologous nucleic acid introduced by a transformation technique well known in the art.
Have. Introduction of nucleic acids into cells directly or indirectly guides them to the cell's precursors.
Injecting, by deliberate genetic manipulation, eg by microinjection
Alternatively, it is carried out by introducing a recombinant virus. The term genetically engineered is a classic crossbred
Seed orin vitroIt does not refer to fertilization, but to the introduction of recombinant DNA molecules.
is there. Genetic transformants contemplated according to the invention include bacteria, cyanobacteria.
There are cteria, fungi and flora and fauna. The isolated DNA of the present invention is known in the art.
By a known method, such as infection, transfection, transformation or transconjugation
It can be introduced into the host. Technology for transferring the DNA of the present invention into such an organism
Are well known and are given in references such as Sambrook et al. (1989) supra.
It The "variant sequence" of a specific nucleic acid sequence is defined as "BLAST 2 Se" of the default parameter setting.
quences "tool Version 2.0.9 (May-07-1999) by blastn, some nuclei
The identity of the particular nucleic acid sequence to a length of acid sequence is determined to be at least 40%.
A defined nucleic acid sequence. Such nucleic acid pairs are
For example, at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 9
2%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
The above homology may be exhibited. One variant sequence is, for example, an "allelic" variant sequence (above
Or “splice” variant sequence, “species” variant sequence, “polymorphic” variant sequence
be able to. The splice variant may have considerable homology to the reference molecule, but the mR
Alternate splicing of exons during NA processing usually results in multiple or
It will have a small number of polynucleotides. The corresponding polypeptide is
It must have a functional domain or be missing a domain present in the reference molecule.
There is. Species variants are polynucleotide sequences that differ from one another. as a result
The resulting polypeptides typically have significant amino acid homology to each other. Polymorphism
Variants differ in the polynucleotide sequence of a particular gene between individuals of a given species
. Polymorphic variant sequences also differ by one nucleotide in the polynucleotide sequence.
It may also include "single nucleotide polymorphism" (SNP). The presence of SNPs can affect, for example, a particular population, medical condition, or
Or may exhibit a propensity for pathology. A "variant" of a particular polypeptide sequence is defined by the default parameter setting "BLAS
Blastp using the "T 2 Sequences" tool Version 2.0.9 (May-07-1999)
The identity of the particular polypeptide sequence to a length of a nucleic acid sequence is low
Both are polypeptide sequences determined to be 40%. Such a polypepti
For example, at least 50%, 60%, 70%, 80
%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98
It may exhibit%, 99% or more sequence identity. (invention) The present invention encodes a novel human cytoskeletal binding protein (CYSKP) and CYSKP
Based on the discovery of polynucleotides, abnormal cell growth using these compositions,
Autoimmune / inflammatory disorders, vesicle transport, neurological disorders, and cell motility, reproductive disorders and
To the diagnosis, treatment, and prevention of muscle disorders. Table 1 is a nomenclature for the nomenclature of full-length polynucleotide and polypeptide sequences of the invention.
Is an abbreviation. Each polynucleotide and its corresponding polypeptide consist of one Incyte
Correlates with the project identification number (Incyte project ID). Distribution of each polypeptide
Columns include polypeptide sequence identification number (Polypeptide SEQ ID NO) and Incyte Polypeptide.
Tide sequence numbers (Incyte Polypeptide ID) are indicated. Each polynucleotide
Sequences include the polynucleotide sequence identification number (Polynucleotide SEQ ID NO) and Incyt
By e-polynucleotide consensus sequence number (Incyte Polynucleotide ID)
Was displayed. Table 2 shows the BLAST analysis against the GenBank protein (genpept) database.
Showing a sequence with homology to a polypeptide of the invention as identified by
It Columns 1 and 2 are each the polypeptide for each polypeptide of the invention.
Sequence identification number (Polypeptide SEQ ID NO :) and its corresponding Incyte polype
The peptide sequence number (Incyte Polypeptide ID) is shown. Row 3 is closest to GenBank
Indicates the GenBank identification number (Genbank ID NO :) of the homologue. Row 4 is each polypepti
The probability score representing the match between the Cand and its GenBank homolog is shown. Column 5 is GwnBank
Annotations of homologues are shown, and appropriate citations are also shown where applicable. this
Are incorporated herein by reference. Table 3 shows various structural features of the polypeptides of the invention. Rows 1 and 2 are
Polypeptide sequence identification number (SEQ ID NO :) of each polypeptide of the present invention
And corresponding Incyte Polypeptide ID
Indicates. Column 3 shows the number of amino acid residues in each polypeptide. Rows 4 and 5 are
The GTI sequence analysis software package MOTIFS program (Genetics Co
phosphorylation and glycosylation as determined by mputer Group, Madison WI)
The possible sites are shown below. Column 6 is the signature array, domain
, And the amino acid residues containing the motif. Row 7 is protein structure / function
The analysis method for the analysis of the
Indicates a valid database. Tables 2 and 3 together summarize the properties of each of the polypeptides of the invention.
The polypeptide whose characteristics are claimed is a cytoskeletal binding protein.
Has established that. For example, SEQ ID NO: 31 is Basic Local Alignment Sea
Mouse Ankyrin (GenBank ID g387 as determined by rch Tool (BLAST)
A nematode protein similar to 9121) has 34% identity (see Table 2). BLAST probability
Is 1.1e-146, which is a coincidence of the observed polypeptide sequence alignment.
Shows the probability that can be obtained. SEQ ID NO: 31 also has an Ank repeat,
 This is a conserved protein family based on the Hidden Markov Model (HMM)
-Determined by searching for statistically significant matches in the domain PFAM database
It was In another example, SEQ ID NO: 34 is the Basic Local Alignment Search Tool (
Human intermediate filament binding protein (GenBank ID
1333846) has 96% identity over 97 amino acids. (See Table 2) BLAST probability score
Is 8.2e-45, which is a coincidence of the observed polypeptide sequence alignment.
Shows the probability that can be obtained. Data from BLAST using PRODOM database
The analysis further confirmed that SEQ ID NO: 34 is a cytoskeletal protein.
Is obtained. SEQ ID NO: 1-30 and SEQ ID NO: 32-33 are solved in a similar way.
Analyzed and annotated. The algorithms and parameters for the analysis of
 SEQ ID NO: 1-34 are described in Table 7. As shown in Table 4, the full-length polynucleotide sequences of the present invention are either cDNA sequences or genomic sequences.
Using the coding (exon) sequence derived from nom DNA, or using these two sequences
It was constructed by arbitrarily combining. Rows 1 and 2 are each polynuclears of the invention.
Reotide polynucleotide sequence identification number (Polynucleotide SEQ ID NO :) and
And corresponding Incyte polynucleotide consensus sequence numbers (Incyte Polynucleotide
nucleotide ID). Column 3 is the length of each polynucleotide sequence in base pairs.
Shows Column 4 identifies, for example, SEQ ID NO: 35-68, or
Hybridization to distinguish O: 35-68 from related polynucleotide sequences
1 shows a fragment of a polynucleotide sequence that is useful for an amplification or amplification technique. Row 5 is cDNA
Sequence, coding sequence (exon) and / or cDNA predicted from genomic DNA and
The identification numbers corresponding to the set of sequences having both genomic DNA are shown. These distributions
The columns were used to construct the full length polynucleotide sequences of the invention. Column 6 of Table 4
And column 7 indicate the start of the cDNA and genomic sequences corresponding to the sequence in column 5, respectively.
The nucleotide (5 ') position and the stop nucleotide (3') position are indicated. The identification number in column 5 of Table 4 is, for example, the Incyte cDNA and its corresponding cDNA live.
You can refer to Lari. For example, 3824958H1 is the identification number for the Incytec DNA sequence and is BRA
XNOT01 is the identification number of the cDNA library from which it was derived. cDNA library shows
Incyte cDNA that is not cloned is a pooled cDNA library (eg, 71263
527V1). Alternatively, the identification number in column 5 is the set of polynucleotide sequences.
In the case of GenBank cDNA used for standing, that is, the identification number of EST (eg, g2276318)
There is also. Alternatively, the identification number in column 5 is the code predicted by Genscan analysis of genomic DNA.
It can be said that it is the region of the world. Edit this Genscan deduced coding sequence before assembling the sequence.
There is a case. (See Example IV). Or the identification number in column 5 is "Exon
CDNA and Gensca combined by "exon-stitching" algorithm
We can query both sets of n predicted exons. (See Example V). Or the identification number in column 5
Issues are linked by the "exon-stretching" algorithm.
Both sets of matched cDNA and Genscan predicted exons can be queried. (See Exa
mple V.) In some cases, confirm final consensus polynucleotide sequence
The coverage of the Incyte cDNA overlaps with the coverage of the sequence as shown in column 5 for
However, the relevant Incyte cDNA identification number was not shown. Table 5 shows the full-length polynucleotide sequences constructed using the Incyte cDNA sequence.
A representative cDNA library for A representative cDNA library is
The Incyte cDNA sequence used to construct and confirm the polynucleotide sequence.
Therefore, it is the most frequently represented Incyte cDNA library. Create a cDNA library
The tissues and vectors used to make are shown in Table 5 and described in Table 6. The present invention also includes variants of CYSKP. Preferred CYSKP mutants have CYSKP function
CYSKP amino acid sequence that has at least one of the following structural or structural features
At least about 80% amino acid sequence identity to, or at least about 90%
Having at least about 95% amino acid sequence identity
It The present invention also provides a polynucleotide encoding CYSKP. Specific examples
In the present invention, the invention relates to a group of SEQ ID NO: 35-68 encoding CYSKP.
A polynucleotide sequence comprising the selected sequence is provided. SEQ ID N shown in the sequence listing
The polynucleotide sequence of O: 35-68 has the nitrogen base thymine replaced with uracil.
, An equivalent RNA array in which the sugar chain backbone is ribose instead of deoxyribose
Contains columns. The invention also includes variant sequences of the polynucleotide sequence encoding CYSKP. Details
In particular, the mutated sequence of such a polynucleotide sequence is a CYSKP-encoding
At least 70% polynucleotide sequence identity with the nucleotide sequence, or
At least 85% polynucleotide sequence identity, and even at least 95%
Has polynucleotide sequence identity. A particular embodiment of the invention is SEQ ID NO:
A nucleic acid sequence selected from the group consisting of 35-68 and at least 70% polynucleosides
Tide sequence identity, or at least 85% polynucleotide sequence identity, and
Has at least 95% polynucleotide sequence identity SEQ ID NO: 35-68
Provide a mutant sequence of a polynucleotide sequence containing a sequence selected from the group consisting of
To do. All of the above-mentioned polynucleotide mutant sequences are functional or structural of CYSKP.
An amino acid sequence can be encoded that has at least one of the characteristics. Different polynucleotids encoding CYSKP that can be created by the degeneracy of the genetic code
The nucleotide sequence includes the polynucleotide sequence of any
Those of ordinary skill in the art will understand that some have only similarities. But
Therefore, the present invention can be produced by selecting a combination based on possible codon choices.
It can cover any and all possible polynucleotide sequence variants. this
These combinations are standard applied to the native CYSKP polynucleotide sequences.
Built on the triplet genetic code, all such mutations are clearly disclosed.
Consider it to be. The nucleotide sequence encoding CYSKP and its variant sequences are generally appropriately selected.
Hybridized to the nucleotide sequence of natural CYSKP under controlled stringent conditions.
Soybean capable, but substantially different, eg, including unnatural codons
Nucleotide sequence encoding CYSKP or its derivative with codons for usage
Can be advantageous. Based on how often the host uses a particular codon,
Codons are selected to increase the expression rate of peptides that occur in a particular eukaryotic or prokaryotic host.
It is possible to choose. CYSKP and its encoded amino acid sequence unchanged.
Another reason for substantially altering the nucleotide sequence encoding the derivative of
RNA transcripts with favorable properties, such as a longer half-life, than transcripts made from sequences.
It is about making a picture. The present invention also provides DNA sequences encoding CYSKP and its derivatives or fragments thereof.
It also includes producing completely by synthetic chemistry. After making this synthetic sequence,
Using many well known reagents in the field, many of the available expression vectors and cell lines
It can be inserted in it. Furthermore, using synthetic chemistry, CYSKP or any of its
It is also possible to introduce mutations into the sequence encoding the fragment. The present invention further provides that the polyphenols described in the claims under various stringent conditions.
Hybridizes to nucleotide sequences, especially SEQ ID NOs: 35-68 and fragments thereof
Possible polynucleotide sequences are included (e.g. Wahl, G.M. and S.L. Berger (
1987) Methods Enzymol. 152: 399-407, Kimmel. A.R. (1987) Methods Enzymol.
 152: 507-511). Hybridization including annealing and washing conditions
The conditions of the code are described in "Definition". Methods of DNA sequencing are well known in the art and are described in any of the embodiments of the invention.
Can also be performed using a DNA sequencing method. DNA sequencing method
Enzymes can be used for, for example, the Klenow fragment of DNA polymerase I, SEQUEN
ASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems
), Thermostable T7 polymerase (Amersham, Pharmacia Biotech, Piscataway NJ)
Can be used. Alternatively, for example, the ELONGASE amplification system (Life Technolo
gies, Gaithersburg MD), polymerase and proofreading exo.
Nucleases can be used in combination. Preferably, the MICROLAB 2200 Liquid Transfer System
(Hamilton, Reno, NV), PTC200 thermal cycler (MJ Research, Waterto
wn MA) and ABI CATALYST 800 thermal cyclers (Applied Biosystems) etc.
The instrument is used to automate array preparation. Then ABI 373 or 377 DNA Sequence
Applied Biosystems, MEGABACE 1000 DNA Sequencing
Systems (Molecular Dynamics, Sunnyvale CA) or well known in the art
Sequencing is performed using other methods. The resulting sequence is
Analyze using various algorithms well known in the art. (Ausubel, F.
M. (1997)Short Protocols in Molecular Biology, John Wiley & Sons, New Y
ork NY, unit 7.7, Meyers, R.A. (1995)Molecular Biology and Biotechnolog y , Wiley VCH, New York NY, pp. 856-853). A partial nucleotide sequence can be generated by various methods based on PCR methods well known in the art.
To extend the nucleic acid sequence encoding CYSKP, and to
Upstream sequence such as a sequence. For example, one of the methods that can be used is
The limited-site PCR method uses universal primers and nested primers.
This is a method of amplifying an unknown sequence from genomic DNA in a rolling vector (Sarka
r, G. (1993) PCR Methods Applic 2: 318-322). Inverse PCR is another method
Yes, this is unresolved from template circularized with primers extended in a wide range of directions.
This is a method of amplifying a known sequence. Templates for known genomic loci and their surroundings
Obtained from restriction enzyme fragments consisting of sequences (Triglia, T. et al. (1988) Nucleic Acids Res
 16: 8186 etc.). The third method is the capture PCR method, which is used in humans and humans.
And a method for PCR amplification of a DNA fragment flanking a known sequence of yeast artificial chromosome DNA.
(See Lagerstrom, M. et al. (1991) PCR Methods Applic 1: 111-119, etc.
). This method uses multiple restriction enzyme digests and ligations before performing PCR.
It is possible to insert a recombinant double-stranded sequence within an unknown sequence region. Also unknown
Are known in the art for other methods that can be used to search for sequences in
rker, J.D. et al. (1991) Nucleic Acids Res. 19: 3055-3060 etc.). Furthermore, PCR
, Nested primers and PromoterFinder ™ libraries (Clontech, Pa
lo Alto CA) can be used to walk genomic DNA. This procedure
, Without the need to screen the library, for intron / exon junctions
Useful for finding. For all PCR-based methods, commercially available software
Software such as OLIGO 4.06 primer analysis software (National Bioscie
nces, Plymouth MN) or another suitable program, with a length of about 22 to 3
0 nucleotides, GC content of about 50% or more, temperature of about 68 ℃ ~ 72 ℃ to the template
Primers can be designed to anneal by annealing. Size-selected to include larger cDNAs when screening full-length cDNAs
It is preferable to use the prepared library. In addition, a random primer library
Often contains sequences with the 5'regions of genes, and oligo d (T) libraries
It is suitable for situations where full-length cDNA cannot be produced. Genome library is 5 '
It may be useful for extension of sequences into nontranscribed regulatory regions. Use a commercially available capillary electrophoresis system to perform sequencing or PCR production.
The size of the object can be analyzed or its nucleotide sequence can be confirmed. Ingredient
Physically, capillary sequencing is a flow-through for electrophoretic separations.
Active with lasers, specific for 4 different nucleotides with reactive polymers
Fluorescent dye, and a CCD camera used to detect the emitted wavelength
. Output / light intensity is measured by appropriate software (GENOTYPER from Applied Biosystems).
, SEQUENCE NAVIGATOR, etc.) can be used to convert the electric signal. Load sample
Computer control of the entire process from computer analysis to electronic data display
It is possible. Capillary electrophoresis is only present in small amounts in certain samples
It is particularly suitable for sequencing such small DNA fragments. In another embodiment of the invention, the polynucleotide sequence encoding CYSKP or a sequence thereof.
The fragment is cloned into a recombinant DNA molecule and CYSKP
It is possible to express a piece or a functional equivalent. Due to the degeneracy inherent in the genetic code
, Another DNA sequence that encodes a substantially identical or functionally equivalent amino acid sequence.
And these sequences are available for expression of CYSKP. It is commonly known in the art to alter the sequence encoding CYSKP for various purposes.
The methods described above can be used to recombine the nucleotide sequences of the invention. This
For these purposes, cloning, processing and / or expression of gene products
Includes but is not limited to regulation. Gene fragment and synthetic origin
D by random fragmentation of gonucleotides and PCR reassembly
NA shuffling can be used to recombine nucleotide sequences. An example
For example, using oligonucleotide-mediated site-directed mutagenesis, novel restriction
Site generation, glycosylation pattern changes, codon preference changes, splice mutations
Mutations may be introduced that produce the body, etc. The nucleotides of the present invention were labeled with MOLECULAR BREEDING (Maxygen Inc., Santa Clara CA;
 US Patent No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17: 793-7.
97; Christians, F.C. et al. (1999) Nat. Biotechnol. 17: 259-264; Crameri, A.
Et al. (1996) Nat. Biotechnol. 14: 315-319).
Shuffling to cause biological or enzymatic activity of CYSKP, or other molecules or chemicals.
It is possible to modify or improve the biological properties of CYSKP, such as its ability to bind compounds.
Wear. DNA shuffling involves gene modification using PCR-mediated recombination of gene fragments.
This is the process of creating a foreign library. The library is then the gene
The mutants are subjected to selection or screening to identify the desired property. next
These preferred mutants are pooled and further iterated for DNA shuffling and selection /
Screening may be performed. Therefore, for artificial breeding and rapid molecular evolution.
Therefore, various genes are created. For example, a single with a random point mutation
Recombine and screen a single gene fragment, then optimize for desired properties
You can shuffle until you are done. Alternatively, a given gene may
Recombine with a homologous gene of the same gene family obtained from either of different species,
Directed control of the genetic diversity of multiple naturally occurring genes
Can be maximized by law. According to another example, the sequence encoding CYSKP may be prepared by chemical methods well known in the art.
Can be used in whole or in part (eg, Caruthers. M.H. et al. (1980
) Nucl. Acids Res. Symp. Ser 7: 215-223; and Horn, T. et al. (1980) Nucl. Acid
s Res. Symp. Ser. 225-232). Alternatively, chemical methods can be used to
It is possible to synthesize the body or fragments thereof. For example, various liquid or solid phases
Technology can be used to perform peptide synthesis (Creighton, T. (1984)Protein s, Structures and Molecular Properties , WH Freeman, New York NY, 55-60
Page, Roberge, J.Y. et al. (1995) Science 269: 202-204, etc.). Automatic synthesis
Can be achieved using the ABI 431A Peptide Synthesizer (Perkin Elmer). Further CY
The amino acid sequence of SKP, or any portion thereof, may be altered during direct synthesis, and / or
Or with sequences from other proteins or any part thereof using chemical methods
A polypeptide or variant having a native polypeptide sequence, depending on the combination
It is possible to make a polypeptide. Peptides can be substantially purified using preparative high performance liquid chromatography
(Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182: 392-421, etc.
reference). The composition of synthetic peptides can be determined by amino acid analysis or sequencing.
Can be confirmed (see Creighton, pages 28-53, etc., above). In order to express biologically active CYSKP, the nucleotide sequence encoding CYSKP is
The row or derivative thereof is inserted into a suitable expression vector. This expression vector
Elements required for regulation of transcription and translation of coding sequences inserted into a suitable host.
Incl. These elements include the vector and the polynucleotide encoding CYSKP.
Enhancers in constitutive sequences, constitutive and expression-inducible promoters,
Regulatory sequences such as 5'and 3'untranslated regions are included. Such elements are
And various peculiarities. Sequence encoding CYSKP with specific initiation signal
It is possible to achieve a more effective translation of. AT for such signals
It includes the G start codon and nearby sequences such as the Kozak sequence. Code CYSKP
Sequence and its initiation codon, upstream regulatory sequences were inserted into a suitable expression vector.
In some cases, additional transcriptional or translational control signals may not be needed.
However, if only the coding sequence or a fragment thereof is inserted, the
Exogenous translational control signals, including the in-frame ATG initiation codon, are included in the expression vector.
You should be rare. Exogenous translational elements and initiation codons are
And of synthetic origin. The efficiency of expression is suitable for the particular host cell line used.
It can be enhanced by the inclusion of various enhancers (Scharf, D. et al. (1
994) Results Probl. Cell Differ. 20: 125-162. The sequence encoding CYSKP, the appropriate transcriptional and translational sequences are determined using methods well known to those skilled in the art.
It is possible to make expression vectors that include node elements. This way, in vitro Recombinant DNA technology, synthetic technology andin vivoThere is gene recombination technology (Sambro
ok, J. et al. (1989)Molecular Cloning. A Laboratory Manual, Cold Spring Har
bor Press, Plainview NY, chapters 4, 8, 16-17, Ausubel, F.M. et al. (1995).Current Protocols in Molecular Biology , John Wiley & Sons, New York NY 9, 13,
(See Chapter 16). A variety of expression vector / host systems may be utilized to retain and express sequences encoding CYSKP.
The reality is possible. Such expression vectors / host systems include, but are not limited to
, Recombinant bacteriophage, plasmid or cosmid DNA expression vector
Transformed bacteria, yeast transformed with yeast expression vector, virus
Insect cell lines infected with expression vectors (eg baculovirus) or viral
The current vector (eg Cauliflower mosaic virus CaMV or tobacco mosaic
Illus TMV) or bacterial expression vector (eg Ti or pBR322 plasmid)
There are microorganisms such as plant cell lines or animal cell lines that have undergone quality conversion (see Sa above).
mbrook, Ausubel, supra, Van Heeke, G. and S.M.Schuster (1989) J. Biol.
Chem. 264: 5503-5509, Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci.
USA 91: 3224-3227, Sandig, V. et al. (1996) Hum. Gene Ther. 7: 1937-1945, Taka
matsu, N. (1987) EMBOJ. 6: 307-311, Coruzzi, G. et al. (1984) EMBOJ. 3: 1671-1
680, Broglie, R. et al. (1984) Science 224: 838-843, Winter, J. et al. (1991) Res.
ults Probl. Cell Differ. 17: 85-105, The McGraw-Hill Science and Technology Yearbook (The McG
raw Hill Yearbook of Science and Technology) (1992) McGraw Hill New York
 NY, pp. 191-196, Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sc
i. USA 81: 3655-3659, Harrington, J.J. et al. (1997) Nat. Genet. 15: 345-355 etc.
See). Retrovirus, adenovirus, herpes virus or vaccinia
Avirus-derived expression vector or expression vector derived from various bacterial plasmids
To deliver a polynucleotide sequence to a target organ, tissue or cell population
(Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5 (6): 350-356, Yu.
, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90 (13): 6340-6344, Buller, R.M.
 Et al. (1985) Nature 317 (6040): 813-815; McGregor, D.P. et al. (1994) Mol. Immun.
ol. 31 (3): 219-226, Verma, I.M. and N. Somia (1997) Nature 389: 239-242.
Etc.). The present invention is not limited by the host cell used.  In bacterial systems, numerous cloning and expression vectors encode CYSKP.
Can be selected depending on the intended use of the polynucleotide sequence. For example, CYSKP
Routine cloning and subcloning of polynucleotide sequences encoding
, PBLUESCRIPT (Stratagene, La Jolla CA) or pSPORT1 plasmid for propagation
(GIBCO BRL)E. coliVectors can be used. Vector
Ligation of the CYSKP-encoding sequence at multiple cloning sites resulted in lac
Colorimetric for identification of transformed bacteria containing a recombinant gene in which the Z gene has been disrupted
A screening method becomes possible. In addition, these vectors were cloned
In the arrayin vitroTranscription, dideoxy sequencing, helper phage
May also be useful in single-stranded rescue by and the creation of nested deletions (Van Heeke, G
And S.M. Schuster (1989) J. Biol. Chem. 264: 5503-5509.
For example, when a large amount of CYSKP is required for antibody production, expression of CYSKP should be suppressed.
Vectors that induce at high levels can be used. For example, the strong induction T5 bacterio
Vectors containing a phage promoter or an inducible T7 bacteriophage promoter
Can be used. Yeast expression systems can be used to generate CYSKP. alpha factor, alcohol oxida
A large number of bases including constitutive or inducible promoters such as
The yeast Saccharomyces cerevisiae orPichia pastorisCan be used for
Noh. Furthermore, such a vector may secrete or express the expressed protein.
In the host genome of the foreign sequence for stable growth.
(Ausubel (1995), Bitter, G.A. et al. (1987) Metho
ds Enzymol. 153: 516544; and Scorer, C.A. et al. (1994) Bio / Technology 12: 181.
-184). Plant systems can also be used to express CYSKP. Transcription of the CYSKP coding sequence is
Ils promoter, eg, alone or TMV (Takamatsu, N. (1987) EMBO J 6: 3
07-311) derived from CaMV as used in combination with the derived omega leader sequence
Promoted by the 35S and 19S promoters. Or, a small sub unit of RUBISCO
A plant promoter such as G. or heat shock promoter may be used (see above).
See Coruzzi, Broglie, and Winter, etc.). These constructs are directly DN
Can be introduced into plant cells by A-transformation or pathogen-mediated transfection
Is. ("Maguro Hill Science and Technology Yearbook" (The McGraw Hill Yearbook of Scien ce and Technology ) (1992) McGraw Hill New York NY, pp. 191-196
. ) In mammalian cells, a number of virus-based expression systems are available. Adeno
When the virus is used as an expression vector, a late promoter and triplex promoter
Sequence encoding CYSKP in the adenovirus transcript / translation complex consisting of a double sequence
Can be combined. By insertion into the nonessential E1 or E3 region of the viral genome
, It is possible to obtain a live virus expressing CYSKP in infected host cells (Lo
gan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81: 3655-3659).
. In addition, a transcription enhancer such as the Rous sarcoma virus (RSV) enhancer is used.
Can increase expression in mammalian host cells. Based on SV40 or EBV
The vectors described above can also be used to express proteins at high levels. Expressed from and contained in a plasmid using a human artificial chromosome (HAC)
It can also transport larger fragments of DNA than those that do. About 6kb for treatment
We prepared 10 Mb HACs and used conventional transport methods (liposome, polycation aminopoly).
Or vesicles). (For example, Harrington. J.J. et al. (1997) Na
t Genet. 15: 345-355.). For long-term production of recombinant proteins in mammalian systems, cell lines should be used.
Stable expression of CYSKP is desirable. For example, use an expression vector to encode PP.
It is possible to transform the cell line into a cell line. Such expression
Is a replication origin of viral origin and / or an endogenous expression element, the same or different
Includes a selectable marker gene above the vector. After transfer of vector, transfer to selective medium
The cells can be grown in enriched medium for about 1-2 days before. Selectable marker
Purpose is to confer resistance to selective medium, and there is a selectable marker
Allows growth and recovery of cells that successfully express the introduced sequences
Becomes The resistant clones of stably transformed cells are the appropriate set for the cell type.
Can be propagated using woven culture technology. Transformed cell lines can be recovered using any number of selection systems. The selection system is
Without limitation, herpes simplex virus thymidine kinase gene and adde.
Contains nin phosphoribosyl transferase gene, each tk⁻Or ap
r⁻Used in cells. (For example, Wigler, M. et al. (1977) Cell 11: 223-232.
And Lowy, I. et al. (1980) Cell 22: 817-823). Also, as a basis for selection
Resistance to anxiety antagonists, antibiotics or herbicides can be used. Eg dhfr
Confers resistance to methotrexate and neo is aminoglycoside neomycosis.
And G-418 resistance, and als or pat is chlorsulfon (chlorsulfon).
uron), phosphinotricin acetyltransferase (phosphinotricin ac
etyltransferase) (for example, Wigler, M. et al. (1980) Proc
Natl. Acad. Sci. 77: 3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Bi
ol. 150: 1-14). (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 7
7: 35673570; Colbere Garapin, F. et al. (1981) J. Mol. Biol. 150: 114.
. ) Other selectable genes, e.g., t that alter the cellular requirements for metabolism.
rpB and hisD are described in the literature (Hartman, S.C. and R.C. Mulligan (
1988) Proc. Natl. Acad. Sci. USA 85: 8047-8051). Visible marker,
For example, anthocyanin, green fluorescent protein (GFP; Clontech), β-glucuroni
Or its substrate β-glucuronide, or luciferase and its substrate lucif
Wellin or the like may be used. Use these markers to identify transformants
Not only the transient or stable protein expression due to the specific vector system
It is also possible to quantify (Rhodes, C.A. (1995) Methods Mol. Biol. 55: 121-
See 131 etc.). The presence / absence of marker gene expression suggests the presence of the gene of interest
In some cases, it is necessary to confirm the presence and expression of the gene. For example, code CYSKP
If the sequence to be inserted is inserted into the marker gene sequence, the sequence encoding CYSKP
Transformed cells containing rows are identifiable by the lack of marker gene function.
It Alternatively, the marker gene encodes CYSKP under the control of one promoter.
It is also possible to arrange them in a line with the array. Markers in response to induction or selection
Expression of the gene usually also indicates expression of the tandem gene. Generally, a host cell that contains a nucleic acid sequence encoding CYSKP and expresses CYSKP is
It can be identified using various methods well known to those skilled in the art. To these methods
Means DNA-DNA or DNA-RNA hybridization, PCR method, nucleic acid or tag
Membrane system, solution-based, or chip-based for protein detection and / or quantification
Includes protein biological tests or immunological assays that include
However, it is not limited thereto. CYSK with either specific polyclonal or monoclonal antibodies
Immunological methods for detecting and measuring P 2 expression are well known in the art. This
Such techniques include enzyme-linked immunosorbent assay (ELISA), radioimmuno
Assay (RIA), flow cytometer (FACS), etc. Two on PKIN
Two-site monochrome using a monoclonal antibody that reacts to non-interfering epitopes
Null-based immunoassay (two-site, monoclonal-based immunoassay)
Of course, competitive binding assays can also be used. These assays and
Other assays are known in the art (Hampton. R. et al. (1990).Serologica l Methods, a Laboratory Manual.  APS Press. St Paul. MN, Sect. IV, Coliga
n, J. E. et al. (1997)Current Protocols in Immunology, Greene Pub. Associat
es and Wiley-Interscience, New York NY, Pound, J.D. (1998)Immunochemica l Protocols , Humans Press, Totowa NJ, etc.). A wide variety of labeling and conjugation methods are known to those of skill in the art and include various nucleic acid assays.
These methods can be used for assay and amino acid assays. The code that encodes CYSKP
Labeled hybridisation for detecting sequences related to the nucleotides
The method for generating an application probe or a PCR probe includes oligo labeling and nicking.
PCR with translation, end-labeling, or labeled nucleotides
Amplification is included. Alternatively, the sequence encoding CYSKP, or any fragment thereof.
Can also be cloned into a vector to generate mRNA probes
. Such vectors are known in the art, are commercially available, and are known as T7
, A suitable RNA polymerase such as T3 or SP6 and labeled nucleotides
,in vitroCan be used to synthesize RNA probes. Such a method is an example
For example, Amersham Pharmacia Biotech, Promega (Madison WI), U.S. Biochemical
It can be carried out using various kits commercially available from J.K. Easy to detect
Suitable reporter molecules or labels that can be used to
Inhibitors, magnetic particles, radionuclides, enzymes, fluorescent agents, chemiluminescent agents, coloring
There are agents, etc. Host cells transformed with a nucleotide sequence encoding CYSKP are
Cultured under conditions suitable for the expression and recovery of this protein. Transformed cell
Whether the protein produced from it is secreted or remains intracellular depends on the sequence used,
Depends on the vector, or both. A polynucleotide encoding CYSKP
An expression vector containing induces CYSKP secretion across prokaryotic and eukaryotic cell membranes
It will be appreciated by those skilled in the art that it can be designed to include a signal sequence that In addition, the choice of host cell line may be dependent on the ability or expression of the inserted sequences to regulate expression.
This can be done by the ability to process the protein into the desired form. Not limited
However, modifications of such polypeptides include acetylation, carboxylation and glycosylation.
There are phosphorylation, phosphorylation, lipidation and acylation. Protein "pre-pro" or
Post-translational processing that cleaves the "pro" form is used to target the target protein, folding
It is possible to specify folding and / or activity. A solid for post-translational activity
A variety of host cells (eg CHO, HeLa, M
DCK, MEK293, WI38, etc.) are American Type Culture Collection (ATCC, Bethesd
a, VA) to ensure correct modification and processing of foreign proteins
Can be selected. In another embodiment of the invention, the natural or modified or group encoding CYSKP is
A heterologous sequence which results in the translation of the fusion protein of any of the host systems described above with alternative nucleic acid sequences.
Bind to. For example, a chimeric CY containing a heterologous moiety that can be recognized by a commercially available antibody.
The SKP protein is a peptide library switch for inhibitors of CYSKP activity.
Facilitates cleaning. Also, heterologous protein moieties and heterologous peptide moieties
Facilitates purification of the fusion protein using commercially available affinity substrates. Limited
This part, though not, contains glutathione S transferase (GST)
, Maltose binding protein (MBP), thioredoxin (Trx), calmodulin
There are binding peptides (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST
On immobilized glutathione, MBP on maltose and Trx on phenylarsineoxy.
, CBP on calmodulin, and 6-His on metal chelate resins.
Enables the purification of the fusion protein of FLAG, c-myc and hemagglutinin (HA)
, A commercially available monoclonal antibody that specifically recognizes these epitope tags.
Allows immunoaffinity purification of fusion proteins using somatic and polyclonal antibodies
To do. In addition, the tamper between the CYSKP coding sequence and the heterologous protein sequence
When CYSKP is engineered so that the fusion protein contains a cleavage site.
It can be cut from dissimilar parts after manufacture. The method of expression and purification of the fusion protein is described in
Ausubel (1995), chapter 10 Uses various commercially available kits
It can also facilitate the expression and purification of the fusion protein. In another embodiment of the invention, TNT rabbit reticulocyte lysate or wheat germ extract
System (Promega)in vitroIt is possible to synthesize CYSKP radiolabeled with. This
These systems are based on protein coding that is functionally linked to the T7, T3 or SP6 promoters.
Couple transcription and translation of the sequence. Translation is for example³⁵Like S-methionine
Occurs in the presence of various radiolabeled amino acid precursors. Using CYSKP of the present invention or a fragment thereof, a compound that specifically binds to CYSKP is screened.
Can be trained. With at least one or more test compounds,
It is possible to screen for specific binding to CYSKP. Of test compound
Examples are antibodies, oligonucleotides, proteins (eg receptors) or small molecules
Is mentioned. In one embodiment, the compound thus identified may be, for example, a ligand or fragment thereof.
CYSKP natural ligand, or natural substrate, structural or functional mimetic
Or closely related to natural binding partners (Coligan, J.E. et al. (1991)Curr ent Protocols in Immunology  (See Chapter 5 of 1 (2)). Similarly, the compound is CYSKP
 The natural receptor to which is bound, or at least the receptor, eg, the ligand binding site
It may be closely related to certain fragments of the body. In each case, this compound is made using known techniques.
Things can be designed reasonably. In one embodiment, for such compounds
For screening, either secreted protein or protein on cell membrane
The production of suitable cells expressing CYSKP is included. Suitable cells include mammalian
Cells from yeast, E. coli. Cells expressing CYSKP or CYSKP
The containing cell membrane fragment is contacted with a test compound to remove either CYSKP or the compound.
Binding, stimulation or inhibition of activity is analyzed. One assay simply binds the test compound experimentally to the polypeptide and
Detected with fluorescent dyes, radioisotopes, enzyme conjugates or other detectable labels
can do. For example, this assay comprises at least one test compound
Combining with CYSKP in solution or immobilized on a solid support;
And detecting the binding of the compound to the compound. Alternatively, labeled competition
Detection and measurement of binding of the test compound in the presence of the compound can be performed. More
Assays for cell-free reconstituted specimens, chemical libraries or natural product mixtures
The test compound can be released in solution or on a solid support.
Fix it. Using the CYSKP of the present invention or a fragment thereof, a compound that regulates the activity of CYSKP is screened.
It is possible to lean. Such compounds include agonists,
This includes gonists, partial or inverse agonists and the like. In one embodiment, CY
Conditions under which CYSKP activity is permissible, in which SKP binds to at least one test compound
The assay was performed under the following conditions and the activity of CYSKP in the presence of the test compound was
Compare with the activity of CYSKP below. Changes in CYSKP activity in the presence of test compounds
, Suggests the presence of compounds that regulate the activity of CYSKP. Alternatively, the test compound is CY
Contains CYSKP under conditions suitable for SKP activityin vitroOr combined with cell-free reconstitution system
And perform the assay. CYSKP activity in either of these assays
The test compound to be prepared can be bound indirectly and directly contacted with the test compound.
No need to touch. Screen at least one to multiple test compounds
be able to. In another example, an animal model using homologous recombination in embryonic stem cells (ES cells)
Within the system, the polynucleotide encoding CYSKP or its mammalian homologue will
"Cook out". Such techniques are well known in the art and include human disease
Useful for making animal models (see US Pat. Nos. 5,175,383 and 5,767,337)
reference). For example, mouse ES cells such as the 129 / SvJ cell line are derived from early mouse embryos and
Can be grown on the ground. These ES cells are neomycin phosphotransfected
Marases such as the lacse gene (neo: Capecchi, M.R. (1989) Science 244: 1288-1292)
Transform with a vector containing the gene of interest disrupted with the Kerr gene. This vector
-Is integrated into the corresponding region of the host genome by homologous recombination. Alternatively,
Homologous recombination is performed using the Cre-loxP system to target tissue-specific or developmental stage-specific
Knocking out a gene (Marth, J.D. (1996) Clin. Invest. 97: 1999-2002;
 Wagner, K.U. et al. (1997) Nucleic Acids Res. 25: 4323-43 30). Transformed
ES cells were identified and subdivided into mouse cell blastocysts collected from, for example, the C57BL / 6 mouse line.
Inject a quantity. Inheritance of chimeric offspring obtained by surgically introducing blastocysts into pseudopregnant females
The traits are determined and crossed to produce a heterozygous or homozygous system.
The genetically modified animals produced in this way are treated with potential therapeutic and toxic agents.
Can be inspected. A polynucleotide encoding CYSKPin vitroFor human blastocyst-derived ES cells
Can be operated. Human ES cells are derived from endoderm, mesoderm and ectoderm cells.
Have the potential to differentiate into at least eight separate cell lineages, including cell types. This
These cell lineages differentiate into, for example, neuronal cells, hematopoietic lineages and cardiomyocytes (Thomso
n, J.A. et al. (1998) Science 282: 1145-1 147). Using a polynucleotide that encodes CYSKP, the
Quinn "humanized animals (pigs) or transgenic animals (mouse or rat)
It is possible to make. Using knock-in technology, poly encoding CYSKP
A region of nucleotides was injected into animal ES cells and the injected sequence
To be incorporated into. The transformed cells are injected into the blastula and the blastula is transplanted as described above.
Study genetically modified offspring or inbred lines and treat with potential drug.
And obtain information on the treatment of human diseases. Alternatively, for example, CYSKP in milk
Mammalian inbred strains that overexpress CYSKP, such as those secreted by Escherichia coli, are a convenient source of protein.
(Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4: 55-74).

[Treatment]

Between the region of CYSKP and the region of cytoskeletal binding protein, for example, the sequence and motif
There are chemical and structural similarities in the content of fu. Furthermore, the expression of CYSKP
Lung, reproductive (including placenta), nerve (including brain), adrenal, endothelium, kidney and spleen tissue
And closely related to ovarian, breast and testicular tumor tissue. Therefore, CYSKP is
Abnormal vesicle proliferation, autoimmune / inflammatory diseases, vesicle transport, neurological disorders, cell motility, reproductive and
And is believed to play a role in muscle disease. Expression or activity of CYSKP
In treating diseases associated with increased sex, decrease CYSKP expression or activity.
Is desirable. In addition, treatment of diseases associated with decreased expression or activity of CYSKP
In, it is desirable to increase the expression or activity of CYSKP. Therefore, in one embodiment, treatment of diseases associated with decreased expression or activity of CYSKP is treated.
Patients may be given CYSKP or a fragment or derivative thereof for treatment or prevention.
It is possible. Such disorders include, but are not limited to, immune system disorders, neurological disorders.
Cell proliferation, including disorders, developmental or developmental disorders, and cell proliferative disorders, including cancer
Abnormalities include actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, liver
Inflammation, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, true
Polycythemia, psoriasis, primary thrombocythemia, and adenocarcinoma and leukemia, lymphoma, melanoma
, Myeloma, sarcoma, and teratocarcinoma, specifically adrenal gland, bladder, bone, bone marrow, brain, breast,
Cervix, gallbladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, epidermis
Cancer of the thyroid gland, penis, prostate, salivary gland, skin, spleen, testis, thymus, thyroid, uterus,
Autoimmune / inflammatory diseases include acquired immune deficiency syndrome (AIDS), Addison's disease, and adult calling.
Cramping syndrome, allergy, ankylosing spondylitis, amyloidosis, anemia, asthma, ate
Loam arteriosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune
Polyglandular endocrine candid ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact
Dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, lymph
Cytotoxic transient lymphopenia, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulus
Somatic nephritis, Goodpasture syndrome, gout, Graves' disease, Hashimoto thyroiditis, hypereosic acid
Polycythemia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardium or pericarditis
, Osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, chronic
Rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis, whole body
Lupus erythematosus, systemic sclerosis, thrombocytopenia, ulcerative colitis, Werner
Syndrome, cancer complications, hemodialysis, extracorporeal circulation, viral infection, bacterial infection, fungal sensation
Staining, parasitic infections, protozoal infections, helminthic infections, trauma, including impaired vesicle transport
, Cystic fibrosis, glucose galactose malabsorption syndrome, high cholesterol
Blood sugar, diabetes mellitus, diabetes insipidus, hyperglycemia, hypoglycemia, Graves' disease, goiter, black
Gastrointestinal, including Singing's disease, Addison's disease, ulcerative colitis, gastroduodenal ulcer, etc.
Other conditions associated with vascular disorders and abnormal vesicular transport include acquired immunodeficiency syndrome (AIDS),
Hay fever, asthma and urticaria (rash), autoimmune hemolytic anemia, proliferative glomerulonephritis, inflammation
Enteric disease, multiple sclerosis, myasthenia gravis, rheumatoid, osteoarthritis, scleroderma
, Chediak-East syndrome, Sjogren's syndrome, systemic lupus erythematosus,
Toxic shock syndrome, traumatic tissue injury, and viral and bacterial infections
Diseases, fungal infections, parasitic infections, protozoal infections, neurological disorders include epilepsy, ischemia
Cerebrovascular disease, stroke, cerebral neoplasm, Alzheimer's disease, Pick's disease, hunting
Disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and
Other motor neuron disorders, progressive neuromuscular atrophy, retinitis pigmentosa, hereditary motor
Ataxia, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess,
Subdural empyema, epidural abscess, purulent intracranial thrombophlebitis, myelitis and radiculitis,
Viral central nervous system diseases, prion diseases (Kuru, Creutzfeld-Jakob
Disease, including Gerstmann-Straussler-Scheinker syndrome), fatal familial insomnia
, Neurotrophic and metabolic diseases, neurofibromatosis, tuberous sclerosis, cerebellar retinal hemangioblastoma (
cerebelloretinal hemangioblastomatosis), trigeminal neurovascular syndrome, Down syndrome
CNS mental retardation and other developmental disorders, including symptoms, cerebral palsy, neuroskeletal abnormalities
, Autonomic nervous system disorders, cranial nerve disorders, spinal cord disorders, muscular dystrophy and other deities
Transmuscular disorders, peripheral nerve diseases, dermatomyositis and polymyositis, hereditary, metabolic, endocrine
, And addictive muscle disease, myasthenia gravis, periodic quadriplegia, psychosis (moodness, anxiety
Sexual disorder, schizophrenic disease), seasonal disorder (SAD), akathisia, amnesia, catatonic disease,
Diabetic neuropathy, extrapyramidal terminal defect syndrome, dystonia, schizophrenia
Divinity, postherpetic neuralgia, and Tourette's disease, progressive supranuclear palsy, cerebral cortex
Basal ganglia degeneration and familial frontotemporal amnesia include
, Ankylosing spondylitis, Chediac East syndrome, Duchenne and Becker muscles
Dystrophy, intrahepatic cholestasis, myocardial hyperplasia (thickening), cardiomyopathy, premature periodontitis
Included cancers such as adenocarcinoma, ovarian cancer and chronic myelogenous leukemia, and bacterial infections
Parasitic infection and reproductive disorders include prolactin production disorder, infertility, fallopian tube disease, and ovulation deficiency.
Deficiency, endometriosis, sexual cycle disorder, menstrual cycle disorder, polycystic ovary syndrome, ovarian hyperpuncture
Acute syndrome, endometrial cancer, ovarian cancer, uterine fibroids, autoimmune disease, ectopic pregnancy, teratology
, Breast cancer, fibrocystic breast disease, milk leakage, spermatogenesis disruption, abnormal sperm physiology, testicular cancer
, Prostate cancer, benign prostatic hypertrophy, prostatitis, Pyronie's disease, incompatibility, male breast cancer,
Gynecomastia, high gonadotropin hypogonadism, hypogonadotropic hypogonadism
, Pseudohermaphroditism, azoospermia, premature ovarian insufficiency, acrosin deficiency, delayed puberty
, Retrograde ejaculation, a ejaculation, and hemangioblastoma, cystsphaeo-chro
mocytomas), paraganglioma, epididymal cystadenoma, and endolymphatic sac tumor, as well as muscle disease.
Patients include myocarditis, Duchenne muscular dystrophy, Becker muscular dystrophy
, Myotonic dystrophy, core disease, nemarin myopathy, central pontine myelination
Necrosis, lipid muscle disease, mitochondrial myopathy, infectious myositis, polymyositis, skin
Myositis, inclusion body myositis, thyroid poisoning myositis and ethanol myopathy, angina,
Naphylaxis shock, arrhythmia, asthma, cardiovascular shock, Cushing's disease
Symptom group, hypertension, hypoglycemia, myocardial infarction, migraine, and pheochromocytoma (brown cell
Cystoma), and cardiomyopathy, brain disease, epilepsy, Kerns-Sayer syndrome, lactate acid
Illness, muscle clonus disease, and ophthalmoplegia. In another example, expression of CYSKP, including but not limited to the diseases listed above.
Or for the treatment or prevention of diseases associated with decreased activity, CYSKP or its
It is also possible to administer a vector capable of expressing a fragment or derivative to a patient. In yet another embodiment, CYSKPs including, but not limited to, the diseases listed above.
Substantially purified for the treatment or prevention of diseases associated with decreased expression or activity
It is also possible to administer the composition containing CYSKP to a patient together with a suitable pharmaceutical carrier.
Is. In yet another embodiment, CYSKPs including, but not limited to, the diseases listed above.
CYSKP activity for the treatment or prevention of disorders associated with decreased expression or activity
It is also possible to administer to the patient an agonist which regulates. In a further embodiment, treatment of a disease associated with increased CYSKP expression or activity or
For prophylaxis, the patient may be administered an antagonist of CYSKP. Limit
Although it is not specified, examples of such diseases include the above-mentioned abnormal cell proliferation and autologous diseases.
Immune / inflammatory disorders, vesicle transport, neurological disorders, cell motility disorders, reproductive disorders and muscle disorders
Is included. In one embodiment, the antibody that specifically binds CYSKP is directly antagonized.
Targeting drug delivery to cells or tissues expressing CYSKP
It can be used indirectly as a ring or transport mechanism. In another example, expression of CYSKP, including but not limited to the diseases listed above.
Alternatively, encode CYSKP for the treatment or prevention of diseases associated with increased activity.
It is also possible to administer a vector expressing a complementary sequence of a polynucleotide
Is. In another embodiment, any of the proteins, antagonists, antibodies, Agonis of the invention
Or a complementary sequence or vector in combination with another suitable therapeutic agent.
I can do it. Suitable therapeutic agents for use in combination therapy are well known to those of ordinary skill in the art according to conventional pharmaceutical principles.
Can be selected. Treatment of various diseases mentioned above in combination with therapeutic agents
Or it may have a synergistic effect on prevention. By using this method small amount of each drug
It is possible to increase the medicinal effect of the drug, thereby reducing the possibility of side effects.
obtain. CYSKP antagonists can be prepared using methods common in the art
Is. For details, use purified PP to make antibodies, and use therapeutic drug libraries.
It is possible to screen the cells to identify those that specifically bind to CYSKP. CY
Antibodies for SKP can also be produced using methods common in the art. this
Such antibodies include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single
Chains, Fab fragments, and fragments produced by Fab expression libraries
included. However, it is not limited to these. Neutralizing antibody (ie dimeric
Antibodies that inhibit formation) are usually suitable for therapy. For the production of antibodies goat, rabbit, rat, mouse, human and others
Various hosts including CYSKP or any fragment, or with immunogenic properties
It can be immunized by injection of the oligopeptide. Depending on the host species, various
Adjuvants can also be used to enhance the immune response. But not limited to
Such adjuvants include Freund's adjuvant and aluminum hydroxide.
Mineral gel adjuvant such as lysolecithin, pluronic polyol, poly
Anion, peptide, oil emulsion, keyhole limpet hemocyanin, dinitro
There are surfactants such as phenol. Among the adjuvants used in humans,
BCG (Cormette-Guerin) and Corynebacterium parvum are particularly preferred
Good Oligopeptides, peptides, or peptides used to induce antibodies to CYSKP
Or fragments consist of at least about 5 amino acids, typically about 10 or more amino acids.
Those consisting of amino acids are preferred. These oligopeptides, peptides or peptides
The fragment is identical to a portion of the amino acid sequence of the native protein and contains small
It is desirable to include the entire amino acid sequence of the offspring. A short stretch of CYSKP amino acids
, KLH and other protein sequences are fused to produce antibodies against the chimeric molecule.
Can be Monoclonal antibodies to CYSKP are produced by continuous cell lines in culture.
It can be made using any technique that produces a molecule. Limited
However, such techniques include hybridoma technology, human B cell hybridoma
Technology and EBV-hybridoma technology (Kohler, G. et al. (1975) Nature 256: 4
95-497, Kozbor, D. et al. (1985) .J. Immunol. Methods 81: 31-42, Cote, R.J.
(1983) Proc. Natl. Acad. Sci. USA 80: 2026-2030, Cole, S.P. et al. (1984).
Mol. Cell Biol. 62: 109-120 etc.). In addition, the human antibody gene developed for the production of "chimeric antibody" is replaced by the mouse antibody gene.
Techniques, such as splicing, have good antigen specificity and biological activity.
(Morrison, S.L. et al. (1984) Proc. Nat.
l. Acad. Sci. 81-4851-4855; Neuberger, M.S. et al. (1984) Nature 312: 604-6.
08; Takeda, S. et al. (1985) Nature 314: 452,454). Alternatively, in the field
Applying the described techniques for the production of single chain antibodies using well known methods, CY
Generates SKP-specific single chain antibodies. Has an idiotypic composition with related specificity
Different antibodies are chained from a random combination of immunoglobulin libraries.
Can also be produced by shuffling (Burton D.R. (1991) Proc. N.
atl. Acad. Sci. USA 88: 10134-10137 etc.). Antibody production in lymphocyte populationsin vivoBy induction of production, or immunity
Screening of globulin libraries or high performance features such as those disclosed in the literature.
It can also be done by screening a panel of heterologous reagents (Orlandi, R. et al. (19
89) Proc. Natl. Acad. Sci. USA 86: 3833-3837, Winter, G. et al. (1991) Natur.
e 349: 293-299 etc.). Antibodies containing specific binding sites for CYSKP can also be obtained. For example, limited
However, such fragments may be generated by digestion of antibody molecules with pepsin.
Made F (ab ')₂ Fragment and F (ab ')₂ By reducing the disulfide bridges of the fragment
Fab fragments produced in this way. Alternatively, to create a Fab expression library
Thus, to quickly and easily identify monoclonal Fab fragments with the desired specificity.
(See Huse, W.D. et al. (1989) Science 256: 1275-1281 etc.). Screen for various immunoassays to find antibodies with the desired specificity.
Can be identified. A polyclonal antibody or model with isolated specificity
Due to competitive binding using any of the monoclonal antibodies, or immunoradioactivity
Numerous protocols are well known in the art. Usually such an immunoasset
B) includes measurement of complex formation between CYSKP and its specific antibody. two
Two parts using a monoclonal antibody reactive against non-interfering CYSKP epitopes
Monoclonal-based immunoassays are commonly used, but competitive binding assays
You can also use essays (Pound, supra). Using various methods such as Scatchard analysis with radioimmunoassay technology, CYS
Assess the affinity of the antibody for KP. Affinity is represented by the binding constant Ka, which is
Under equilibrium, the molar concentration of the CYSKP antibody complex is the molar concentration of free antibody and free antigen.
It is the value obtained by dividing. Possessing heterogeneous affinity for many CYSKP epitopes
Ka of the reclonal antibody drug is the average affinity or binding activity of the antibody to CYSKP.
Represents sex. K, a monoclonal antibody drug specific for a specific CYSKP epitope
a represents a true measure of affinity. Ka value is 10⁹-10¹²L / mol high affinity
Antibody drugs are immunoassays in which the CYSKP antibody complex must withstand intense manipulation.
It is preferably used for essays. Ka value is 10⁶-10⁷L / mol low affinity antibody
The drug is an immunopurified (i) in which CYSKP must ultimately dissociate from the antibody in an activated state.
mmunopurification) and similar treatments. (Catty, D. (1988
)Antibodies, Volume I: A Practical ApproachIRL Press, Washington, DC;
 Liddell, J.E. and Cryer, A. (1991)A Practical Guide to Monoclonal Antibodies , John Wiley & Sons, New York NY). The antibody titer and binding activity of the polyclonal antibody reagent are further evaluated and some suitable
The quality and suitability of such reagents for an application can be determined. For example,
At least 1-2 mg / ml of specific antibody, preferably 5-10 mg / ml
Polyclonal antibody drugs containing specific antibodies generally precipitate CYSKP antibody complexes.
Used for processing that must be done. Antibody specificity, antibody titer, binding activity,
Guidelines for antibody quality and use in various applications are publicly available
Is. (See Catty's reference, Coligan's reference, etc.). In another embodiment of the present invention, a polynucleotide encoding CYSKP, or its option.
Any fragment or complementary sequence can be used for therapeutic purposes. In one embodiment,
Sequences and amplicons complementary to the coding and regulatory regions of the gene encoding CYSKP
Alter gene expression by designing thisense molecules (DNA and RNA, modified nucleotides)
You can Such techniques are well known in the art and may be sense or antisense.
Control of the CYSKP-encoding sequence by a sense oligonucleotide or large fragment
It can be designed from a region or from various locations along the code region (Agra
wal, S., ed. (1996)Antisense TherapeuticsHumana Press Inc., Totawa NJ
Etc.). When used in therapy, it is suitable to introduce the antisense sequence into suitable target cells.
Any gene delivery system can be used. Antisense sequences are targeted at the target during transcription.
Expression expressing a sequence complementary to at least a part of the cell sequence encoding the protein
It can be delivered intracellularly in the form of a plasmid (Slater, J.E. et al. (1998)
 J. Allergy Clin. Immunol. 102 (3): 469-475 and Scanlon, K.J. et al. (1995) 9 (
13): 1288-1296.). Antisense sequences also include, for example, retroviruses and
It can be introduced into cells using viral vectors such as adeno-associated viral vectors.
Can also do (Miller, A.D. (1990) Blood 76: 271, Ausubel, Uckert, W.
And W. Walther (1994) Pharmacol. Ther. 63 (3): 323-347 etc.). That
Other genetic delivery mechanisms include liposome systems, artificial viral envelopes and for the time being.
Other systems known in the field are included (Rossi, J.J. (1995) Br. Med. Bull. 51 (1):
217-225; Boado, R.J. et al. (1998) J. Pharm. Sci. 87 (11): 1308-1315, Morris, M.
C. et al. (1997) Nucleic Acids Res. 25 (14): 2730-2736. In another embodiment of the present invention, a polynucleotide encoding CYSKP is added to somatic cells or
Alternatively, it can be used for gene therapy of germ cells. To do gene therapy
From (i) gene deficiency (eg, X chain chain inheritance (Cavazzana-Calvo, M. et al. (20
00) Science 288: 669-672).
) -X1), severe adenosine deaminase (ADA) deficiency associated with severe
Combined immunodeficiency (Blaese, R.M. et al. (1995) Science 270: 475-480, Bordignon,
 C. et al. (1995) Science 270: 470-475), cystic fibrosis (Zabner, J. et al. (1993) Ce.
ll 75: 207-216: Crystal, R.G. et al. (1995) Hum. Gene Therapy 6: 643-666, Crys
tal, R.G. et al. (1995) Hum. Gene Therapy 6: 667-703), Thalassam (thalassam).
ia), familial hypercholesterolemia, factor VIII or IX deficiency
Hemophilia (Crystal, 35 R.G. (1995) Science 270: 404-410, Verma, I.M.
And Somia. N. (1997) Nature 389: 239-242), and (ii) conditional lethal inheritance.
Express offspring products (eg in case of cancer due to uncontrolled cell growth), (iii)
Intracellular parasites such as human immunodeficiency virus (HIV) (Baltimore, D. (1988)
Nature 335: 395-396, Poescbla, E. et al. (1996) Proc. Natl. Acad. Sci. USA. 93.
: 11395-11399), hepatitis B or C virus (HBV, HCV),Candida albicans as well asParacoccidioides brasiliensisFungal parasites such asPlasmodium falciparum
insectas well asCruise trypanosomaTan with a protective function against protozoan parasites such as
The protein can be expressed. Of genes required for expression or regulation of CYSKP
If the deficiency causes disease, express CYSKP from a suitable population of introduced cells.
Thus, it is possible to mitigate the onset of symptoms caused by the genetic defect. In a further embodiment of the invention, the disease or disorder due to CYSKP deficiency is a CYSKP
Mammalian expression vectors to be constructed and used to drive these vectors by mechanical means.
Treatment by introducing into CYSKP-deficient cells.in vivoOrex vitroof
Mechanical transfer technology used for cells includes (i) direct DNA microinjection into individual cells.
Method, (ii) gene gun, (iii) liposome-mediated transfection, (iv) receptor-mediated
Gene transfer and (v) use of DNA transposons (Morgan, R.A. and W.
F. Anderson (1993) Annu. Rev. Biochem. 62: 191-217, Ivics, Z. (1997) Cell
 91: 501-510; Boulay, J-L. And H. Recipon (1998) Curr. Opin. Biotechno
l. 9: 445-450). Expression vectors that can influence the expression of CYSKP include, but are not limited to,
PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vector (Invitrogen, Carlsbad CA)
, PCMV-SCRIPT, PCMV-TAG, PEGSH / PERV (Stratagene, La Jolla CA), PTET-OFF
, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA)
. In order to express CYSKP, (i) a constitutively active promoter (for example,
Tomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, Thimeji
Kinase (TK) or β-actin gene, etc.), (ii) inducible promoter
(For example, the T-REX plasmid (Invitrogen), which is commercially available,
Tracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Pro
c. Natl. Acad. Sci. U.S.A. 89: 5547-5551; Gossen, M. et al. (1995) Science 26.
8: 1766-1769; Rossi, F.M.V. and H.M.Blau (1998) Curr. Opin. Biotechno
l. 9: 451-456)), ecdysone inducible promoter (commercially available plasmid
Included in PVGRXR and PIND: Invitrogen), FK506 / rapamycin-induced
Romotor, or RU486 / Mifepristone inducible promoter (Rossi, F.
M.V. and H.M. Blau, supra), or (iii) CYSKP derived from normal individuals
Natural or tissue-specific promoter of the encoding endogenous gene
Can be used. Commercially available liposome transformation kit (eg, PerFect Lipid Transf from Invitrogen)
Section Kit) allows those skilled in the art to rely on experience
Can be introduced into target cells in culture. Alternatively, calcium carbonate
Method (Graham. F.L. and A.J. Eb (1973) Virology 52: 456-467) or electric
Transformation was performed using the air-poration method (Neumann, B. et al. (1982) EMBO J. 1: 841-845).
U For the introduction of DNA into primary cells, standardized mammalian transfection
Protocol modification is required. In another embodiment of the invention, a disease or disorder caused by a gene deficiency associated with expression of
Parasitism is due to (i) retroviral terminal repeat (LTR) promoter or independent
A polynucleotide encoding CYSKP under the control of a promoter,
ii) a suitable RNA packaging signal and (iii) additional retroviral cis
Rev with functional RNA sequences and coding sequences required for efficient vector propagation
Generating and treating retroviral vector consisting of responsive element (RRE)
You can Retroviral vectors (eg PFB and PFB NEO) are Stratagen
It is commercially available from e-Company and published data (Riviere, I. et al. (1995) Proc. Natl. Ac
Ad. Sci. U.S.A. 92: 6733-6737). Citing the above data
Are made a part of this specification. The vector is a suitable vector-producing cell line (VPCL
), VPCL is engulfed in an envelope with tropism for receptors on target cells.
Expression of promiscuous envelope proteins such as the rope gene or VSVg (Arment
ano, D. et al. (1987) J. Virol. 61: 1647-1650, Bender, M.A. et al. (1987) J. Viro.
61.1639-1646, Adam, M.A. and A.D. Miller (1988) J. Virol. 62: 3802-
3806, Dull, T. et al. (1998) J. Virol. 72: 8463-8471, Zufferey, R. et al. (1998).
J. Virol. 72: 9873-9880). No. 5,910,434 to RIGG ("Metho
d for obtaining retrovirus packaging cell lines producing high transduci
ng efficiency retroviral supernatant ”)
A method for obtaining a Zing cell line is disclosed and is hereby incorporated by reference.
As part of the book. Propagation of retroviral vectors, cell population (eg CD4⁺ T-thin
Cells) and the return of the transduced cells to the patient in the field of gene therapy.
Is a method known to those skilled in the art and has been described in numerous documents (Ranga, U. et al. (1
997) J. Virol. 71: 7020-7029, Bauer, G. et al. (1997) Blood 89: 2259-2267, Bon.
yhadi, M.L. (1997) J. Virol. 71: 4707-4716, Ranga, U. et al. (1998) Proc. Nat
L. Acad. Sci. U.S.A. 95: 1201-1206, Su, L. (1997) Blood 89: 2283-2290). Alternatively, an adenovirus-based gene therapy delivery system could be used to correlate expression of CYSKP.
Polynucleotide encoding CYSKP in cells with one or more genetic abnormalities
Deliver Ochid. For the production and packaging of adenovirus-based vectors
Are known to those skilled in the art. Replication-defective adenovirus vectors are immunoregulatory
Variable to introduce a protein-encoding gene into intact pancreatic islets
(Csete, M.E. et al. (1995) Transplantation 27: 263-26)
8). A possible adenovirus vector was provided to Armentano.
Described in US Pat. No. 5,707,618 ("Adenovirus vectors for gene therapy").
Which is incorporated herein by reference. Adenovirus vector
Regarding Antinozi, P.A. et al. (1999) Annu. Rev. Nutr. 19: 511-544 and
 See also Verma, I.M. and N. Somia (1997) Nature 18: 389: 239-242.
. Both documents are incorporated herein by reference. Alternatively, a herpes-based gene therapy delivery system may be used to link CYSKP expression 1
Polynucleotides Encoding CYSKP in Target Cells with One or Multiple Gene Abnormalities
Deliver tide. Herpes simplex virus (HSV) -based vectors are the central god of HSV affinity.
It is especially important when introducing CYSKP into transcellular cells. Preparation of herpes vector and
Packaging is known to those skilled in the art. Replication competent herpes simplex virus (H
SV) Type I vector is used to deliver the reporter gene to the primate eye.
(Liu, X. et al. (1999) Exp. Eye Res. 169: 385-395). HSV-1 virus
Also for the construction of the vector, US Pat. No. 5,804,413 ("Herp
es simplex virus swains for gene transfer ").
Is used as a part of this specification. U.S. Pat.No. 5,804,413 describes human gene therapy.
A small amount that is introduced into cells under the control of a suitable promoter for purposes including
Description of recombinant HSV d92 containing a genome with at least one exogenous gene
There is a listing. The above patent also claims that recombinant HS removed for ICP4, ICP27 and ICP22.
It discloses the production and use of the V strain. Goins, W for HSV vectors
.F. Et al. (1999) J. Virol. 73: 519-532 and Xu, H. et al. (1994) Dev. Biol. 163:
See also 152-161. Both documents are incorporated herein by reference. Black
Manipulated herpesvirus sequences, different giant herpesvirus genomes
Production of recombinant virus after transfection of multiple plasmids containing
Those skilled in the art are familiar with the
Is a known technique. Alternatively, the α virus (positive single-stranded RNA virus) vector is used to clone CYSKP.
The target polynucleotide is delivered to the target cells. Prototype alpha virus
A biological study of a Semliki Forest Virus (SFV)
It is widely practiced and it has been found that gene transfer vectors are based on the SFV genome.
It was (Garoff, H. and K.-J. Li (1998) Cun. Opin. Biotech. 9: 464-469.
). During replication of alphavirus RNA, viral capsid proteins are usually
Sub-genomic RNA is created. This subgenomic RNA is a full-length genome
It replicates to a higher level than RNA, and therefore enzymatic activity (such as protease and poly
Excess capsid protein compared to viral protein with merase)
Produced. Similarly, the sequence encoding CYSKP was replaced with the capsid of the alphavirus genome.
Introduced into the coding region, many CYSK
RNA encoding P is produced and CYSKP is synthesized at high levels. Normally α
While infection with Ils is associated with cell lysis within days, Sindbis virus (
Confirmed persistent infection of hamster normal kidney cells (BHK-21) carrying SIN) variants
The ability to establish is suitably modified so that lytic replication of alphavirus can be applied to gene therapy.
Suggesting that it is possible (Dryga, S.A. et al. (1997) Virology 228: 74-
83). Since α virus can be introduced into various hosts, CY can be introduced into various types of cells.
SKP can be introduced. Specific transduction of a subset of cells in a population
May require sorting of cells prior to transduction. α virus infectious cDNA clone
Treatment method, α virus cDNA and RNA transfection method, and α virus infection method
Methods are known to those of skill in the art. Gene expression can also be inhibited using oligonucleotides derived from the transcription start site.
Noh. The transcription initiation site is, for example, about −10 and about +10 counted from the initiation site.
In between. Similarly, inhibition is possible using the triple helix base pair formation method. three
Heavy helix base pairing is due to the binding of polymerases, transcription factors or regulatory molecules.
Triple helix base pairing is possible because it inhibits the ability of the double helix to open sufficiently.
It is for. Recent advances in therapy with triple helix DNA have been documented in the literature.
(Gee, J.E. et al. (1994) in: Huber, B.E. and B.I.Carr,Molecular and I mmunologic Approaches , Futura Publishing Co., Mt. Kisco, NY, 163-177 page
See the page etc.). Complementary sequences or antisense molecules also direct transcripts to ribosomes.
Can be designed to block translation of mRNA by blocking binding
it can. Ribozymes are enzymatic RNA molecules that are used to catalyze the specific cleavage of RNA.
Can also be used. The mechanism of ribozyme action is nucleotide chain scission
-Specific hybridization of ribozyme molecules to complementary target RNA prior to
Are involved in For example, a nucleotide strand break in the sequence encoding CYSKP
Contains a recombinant hammerhead ribozyme molecule that catalyzes specifically and effectively
Be done. Specific ribozyme cleavage sites within any RNA target include ribozymes, including GUA, GUU, and GUC sequences.
First identified by scanning the target molecule against the bozyme cleavage site
. Once identified, secondary structure features that render the oligonucleotide dysfunctional.
15-20 ribonucleosides corresponding to the region of the target gene containing the cleavage site for
It makes it possible to evaluate short RNA sequences of tide. Evaluation of suitability of candidate targets
Hive with complementary oligonucleotides using a ribonuclease protection assay
This can be done by testing the feasibility of redistribute. Complementary ribonucleic acid molecules and ribozymes of the invention may be used in the art for nucleic acid molecule synthesis.
It can be prepared using any well-known method. The solid phase phosph
There is a method of chemically synthesizing an oligonucleotide such as a amide compound. Some
Or, of the DNA sequence encoding CYSKPin vitroas well asin vivoTranscription of RNA molecules
Can produce. Such a DNA sequence may be constructed using a suitable RNA polymerase promoter such as T7 or SP6.
It can be incorporated into various vectors using a vector. Or complementary
These cDNA products, which synthesize RNA either constitutively or inducibly, are introduced into cell lines or cells.
Or within the organization. RNA molecules can be modified to increase intracellular stability and half-life
It Possible modifications include, but are not limited to, the 5'end of the molecule, the 3'end, or
Add flanking sequences in both, or in the backbone of the molecule.
Use phosphorothionate or 2'O-methyl instead of hodiesterase coupling
It is included. This concept is unique to the production of PNAs, and these
It can be extended to all molecules. To do this, the endogenous endonuclease
Therefore, adenine, cytidine, guanine, thymine, and uri which are not easily recognized
Zinc with acetyl-, methyl-, thio- and similar modifications, as well as non-conventional
Bases such as inosine, queosine, wybutosine
Etc. can be added. A further embodiment of the invention relates to altered expression of polynucleotides encoding CYSKP.
Methods for screening for effective compounds are included. Without limitation
Compounds that are effective in expressing mutant nucleotides include oligonucleotides and antisense compounds.
Sense oligonucleotides, triple helix-forming oligonucleotides, transcription factors, etc.
Polypeptide Transcription Regulator, and Can Interact with Specific Polynucleotide Sequences
There are non-polymeric chemical entities. An effective compound is an inhibitor of polynucleotide expression.
Polynucleotides by acting as either
Expression can be mutated. Therefore, treatment of diseases associated with increased expression or activity of CYSKP
In treatment, compounds that specifically inhibit the expression of the polynucleotide encoding PP
Is useful therapeutically and is useful in the treatment of diseases associated with decreased expression or activity of PP.
For example, compounds that specifically promote the expression of polynucleotides encoding CYSKP
Can be therapeutically useful. From at least one for efficacy in mutating expression of specific polynucleotides
Multiple test compounds can be screened. Test compounds are commonly known in the art.
It can be obtained by any of the available methods. Such methods include polynucleotides
When mutating the expression of, and when existing, commercial or proprietary, natural or unnatural
When selecting from a compound library, the chemical and / or
Or rational design of compounds based on structural properties,
Are known to be useful when selecting from a library of randomly generated compounds.
There are chemical modifications of the compound as described. Polynucleotide encoding CYSKP
A sample containing a probe is obtained by exposure to at least one test compound. To sample
Are, for example, intact cells, permeabilized cells, cell-free reconstitution systems or reconstitution biochemistry.
There can be a system. Changes in the expression of the polynucleotide encoding CYSKP may be
Assay by any method commonly known in the art. Usually code CYSKP
Using a probe with a nucleotide sequence complementary to the polynucleotide sequence
Expression of specific nucleotides is detected by hybridization. Hybrid
The amount of quantification is quantified, which results in exposure to one or more test compounds.
And may form the basis for comparison of expression of unexposed polynucleotides. test
Detection of changes in expression of polynucleotides exposed to a compound is
It is shown that the test compound is effective in mutating the expression of octide. Singularity
For compounds effective for mutant expression of a nucleotide, for example,Schizosaccharomyce s pombe Gene expression system (Atkins, D. et al. (1999) US Pat. No. 5,932,435, Arndt,
G.M. et al. (2000) Nucleic Acids Res. 28: E15) or human cell lines such as HeLa cells (
Clarke, M.L. et al. (2000) Biochem. Biophys. Res. Commun. 268: 8-13).
Perform screening. Particular embodiments of the present invention include specific polynucleotides.
Oligonucleotides for antisense activity against sequences (deoxyribonucleic acid
Reotide, ribonucleotide, peptide nucleic acid, modified oligonucleotide)
Involved in screening combinatorial libraries (Bruice, T.W. et al.
(1997) U.S. Pat.No. 5,686,242, Bruice, T.W. et al. (2000) U.S. Pat.No. 6,022,691
issue). Numerous methods are available for introducing vectors into cells or tissues,in vivo,i n vitro as well asex vivoIs equally suitable for use.ex vivoFor treatment,
The vector was introduced into stem cells collected from the patient, cloned and propagated, and the same patient
It can be returned to home by autologous transplant. Transfection, ribosome injection or
Delivery by polycationic amino polymers uses methods well known in the art.
(Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:46.
See 2-466.). Any of the above treatment methods, for example, human, dog, cat, cow, horse, rabbit,
It can be applied to all subjects in need of treatment including mammals such as monkeys. Additional embodiments of the present invention include active ingredients that are typically formulated with pharmaceutically acceptable excipients.
Related to the administration of ingredients having Excipients include, for example, sugar, starch, cellulose
, Gum and protein. Various prescriptions are commonly known and details areRemingto n's Pharmaceutical Sciences Listed in the latest version of (Maack Publishing, Easton PA)
Has been done. Such compositions include CYSKP, CYSKP antibodies, mimetics, agonists,
Constituents or inhibitors of CYSKP. The components used in the present invention can be administered by any number of routes,
Routes that do not include oral, intravenous, intramuscular, intraarterial, intramedullary, and arachnoid.
Intracavitary, Intraventricular, Lung, Percutaneous, Subcutaneous, Intraperitoneal, Intranasal, Intestinal, Topical, Sublingual or direct
I have an intestine. Pulmonary administration ingredients may be prepared in liquid or dry powder form. Such ingredients are
It is usually aerosolized immediately before inhalation by the patient. Small molecules (eg traditional small molecules)
Aerosol delivery of fast-acting formulations is known in the art. High
In the case of molecules (eg larger peptides and proteins)
The recent improvements in pulmonary delivery through the alveolar region of the lungs have led to drugs such as insulin.
It made it possible to deliver the drug to the blood circulation substantially (Patton, J.S. et al., US patent).
See Xu 5,997,848). Pulmonary delivery is superior in that it can be administered without needle injection.
Eliminates the need for potentially toxic penetration enhancers. Ingredients suitable for use in the present invention include only the amount necessary to achieve a given purpose.
Ingredients that include the active ingredients of Determination of the effective dosage is within the ability of one of ordinary skill in the art.
Do it in the circle. In order to deliver macromolecules containing CYSKP or its fragments directly into cells, it has a special form.
Preferably the composition is prepared. For example, a liposome containing a cell-impermeable polymer
Formulations may facilitate cell fusion and intracellular delivery of macromolecules. Alternatively, CYSKP or
Alternatively, the fragment can be linked to the cationic N-terminal part of the HIV Tat-1 protein.
. The fusion protein produced in this way is compatible with all mouse model system brains.
It is known to transduce cells of the tissues of Schwarzee (Schwarze, S.R. et al. (1999) S
cience 285: 1569-1572). For any compound, a cell culture assay, such as a neoplastic cell culture assay.
Or in animal models such as mice, rabbits, dogs or pigs
In, first, the effective dosage of treatment can be estimated. Animal models also
It can also be used to determine the appropriate concentration range and route of administration. Use such information
The beneficial dosage and administration route for humans can then be determined. A medically effective dose will improve the symptoms or condition, for example CYSKP or
Fragment, CYSKP antibody, CYSKP agonist or antagonist, inhibitor
Related to the amount of active ingredients such as. Medicinal efficacy and toxicity depend on cell culture or animal
By standard drug techniques in physical experiments, eg ED₅₀(50% of the population is medical
Effective amount) or LD₅₀Determine by measuring (lethal dose of 50% of the population)
You can The dose ratio of toxic to therapeutic effects is the therapeutic index, LD₅₀/ E
D₅₀It can be expressed as a ratio. Ingredients that exhibit high therapeutic indices are desirable. Fine
The data obtained from cell culture assays and animal studies are used as doses for human use.
Used to dispense a range of Dosages containing such compositions are toxic
With little or no₅₀It is preferable that the blood concentration range includes
Yes. Depending on the dosage form used, the sensitivity of the patient and the route of administration, the dose
It varies within the range. The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment.
Will be decided. Gives effective levels of active ingredient or maintains desired effect
Dosage and administration are adjusted accordingly. Factors related to the subject include the severity of the disease.
Severity, general health of the patient, age, weight and sex of the patient, time and frequency of administration
, Consideration of drug composition, reaction sensitivity, response to treatment, etc. Long duration of action
Ingredients should be taken once every 3-4 days, depending on the half-life and clearance rate of the particular formulation.
It may be administered once a week, or once every two weeks. The usual dose is about 0.1 to 100,000 μg, depending on the route of administration.
The total amount is about 1g. Guidance on specific dosages and delivery methods can be found in the literature.
It has been described and is usually available to the on-site physician. Those skilled in the art
For nucleotides, which is different from the formula for proteins or inhibitors
The prescription will be used. Similarly, delivery of polynucleotides or polypeptides
They are specific to particular cells, conditions, locations, etc. (Diagnosis) In another example, an antibody that specifically binds to CYSKP is characterized by the expression of CYSKP.
Diagnosis of diseases attached to CYSKP or CYSKP agonist or antagonis
Used in assays to monitor patients being treated with inhibitors
To be Antibodies useful for diagnostic purposes are prepared in the same manner as described in the treatment section above.
Is prepared in. CYSKP diagnostic assays use antibodies and labels to
Or methods for detecting CYSKP in cells or tissues. antibody
Is used with or without modification, depending on the covalent or non-covalent nature of the reporter molecule.
It may be labeled by covalent attachment. Diverse reporter molecules in this technical field
Known and can be used. For some reporter molecules
As mentioned above. Various protocols, including ELISA, RIA, and FACS for measuring CYSKP are available for the time being.
It is well known in the field and is the basis for diagnosing abnormal or abnormal levels of CYSKP expression.
To provide Normal or normal CYSKP expression levels are associated with complex formation.
Body fluid collected from a subject, such as a normal mammal, for example a human, under suitable conditions.
Or by binding the cells to an antibody against CYSKP. Standard composite
The amount of body formation can be quantified by various methods such as photometry. Of subjects, controls and disease
The amount of CYSKP expression in samples from biopsy tissue is compared to a reference value. Standard value and
Deviation from the examiner is a parameter for diagnosing the disease. According to another embodiment of the present invention, a polynucleotide encoding CYSKP is used for diagnosis.
It can also be used for The polynucleotides that can be used include
Includes gonucleotide sequences, complementary RNA and DNA molecules, and PNAs. This poly
Nucleotides have been used in biopsied tissues expressing CYSKP that may be correlated with disease.
Gene expression is detected and quantified. Presence or absence of CYSKP using this diagnostic assay
, For further overexpression and monitoring regulation of CYSKP levels during treatment. In one embodiment, a polynucleotide comprising a gene sequence encoding CYSKP or a closely related molecule.
By hybridization with a PCR probe that can detect the cleotide sequence
Thus, it is possible to identify the nucleic acid sequence encoding CYSKP. For example, 5 '
A highly specific region that is a node region or, for example, a conserved motif that is somewhat unique
Specificity of the probe whether it is made from a less
Or the stringency of amplification is the natural sequence in which the probe encodes CYSKP.
Whether to identify only the sequence, or the natural sequence encoding alleles or related sequences
It will depend on whether or not you identify only one. The probe can also be used to detect related sequences and can be used in any array that encodes CYSKP.
It may have at least 50% sequence identity with the column. The hybridise of the invention of interest
The DNA probe or the RNA can be used as the sequence probe, and the sequence of SEQ ID NO: 35-68 is used.
, Or the genome containing the CYSKP gene promoter, enhancer and intron
It can be derived from the sequence. Construction of hybridization probe specific for CYSKP-encoding DNA
As a method of production, a polynucleotide sequence encoding CYSKP and a CYSKP derivative was used as mRNA.
There is a method for cloning into a vector for producing a probe. mRNA probe
Vectors for production are known to those of skill in the art, are commercially available, and are suitable RNA.
By adding a polymerase and a suitable labeled nucleotide,in vit ro Can be used to synthesize RNA probes. Hybridization pro
A probe can be labeled with a population of different reporters. Reporter group example
as,³²P or³⁵Via radionuclides such as S or avidin / biotin binding system
And enzyme labels such as alkaline phosphatase bound to the probe.
It A polynucleotide sequence encoding CYSKP is used to determine the pathology associated with CYSKP expression.
The patient can be diagnosed. For example, but not by way of limitation,
, Cell dyskeratosis actinic keratoses, arteriosclerosis, atherosclerosis, synovial bursa
Inflammation, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobin
Vinuria, polycythemia vera, psoriasis, primary thrombocythemia, and adenocarcinoma and leukemia, phosphorus
Pamas, melanomas, myelomas, sarcomas, and teratocarcinomas, specifically adrenal gland, bladder, bone
, Bone marrow, brain, breast, cervix, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle
, Ovary, pancreas, parathyroid gland, penis, prostate, salivary gland, skin, spleen, testis, thymus, instep
Cancer of the thyroid gland, uterus, and autoimmune / inflammatory diseases include acquired immunodeficiency syndrome (AIDS).
), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloid
, Anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmunity
Thyroiditis, autoimmune polyglandular endocrine candid ectodermal dystrophy (APECED),
Bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, genuine
Diabetes mellitus, emphysema, lymphotoxin-induced temporary lymphopenia, fetal erythroblastosis, nodular
Erythema, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease
, Hashimoto thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis
Vitiligo, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis
, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic
Anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenia, ulcer
Ulcerative colitis, Werner syndrome, cancer complications, hemodialysis, extracorporeal circulation, virus infection
Disease, bacterial infection, fungal infection, parasitic infection, protozoal infection, helminth infection, trauma
Includes impaired vesicular transport, cystic fibrosis, glucose galactose malabsorption
Group, hypercholesterolemia, diabetes mellitus, diabetes insipidus, hyperglycemia, hypoglycemia, gray
Orb's disease, goiter, Cushing's disease, and Addison's disease, ulcerative colitis, gastroduodenum
Other pathologies associated with gastrointestinal tract disorders, including digital ulcers, and abnormal vesicular transport include acquired
Immunodeficiency syndrome (AIDS), hay fever, asthma and hives (rash), autoimmune hemolytic poverty
Blood, proliferative glomerulonephritis, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, rheumatism
Id, osteoarthritis, scleroderma, Chediak-East syndrome, Sjogren's syndrome, all
Somatic lupus erythematosus, toxic shock syndrome, traumatic tissue injury, and
Illus infection, bacterial infection, fungal infection, parasitic infection, protozoal infection, neurological disease
Among the patients are epilepsy, ischemic cerebrovascular accident, stroke, cerebral neoplasm, Alzheimer's disease.
, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders,
Amyotrophic lateral sclerosis and other motor neuron disorders, progressive neuromuscular atrophy, color
Predisposition to retinitis, hereditary ataxia, multiple sclerosis and other demyelinating diseases, bacterial and viral
Lupus meningitis, brain abscess, subdural empyema, epidural abscess, purulent intracranial thrombophlebitis
, Myelitis and radiculitis, viral central nervous system diseases, prion diseases (Kuru, Kur
Includes Leutzfeldt-Jakob disease and Gerstmann-Straussler-Scheinker syndrome
), Fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis
, Cerebellar retinal hemangioblastomatosis, trigeminal brain
Central nervous system mental retardation and other developmental disorders, including transvascular syndrome, Down's syndrome,
Cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord disorders, muscular dystrophy
Loffey and other neuromuscular disorders, peripheral nerve diseases, dermatomyositis and polymyositis, remnants
Conductive, metabolic, endocrine, and toxic myopathy, myasthenia gravis, periodic quadriplegia
, Psychosis (moodness, anxiety disorder, schizophrenia), seasonal disorder (SAD), quiz
Disability, amnesia, catatonia, diabetic neuropathy, extrapyramidal terminal defect syndrome,
With dystonia, schizophrenia, postherpetic neuralgia, and Tourette's disease,
Includes familial supranuclear palsy, corticobasal degeneration, and familial frontotemporal amnesia.
Rare, cell dyskinesias include ankylosing spondylitis, Chediak East syndrome, Duchen
Nu-type and Becker muscular dystrophy, intrahepatic cholestasis, myocardial hyperplasia (thickening), heart
Myopathy, early-onset periodontitis, including adenocarcinoma, ovarian cancer and chronic myelogenous leukemia
Cancer, and bacterial and parasitic infections and reproductive disorders, impaired prolactin production,
Fertility, fallopian tube disease, ovulation deficiency, endometriosis, sexual cycle disorder, menstrual cycle disorder, polycystic
Ovarian syndrome, ovarian hyperstimulation syndrome, endometrial cancer, ovarian cancer, uterine fibroids, autoimmune disease
Patients, ectopic pregnancy, teratogenesis, breast cancer, fibrocystic breast disease, milk leakage, spermatogenic destruction
, Sperm physiology, testicular cancer, prostate cancer, benign prostatic hypertrophy, prostatitis, Pyronie's disease
, Incompatibility, male breast cancer, gynecomastia, high gonadotropin hypogonadism, hypogona
Dotropin hypogonadism, pseudohermaphroditism, azoospermia, premature ovarian failure, acro
Syn deficiency, delayed puberty, retrograde ejaculation, ejaculation, and hemangioblastoma, cystic brown
Cyst sphaeochromocytomas, paraganglioma, cystadenoma of the epididymis, and internal
Sac tumors, and muscle disorders include myocarditis, Duchenne muscular dystrophy,
Becker muscular dystrophy, myotonic dystrophy, core disease, nemarin
Opathy, central pontine myelinolysis, lipid muscle disease, mitochondrial myopathy, infection
Myositis, polymyositis, cutaneous myositis, inclusion body myositis, thyrotoxic myositis and ethano
Lumiopathy, angina, anaphylactic shock, arrhythmia, asthma, cardiovascular
Shock, Cushing's syndrome, hypertension, hypoglycemia, myocardial infarction, migraine, and
Pheochromocytoma, and cardiomyopathy, brain disease, epilepsy, Kerns-Say
Includes Yer syndrome, lactic acidosis, muscular clonic disease, and ophthalmoplegia
It The polynucleotide sequence encoding CYSKP can be cloned using the Southern method, Northern method,
Blot, or other membrane-based technology, PCR, dipstick
stick, pin, ELISA assay, and detect expression of mutant CYSKP
For use in microarrays that utilize body fluids or tissues collected from patients for
Is possible. Such qualitative or quantitative methods are known in the art. In one embodiment, the CYSKP-encoding nucleotide sequence is associated with a disorder associated with the disease.
It may be useful in assays to detect the diseases mentioned above. Code CYSKP
Nucleotide sequences are labeled by standard methods and labeled with hybridization complexes.
In addition to a fluid or tissue sample taken from a patient under conditions suitable for body formation
Could be Sample after a suitable incubation period
Are washed and the signal is quantified and compared to standard values. Signal of patient sample
If the amount is significantly different from the control sample, change the CYSKP in the sample.
Mutation levels in the encoding nucleotide sequence reveal the presence of related diseases
become. Such an assay can demonstrate specific therapeutic effects in animal studies and clinical trials.
It can also be used to estimate, or to monitor the treatment of individual patients.
It To provide a basis for the diagnosis of diseases associated with the expression of CYSKP, expression of
A normal or standard profile is established. This is the hybridization
From normal subjects, either animals or humans, under suitable conditions for amplification or amplification.
Combines extracted body fluids or cells with CYSKP-encoding sequences or fragments thereof
Can be achieved. A known amount of substantially purified polynucleotide
By comparing the values obtained from the experiments performed with
Quasi-hybridization can be quantified. Standard value obtained in this way
Can be compared to values obtained from samples obtained from patients showing signs of disease
. Deviation from standard values is used to establish the presence of disease. Once the presence of the disease is established and the treatment protocol is initiated, the patient's expression level is positive.
The hive is used to determine if it has begun to approach the levels observed in normal subjects.
The redidation assay can be repeated on a regular basis. Obtained from a continuous assay
The results obtained can be used to show the effect of treatment over a period of days to months. For cancer, abnormal amounts of transcripts (underexpression or
The presence of (overexpression) indicates the predisposition to develop the disease or the actual clinical symptoms.
May provide a method for detecting a disease. A clearer diagnosis of this kind may
Medical professionals early access to preventative or aggressive therapies, which may lead to cancer
It is possible to prevent the occurrence of or further progress. Further diagnostics of oligonucleotides designed from CYSKP coding sequences
Utilization can include utilization of PCR. These oligomers are chemically synthesized
Or produced enzymatically, orin vitroCan be produced at. Oligomer preferred
Or a fragment of a polynucleotide encoding CYSKP, or a polynucleotide encoding CYSKP.
It contains a polynucleotide fragment that is complementary to the polynucleotide and is
It is used to identify certain genes and conditions. Also, the oligomers are rather loose
Under stringent conditions, detection, quantification, or detection of closely related DNA or RNA sequences
It can be used for both. In some embodiments, oligos derived from a polynucleotide sequence encoding CYSKP.
Nucleotide primers can be used to detect single nucleotide polymorphisms (SNPs). SNP is
Substitutions, insertions and insertions that may cause congenital or acquired genetic disease in humans in
It is a deletion. A method for detecting SNP includes, but is not limited to, SSCP (single-str
anded conformation polymorphism) and fluorescent SSCP (fSSCP). C in SSCP
Oligonucleotide primers derived from the polynucleotide sequence encoding YSKP
Amplify DNA by polymerase chain reaction (PCR). DNA is, for example,
It may be derived from woven or normal tissue, biopsy samples, body fluids and the like. SNPs in DNA are
It makes a difference in the secondary and tertiary structure of the PCR product in the single-stranded form. The difference is
It can be detected by gel electrophoresis in a gel. For fSCCP, oligonucleo
The tide primer is fluorescently labeled. This allows for DNA sequencing machines, etc.
It is possible to detect amplimer with high processing equipment. In addition,
Sequence database analysis method called co-SNP (in silico SNP, isSNP) is commonly used
Compare sequences of individual overlapping DNA fragments that are arranged in different consensus sequences
By doing so, polymorphism can be identified. These computer-based methods are DN
Using A's laboratory adjustments and statistical models and automated analysis of DNA sequence chromatograms
Filtered out sequence variations due to sequencing errors
It In another embodiment, for example, a high throughput MASSARRAY system (Sequenom, Inc., San Die
SNPs are detected and characterized by mass spectrometry using go CA). Methods that can be used to quantify CYSKP expression include radiolabeling of nucleotides.
Alternatively, biotin labeling, coamplification of regulatory nucleic acids, and standard
Includes curves plus results (eg Melby, P.C. et al. (1993) J. Imm
unol. Methods, 159: 235-44; Duplaa, C. et al. (1993) Anal. Biochem. 229-236.
See). Oligomers of interest are present in various dilutions and can be used for spectrophotometric or colorimetric reactions.
By performing a high-throughput format assay that results in rapid quantitation.
Therefore, the quantification rate of a plurality of samples can be accelerated. In yet another example, an oligonucleotide derived from any of the polynucleotide sequences described herein is used.
Rigonucleotides or longer fragments are used as elements in microarrays.
Can be used. Monitor related expression levels of multiple genes simultaneously
It is possible to use microarrays for transfer imaging technology. This
Will be described below. Microarrays also include genetic variants, mutations and polymorphisms.
It can be used for form identification. Use this information to determine gene function
, Understanding the genetic basis of the disease, diagnosing the disease, and treating the disease as a function of gene expression.
To monitor the progress / regression of drugs and to develop and monitor drug activity in disease treatment
You can In particular, select the most appropriate and effective treatment for the patient.
In order to develop a pharmacogenomic profile of a patient, this information can be used to:
Wear. For example, based on the patient's pharmacogenomic profile, the
It is possible to select a therapeutic drug that is effective and shows few side effects. In another embodiment, CYSKP, CYSKP fragments, and CYSKP-specific antibodies are microarrayed.
It can be used as the upper element. Using a microarray,
Such protein-protein interactions, drug-target interactions and gene expression
It is possible to monitor or measure the feel. Certain embodiments may be designed to generate a transcriptional image of a tissue or cell type.
Related to the use of the light polynucleotide. The transferred image can be specific tissue or
Cell types represent global patterns of gene expression. Comprehensive gene expression pattern
Quantifies the number and relative abundance of genes expressed at a given time under given conditions
(Seilliamer et al., US Patent No. 5,840,484, "Comparative").
 Gene Transcript Analysis ", which is hereby incorporated by reference for this patent.
As part of the book). Therefore, total transcription or reverse transcription of a particular tissue or cell type.
By hybridizing the polynucleotide of the present invention or its complement to the body
, Can produce a transfer image. In one embodiment, the polynucleotide of the invention or
Is the complement whose complement contains multiple subsets of the elements on the microarray.
Hybridization occurs in physical format. The resulting roll
Photographic images can provide a profile of gene activity. Transcriptional images are isolated from tissues, cell lines, biopsies or biological samples thereof.
Generated transcripts. The transfer image is therefore a tissue or biopsy sample
In Case ofin vivo, Or in the case of cell linesin vitroReflects gene expression in
To do. The transcription image that produces the expression profile of the polynucleotide of the invention is also
, Not only toxicity tests of industrial or natural environmental compounds,in vitroModel system and
It may be used in connection with preclinical evaluation of drugs. All compounds have a mechanism of action and toxicity.
Often referred to as a molecular fingerprint or toxicity signature that implies nysmism
(Nuwaysir, E.F. et al. (19
99) Mol. Carcinog. 24:15 3-159, Steiner, S. and N.L. Anderson (2000).
Toxicol. Lett. 112-113: 467-471, which is specifically incorporated by reference herein.
Part of). Test compound has the same signature as a compound with known toxicity
May have shared toxicological properties. fin
Garprints or signatures originate from many genes and gene families.
Most useful and accurate if it contains current information. Ideally, the expression
Measurements across the nom provide the highest quality signature. Any expression of self
Even if genes that are unchanged by the compound tested are equally important,
The expression level of the gene is used to normalize the remaining expression data. The standardization procedure is different.
It is useful for comparison of expression data after treatment with other compounds. To the elements of the toxicity sign
Assigning the gene function of Escherichia coli helps prevent toxic mechanisms, but predicts toxicity
Knowledge of gene function is required to statistically match signatures leading to
No (for example, February 29, 2000, National Institute of Environmental He
See Press Release 00-02 published by alth Sciences. About this
Are available at http://www.niehs.nih.gov/oc/news/toxchip.htm). Obey
Therefore, using the toxicity signature during the toxicological screening, all expressed residues were
The inclusion of a gene array is important and desirable. In some embodiments, treating a biological sample containing nucleic acids with a test compound.
Calculate the toxicity of the test compound by. Nuclei expressed in processed biological samples
Acids are labeled with one or more probes specific for the polynucleotides of the invention.
To produce a transcription level corresponding to the polynucleotide of the present invention.
It can be quantified. Transcription levels in treated biological samples can be determined using untreated biological samples.
Compare with the level being pulled. The difference in transcription levels between the two samples is the processed sample.
The toxic reaction caused by the test compound is shown in the table. Another example is the use of the polypeptide sequences of the invention to analyze tissue or cell type profiles.
Related to analyzing the themes. The term proteome refers to a specific organization or
Refers to the global pattern of protein expression by cell type. Each tamper of the proteome
The mineral components can be individually subjected to further analysis. Proteome expression pattern
Or profile is the number of proteins expressed under a given condition at a given time.
And relative abundance can be quantified for analysis. So of the cellular proteome
Profiles isolate and analyze polypeptides of a specific tissue or cell type.
It can be created by In one embodiment, the sample is sampled by one-dimensional isoelectric focusing.
Protein from the gel and two-dimensional sodium dodecyl sulfate slab gel electrophoresis
Separation is achieved by two-dimensional gel electrophoresis which separates according to the molecular weight.
(Steiner and Anderson, above). Protein is usually Coomassie blue
Or by staining with a gel using a substance such as silver or fluorescent stain,
It is visualized in the gel as dispersed, uniquely located points. Each protein
The optical density of cells is usually proportional to the protein level in the sample. Different sun
Pull, eg, biologically treated or untreated with test compound or therapeutic agent
Compare the optical densities of the protein spots obtained from the sample and
Identify changes in protein spot density. The proteins in the spot are, for example,
Use standard methods with chemical or enzymatic cleavage followed by mass spectrometry.
Sequentially sequence. The identity of the proteins within a spot is its partial distribution
A string, preferably at least 5 contiguous amino acid residues, is designated as a polypeptide of the invention.
It can be determined by comparison with the sequence. In some cases, the definitive protein
Additional sequences are obtained for identification. The proteome profile was determined using a CYSKP-specific antibody.
It can also be created by quantifying In one embodiment, a microarray
Using an antibody as the element above, expose the microarray to the sample for each array.
Determine protein expression levels by detecting protein binding levels to elements
(Lueking, A. et al. (1999) Anal. Biochern. 270: 103-111, Mendoze, L.G.
(1999) Biotechniques 27: 778-788). Detection is a variety of methods known in the art
Can be performed with, for example, a thiol or amino reactive fluorescent compound.
The amount of fluorescent binding in each array element by reacting the proteins in the sample
Can be detected. The toxicity signature at the proteome level is also useful for toxicological screening and
It should be analyzed in parallel with the phototoxicity signature. A protein in a tissue
For quality, there may be poor correlation between transcription and protein abundance (Ande
rson, N.L. and J. Seilhamer (1997) Electrophoresis 18: 533-537), transcription
It does not affect the image so much, but it changes the protein profile.
The proteome toxicity signature can be useful in the analysis of compounds. Furthermore, in body fluids
Since the analysis of the transcripts of the protein is difficult due to the rapid degradation of mRNA, the protein profile
The rulemaking is more reliable and informative in such cases. In another example, a biological sample containing proteins is treated with a test compound.
To calculate the toxicity of the test compound. Emitted in processed biological samples
The revealed proteins are separated so that the amount of each protein can be quantified. Each tamper
The amount of protein is compared to the amount of the corresponding protein in the untreated biological sample.
The difference in the amount of protein between the two samples is the response to the test compound in the treated sample.
Show. Individual proteins sequence amino acid residues of individual proteins
And identify these subsequences by comparison with the polypeptides of the invention.
In another example, a biological sample containing proteins is treated with a test compound.
To calculate the toxicity of the test compound. Proteins from biological samples
Quality is incubated with an antibody specific for the polypeptide of the invention. antibody
The amount of protein recognized by is quantified. In the processed biological sample
The amount of protein is compared to the amount of protein in the untreated biological sample. Both
Differences in the amount of protein in the sample indicate the response to the test compound in the treated sample.
You Microarrays are prepared and used using methods well known in the art.
, And analyze (Brennan, T.M. et al. (1995) US Pat. No. 5,474,796, Schen.
a, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93: 10614-10619, Baldeschweile.
(1995) PCT application No. WO95 / 251116, Shalon, D. et al. (1995) PCT application No. WO9.
5/35505, Heller, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94: 2150-215.
5, Heller, M.J. et al. (1997) US Pat. No. 5,605,662). Various thailand
Microarrays are well known, and for details, seeDNA Microarrays: A Pract ical Approach , M. Schena, Editor. (1999) Oxford University Press, London
Have been described. The document is made a part of this specification by citation in particular.
. In another embodiment of the invention, a nucleic acid sequence encoding CYSKP is also used to produce a native
Creates a hybridization probe useful for mapping nom sequences
It is possible to Can use either coding or non-coding sequences
Yes, and in some instances, non-coding sequences are preferred over coding sequences. For example, multiple relics
During chromosome mapping due to the conservation of coding sequences within members of the gene family
Undesirable cross-hybridization can occur. Nucleic acid sequence
Is a specific chromosome, a specific region of a chromosome or an artificially formed chromosome, for example, a human
Engineered chromosome (HAC), yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), bacterial P1 production
Or to a single-chromosome cDNA library (Harrington,
J.J. et al. (1997) Nat Genet. 15: 345-355, Price, C.M. (1993) Blood Rev. 7:12.
7-134, Trask, B.J. (1991) Trends Genet. 7: 149-154 etc.). Once Mappy
When the nucleic acid sequence of the present invention is used, for example, the inheritance of pathological conditions and specific chromosomal regions
A gene linkage map that correlates with the inheritance of restriction fragment length polymorphism (RFLP)
(Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad
Sci. USA 83: 7353-7357). Fluorescence in situ hybridization (FISH) is compatible with other physical and genetic map data.
(See Heinz-Ulrich, et al. (1995) in Meyers, pp. 965-968.
See). Examples of genetic map data are available in various scientific journals or the Online Mendelian Inher
It can be found on the itance in Man (OMIM) website. Physical chromosome
Correlation between the position of the gene encoding CYSKP on the map and a specific disease, or a specific
Because the predisposition to disease helps determine the DNA regions associated with such disease,
Cloning is performed to determine additional location. Physical mapping techniques such as binding analysis using defined chromosome markers and chromosomes
Specimen in situ hybridization can be used to extend the genetic map. An example
By placing the gene on the chromosome of another mammal, such as a mouse,
Reveal relevant markers even when chromosomal loci are unknown
. This information can be used to identify disease genes using positional cloning and other gene discovery techniques.
It is valuable to the investigators looking for. The disease or syndrome is 11q2 with ataxia telangiectasia
2-23 Regions, etc. are not positioned roughly due to genetic linkage to specific gene regions.
, The relevant gene for further investigation by any mapping to the region.
Alternatively, it can represent a regulatory gene (Gatti, R.A. et al. (1988) Nature 336: 577-
See 580). Between healthy persons, carriers, and infected persons due to translocation, inversion, etc.
Using the nucleotide sequences of the invention to discover differences in chromosomal location in
Good. In another embodiment of the invention, CYSKP, its catalytic or immunogenic fragments or fragments thereof.
Of oligopeptides from compounds in a variety of arbitrary drug screening techniques.
It can be used for ibullery screening. Used for drug screening
The fragments are either free in solution, fixed on a solid support, or kept on the cell surface.
Will be carried or will be located intracellularly. For binding CYSKP to the drug being tested
The formation of the complex according to may be measured. Another method of drug screening has a suitable binding affinity for the protein of interest.
Used to screen compounds with high throughput (Geysen,
(1984) PCT application number WO84 / 03564, etc.). In this way,
Different small test compounds are synthesized on a solid substrate. The test compound is CYSKP,
Alternatively, it is reacted with the fragments and then washed. Next, the combined CYSKP is
It is detected by a known method in the field. Purified CYSKP also screens for the drugs listed above.
It is also possible to coat directly on the plates used in the leaning technique. Alternative method
Uses non-neutralizing antibodies to capture the peptide and immobilize it on a solid support
You can also In another example, a neutralizing antibody capable of binding to CYSKP binds to CYSKP and thus is
Competitive drug screening assays can be used that are particularly competitive with
. In this method, the antibody is used to determine which peptide shares one or more antigenic determinants with CYSKP.
It also detects the presence of tide. In another embodiment, CYSKP-encoding nucleotides are used in developing molecular biology techniques.
Arrays can be used, including, but not limited to, currently known triplet ciphers and singularities.
Provides new technologies that rely on nucleotide sequence properties such as specific base-pair interactions
can do. Without further detailed explanation, those skilled in the art can use the above description to maximize the present invention.
Would be available for. Therefore, the examples described below are for illustrative purposes only.
Nor is it intended to limit the invention in any way. Without further detailed explanation, those skilled in the art can use the above description to maximize the present invention.
Would be available for. Therefore, the specific preferred embodiments described below are
It is for the purpose of illustration and not limitation of the invention. All patent applications, patents, publications, especially US patent applications, mentioned above and below.
No. 60 / 201.960, No. 60 / 202.729, No. 60 / 209.705, No. 60 / 210.149,
And 60 / 213.215 are incorporated herein by reference. (Example)1 Preparation of cDNA library Incyte cDNA is in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA)
It is derived from the cDNA library described in Table 4 and is listed in column 5 of Table 4.
Some tissues were homogenized and dissolved in guanidinium isothiocyanate solution.
, Homogenized and dissolved in a mixture of phenol or a suitable denaturant
There is also woven. The mixture of denaturants is, for example, phenol and guanidinium isothiocyanate.
TRIZOL (Life Technologies), which is a single-phase solution of anate, and the like. as a result
The lysate obtained is centrifuged in cesium chloride or extracted with chloroform.
It was Isopropanol, sodium acetate and ethanol, either, or
Another method was used to precipitate RNA from lysates. To enhance RNA purity, RNA extraction with phenol and precipitation are repeated as often as necessary.
I returned. In some cases RNA was treated with DNase. In most libraries,
Oligo d (T) -linked paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Val
encia CA) or OLIGOTEX mRNA purification kit (QIAGEN)
Was isolated. Alternatively, use another RNA isolation kit, such as the POLY (A) PURE mRNA purification kit.
RNA was isolated directly from tissue lysates using a kit (Ambion, Austin TX). In some cases, RNA will be provided to Stratagene and the corresponding cDNA library
It was sometimes made by atagene. If not, the UNIZAP vector system
(Stratagene) or SUPERSCRIPT plasmid system (Life Technologies)
CDNA is synthesized using the recommended method known in the art or a similar method using
Ibrari was prepared (see Ausubel, 1997, unit 5.1-6.6, etc., cited above). Reverse transcription
Were started with oligo d (T) or random primers. Synthetic oligo nucleotide
The octido adapter is ligated to the double-stranded cDNA, and the cDNA is digested with a suitable restriction enzyme.
It was For most libraries, cDNA size (300-1000 bp) selection is
SEPHACRYL S1000, SEPHAROSE CL2B or SEPHAROSE CL4B column chromatograph
(Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis
Was performed using. Ligation of synthetic oligonucleotide adapter to double-stranded cDNA
And digested the cDNA with a suitable restriction enzyme or enzyme. Suitable plasmids are eg
For example, PBLUESCRIPT plasmid (Stratagene), pSPORT1 plasmid (Life Technol
ogies) or plNCY (Incyte Pharmaceuticals, Palo Alto CA). Recombination
The plasmid is XL1-Blue, XL1-BIueMRF or SOLR of Stratagene, or Lif
Formed into E. coli cells containing DH5α, DH10B or ELECTROMAX DH10B from e Technologies.
The quality has changed.2 Isolation of cDNA clone Using the UNIZAP vector system (Stratagene)in vivoBy excision, or
By cell lysisExample 1The plasmid obtained as above was recovered from the host cell
It was Magic or WIZARD Minipreps DNA Purification System (Promega), AGTC Minipre
p Purification Kit (Edge Biosystems, Gaithersburg MD), QIAWELL 8 Pla from QIAGEN
smid, QIAWELL 8 Plus Plasmid and QIAWELL 8 Ultra Plasmid Purification Systems, R.
Use at least one of the E.A.L.Prep 96 plasmid kits to
Was purified. After precipitation, resuspend in 0.1 ml distilled water, freeze-dry or
It was stored at 4 ° C without freeze-drying. Alternatively, host cell lysates may be used in a high throughput format using direct ligation PCR.
The plasmid DNA was amplified from (Rao, V.B. (1994) Anal. Biochem. 216: 1-14).
. Host cell lysis and thermal cycling processes were performed in a single reaction mixture. Sun
The pull was processed and stored in a 384-well plate for amplification of the plasmid DNA.
Concentration of PICOGREEN dye (Molecular Probes, Eugene OR) and Fluoroskan II fluorescence
Fluorometrically quantified using a scanner (Labsystems Oy, Helsinki, Finland)
.3 Sequencing and analysis Example 2 The Incyte cDNA recovered from the plasmid as described in
Sequenced as shown in. The cDNA sequencing reaction can be performed using standard methods or
Or high processing equipment, such as the ABI CATALYST 800 thermal cycler (Applied Biosy
stems) or PTC-200 thermal cycler (MJ Research)
Spencer (Robbins Scientific) or MICROLAB 2200 (Hamilton) liquid transfer
Processed in combination with transfer system. The cDNA sequencing reaction is performed by Amersham Pharma
reagents or ABI sequencing kits provided by cia Biotech, eg ABI PR
ISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosy
stems) was prepared using the reagents given. Electrophoresis of cDNA sequencing reaction
MEGABACE 1000 DNA seeks for dynamic separation and detection of labeled polynucleotides.
Encing system (Molecular Dynamics) or standard ABI protocol and base pairs
ABI PRISM 373 or 377 sequencing system with calling software
(Applied Biosystems) or other system well known in the art.
A column analysis system was used. Reading frame within the cDNA sequence is standard
(Summary of Ausubel, 1997, unit 7.7, supra). how many of the cDNA sequences
Or selectExample 8The sequence was extended as described in. Polynucleotide sequences derived from the Incyte cDNA sequence include vectors, linkers and
Effectiveness is confirmed by removing poly (A) sequences and masking ambiguous base pairs.
I confirmed. BLAST, dynamic programming and flanking dinucleotide frequency analysis
Based on the algorithm and program used. Incyte cDNA sequence, or its
Translate translations into public databases (eg GenBank primates and rodents, mammals,
Database of vertebrates and eukaryotes, BLOCKS, PRINTS, DOMO, PRODOM) and PF
Hidden Markov model (HMM) based protein family database such as AM
(HMM analyzes the consensus primary structure of gene families
Is a probabilistic approach. For example, Eddy, S.R. (1996) Curr. Opin. Struct
See Biol. 6: 361-365. ) Inquiries are BLAST, FASTA, BLIMPS and HMMER
Was performed using a program based on. The Incyte cDNA sequence is a full-length polynucleotide.
It was constructed to yield an octido sequence. Alternatively, GenBank cDNA, GenBank EST,
Stitched, stretched or Genscan predicted code sequences (Fruit
Examples 4 and 5(See above) was used to extend the population of Incyte cDNA to full length. Phr
Built using programs based on ed, Phrap and Consed, GenMark, BLAST and F
Opening reading frames of cDNAs using ASTA-based programs
It was screened. Complete to derive the corresponding full-length polypeptide sequence
The full length polynucleotide sequence was translated. Alternatively, the polypeptides of the invention are full length
It can start at any methionine residue of the translated polypeptide. Then, GenBank
Protein database (genpept), SwissProt, BLOCKS, PRINTS, DOMO, PROD
Based on databases such as OM and Prosite, Hidden Markov Models (HMM) such as PFAM
Full-length polypeptide by querying the protein family database
The sequences were analyzed. The full-length polynucleotide sequence is also available in the MACDNASIS PRO software.
(Hitachi Software Engineering, South San Francisco CA) and LASE
Analysis was performed using RGENE software (DNASTAR). Polynucleotides and polypeptides
Peptide sequence alignment also calculates the percent match between aligned sequences.
Embedded in the MEGALIGN multi-sequence alignment program (DNASTAR)
Default parameters specified by the CLUSTAL algorithm as they are rare
It is generated using Tools and programs used to analyze and assemble Incyte cDNA and full-length sequences
Outlines the algorithms and algorithms with applicable explanations, references, and threshold parameters.
7 shows. The tools, programs and algorithms used are listed in column 1 of Table 7,
A brief description of is given in column 2. Column 3 is the preferred reference and all references are
It will be referred to as it is as a part of this specification. Row 4 if applicable
Is the score, probability value, or other parameter used to evaluate the strength of matching of the two sequences.
Parameter (higher score indicates higher homology between two sequences). Used for assembly and analysis of full-length polynucleotide and polypeptide sequences
The above program for identification of polynucleotide sequence fragments of SEQ ID NO: 35-68
Is also available. About 20 to about useful for hybridization and amplification techniques
The 4000 nucleotide fragment is shown in Table 4, column 4.4 Identification and editing of coding sequences from genomic DNA Putative cytoskeletal-binding proteins are available in public genomic sequence databases (eg,
gbpri and gbhtg) and run the Genscan gene identification program to identify first
It was Genscan is a universal gene identification program that analyzes genomic DNA sequences from various organisms.
Lamb (Burge, C. and S. Karlin (1997) J. Mol. Biol. 268: 78-94 and
Burge, C. and S. Karlin (1998) Cuff. Opin. Struct. Biol. 8: 346-354.
See). The program links predicted exons and spans methionine to the stop codon.
Form the constructed cDNA sequence. The output of Genscan is
It is a FASTA database of peptide sequences. Maximum range of sequences that Genscan analyzes at one time
The box was set to 30 kb. Which of these Genscan putative cDNA sequences is the cell
To determine whether it encodes a backbone-binding protein, the encoded polypeptide
Were interrogated and analyzed for cytoskeletal binding proteins in the PFAM model. Diving
An existing cytoskeletal binding protein is annotated as a cytoskeletal binding protein.
It was identified based on its homology to the tagged in-site cDNA sequence. Thus
Selected Genscan predicted sequences were then analyzed by BLAST analysis in the public database gbpri
And gbhtg. If needed, compare to the top BLAST hits from genpept
Edit the Genscan predicted sequence by comparing and removing the extra or removed exo
Correct errors in the sequence predicted by Genscan, such as BLAST analysis also
Used to discover the public cDNA coverage of any Incyte cDNA or Genscan predicted sequence
Therefore, we provide evidence of transcription. If Incyte cDNA coverage is available,
This information was used to correct or confirm the Genscan predicted sequence. Full length polynucleo
The tide array isExample 3Incyte cDNA sequence and
And / or obtained by constructing a Genscan predicted coding sequence with the public cDNA sequence
. Alternatively, the full-length polynucleotide sequence may be edited or unedited in the Genscan prediction code.
Completely derived from the D sequence.5 Assembly of genomic sequence data into cDNA sequence data Stitched Sequence The partial cDNA sequence isExample 4Predicted by the Genscan gene identification program described in
The extended exon was used for extension.Example 3Part built as described in
A cDNA maps to genomic DNA to associate the related cDNA and one or more geno
Clusters containing the Genscan exons predicted from the sequence. cDNA and
Algorithms based on graph theory and dynamic programming to integrate nom information
To analyze each cluster and then view, edit, or extend it for full-length distribution.
Potential splice variants were generated that produced the sequence. The length of the entire interval is
Between sequences identified as present in more than one sequence in the
The intervals were considered to be equal by the transition. For example, one cDNA and two genomic sequences
If there is an interval at, then all three intervals are considered equal. This process
, Irrelevant but contiguous genomic sequences can be linked and bridged by a cDNA sequence
. The intervals identified in this way appear along the parent sequence.
Stitched with stitching algorithm as shown, longest possible sequence and mutation
Create an array. Intervals generated along one kind of parent sequence and the linkage of the intervals (cDNA-
cDNA or genomic sequence-genomic sequence is a sequence that changes the type of parent (cDNA-genomic sequence).
Sequence). The resulting stitch sequence is publicly available by BLAST analysis.
Translated into the databases genpept and gbpri and compared. Predicted by Genscan
Inaccurate exons compared to top BLAST hits from genpept
Fixed by. Use additional cDNA sequences or test genomic DNA if necessary
The sequence was further extended.Stretched Sequence The partial DNA sequence was extended to full length by an algorithm based on BLAST analysis.
First, using the BLAST program, GenBank primates, rodents, mammals, spine
For public databases such as animal and eukaryotic databases,Example 3In
The partial cDNA constructed as listed was queried. Next, the closest GenBank
Protein homologues by BLAST analysis for the Incyte cDNA sequence orExample 4GenS described in
The can exons were compared to any of the predicted sequences. High scoring results
The chimeric protein was produced using a pair of segment segments (HSP), and the translated sequence was converted into Gen
Mapped onto the Bank protein homolog. Related to the original GenBank protein homolog
In turn, insertions or deletions can occur within the chimeric protein. GenBank protein
Using homologues, chimeric proteins or both as probes,
The homologous genomic sequence was searched from the genomic database. In this way, partial
The DNA sequence was stretched by the addition of homologous genomic sequences. As a result
The resulting stretch sequence was examined to determine whether it contained the complete gene
.Chromosome mapping of polynucleotide encoding 6 CYSKP The sequences used to assemble SEQ ID NOs: 35-68 were derived from BLAST and Smith-Waterman.
Incyte LIFESEQ database and public domain data using algorithms
Compared to the base sequence. Of these databases consistent with SEQ ID NO: 35-68
Sequences are sequenced and duplicated using construction algorithms such as Phrap.
The clusters of rows were constructed (Table 7). Stanford Human Genome Center (SH
From public sources such as GC), Whitehead Genome Research Institute (WIGR), Genethon, etc.
Clustered using available radiation hybrid and genetic map data
It was determined if the sequences were previously mapped. The mapped array is a class
As a result, the entire sequence of the cluster including the individual sequence numbers is included in the map.
Assigned to the position. Locations on the map are represented as ranges or intervals of human chromosomes. Centi-morgan
Spatial map positions are measured relative to the ends of the p-arms of the chromosome. (Centimeter
Morgan (cM) is a unit of measurement based on the frequency of recombination between chromosomal markers. flat
On average, 1 cM is approximately equal to 1 megabase (Mb) of DNA in humans. Well, this
Values vary widely due to recombination hotspots and coldspots.
It The cM distance is a radiation hybrid marker such that the sequence is contained within each cluster.
Genetic markers mapped by Genethon to provide boundaries for
Based on NCBI "GeneMap99" (http: //www.ncbi.nlm.nih.gpv/genemap) etc.
Using the human genetic map and other sources available to the general public in
Whether the section is located in or near the already identified disease gene map.
I can decide. Thus, SEQ ID NO: 44 stained within the 62.90-64.20 centimorgan interval.
In body 17, SEQ ID NO: 49 is located on chromosome 14 within the 73.70 to 76.40 centimorgan interval.
, SEQ ID NO: 50 is located on chromosome 8 within the 25.80 to 40.30 centimorgan interval, SEQ ID NO: 50
 NO: 54 is located on chromosome 1 within the interval 117.6 to 132.4 centimorgans, SEQ ID NO: 64
Is on chromosome 4 within the 56.7 to 60.5 centimorgan interval, SEQ ID NO: 65 is 141.40?
Each of them was mapped to chromosome 5 within the interval of 142.60 cmorgan.7 Polynucleotide expression analysis Northern analysis is an experimental technique used to detect the presence of transcribed genetic information.
Is a procedure in which labeled RNA is attached to the membrane to which RNA from specific cell types or tissues is bound.
It is involved in the hybridization of cleotide sequences. (Sambrook,
See Chapter 7, Ausubel. F.M. et al., Chapters 4 and 16, etc. ) Using similar computer technology applied to BLAST, GenBank and LifeSeq (Incyte
Identical or related molecules in nucleotide databases such as Pharmaceuticals)
To search. Northern analysis is much more convenient than many membrane-based hybridizations.
fast. In addition, decide whether to classify a particular identity as strict or homologous.
Computer search sensitivity can be changed. Search criteria is
It is a product score and is defined by the following equation.

[Equation 1] The product score takes into account both the similarity between the two sequences and the length of the sequence match. The product score is a normalized value of 0 to 100 and is obtained as follows. Multiply the BLAST score by the sequence identity of the nucleotides and multiply the product by 2
Divide by 5 times the length of the shorter of the two sequences. High Scoring Segment Pair (HSP)
The BLAST score is calculated by assigning a score of +5 to each base that matches and a −4 to each mismatch. Two sequences can share more than one HSP (separated by a gap). If there is more than one HSP, calculate the product score using the base pair with the highest BLAST score. The product score represents the balance between fragmentary convolution and quality of BLAST alignment. For example, a product score of 100 is only obtained if there is 100% match over the shorter length of the two sequences compared. The product score 70 has a 100% match at one end and 70%
It is obtained in either case of overlapping, or the other ends are 88% coincident and 100% overlapped. The product score 50 is obtained when either one end is 100% coincident and has 50% overlap, or the other end is 79% coincident and has 100% overlap. Alternatively, the polynucleotide sequence encoding CYSKP is analyzed against the tissue of origin. For example, one full length sequence is constructed to at least partially overlap the Incyte cDNA sequence (see Example 3 ). Each cDNA sequence is derived from a cDNA library made from human tissue. Human tissues include cardiovascular system, connective tissue, digestive system, embryonic structure, endocrine system, exocrine gland, female reproductive system, male reproductive system, germ cells, blood and immune system, lung, musculoskeletal system, nervous system, pancreas, respiratory system. It is classified into one organism / tissue category such as organ system, sensory organ, skin, stomatognathic system, unclassifiable / mixed, or ureter.
Count the number of libraries in each category and divide by the total number of libraries in all categories. Similarly, each human tissue has the following disease / condition categories: cancer, cell line, development,
It is classified as one of inflammation, nervousness, trauma, cardiovascular, congestion. Count the number of libraries in each category and divide by the total number of libraries in all categories. The resulting percentages reflect disease-specific expression of the cDNA encoding CYSKP. cD
NA sequence and cDNA library / tissue information can be found in the LIFESEQ GOLD database (Inc
yte Genomics, Palo Alto CA). Extension of Polynucleotides Encoding 8 CYSKP Full length polynucleotide sequences were also generated by extending the fragments using oligonucleotide primers designed from the appropriate fragment of the full length molecule. One primer was synthesized to initiate the 5'extension of the known fragment and the other primer was synthesized to initiate the 3'extension of the known fragment. The starting primer has a length of about 22-
30 nucleotides, a GC content of about 50% or more, and the OLIGO 4.06 software (National Bioscie
nces) or another suitable program. Hairpin structures and extension of nucleotides resulting in primer-primer dimers were all avoided. A selected human cDNA library was used to extend the sequence. Additional primers or nested sets of primers were designed when more than one extension was required or desired. High fidelity amplification was obtained by PCR utilizing methods well known to those of skill in the art. PCR was performed in a 96-well plate using a PTC-200 thermal cycler (MJ Research, Inc.). The reaction mixture consisted of template DNA and 200 nmol of each primer, a buffer containing Mg ²⁺ , (NH ₄ ) ₂ SO ₄ and β-mercaptoethanol, Taq DNA.
Polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies
), Pfu DNA polymerase (Stratagene). Primer set, PCI A and PCI B
Was amplified with the following parameters. Step 1: 94 ℃, 3 minutes, step
2: 94 ° C, 15 seconds, Step 3: 60 ° C, 1 minute, Step 4: 68 ° C, 2 minutes, Step 5: Repeat steps 2, 3 and 4 20 times. Step 6: 68 ℃, 5 minutes, Step 7: Store at 4 ℃. Alternatively, amplification was performed on the primer set, T7 and SK +, with the following parameters. Step 1: 94 ℃, 3 minutes, Step 2: 94 ℃, 15 seconds,
Step 3: 57 ℃, 1 minute, Step 4: 68 ℃, 2 minutes, Step 5: Step 2,
Repeat 3 and 4 20 times. Step 6: 68 ℃, 5 minutes, Step 7: Store at 4 ℃. The DNA concentration in each well was dissolved in 1X TE and 0.5 μl of undiluted PCR product.
0 μl PICOGREEN quantification reagent (0.25 (v / v) PICOGREEN; Molecular Probes, Eugene OR
) Is distributed to each well of an opaque fluorometer plate (Coming Costar, Acton MA) to allow the DNA to bind to the reagent. Fluoroskan II (Labsystems Oy, Helsinki, Fin) was used to measure the fluorescence of the sample and quantify the DNA concentration.
land). Aliquots of 5-10 μl of the reaction mixture were analyzed by electrophoresis on a 1% agarose minigel to determine which reaction was successful in extending the sequence. The extended nucleotide was desalted and concentrated, transferred to a 384-well plate, and subjected to CviJI.
Cholera virus endonuclease (Molecular Biology Research, Madison WI
) And was sonicated or sheared before the religation reaction into pUC 18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on a low concentration (0.6-0.8%) agarose gel, the fragments excised and the agar digested with Agar ACE (Promega). The extended clones were religated into pUC 18 vector (Amersham Pharmacia Biotech) using T4 ligase (New England Biolabs, Beverly MA) and Pfu DNA polymerase (Stratagen
E. coli cells were transfected by treatment with e) to fill the overhang of the restriction site. The transfected cells were selected and transferred to a medium containing antibiotics, each colony was cut off, and cultured in a 384-well plate of LB / 2X carbenicillin culture solution at 37 ° C. overnight. Lyse cells and lyse with Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu D
DNA was PCR amplified using NA polymerase (Stratagene) by the following procedure. Step
1: 94 ℃, 3 minutes, step 2: 94 ℃, 15 seconds, step 3: 60 ℃, 1 minute, step
4: 72 ° C, 2 minutes, step 5: Repeat steps 2, 3 and 4 29 times. Step 6: 72 ℃, 5 minutes, Step 7: Store at 4 ℃. DNA was quantified with PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recovery were reamplified using the same conditions as above. The sample is 20% dimethyl sulfoxide (1:
2, v / v), DYENAMIC Energy Transfer Sequencing Primer, and DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or ABI
PRISM BIGDYE Terminator Cycle Sequencing Reaction Kit (Termina
Sequencing was performed using the tor cycle sequencing ready reaction kit) (Applied Biosystems). Similarly, the full length nucleotide sequences are verified using the procedures described above, or 5'regulatory sequences are obtained using oligonucleotides designed for such extensions and appropriate genomic libraries. 9. Labeling and Use of Individual Hybridization Probes Using the hybridization probes from SEQ ID NO: 35-68, cDNA, mRNA
, Or screen genomic DNA. Although the labeling of oligonucleotides consisting of about 20 base pairs is specifically described, virtually the same procedure is used for larger nucleotide fragments. Oligonucleotides were designed using state-of-the-art software, such as OLIGO 4.06 software (National Bioscience), with 50 pmol of each oligomer and 250 μCi [γ ³² P] adenosine triphosphate (Amersham).
Pharmacia Biotech),) and T4 polynucleotide kinase (DuPont NEN, B
oston MA) and used in combination for labeling. Labeled oligonucleotides are labeled SEPHADEX G-25 ultrafine molecular size exclusion dextran bead column (Am
ersham Pharmacia Biotech). Ase I, Bgl II, Eco RI
, Pst I, Xba1 or Pvu II (DuPont NEN) in a typical membrane-based hybridization analysis of human genomic DNA digested with one endonuclease, an aliquot containing 10 ⁷ counts per minute of labeled probe To use. The DNA obtained from each digest was fractionated on a 0.7% agarose gel and subjected to nylon membrane (Nytr
an Plus, Schleicher & Schuell, Durham NH). Hybridization is performed at 40 ° C. for 16 hours. To remove non-specific signals, eg 0.1 ×
Blots are washed sequentially at room temperature under conditions consistent with sodium citrate saline and 0.5% sodium dodecyl sulfate. The hybridization patterns are visualized and compared using autoradiography or an alternative imaging tool. 10 Microarrays Chaining or synthesizing array elements on the surface of a microarray should be accomplished using photolithography, piezoelectric printing (inkjet printing, see Baldeschweiler et al., Supra), mechanical microspotting techniques and their derivatives. Is possible. In each of the above techniques, the substrate should be a solid, non-porous solid (Schena (1999) supra). Recommended substrates include silicon, silica, glass slides, glass chips and silicon wafers. Alternatively, an array similar to dot blotting or slot blotting may be utilized to place and bond the elements to the surface of the substrate using thermal, UV, chemical or mechanical bonding procedures. Regular arrays can be made manually or using available methods and machines and can have any suitable number of elements (Schena, M. et al. (1995) Science.
270: 467-470, Shalon. D. et al. (1996) Genome Res. 6: 639-645, Marshall, A.
And J. Hodgson (1998) Nat. Biotechnol. 16: 27-31.). The full-length cDNA, expressed sequence tag (EST), or fragment or oligomer thereof can be a microarray element. Suitable fragments or oligomers for hybridization can be selected using software known in the art such as Laser GENE software (DNASTAR). Array elements are hybridized with a polynucleotide in a biological sample. The polynucleotide in the biological sample is conjugated to a fluorescent label or other molecular tag to facilitate detection. After hybridization, unhybridized nucleotides are removed from the biological sample and hybridization is detected at each array element using a fluorescence scanner. Alternatively, laser desorption and mass spectrometry can also be used to detect hybridization. The degree of complementarity and relative abundance of each polynucleotide that hybridizes to the elements on the microarray can be calculated. The preparation and use of the microarray in one embodiment is detailed below. Preparation of Tissue or Cell Samples Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly (A) ⁺ RNA is purified using the oligo (dT) cellulose method. Each poly (A) ⁺ RNA sample contained MMLV reverse transcriptase, 0.05 pg / μl oligo (dT) primer (21mer), 1x first
Chain buffer, 0.03 unit / μl RNase inhibitor, 500 μM dATP, 500 μM
DGTP, 500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dC
Reverse-transcribe using TP-Cy5 (Amersham Pharmacia Biotech). Reverse transcription reaction is GE
Use the MBRIGHT kit (Incyte) in 25 volume ml containing poly (A) ⁺ RNA. Specific control poly (A) ⁺ RNA is synthesized by in vitro transcription from non-coding yeast genomic DNA.
Each reaction sample (one labeled with Cy3 and the other labeled with Cy5) was treated with 2.5 ml of 0.5 M sodium hydroxide and incubated at 850 ° C for 20 minutes to stop the reaction.
Decompose RNA. Samples are purified using two consecutive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc. (CLONTECH), Palo Alto CA). After conjugation, the two reaction samples are ethanol precipitated with 1 ml glycogen (1 mg / ml), 60 ml sodium acetate and 300 ml 100% ethanol. Samples of SpeedVAC (Savant Instruments Inc., Holbr
ook NY) to finish and resuspend in 14 μl of 5 × SSC / 0.2% SDS. Microarray Preparation The arrays of the invention are used to generate array elements. Each array element is
Amplify from vector-containing bacterial cells with a cloned cDNA insert. PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert. Array elements are amplified by PCR for 30 cycles from an initial amount of 1-2 ng to a final amount of more than 5 μg. The amplified array element is SEPHACRYL-
Purified using 400 (Amersham Pharmacia Biotech). The purified array element is fixed on a polymer-coated glass slide. Microscope glass slides (Corning) are cleaned by sonication in 0.1% SDS and acetone, and very well with distilled water during and after treatment. 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), Wes)
Chester PA), washed extensively in distilled water, 95%
Coat with 0.05% aminopropylsilane (Sigma) in ethanol. The coated glass slide is cured by heating at 110 ° C. Array elements are added to the coated glass substrate using the method described in US Pat. No. 5,807,522. References to this patent are incorporated herein by reference. 1 μl of the array element DNA with an average concentration of 100 ng / μl was opened using an open capillary printing element (open capillary) using a high-speed mechanical device.
Fill the printing element). The instrument now adds about 5 nl of array element sample per slide. The microarray is UV crosslinked using STRATALINKER UV crosslinker (Stratagene). The microarray was washed once with 0.2% SDS at room temperature and then washed with distilled water 3 times.
Wash once. 0 in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA)
Non-specific binding sites are blocked by incubating the microarray in 0.2% casein for 30 minutes at 60 ° C., followed by washing with 0.2% SDS and distilled water as done previously. Hybridization The hybridization reaction has 9 μl of sample mix containing 0.2 μg each of the Cy3 and Cy5 labeled cDNA synthesis products in 5 × SSC, 0.2% SDS hybridization buffer. The sample mixture is heated to 65 ° C. for 5 minutes, aliquoted on the microarray surface and covered with a 1.8 cm ² coverslip. The array is transferred to a waterproof chamber with a cavity slightly larger than the microscope slide. The inside of the chamber is kept at a humidity of 100 by adding 140 μl of 5 × SSC to the corner of the chamber. The chamber containing the array is incubated at 60 ° C. for approximately 6.5 hours. Arrays were washed in the first wash buffer (1 × SSC, 0.1% SDS) for 10 minutes at 45 ° C. and in the second wash buffer (0.1 × SSC) at 45 ° C. for 10 minutes each 3 Wash once and dry. Detection Reporter-labeled hybridization complex is 488n for excitation of Cy3
For excitation of m, Cy3 is detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating a spectral line at 632 nm.
A 20 × microscope objective (Nikon, Inc., Melville NY) is used to focus the pump laser light onto the array. The slide containing the array is placed on the computer-controlled XY stage of the microscope and raster scanned through the objective lens. The 1.8 cm × 1.8 cm array used in this example was scanned at a resolution of 20 μm. Of the two different scans, the mixed gas multi-line laser continuously excites the two fluorochromes. The emitted light is separated based on wavelength, and two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems) corresponding to two fluorescent dyes are used.
, Bridgewater NJ). The signal is filtered using a suitable filter placed between the array and the photomultiplier tube. The maximum emission wavelength of the fluorescent dye used is 565 nm for Cy3 and 650 nm for Cy5. The instrument can record spectra from both fluorochromes at the same time, but with a suitable filter in the laser source, one scan per fluorochrome and each array is typically scanned twice. The sensitivity of the scan is usually calibrated with the signal intensity produced by the cDNA control species added to a sample mixture of known concentration. Complementary to a specific position on the array
A DNA sequence is included and the intensity of the signal at that position is correlated to a weight ratio of hybridizing species of 1: 100,000. When two samples from different sources (eg cells to be tested and control cells) are each labeled with a different fluorescent dye and hybridized to a single array to identify differentially expressed genes The calibration is performed by labeling a sample of the cDNA to be calibrated with two fluorescent dyes and adding equal amounts of each to the hybridization mixture. The output of the photomultiplier tube is a 12-bit RTI-835H analog-to-digital (A / D) conversion board (Analog Devices, installed in an IBM compatible PC computer).
Inc., Norwood MA). The digitized data is displayed as an image where the signal intensities are mapped using a linear 20 color conversion from the blue (low signal) to the red (high signal) pseudo-color range. The data are also analyzed quantitatively. When two different fluorescent dyes are excited and measured simultaneously, the emission spectra of each fluorescent dye is used, and the data is first corrected for optical crosstalk between the fluorescent dyes (due to the overlap of the emission spectra). A grid is overlaid on the fluorescent signal image, whereby the signal from each spot is collected at each element of the grid. The fluorescent signals within each element are integrated and a number is obtained depending on the average intensity of the signal. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte). 11 The sequence complementary to the sequence encoding the complementary polynucleotide CYSKP, or any part thereof, is a natural C
It is used to reduce or inhibit the expression of YSKP. Although the use of oligonucleotides containing about 15-30 base pairs is described, essentially the same method can be used for fragments of smaller or larger sequences. Using Oligo4.06 software (National Biosciences) and CYSKP coding sequence,
Design appropriate oligonucleotides. The most unique 5 to inhibit transcription
A complementary oligonucleotide is designed from the 'sequence and used to prevent the promoter from binding to the coding sequence. To inhibit translation, complementary oligonucleotides are designed to prevent the ribosome from binding to the CYSKP-encoding transcript. 12 Expression of CYSKP Expression and purification of CYSKP can be performed using a bacterial or virus-based expression system. For expression of CYSKP in bacteria, the cDNA is subcloned into a suitable vector containing an inducible promoter that enhances antibiotic resistance and the transcription level of the cDNA. Such promoters include, but are not limited to, the T5 or T7 bacteriophage promoter associated with the lac operator regulatory element and the trp-lac (tac) hybrid promoter. The recombinant vector is transformed into a suitable bacterial host such as BL21 (DE3). Bacteria with antibiotic resistance express CYSKP when induced with isopropyl β-D thiogalactopyranoside (IPTG). Expression of CYSKP in eukaryotic cells is achieved by infecting insect or mammalian cell lines with the Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as Baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with CYSKP-encoding cDNA by either homologous recombination or bacterial-mediated gene transfer involving transfer plasmid mediated. Viral infectivity is maintained, and strong polyhedron promoters drive high levels of cDNA transcription. Recombinant baculovirus is often used to infect Spodoptera frugiperda (Sf9) insect cells, but can also be used to infect human hepatocytes. In the case of the latter infection, further genetic modification of the baculovirus is required. (Enge
lhard. EK et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3224-3227, Sandig, V
(1996) Hum. Gene Ther. 7: 1937-1945. In most expression systems, CYSKP is used, for example, glutathione S transferase (GST).
, Or a fusion protein synthesized with a peptide epitope tag such as FLAG or 6-His, the affinity-based purification of the recombinant fusion protein from crude cell lysate can be performed rapidly in one step. GST is a 26 kDa enzyme from Schistosoma japonicum, which allows purification of fusion proteins on immobilized glutathione while maintaining protein activity and antigenicity (Amersham Pharmacia Biotech). After purification, the GST moiety can be proteolytically cleaved from CYSKP at specific engineering sites. FLAG
Is an 8-amino acid peptide that allows immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, which is a continuous extension of 6 histidine residues, allows purification on a metal chelate resin (QIAGEN). The method of protein expression and purification is described in Ausubel (1995), supra.
) Described in Chapters 10 and 16. CYSKP purified by these methods can be used directly to carry out the assays of Examples 16 and 17 below. 13 Functional Assays CYSKP function is a function of CYSKP at physiologically elevated levels in mammalian cell culture systems.
Evaluation is by expression of the SKP coding sequence. The cDNA is subcloned into a mammalian expression vector containing a strong promoter that expresses the cDNA at high levels. The vectors of choice were pCMV SPORT plasmid (Life Technologies) and pCR 3
.1 plasmid (Invitrogen), both containing the cytomegalovirus promoter. Human cell lines, eg, endothelial-derived or hematopoietic-derived cell lines, are transiently transfected with 5-10 μg of the recombinant vector using liposome formulations or electroporation. In addition, 1-2 μg of plasmid containing the sequence encoding the labeled protein is co-transfected. Expression of the labeled protein provides a means of distinguishing between transfected and untransfected cells. Moreover, the expression of the cDNA from the recombinant vector can be accurately predicted by the expression of the labeled protein. The labeled protein is
For example, it can be selected from green fluorescent protein (GFP; Clontech), CD64 or CD64-GFP fusion protein. Use automated, laser-optically-based technology, flow cytometry (FCM) to identify transfected cells expressing GFP or CD64-GFP and assess their apoptotic status and other cellular characteristics To do. FCM is
The uptake of fluorescent molecules, which diagnoses phenomena that precede or coincide with cell death, is detected and quantified. Such phenomena include changes in nuclear DNA content measured by DNA staining with propidium iodide, changes in cell size and granularity measured by forward light scattering and 90 ° side light scattering, Down-regulation of DNA synthesis as measured by reduced uptake of bromodeoxyuridine, alteration of protein expression on the cell surface and intracellularly as measured by reactivity with specific antibodies, and cells of fluorescent complex annexin V protein There is a change in plasma membrane composition as measured by binding to the surface. For flow cytometry, see Ormerod, MG (19
94) Flow Cytometry Oxford, New York, NY. The effect of CYSKP on gene expression depends on the sequence encoding CYSKP and CD64 or CD64.
-A highly purified cell population transfected with either GFP can be evaluated. CD64 or CD64-GFP is expressed on the surface of transformed cells and binds to the conserved region of human immunoglobulin G (IgG). Transformed and non-transformed cells can be separated using magnetic beads coated with either human IgG or antibodies against CD64 (DYNAL. Lake Success. NY). mRNA can be purified from cells by methods well known in the art. The expression of mRNA encoding CYSKP and other genes of interest can be analyzed by Northern analysis or microarray technology. Preparation of 14 CYSKP specific antibody Polyacrylamide gel electrophoresis (PAGE; eg, Harrington, MG (1990) M
ethods Enzymol. 1816-3088-495) or substantially purified by other purification techniques, and CYSKP is used to immunize rabbits using standard protocols to generate antibodies.
Alternatively, the CYSKP amino acid sequence can be analyzed using LASERGENE software (DNASTAR) to determine highly immunogenic regions, the corresponding oligopeptides synthesized and used to generate antibodies using methods well known to those of skill in the art. Produce. Selection of suitable epitopes, such as those near the C-terminus or within adjacent hydrophilic regions, is well known in the art (see, eg, Ausubel, 1995, supra, Chapter 11). Usually, an oligopeptide of about 15 residues in length is prepared using ABI 431A using Fmoc chemistry.
Synthesized using a peptide synthesizer (Applied Biosystems) and by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
It is bound to KLH (Sigma-Aldrich, St. Louis MO) to enhance immunogenicity (see Ausubel, 1995, supra). Rabbits are immunized with the oligopeptide-KLM complex in complete Freund's adjuvant. To test the anti-peptide activity and anti-PP activity of the obtained antiserum, peptide or CYSKP was bound to the substrate and 1% BSA was added.
Blocking treatment is carried out using the above method, and the resultant is reacted with a rabbit antiserum, washed, and further reacted with a radioiodine-labeled goat anti-rabbit IgG. 15 Purification of native CYSKP using specific antibody Native PP or recombinant CYSKP is substantially purified by immunoaffinity chromatography using an antibody specific for CYSKP. The immunoaffinity column is formed by covalently linking an anti-CYSKP antibody with a resin for activation chromatography such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech).
After binding, the resin is blocked and washed according to the manufacturer's instructions. A culture solution containing CYSKP is passed through an immunoaffinity column, and the column is washed under conditions capable of preferentially adsorbing CYSKP (for example, in a buffer having a high ionic strength in the presence of a surfactant). The column is eluted under conditions that break the binding between the antibody and CYSKP (for example, with a buffer of pH 2-3 or with a high concentration of chaotropic ion such as urea or thiocyanate ion), and CYSKP is recovered. 16 Identification of molecules that interact with CYSKP CYSKP or biologically active fragments thereof are labeled with ¹²⁵ I Bolton-Hunter reagent (see, eg, Bolton AE and WM Hunter (1973) Biochem. J. 133: 529). . A candidate CYCLE pre-arranged in a multi-well plate was labeled with CY.
Incubate with SKP, wash and assay all wells with labeled CYSKP complex. The data obtained at various CYSKP concentrations are used to calculate values for the number, affinity and association of CYSKP bound to the candidate molecule. Alternatively, a molecule that interacts with CYSKP can be isolated from fields, S. and O. Song (1989, Nature
340: 245-246). Yeast two-hybrid syst
em) and MATCHMAKER system (Clontech) for analysis using a commercially available kit based on a 2-hybrid system. CYSKP is also a PA that uses the high-throughput yeast two-hybrid system.
The THCALLING process (CuraGen Corp., New Haven CT) can be used to determine all interactions between proteins encoded by two large libraries of genes (Nandabalan, K. et al. (2000) US Patent No. No. 6,057,101). 17 Demonstration of CYSKP activity Motility protein activity can be measured by the CYSKP microtubule motility assay. In this assay, recombinant CYSKP is immobilized on a glass slide or similar substrate. Taxol-stabilized bovine brain microtubules (commercially available) in a solution containing ATP and cytosolic extract are poured over the slide. Microtubule movements driven by CYSKP motor activity can be visualized and quantified using video light microscopy and image analysis techniques. CYSKP activity is directly proportional to the frequency and rate of microtubule movement.
Alternatively, the CYSKP assay can measure protein filament formation in vitro . To make the polymer, a CYSKP solution with a concentration above the "limit concentration" is used on the carbon coated grid. Place the appropriate nucleation site in the solution. Grids are negatively stained with 0.7% (W / v) water soluble uranyl acetate and examined by electron microscopy. About 2
The appearance of filaments at 5 nm (microtubules), 8 nm (actin) or 10 nm (intermediate filaments) proves to be protein active. Or Hammell, RL and Hitchcock-DeGregori, SE (1997, J. Biol. C
hem. 272: 22409-22416), the binding affinity of CYSKP for actin can be measured by CYSKP assay. CYSKP and actin are generated from an in vitro recombinant cDNA expression system, and the N-terminal of CYSKP is acetylated by a method well known in the art. Binding of N-terminal acetyl-CYSKP to actin is described by Beckman TL-100.
Co-precipitation at 25 ℃ in a centrifugal centrifuge. Bound and free CYSKP are measured by quantitative densitometry on Coomassie blue stained SDS polyacrylamide gels. Appropriate coupling constants (K _app ) using methods well known in the art for applying data to the equation described by Hammell and Hitchcock-DeGregori (1997, supra ).
And the Hill coefficient (H) is determined. Normalize the ratio of CYSKP and actin measured using densitometry. Hammell and Hitchcock-DeGregori (1997, supra ) showed that the saturation of binding corresponded to a stoichiometry of 0.14 for CYSKP and actin, ie 1 for CYSKP and 7 for actin. The binding of CYSKP to actin is CYS
It is proportional to the activity of KP. Alternatively, CYSKP activity is measured as the ability to bind microtubules. Reversible assembly (Vallee, RB (1982) Methods Enzymol. 134: 89-104), or PEM
Microtubules are purified from adult rat brain by the taxol method (Vallee, RB (1982) J. Cell Biol. 92: 435-442) using a buffer (100 mM PIPES, pH 6.6, 1 mM EGTA, 1 mM MgSO ₄ ). Two cycles of microtubule pellets were resuspended in PEM buffer to separate MAP from tubulin, and Sloboda, RD and Rose
Pass through a 0.1 M magnesium sulfate saturated phosphocellulose column as described by nbaum, JL ((1982) Methods Enzymol. 85: 409-416). The protein-containing fraction is passed through a second phosphocell force column. 20 ml CYSKP (250 mg /
ml) to 80 ml of microtubules (450 mg / ml) or tubulin (300 mg / ml) to bring the total volume to 100 ml and incubate at 37 ° C for 10 minutes in the presence of 1 mM GTP and 50 mM taxol. . The suspension is centrifuged to remove the supernatant and resuspend the microtubule pellet in PEM buffer to the original reaction volume. To calculate the partition of CYSKP between the supernatant and the fractions of the pellet, an equal volume of supernatant and resuspended pellet was placed in SDS sample buffer and stained with Coomassie Brilliant Blue 5-20%. Quantify on a gradient SDS polyacrylamide gel. CYSKP in pellet fraction
Is proportional to the binding of CYSKP to microtubules. Alternatively, CYSKP activity is associated with the ability to complex proteins with proteins and is measured by the ability to regulate growth characteristics of NIH3T3 mouse fibroblasts. The cDNA encoding CYSKP is subcloned into a suitable eukaryotic expression vector. This vector is used to transfect NIH3T3 cells using methods well known in the art.
Transfected cells are compared to non-transfected cells for the following quantifiable properties. That is, properties such as high density growth in culture medium, reduction of adhesion of cells to a substrate, alteration of cell morphology and ability to induce tumor when injected into immunodeficient mice are compared. The activity of CYSKP is proportional to the frequency of cell morphology changes and the extent of their proliferation in CYSKP-transfected NIH3T3 cells. One skilled in the art can make various modifications of the described methods and systems of the present invention without departing from the scope and spirit of the invention. Although the invention has been described with reference to particular preferred embodiments, it should be understood that the scope of the invention should not be unduly limited to such particular embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims. Brief Description of Tables Table 1 outlines the nomenclature for full-length polynucleotide and polypeptide sequences of the invention. Table 2 shows the GenBank identification number and the annotation of the GenBank homolog closest to the polypeptide of the invention. The probability score for matching each polypeptide and its GenBank homolog is also shown. Table 3 sets forth the structural features of the polynucleotide sequences of the invention containing the predicted motifs and domains, along with methods, algorithms and searchable databases for use in analyzing the polypeptides. Table 4 shows the cDNA and genome used to construct the polynucleotide sequences of the invention.
DNA fragments are shown along with selected fragments of the polynucleotide sequence. Table 5 shows a representative cDNA library of the polynucleotide of the present invention. Table 6 is an attached table explaining the tissues and vectors used for the preparation of the cDNA library shown in Table 5. Table 7 shows the tools, programs and algorithms used to analyze the polynucleotides and polypeptides of the present invention, along with applicable descriptions, references and threshold parameters.

[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

[Table 7]

[Table 8]

[Table 9]

[Table 10]

[Table 11]

[Table 12]

[Table 13]

[Table 14]

[Table 15]

[Table 16]

[Table 17]

[Table 18]

[Table 19]

[Table 20]

[Table 21]

[Table 22]

[Table 23]

[Table 24]

[Table 25]

[Table 26]

[Table 27]

[Table 28]

[Table 29]

[Table 30]

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 1/18 A61P 3/10 4C084 3/06 5/14 4H045 3/10 7/00 5/14 7 / 04 7/00 7/06 7/04 9/00 7/06 9/10 101 9/00 9/12 9/10 101 11/00 9/12 11/06 11/00 13/02 11/06 13 / 12 13/02 15/00 13/12 15/08 15/00 17/06 15/08 17/12 17/06 19/02 17/12 19/06 19/02 19/10 19/06 21/00 19 / 10 21/04 21/00 25/00 21/04 25/06 25/00 25/14 25/06 25/16 25/14 25/18 25/16 25/28 25/18 29/00 25/28 101 29/00 31/04 101 31/10 31/04 31/12 31/10 33/00 31/12 35/00 33/00 35/02 35/00 37/02 35/02 37/08 37/02 43/00 105 37/08 111 43/00 105 C07K 14/78 111 16/18 C07K 14/78 16/46 16/18 C12N 1/15 16/46 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 21/08 5/10 C12Q 1/0 2 C12P 21/02 1/68 A 21/08 G01N 33/15 Z C12Q 1/02 33/50 Z 1/68 33/53 D G01N 33/15 M 33/50 33/566 33/53 C12N 15/00 ZNAA 5/00 A 33/566 A61K 37/02 (31) Priority claim number 60 / 209,705 (32) Priority date June 5, 2000 (2000.6.5) (33) Priority claim country United States (US) (31) Priority claim number 60/210, 149 (32) Priority date June 7, 2000 (2000.6.7) (33) Priority claim country United States (US) (31) Priority Right claim number 60 / 213,215 (32) Priority date June 21, 2000 (June 21, 2000) (33) Priority claiming country United States (US) (81) Designated country EP (AT, BE, CH) , CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA , GN, GW, ML, MR, NE, SN, TD, TG ), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM) , AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT , LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Oh- Janus, Janice United States California 94005 Brisbane Golden Eagle Lane 233 (72) Inventor Ryu, Dung Aina Em United States California 95123 San Jose Coy Drive 233 (72) Inventor Bogun, Mariah Earl United States California California 94577・ San Leandro Santiago Road 14244 (72) Inventor Hillman, Jennifer El, USA 94040 ・ Mountain View # 17 ・ Monrode Live 230 (72) Inventor Azimu Zai, Yalda, California 94552 ・ Castro Valley Boulder Canyon Drive 5518 (72) Inventor Lar, Pretty, California 95056, Santa Clara Peaux Box 5142 (72) Inventor Yao, Monique Zie Ameri United States California 94043 Mountainview Frederick Court 111 (72) Inventor Bandman, Olga United States California 94043 Mountainview Anna Ave 366 (72) Inventor Burford, Neil Connecticut United States 06422 Durham Wildwood Circle 105 (72) Inventor Batra, Sagebbe United States California 94087, Sunnyvale # 709, El Camino Real 555 (72) Inventor Kearney, Ryan United States California 94127, San Francisco Woodside Avenue 50 (72) Inventor Polky , Jennifer El California 95118 San Jose Jarvis Court 1511 F Term (reference) 2G045 AA40 DA12 DA13 DA14 DA36 FB02 FB03 4B024 AA01 AA11 CA04 DA02 DA05 EA02 EA04 GA11 HA14 HA15 4 B063 QA18 QA19 QQ20 QQ42 QQ52 QQ79 QR56 QR59 QR80 QR82 QS05 QS24 QS25 QS28 QS34 QS38 4B064 AG27 CA10 CA20 CC24 CE12 DA01 DA13 4B065 AA01X AA9090 AA93 ZA52A2 ZA 520 CAA4 CA22 CA22 CA22 CA02 4A08 ZA25 AA93A02 ZA512 ZA532 ZA552 ZA592 ZA662 ZA682 ZA752 ZA812 ZA892 ZA942 ZA962 ZA972 ZB052 ZB072 ZB112 ZB132 ZB152 ZB212 ZB262 ZB272 ZB332 ZB352 ZB372 ZB382 ZC022 ZC062 ZC312 ZC332 ZC352 4H045 AA10 AA11 AA20 AA30 BA10 BA41 CA40 DA76 DA86 EA20 EA50 FA71 FA74 GA26

Claims (112)

[Claims] 1. A substantially isolated polypeptide selected from the group comprising the following (a) to (d): (A) A polypeptide having an amino acid sequence selected from the group having SEQ ID NOs: 1 to 34 (SEQ ID NO: 1-34). (B) a naturally occurring polypeptide having an amino acid sequence which is at least 90% identical to an amino acid sequence selected from the group having SEQ ID NO: 1-34 (c) from the group having SEQ ID NO: 1-34 Biologically active fragment of a polypeptide having a selected amino acid sequence (d) An immunogenic fragment of a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34. 2. The isolated polypeptide of claim 1 selected from the group having SEQ ID NO: 1-34. 3. An isolated polynucleotide encoding the polypeptide of claim 1. 4. An isolated polynucleotide encoding the polypeptide of claim 2. 5. The isolated polynucleotide of claim 4, selected from the group having SEQ ID NO: 35-68. 6. A recombinant polynucleotide comprising a promoter sequence operably linked to the polynucleotide of claim 3. 7. A cell transformed with the recombinant polynucleotide according to claim 6. 8. A genetic transformant containing the recombinant polynucleotide according to claim 6. 9. A method for producing the polypeptide according to claim 1, comprising the step of: (a) culturing cells transformed with the recombinant polynucleotide under conditions suitable for expression of the polypeptide. And (b) a step of receiving the polypeptide so expressed, wherein the recombinant polynucleotide is a promoter sequence operably linked to the polynucleotide encoding the polypeptide of claim 1. A method comprising: 10. An isolated antibody which specifically binds to the polypeptide of claim 1. 11. A substantially isolated polynucleotide selected from the group comprising the following (a) to (d): (A) a polynucleotide having a polynucleotide sequence selected from the group having SEQ ID NO: 35-68 (b) at least 90% identical to a polynucleotide sequence selected from the group having SEQ ID NO: 35-68 Polynucleotides (e) (a) to (d) complementary to the polynucleotides (d) and (b) which are complementary to the polynucleotides of the natural polynucleotide (c) (a) having such a polynucleotide sequence. ) RNA equivalent 12. An isolated polynucleotide comprising at least 60 contiguous nucleotides of the polynucleotide of claim 11. 13. A method for detecting a target polynucleotide having the sequence of the polynucleotide according to claim 11 in a sample, comprising: (a) at least having a sequence complementary to the target polynucleotide in the sample. A step of hybridizing the sample using a probe containing 20 consecutive nucleotides, and (b) detecting the presence / absence of the hybridization complex, and optionally the amount of the complex, if any. Wherein the probe specifically hybridizes to the target polynucleotide under conditions such that a hybridization complex is formed between the probe and the target polynucleotide or fragment. And how to. 14. The method of claim 13, wherein the probe comprises at least 60 contiguous nucleotides. 15. A method for detecting a target polynucleotide having the sequence of the polynucleotide according to claim 11 from a sample, comprising: (a) amplifying the target polynucleotide or a fragment thereof using polymerase chain reaction amplification. And (b) detecting the presence / absence of the target polynucleotide or its fragment, and optionally detecting the amount of the target polynucleotide or its fragment, if any. how to. 16. A component comprising an effective amount of the polypeptide of claim 1 and a pharmaceutically acceptable excipient. 17. The component of claim 16, wherein the polypeptide comprises an amino acid sequence selected from the group having SEQ ID NO: 1-34. 18. A method for treating a disease or medical condition associated with functional downregulation of CYSKP, comprising the step of administering the component of claim 16 to a patient in need of such treatment. How to treat. 19. A method for screening a compound for confirming its effectiveness as an agonist of the polypeptide according to claim 1, comprising the step of: (a) exposing a sample containing the polypeptide according to claim 1 to the compound. And a step of (b) detecting the agonist activity in the sample. 20. An agonist compound identified by the method according to claim 19,
A component comprising a pharmaceutically acceptable excipient. 21. A method of treating a disease or medical condition associated with reduced expression of functional CYSKP, comprising administering the component of claim 20 to a patient in need of such treatment. How to treat. 22. A method of screening a compound to confirm its efficacy as an antagonist of the polypeptide of claim 1, comprising: (a) exposing a sample containing the polypeptide of claim 1 to the compound. And a step of (b) detecting antagonist activity in the sample. 23. A component comprising an antagonist compound identified by the method of claim 22 and a pharmaceutically acceptable excipient. 24. A method for treating a disease or medical condition associated with functional CYSKP overexpression, comprising the step of administering the component according to claim 23 to a patient in need of such treatment. And treatment method. 25. A method for screening a compound that specifically binds to the polypeptide of claim 1, comprising: (a) binding the polypeptide of claim 1 to at least one test compound under suitable conditions. And a step of (b) detecting the binding of the polypeptide of claim 1 to a test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1. And how to. 26. A method for screening a compound that regulates the activity of the polypeptide according to claim 1, comprising: (a) a condition under which the activity of the polypeptide according to claim 1 is allowed. Binding the polypeptide of claim 1 to at least one test compound; (b) calculating the activity of the polypeptide of claim 1 in the presence of the test compound; and (c) the presence of the test compound. Comparing the activity of the polypeptide of claim 1 below with the activity of the polypeptide of claim 1 in the absence of the test compound, wherein the activity of the polypeptide of claim 1 in the presence of the test compound. A method wherein the altered activity of the polypeptide of claim 1 is indicative of a compound that modulates the activity of the polypeptide of claim 1. 27. A method of screening a compound for confirming the effectiveness of mutant expression of a target polynucleotide having the sequence according to claim 5, comprising: (a) conditions suitable for expression of the target polynucleotide. Exposing a sample containing the target polynucleotide to a compound, (b) detecting mutated expression of the target polynucleotide, and (c) in the presence of a variable amount of the compound and the absence of the compound. Comparing the expression of the target polynucleotide in the presence thereof. 28. A method of calculating toxicity of a test compound, comprising: (a) treating a biological sample containing a nucleic acid with the test compound; and (b) treating the nucleic acid of the biological sample. A process of hybridizing a probe comprising at least 20 contiguous nucleotides of the polynucleotide of claim 11, wherein the hybridization is between the probe and a target polynucleotide of the biological sample. Wherein said target polynucleotide is a polynucleotide comprising a polynucleotide sequence of the polynucleotide of claim 11 or a fragment thereof, wherein the target polynucleotide is formed under conditions that form a specific hybridization complex; c) quantifying the yield of the hybridization complex, and (d) above. Comparing the amount of said hybridization complex in the treated biological sample with the amount of said hybridization complex in the untreated biological sample, wherein said treated biological complex A method wherein the difference in the amount of said hybridization complex in the sample is indicative of the toxicity of said test compound. 29. A diagnostic test method for a condition or disease associated with the expression of CYSKP in a biological sample, comprising: (a) the antibody binding to the polypeptide, and the complex of the antibody and the polypeptide. The presence of said complex comprising the steps of: binding said biological sample to an antibody of claim 10 under conditions suitable to form; and (b) detecting said complex. , A method of correlating with the presence of said polypeptide in said biological sample. 30. The antibody is any one of (a) chimeric antibody, (b) single chain antibody, (c) Fab fragment, (d) F (ab ') ₂ fragment, and (e) humanized antibody. The antibody according to claim 10. 31. A compound comprising the antibody of claim 10 and an acceptable excipient. 32. A method of diagnosing a condition or disease associated with CYSKP expression in a subject, comprising the step of administering to the subject an effective amount of the compound according to claim 31. how to. 33. The compound according to claim 31, wherein the antibody is labeled. 34. A method for diagnosing a condition or disease associated with CYSKP expression in a subject, the method comprising the step of administering to the subject an effective amount of the compound of claim 33. how to. 35. A method for preparing a polyclonal antibody having the specificity of the antibody of claim 10, which comprises (a) a group having SEQ ID NO: 1-34 under conditions that induce an antibody response. A step of immunizing an animal with a polypeptide containing a selected amino acid sequence or an immunogenic fragment thereof, (b) a step of isolating an antibody from the animal, and (c) a polypeptide of the isolated antibody Screened by
Identifying a polyclonal antibody that specifically binds to a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34. 36. An antibody produced by the method of claim 35. 37. A compound comprising the antibody of claim 36 and a suitable carrier. 38. A method for producing a monoclonal antibody using the antibody having the specificity of the antibody according to claim 10, comprising: (a) SEQ ID NO: 1-34 under conditions that induce an antibody reaction. Immunizing an animal with a polypeptide containing an amino acid sequence selected from the group having: or an immunogenic fragment thereof, (b) a step of isolating antibody-producing cells from the animal, and (c) an immortalized cell Fusion of the antibody-producing cells with to form a hybridoma cell producing a monoclonal antibody, (d) culturing the hybridoma cell, and (e) a group having SEQ ID NO: 1-34 And a step of isolating from the cultured monoclonal antibody that specifically binds to a polypeptide having an amino acid sequence selected from. 39. A monoclonal antibody produced by the method of claim 38. 40. A compound comprising the antibody of claim 39 and a suitable carrier. 41. The antibody according to claim 10, which produces the antibody by screening a Fab expression library. 42. The antibody according to claim 10, which produces the antibody by screening a recombinant immunoglobulin library. 43. A method for detecting a polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34, which comprises: (a) under a condition that allows specific binding between the antibody and the polypeptide. , A step of incubating the antibody of claim 10 with a sample, and (b) a step of detecting specific binding, wherein the specific binding is an amino acid selected from the group having SEQ ID NO: 1-34. A method characterized by indicating that a polypeptide having a sequence is present in a sample. 44. A method for purifying a polypeptide having an amino acid sequence selected from the group comprising SEQ ID NO: 1-34, which comprises: (a) under conditions permitting specific binding between the antibody and the polypeptide. A step of incubating the antibody of claim 10 with a sample, (b) separating the antibody from the sample and having a purified polypeptide having an amino acid sequence selected from the group having SEQ ID NO: 1-34 And a step of obtaining. 45. The polypeptide of claim 1, having the amino acid sequence of SEQ ID NO: 1. 46. The polypeptide of claim 1, having the amino acid sequence of SEQ ID NO: 2. 47. The polypeptide of claim 1, having the amino acid sequence of SEQ ID NO: 3. 48. The polypeptide of claim 1, having the amino acid sequence of SEQ ID NO: 4. 49. The polypeptide of claim 1, having the amino acid sequence of SEQ ID NO: 5. 50. The polypeptide of claim 1, having the amino acid sequence of SEQ ID NO: 6. 51. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 7. 52. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 8. 53. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 9. 54. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 10. 55. The polypeptide of claim 1, having the amino acid sequence of SEQ ID NO: 11. 56. The polypeptide of claim 1, having the amino acid sequence of SEQ ID NO: 12. 57. The polypeptide of claim 1, having the amino acid sequence of SEQ ID NO: 13. 58. The polypeptide of claim 1, having the amino acid sequence of SEQ ID NO: 14. 59. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 15. 60. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 16. 61. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 17. 62. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 18. 63. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 19. 64. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 20. 65. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 21. 66. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 22. 67. The polypeptide of claim 1, having the amino acid sequence of SEQ ID NO: 23. 68. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 24. 69. The polypeptide of claim 1, having the amino acid sequence of SEQ ID NO: 25. 70. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 26. 71. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 27. 72. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 28. 73. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 29. 74. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 30. 75. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 31. 76. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 32. 77. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 33. 78. The polypeptide of claim 1 having the amino acid sequence of SEQ ID NO: 34. 79. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 35. 80. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 36. 81. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 37. 82. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 38. 83. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 39. 84. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 40. 85. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 41. 86. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 42. 87. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 43. 88. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 44. 89. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 45. 90. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 46. 91. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 47. 92. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 48. 93. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 49. 94. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 50. 95. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 51. 96. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 52. 97. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 53. 98. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 54. 99. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 55. 100. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 56. 101. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 57. 102. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 58. 103. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 59. 104. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 60. 105. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 61. 106. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 62. 107. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 63. 108. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 64. 109. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 65. 110. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 66. 111. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 67. 112. The polynucleotide of claim 11 having the polynucleotide sequence of SEQ ID NO: 68.