JP2003505029A - GTP binding protein - Google Patents

Abstract

  (57) [Summary] The present invention provides GTP binding-related proteins (GBAPs) and polynucleotides that identify and encode GBAPs. The present invention also provides expression vectors, host cells, antibodies, agonists and antagonists. Furthermore, the present invention also provides a method for diagnosing, treating or preventing a disease associated with the expression of GBAP.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to nucleic acid sequences and amino acid sequences of GTP-binding-related proteins, and uses of these sequences in the diagnosis, treatment and prevention of immune system diseases, reproductive disorders, nervous system diseases and cell signal transduction disorders. Regarding

BACKGROUND OF THE INVENTION Guanine nucleotide binding proteins (GTP binding proteins) are present in all eukaryotic cells and are involved in substance metabolism, cell growth, differentiation, signal transduction, cytoskeletal organization, intracellular vesicle transport and secretion. Functions in processes that include. In higher organisms, GTP-binding proteins are associated with immune responses (Aussel, C. et al. (1988) J. Immunol. 140: 215-220),
Involved in signal transduction controlling processes such as cell proliferation including apoptosis, differentiation and tumor formation (Dhanasekaran, N. et al. (1998) Oncogene 17: 1383-1394)
.

The superfamily of GTP-binding proteins includes translation factors, heterotrimeric GTP-binding proteins (also called G proteins) involved in transmembrane signaling processes, proto-oncogene Ras proteins, and other low molecular weight GTP-binding proteins,
For example, it can be subdivided into groups such as rab, rap, rho, rac, smg21, smg25, YPT, SEC4 and ARE gene products, tubulin, etc. (Kaziro, Y. et al. (1991) Annu.
Rev. Biochem. 60: 349-400).

GTP-binding proteins are involved in protein biosynthesis and are found in prokaryotes, initiation factor 2 (IF-2), elongation factor 2 (EF-Tu) and elongation factor G (EF-G). Initiation factor 2 (eIF-2), elongation factor Iα (EF-Iα), elongation factor 2 (EF
-2) and termination factor 3 (eRF3) (Kaziro, supra). IF-2 facilitates the GTP-dependent binding of tRNA to the small subunit of the ribosome, a step that initiates protein translation. Elongation factors interact with tRNA and GTP as protein biosynthesis progresses.
Promotes the binding of and the displacement of GDP after hydrolysis. eRF3 is involved in the recognition of stop codons and the release of nascent proteins from the ribosome.

Heterotrimeric GTP-binding proteins are composed of three subunits (α, β, γ), which associate as trimers on the inner surface of the plasma membrane during quiescence. Heterotrimeric G proteins are characterized by Gs, Gi, G
It can be classified into q and G12 subgroups. In the quiescent state, the α subunit binds to guanosine diphosphate (GDP) and directs the stimulation of G proteins by activating receptors to replace GDP with guanosine triphosphate (GTP). This substitution results in the separation of the α subunit from the β and γ subunits. The β and γ subunits remain in close association as a dimer. Both the α subunit and the β-γ dimer can interact with effectors individually or cooperatively. The α-subunit-specific GTPase activity hydrolyzes bound GTP to GDP. This restores the subunit to an inactive conformation and reassociates it with the β-γ complex, thereby restoring quiescence (Kaziro, supra). Some subunits also exhibit tissue-specific expression, which has an intrinsic signaling role (Dhanasekaran, supra).

[0006] The α-s class of G protein subunits is susceptible to ADP-ribosylation by pertussis toxin that uncouples the receptor (ie, G protein interactions). This uncoupling inhibits signaling to receptors that reduce cAMP levels, which control ion channels to activate phospholipases. The inhibitory α-I class is also sensitive to mutations by pertussis toxin, which prevents α-I from lowering cAMP levels. Two subunits that do not respond to pertussis toxin mutations
One novel class is α-12, which has sequence homology with α-q that activates phospholipase C and the Drosophila gene concertina and may contribute to the regulation of embryonic development (Simon, MI (1991) Science 252 :. 802-808).

The mammalian G protein β and γ subunits (each about 340 amino acids long) are
Share 80% or more homology. The β subunit (also called β transducin) contains 7 repeat units (each about 43 amino acids long). This WD iteration ie
The G-β repeat motif has regulatory functions such as the yeast WD repeat protein Sec13 involved in vesicle transport, the mammalian WD repeat protein coronin-2 involved in the actin cytoskeleton, and the mammalian WD repeat protein Bop1 involved in growth suppression. Found in various proteins (Garcia-Higuera, I. et al. (1998) J. Biol. Chem. 273: 9041-9049, Okumura, M. et al. (1998) DNA Cell Biol. 17: 779-787. , Pestov, DG et al. (1998) O
ncogene 17: 3187-3197). The activity of the β and γ subunits can be regulated by other proteins such as calmodulin, phosducin, the neuronal protein GAP43 (Clapham, DE and EJ Neer (1993) Nature 365: 403-406). The β subunit sequence is highly conserved among species, suggesting that it plays an essential role in the organization and function of the G protein-coupled system (Van der Voorn, L. and
HL Ploegh (1992) FEBS Lett. 307: 131-134).

Mutations and variant expression of β-transducin protein have been linked to various diseases. Mutations in LIS1, a subunit of human platelet activating factor acetylhydrolase, cause Miller-Dicker gyrus defects. RACK1 binds to activated protein kinase C and RbAp48 binds to retinoblastoma protein. CstF is required for polyadenylation of mammalian pre-mRNA in vitro and is associated with a split stimulator factor subunit. Defects in regulatory β-catenin contribute to neoplastic transformation of human cells. Human F-box protein βTrCP W
The D40 repeat mediates binding to β-catenin, and thus ubiquitin ligase
Regulate catenin degradation (Neer, EJ et al. (1994) Nature 371: 297-300, Hart.
, M. et al. (1999) Curr. Biol. 9: 207-210).

The gamma subunit sequence is more variable than the beta subunit sequence. The gamma subunit sequence is often post-translationally modified from the C-terminus by isoprenylation and carboxymethylation of 4 amino acid cysteine residues. These modifications appear to be required for the interaction of β-γ dimers with membranes and β-γ dimers with other GTP-binding proteins. The β-γ dimer has been shown to regulate the activity of adenylyl cyclase isoforms, phospholipase C and some ion channels.
β-γ dimers have been implicated in receptor-mediated phosphorylation of receptors and have been linked to p21ras-dependent activation of the MAP kinase cascade and recognition of intrinsic receptors by GTP-binding proteins (Clapham, supra). and Neer).

The G protein interacts with various effectors, including adenylyl cyclase (Clapham and Neer, supra). The signaling pathway mediated by cAMP is
It is mitogenic in hormone-dependent endocrine tissues such as the adrenal cortex, thyroid, ovary, pituitary, and testis. Cancer in these tissues has been associated with a mutated activated form of Gα _S known as the gsp (Gs protein) oncogene (see Dhanas, supra).
ekaran). Another effector is the retinal phosphoprotein, phosducin, which forms a specific complex with the retinal G protein β and γ subunits, and the β-γ dimer interacts with the retinal α subunit. The ability to do (Clapham and Neer, supra). For additional G protein effectors, RIN1 (Ras interaction / interference) acts as an effector of H-Ras and interferes with the Ras signaling pathway
, Ras3, which is associated with Ras-like GTPase Rab3A, and Rhoekin, a protein that binds to and inhibits Rho GTPase activity (Han, L. and J. Colicelli (
1995) Mol. Cell Biol. 15: 1318-1323, Brondyk, WH et al. (1995) Mol. Cell Bi
15: 1137-1143, Reid, T. et al. (1996) J. Biol. Chem. 27: 13556-13560).

Small GTP-binding proteins regulate cell growth, cell cycle regulation, protein secretion and intracellular vesicle interactions. Low molecular weight GTP-binding protein responds to
By transducing extracellular signals s from receptors s and activating proteins s by mitogenic signals (Tavitian, A. (1995) CR Seances Soc. Biol. Fi.
l. 189: 7-12). The low molecular weight GTP-binding protein has a single polypeptide of 21 to 30 kD, which can bind and hydrolyze GTP like a subunit of a heterotrimeric GTP-binding protein. , Thus returning from the inactive state to the active state. The intrinsic rate of GTP hydrolysis of GTP-binding proteins is usually very slow, but it can be stimulated by several magnitude measures by GTPase-activating proteins (GAPs) such as β2-chimaerin (Ceyer, M. and Wi
ttinghofer, A. (1997) Cuff. Opin. Struct. Biol. 7: 786-792, Caloca, MJ
(1997) J. Biol. Chem. 272: 26488-26496).

[0012] Small GTP-binding proteins play a critical role in cellular protein transport events such as cytosolic to membrane translocation of proteins and soluble complexes by exchanging GDP for GTP (Ktistakis, NT (1998). BioEssays 20: 495-504)
. In vesicle transport, vesicle-specific identifiers (v-SN)
The interaction between ARE and tSNARE) causes the vesicles to dock to the receptor membrane. The budding process is regulated by GTPases, such as the closely related ADP ribosylation factor (ARF) and SAR proteins, which allow the assembly of complexes and eliminate defective complexes. Can play a role in (Rothrnan, JE and FT Wiela
nd (1996) Science 272: 227-234). Rab proteins control vesicle translocation, protein processing and secretion from and into the membrane for protein localization.
Rho GTP-binding proteins control signaling pathways that link growth factor receptors to actin polymerization required for normal cell growth and differentiation. The ran GTP-binding protein is located in the nucleus of cells and plays a key role in nuclear protein import, regulation of DNA synthesis and cell cycle progression (Hall, A. (1990) Science 249: 635-
640; Scheffzek, K. et al. (1995) Nature 374: 378-381).

The Ras proteins Ras1, Ras2 and G _S α stimulate adenylyl cyclase to affect a wide array of cellular processes, including the determination of whether cells sustain growth or terminal differentiation (previously Out of Kaziro). Stimulation of cell surface receptors activates Ras,
This in turn activates cytoplasmic kinase. Cytoplasmic kinases translocate to the nucleus and activate key transcription factors that regulate gene expression and protein synthesis (
Barbacid, M. (1987) Annu. Rev. Biochem. 56: 779-827, Treisman, R. (1994).
Curr. Opin. Genet. Dev. 4: 96-101). Mutant Ras family proteins that bind to GTP but are unable to hydrolyze GTP are persistently activated and thereby conferred carcinogenicity (Drivas, GT et al. (1990) Mol. Cell. Biol. 10: 1793- 1798)
.

Ras-like proteins have also been linked to tumor suppression. For example, the novel gene NOEY2 encoding a Ras-like protein is expressed in normal ovarian and mammary epithelial cells. However, NOEY2 expression was reduced or suppressed in ovarian and breast tumors, suggesting a role for the NOEY2 gene product in tumor suppression (Yu,
Y. et al. (1999) Proc. Natl. Acad. Sci. USA 96: 214-219).

Irregularities in the GTP-binding protein signaling cascade lead to abnormal activation of leukocytes and lymphocytes, leading to many inflammatory diseases such as rheumatoid arthritis, biliary cirrhosis, hemolytic anemia, lupus erythematosus and thyroiditis It can lead to the tissue damage and destruction seen in immune disorders. Cyclic AM for brain, thyroid, adrenal and gonadal tissue proliferation
Abnormal cell proliferation, including P-mediated stimulation, is G protein-controlled. Mutations in the Gα subunit are found in growth hormone-secreting pituitary growth hormone-secreting cell tumors, hyperactive thyroid adenomas, ovarian neoplasms and adrenal neoplasms (Meij, JTA (1996)
Mol. Cell. Biochem. 157: 31-38, Aussel, supra).

The discovery of new GTP-binding related proteins and polynucleotides encoding them provides new compositions that are useful in the diagnosis, treatment and prevention of immune system disorders, reproductive disorders, nervous system disorders and cell signaling disorders. To meet the demands in the field.

(Outline of the Invention) The present invention collectively refers to “GBAP”, and individually to “GBAP-1”, “GBAP-2”, “GBAP-3”, and “GAP”.
"BAP-4", "GBAP-5", "GBAP-6", "GBAP-7", "GBAP-8", "GBAP-9", "GBAP-10", "GB"
"AP-11", "GBAP-12", "GBAP-13", "GBAP-14", "GBAP-15", "GBAP-16", "GBAP-17"
, "GBAP-18", "GBAP-19", "GBAP-20", "GBAP-21", "GBAP-22", "GBAP-23", "GBA"
P-24, GBAP-25, GBAP-26, GBAP-27, GBAP-28, GBAP-29, GBAP-30
, "GBAP-31", "GBAP-32", "GBAP-33", "GBAP-34", "GBAP-35", "GBAP-36", "GBA"
P-37, GBAP-38, GBAP-39, GBAP-40, GBAP-41, GBAP-42, GBAP-43
, "GBAP-44", "GBAP-45", "GBAP-46", "GBAP-47", "GBAP-48", "GBAP-49", "GBA"
P-50, GBAP-51, GBAP-52, GBAP-53, GBAP-54, GBAP-55, GBAP-56
, "GBAP-57", "GBAP-58", "GBAP-59", "GBAP-60", "GBAP-61", "GBAP-62", "GBA"
It is characterized by intracellular signaling molecules that are substantially purified polypeptides, such as called P-63 "," GBAP-64 "," GBAP-65 "and" GBAP-66 ". In certain embodiments, the invention provides at least 90% homology with (a) an amino acid sequence selected from the group having SEQ ID NOS: 1-66, and (b) an amino acid sequence selected from the group having SEQ ID NOS: 1-66. Having a naturally occurring amino acid sequence, (c) a biologically active fragment of an amino acid sequence selected from the group having SEQ ID NOS: 1 to 66, or (d) an immunogenicity of an amino acid sequence selected from the group having SEQ ID NOS: 1 to 66 Provided is a substantially isolated polypeptide, including fragments. In one embodiment, there is provided a substantially isolated polypeptide comprising an amino acid sequence selected from the group having SEQ ID NOs: 1-66.

The present invention also has at least 90% homology with (a) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 66 and (b) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 66. A natural amino acid sequence, (c) a biologically active fragment of an amino acid sequence selected from the group comprising SEQ ID NOs: 1 to 66, or (d) SEQ ID NO: 1
There is provided a substantially isolated polynucleotide encoding a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group having In one embodiment, the polynucleotide encodes a polypeptide selected from the group having SEQ ID NOs: 1-66. In another embodiment, the polynucleotide is selected from the group having SEQ ID NOs: 67-132.

The present invention further has at least 90% homology with (a) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 66 and (b) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 66. A natural amino acid sequence, (c) a biologically active fragment of an amino acid sequence selected from the group comprising SEQ ID NOs: 1 to 66, or (d) SEQ ID NO: 1
Recombinant polynucleotides having a promoter sequence operably linked to a substantially isolated polynucleotide encoding a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group comprising 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.

The present invention also has at least 90% homology with (a) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 66 and (b) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 66. A natural amino acid sequence, (c) a biologically active fragment of an amino acid sequence selected from the group comprising SEQ ID NOs: 1 to 66, or (d) SEQ ID NO: 1
There is provided a method of producing a substantially isolated polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group having The production method comprises: (a) culturing cells transformed with a recombinant polynucleotide under conditions suitable for expression of the polypeptide; and (b) receiving the polypeptide thus expressed. And the recombinant polynucleotide has a promoter sequence operably linked to the polynucleotide encoding the polypeptide.

The present invention further has at least 90% homology with (a) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 66 and (b) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 66. A natural amino acid sequence, (c) a biologically active fragment of an amino acid sequence selected from the group comprising SEQ ID NOs: 1 to 66, or (d) SEQ ID NO: 1
There is provided a substantially isolated antibody which specifically binds to a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group having

The invention further provides at least 70% homology with (a) a polynucleotide sequence selected from the group having SEQ ID NOS: 67 to 132 and (b) a polynucleotide sequence selected from the group having SEQ ID NOS: 67 to 132. A natural polynucleotide sequence having: (c) a polynucleotide sequence complementary to (a), (d) a polynucleotide sequence complementary to (b), or RNA equivalents of (e) (a) to (d). A substantially isolated polynucleotide comprising the same is provided. 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 has at least 70% homology with (a) a polynucleotide sequence selected from the group having SEQ ID NOS: 67 to 132 and (b) a polynucleotide sequence selected from the group having SEQ ID NOS: 67 to 132. A natural polynucleotide sequence having, (c) a polynucleotide sequence complementary to (a), (d) (
Provided is a polynucleotide sequence complementary to b), or a substantially isolated polynucleotide comprising (e) (a)-(d) RNA equivalents. The detection method includes (a) a step of hybridizing the sample with a probe containing at least 20 consecutive nucleotides having a sequence complementary to the target polynucleotide in the sample, and (b) the presence of a hybridization complex. A probe is provided under conditions that detect the absence and optionally detect the amount of complex, if present, under conditions such that a hybridization complex is formed between the probe and the target polynucleotide. It 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 has at least 70% homology with (a) a polynucleotide sequence selected from the group having SEQ ID NOS: 67 to 132 and (b) a polynucleotide sequence selected from the group having SEQ ID NOS: 67 to 132. A natural polynucleotide sequence having, (c) a polynucleotide sequence complementary to (a), (d) (
Provided is a polynucleotide sequence complementary to b), or a substantially isolated polynucleotide comprising (e) (a)-(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 pharmaceutical ingredient comprising an effective amount of the polypeptide and a pharmaceutically acceptable excipient. An effective amount of a polypeptide is a natural product having at least 90% homology with (a) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 66 and (b) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 66. Or (c) a biologically active fragment of the amino acid sequence selected from the group having SEQ ID NOS: 1 to 66, or (d) an immunogenic fragment of the amino acid sequence selected from the group having SEQ ID NOS: 1 to 66. Including. In one embodiment, the pharmaceutical ingredient comprises an amino acid sequence selected from the group having SEQ ID NOs: 1-66. The present invention further provides a method for treating a disease or medical condition associated with reduced expression of functional GBAP, the method comprising the step of administering a pharmaceutical ingredient to a patient in need of such treatment.

The present invention also has at least 90% homology with (a) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 66 and (b) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 66. A natural amino acid sequence, (c) a biologically active fragment of an amino acid sequence selected from the group comprising SEQ ID NOs: 1 to 66, or (d) SEQ ID NO: 1
A method for screening a compound for confirming the effectiveness as a agonist of a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group having Nos. The screening method includes (a) exposing a sample having a polypeptide to a compound, and (b) detecting an agonist activity in the sample. In one embodiment, the invention provides a method of treating a disease or condition associated with reduced expression of functional GBAP, the method comprising the step of administering a pharmaceutical ingredient to a patient in need of such treatment. provide.

The invention further has at least 90% homology with (a) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 66 and (b) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 66. A natural amino acid sequence, (c) a biologically active fragment of an amino acid sequence selected from the group comprising SEQ ID NOs: 1 to 66, or (d) SEQ ID NO: 1
Provided is a method of screening a compound for confirming the effectiveness as a antagonist of a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group having Nos. The screening method includes (a) exposing the sample containing the polypeptide to the compound, and (b) detecting the antagonist activity in the sample. In one embodiment, the invention provides a pharmaceutical ingredient comprising an antagonist compound identified by this method and a pharmaceutically acceptable excipient. In another embodiment, there is provided a method of treating a disease or condition associated with overexpression of functional GBAP comprising administering a pharmaceutical ingredient to a patient in need of such treatment. .

The invention further has at least 90% homology with (a) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 66 and (b) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 66. A natural amino acid sequence, (c) a biologically active fragment of an amino acid sequence selected from the group comprising SEQ ID NOs: 1 to 66, or (d) SEQ ID NO: 1
A method for screening a compound that specifically binds to a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group having Nos. 66 to 66 is provided. The screening method comprises: (a) a process of binding the polypeptide to at least one test compound under appropriate conditions; and (b) a compound that detects the binding of the polypeptide to the test compound and thereby specifically binds to the polypeptide. And a process of identifying.

The invention further has at least 90% homology with (a) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 66 and (b) an amino acid sequence selected from the group having SEQ ID NOS: 1 to 66. A natural amino acid sequence, (c) a biologically active fragment of an amino acid sequence selected from the group comprising SEQ ID NOs: 1 to 66, or (d) SEQ ID NO: 1
Provided is a method of screening for a compound that regulates the activity of a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group having: The screening method includes (a) a step of binding the polypeptide to 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. Indicates compounds that modulate the activity of the polypeptide.

The present invention further provides methods of screening compounds to confirm the efficacy of mutated expression of a target polynucleotide. The target polynucleotide comprises a sequence selected from the group having SEQ ID NOs: 67-132. The screening method is (
a) exposing a sample containing the target polynucleotide to a compound, and (b) detecting the mutated expression of the target polynucleotide.

The present invention further provides a method of assessing the toxicity of a test compound. The toxicity evaluation method includes (a) a step of treating a biological sample containing a nucleic acid with a test compound, and (b) (
i) a polynucleotide sequence selected from the group comprising SEQ ID NOs: 67 to 132, (i
i) a natural polynucleotide sequence having at least 70% homology with a polynucleotide sequence selected from the group comprising SEQ ID NOs: 67 to 132, (iii) a polynucleotide sequence complementary to (i), (iv) (ii) Or a probe comprising at least 20 contiguous nucleotides of a polynucleotide sequence having a polynucleotide sequence complementary to (a), or (v) a polynucleotide sequence selected from the group having RNA equivalents of (i) to (iv) Hybridizing the nucleic acids of the treated biological sample. Hybridization occurs under conditions such that a unique hybridization complex is formed between the probe and the target polynucleotide in the biological sample, and the target polynucleotide includes (i) SEQ ID NO: 67.
A polynucleotide sequence selected from the group consisting of:
To a natural polynucleotide sequence having at least 90% homology with a polynucleotide sequence selected from the group comprising: iii to 132; (iii) a polynucleotide sequence complementary to (i); and (iv) a complementary sequence to (ii). A polynucleotide sequence, and (v) (i)
~ (Iv) RNA equivalents are included. Alternatively, the target polynucleotide comprises a fragment of the above polynucleotide sequence, (c) a step of quantifying the amount of hybridization complex, and (d) an amount of the hybridization complex in the treated biological sample that has not been treated. The difference in the amount of hybridization complex in the treated biological sample is indicative of the toxicity of the test compound.

(Mode for Carrying Out the Invention) The protein, nucleotide sequence and method of the present invention will be described, but the present invention is not limited to the specific devices, materials and methods described above. It should be understood that modifications can be made. 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.

The singular forms “a” and “the (this)” as used in the claims and specification
It should be noted that the notation of may refer to more than one, unless the context clearly indicates otherwise. Thus, for example, where the phrase "a host cell" is used, there may be more than one such host cell, and "a antibody"
When it is described, it also refers to one or more antibodies, and antibody equivalents known to those skilled in the art.

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 that were reported in the publications and which may be relevant to the invention. . Nothing herein is to be admitted that the present invention is not entitled to antedate such disclosure by virtue of prior art.

The definition "GBAP" is the amino acid sequence of substantially purified GBAP, obtained from any species, particularly mammalian species including bovine, ovine, porcine, murine, equine and human, It refers to any natural, synthetic, semi-synthetic or recombinant origin.

The term “agonist” refers to a molecule that enhances or mimics the biological activity of GBAP. Examples of agonists include proteins, nucleic acids, carbohydrates, small molecules and any other compounds or components, which are either directly interacting with GBAP or are involved in the formation of biological pathways involving GBAP. Acts on elements to regulate the activity of GBAP.

“Allelic variants” are another form of the gene encoding GBAP. 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 one or more times within a given sequence.

A “altered” nucleic acid sequence that encodes GBAP has a nucleic acid sequence that deletes, inserts, or replaces various nucleotides, and is a polypeptide that has the same or at least one functional characteristic of GBAP. Produce. Included in this definition are polymorphisms that are easily or difficult to detect using specific oligonucleotide probes for polynucleotides encoding GBAP, and normal chromosomal genes for polynucleotide sequences encoding GBAP. Inappropriate or unexpected hybridization to allelic variants that occupy loci other than loci. The encoded protein may also be "mutated" and may contain deletions, insertions or substitutions of amino acid residues which result in silent changes, resulting in a functionally equivalent GBAP. The deliberate amino acid substitution is based on the similarity of the polarities, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic properties of the residues, as long as the biological or immunological activity of GBAP is retained. obtain. 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 term “amino acid” or “amino acid sequence” refers to a sequence of oligopeptides, peptides, polypeptides or proteins or fragments thereof, which refers to natural or synthetic molecules. As used herein, "amino acid sequence" refers to the amino acid sequence of a naturally occurring protein molecule, and "amino acid sequence" and similar terms limit an amino acid sequence to the complete native amino acid sequence that associates with the listed protein molecules. Not what you are trying to do.

“Amplification” refers to the additional replication 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” refers to a molecule that inhibits or attenuates the biological activity of GBAP. Antagonists can include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or moiety, which are either directly interacting with GBAP or biological in which GBAP is involved. It regulates the activity of GBAP by acting on components of the pathway.

The term “antibody” refers to intact immunoglobulins and fragments thereof, such as Fa, F (ab ′) ₂ and F.
v Fragments, which are capable of binding epitope determinants. Antibodies that bind GBAP polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. 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 bond with 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 of the molecule (ie, the epitope) that is in contact with a particular antibody. When a host animal is immunized with a protein or protein fragment, a large number of regions of the protein are associated with antigenic determinants (specific regions of the protein or 3
Can induce the production of antibodies that specifically bind to (dimensional structure). An antigenic determinant can compete with an intact antigen for binding to an antibody (ie, an immunogen used to induce an immune response).

The term “antisense” refers to the “sense” strand (coding strand) of a particular nucleic acid sequence.
Refers to any component capable of forming base pairs with. For the antisense component, DN
A, 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'-methoxyethoxy sugar There are oligonucleotides having modified sugars, 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. Once introduced into a cell, the complementary antisense molecule base pairs with the native nucleic acid sequence formed by the cell to form a duplex that interferes with transcription or translation. The term "negative" or "minus (-)" may refer to the antisense strand, and "positive" or "plus (+)" may refer to the sense strand.

The term “biologically active” refers to a protein having the structural, regulatory or biochemical functions of a naturally occurring molecule. Similarly, "immunologically active" is the ability of natural, recombinant or synthetic GBAP, or any oligopeptide thereof, to induce a particular immune response in a suitable animal or cell to induce a specific antibody. Refers to the ability to combine.

The terms “complementary” or “complementarity” describe the relationship between two single-stranded molecules that anneal by base pairing. As an example, "5'AG-T3 '" and its complementary sequence "3'T
-There is a C-A5 '.

“Component consisting of a given polynucleotide sequence” and “component consisting of a given amino acid sequence” refer broadly to any component having the given polynucleotide or amino acid sequence. This component may include a dry formulation or an aqueous solution. GBAP
Alternatively, a component consisting of a polynucleotide encoding the GBAP fragment can be used as a hybridization probe. This probe can be stored in a freeze-dried state and can be associated with a stabilizer such as sugar. In hybridization, salt (eg NaCl), detergent (eg sodium dodecyl sulfate; SD
The probe can be dispersed in an aqueous solution containing S) and other constituent elements (eg Denhardt's solution, skim milk powder, salmon sperm DNA, etc.).

The “consensus sequence” is rearranged to separate unnecessary bases, extended in 5 ′ direction and / or 3 ′ direction using XL-PCR kit (PE Biosystems, Foster City CA), It also refers to rearranged nucleic acid sequences. Alternatively, it refers to nucleic acid sequences assembled from overlapping sequences of one or several Incyte clones, optionally one or several public domain ESTs, using a fragment assembly computer program. Examples of computer programs include the GELVIEW fragment assembly system (GCG, Madison WI) and Phrap (University of Washington,
Seattle WA). Some sequences are extended and assembled together to determine consensus sequences.

A “conservative amino acid substitution” is a substitution that does not substantially deteriorate the properties of the original protein when the substitution is made, that is, the structure and particularly the function of the protein are preserved, and there is no significant change due to such substitution. Refers to substitution. 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. Original residue Conservative substitution Ala Gly, Set Arg His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln , Glu Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile , Leu, Thr Conservative amino acid substitutions usually involve (a) a backbone structure of the polypeptide in the substitution region, such as β-sheet or α-helical structure, (b) charge or hydrophobicity of the molecule at the substitution site, and / or (c) side. Holds most of the chains.

“Deletion” refers to a change in an amino acid or nucleotide sequence that results in the loss of one or several amino acids or nucleotides.

The term “derivative” refers to a chemical modification of a polypeptide or polynucleotide sequence. For example, replacement of hydrogen by an alkyl, acyl, hydroxyl or amino group can be included in the chemical modification of the polynucleotide sequence. A polynucleotide derivative encodes a polypeptide that retains at least one biological or immunological function of the natural molecule. A polypeptide derivative is modified by glycosylation, polyethylene pegylation, or any similar process that retains at least one biological or immunological function from the polypeptide of derived origin. Is a polypeptide that has been deleted.

“Detectable label” refers to a reporter molecule or enzyme that is capable of producing a measurable signal and is covalently or non-covalently attached to a polynucleotide or polypeptide.

“Fragment” refers to a unique portion of GBAP or a polynucleotide encoding GBAP that is identical to the parent sequence but shorter in length than the parent sequence. Fragments can have a length that is less than the total length of the defined sequence minus one nucleotide / amino acid residue. For example, a fragment may have 5 to 1000 consecutive nucleotides or amino acid residues. Fragments used as probes, primers, antigens, therapeutic molecules or for other purposes should be at least 5,10,15
, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250
Alternatively, it may be at least 500 contiguous nucleotides or amino acid residues in length. Fragments can be preferentially selected from specific regions of the molecule. For example, a polypeptide fragment may have a length of contiguous amino acids selected from the first 250 or 500 amino acids (or the first 25% or 50% of the polypeptide) as shown in the given sequence. These lengths are explicitly given by way of example, and in the embodiments of the present invention they may be of any length supported by the specification including the sequence listings, tables and figures.

The fragments of SEQ ID NOs: 67-132 contain unique polynucleotide sequence regions. This region uniquely identifies SEQ ID NOS: 67 to 132, and is different from sequences other than SEQ ID NOS: 67 to 132 in the same genome, for example.
Fragments of SEQ ID NOs: 67-132 are provided, for example, in hybridization and amplification techniques or from related polynucleotide sequences.
Is useful in a similar way to distinguish The exact length of a fragment of SEQ ID NO: 67-132 and the region of SEQ ID NO: 67-132 corresponding to the fragment can be routinely determined by one of skill in the art based on the intended purpose for the fragment.

The fragments of SEQ ID NOs: 1 to 66 are encoded by the fragments of SEQ ID NOs: 67 to 132. The fragments of SEQ ID NOS: 1 to 66 include unique amino acid sequence regions that specifically identify SEQ ID NOS: 1 to 66. For example, the fragments of SEQ ID NOS: 1 to 66 are useful as immunogenic peptides for producing antibodies that specifically recognize SEQ ID NOS: 1 to 66. SEQ ID NOS: 1 to 66 and SEQ ID NO: 1 corresponding to the fragments
The exact lengths of the regions 66 to 66 can be routinely determined by those skilled in the art based on the intended purpose for the fragment.

A “full length” polynucleotide sequence is a sequence that has at least one translation initiation codon (eg, methionine), an open reading frame and a translation stop codon. A "full length" polynucleotide sequence encodes a "full length" polypeptide sequence.

The term “homology” refers to sequence similarity, that is, two or more polynucleotide sequences or two
Compatible sequence identity between sequences of two or more polypeptide sequences.

The term “match rate” or “% match” as applied to a polynucleotide sequence refers to the percentage of residues that match between at least two or more polynucleotide sequences aligned using a standardized algorithm. means. Such algorithms insert gaps in the sequences being compared in order to optimize the alignment between the two sequences in a standardized and reproducible manner, thus allowing more significant comparisons of the two sequences.

The percent match between polynucleotide sequences can be determined using the default parameters of the CLUSTAL V algorithm as incorporated in the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package and is a suite of molecular biology analysis programs (DNASTAR, Madison WI).
Is. For CLUSTAL V, Higgins, DG and PM Sharp (1989) CABIOS
5: 151-153 and Higgins, DG et al. (1992) CABIOS 8: 189-191. For pairwise alignments of polynucleotide sequences, the default parameters are set as Ktuple = 2, gap penalty = 5, window = 4, "diagonals saved" = 4. Select the "weighted" residue weighting table as the default. CLUS
TAL V reports concordance as the "similarity" between aligned polynucleotide sequence pairs.

Alternatively, a set of commonly used and freely available sequence comparison algorithms can be found in the Basic Local Alignment Search of the National Center for Biotechnology Information (NCBI).
Tool (BLAST) (Altschul, SF et al. (1990) J. Mol. Biol.
215: 403-410). The algorithm is available from several sources, including NCBI in Bethesda, Maryland, and can be found on the Internet (http://www.ncbi.nl
It is also available on m.nih.gov/BLAST/). The BLAST suite of software includes a variety of sequence analysis programs, one of which is "blastn," which aligns a known polynucleotide sequence with another polynucleotide sequence obtained from a variety of databases. In addition, a tool called "BLAST 2 Sequences" used for directly comparing two nucleotide sequences in pairs is also available. "BLAST 2
"Sequences" can be used interactively by accessing http://www.ncbi.nlm.nih.gov/gorf/bl2.html. "BLAST 2 Sequences" tool, blast
It can be used for both n and blastp (described later). The BLAST program is typically used with gaps and other parameters set to default settings.
For example, blastn may be performed using the "BLAST 2 Sequences" tool Version 2.0.12 (April 21, 2000) set as default parameters to compare two nucleotide sequences. An example of setting default parameters 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 Match rate is fully defined It can be measured relative to the sequence length (eg defined by a particular SEQ ID NO). Alternatively, fragments derived from shorter lengths, eg, larger defined sequences (eg, at least 20, 30, 40, 50, 70, 100).
Alternatively, the concordance rate may be determined by comparison with the length of 200 consecutive nucleotide fragments). The lengths listed here are merely exemplary and are used to determine the percent match using fragments of any sequence length supported by the sequences described herein, including tables, figures and sequence listings. It should be understood that this may account for the length that can be measured.

Nucleic acid sequences that do not exhibit a high degree of identity may nonetheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is to be understood that this degeneracy can be utilized to produce changes in a nucleic acid sequence to produce multiple nucleic acid sequences such that all nucleic acid sequences encode substantially the same protein.

The term “match rate” or “% match” as applied to a polypeptide sequence means the percentage of matching residues between at least two or more polypeptide sequences aligned using a standardized algorithm. To do. Methods for polypeptide sequence alignment are known. There are also alignment methods that consider conservative amino acid substitutions.
Such conservative substitutions detailed above generally preserve the acidity and hydrophobicity of the site of substitution and thus the structure (and thus function) of the polypeptide.

The degree of concordance between polypeptide sequences can be determined using the default parameters of the CLUSTAL V algorithm as incorporated in the MEGALIGN version 3.12e sequence alignment program (see it previously described). CLUSTAL
The default parameters for aligning two pairs of polypeptide sequences using V are set as Ktuple = 1, gap penalty = 3, window = 5, and “diagonals saved” = 5. Select the PAM250 matrix as the default residue weight table. Like polynucleotide alignments, CLUSTAL V reports concordance as the "similarity" between aligned polypeptide sequence pairs.

Alternatively, the NCBI BLAST software suite may be used. For example, when comparing polypeptide sequences in pairs, the blastp set as a default parameter
The "BLAST 2 Sequences" tool Version 2.0.12 (April 21, 2000) may be used with. 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 It can be measured relative to the length of the polypeptide sequence. The concordance rate is a sequence or shorter length,
For example, a fragment obtained from a larger defined polypeptide sequence (eg, a fragment of at least 15, 20, 30, 40, 50, 70 or 150 contiguous residues).
The match rate may be measured by comparing with the length of the. The lengths given here are merely exemplary and may be used with fragments of any sequence length backed by the sequences described herein, including tables, figures and sequence listings. It should be understood that it can be described as a length, for which a match rate can be measured.

“Human Artificial Chromosome” (HAC) is a linear, small chromosome containing a DNA sequence of 6 kb to 10 Mb in size and containing all elements necessary for stable segregation and maintenance of mitotic chromosomes. Has been.

The term “humanized antibody” refers to an antibody molecule in which the amino acid sequence in the non-antibody binding region has been mutated so that it is closer to that of a human antibody, and the original binding ability is retained. Point to.

“Hybridization” refers to the process by which a single-stranded polynucleotide anneals with a complementary strand by forming base pairs under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share high homology. The specific hybridization complex is formed under acceptable annealing conditions and remains hybridized after the "wash" step. The wash step is particularly important in determining the stringency of the hybridization process, and more stringent conditions reduce non-specific binding (ie, pair binding between inconsistent nucleic acid strands).
Permissive conditions for the annealing of nucleic acid sequences are routinely determined by those of ordinary skill in the art. Permissive conditions may be constant during the hybridization experiment, but wash conditions can be varied during the experiment to obtain the desired stringency, and thus hybridization specificity. The annealing permissive conditions are satisfied at a temperature of 68 ° C. in the presence of, for example, about 6 × SSC, about 1% (w / v) SDS and about 100 μg / ml denatured salmon sperm DNA.

In general, hybridization stringency can be expressed in part to the temperature at which the wash step is performed. Such washing temperatures are usually selected to be about 5 to 20 ° C. below the melting point (Tm) of the specific sequence at a given ionic strength and pH. This Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe under a given ionic strength and pH. The formula for calculating the Tm and the hybridization conditions for nucleic acids are well known and are described by Sambrook et al.
(1989) Molecular Cloning: A Laboratory Manual , 2nd edition, Volume 1-3, Cold Spring
See Harbor Press, Plainview NY, see in particular Volume 2, Chapter 9.

High stringency conditions for hybridization between polynucleotides of the invention include 1 at about 68 ° C. in the presence of about 0.2 × SSC and about 1% SDS.
Time wash conditions are included. Alternatively, it may be performed at a temperature of 65 ° C, 60 ° C, 55 ° C or 42 ° C. The SSC concentration can vary in the range of about 0.1 to 2 × SSC in the presence of about 0.1% SDS. Blockers are usually used to block non-specific hybridization. Such blocking agents include, for example, about 100-200 μg / ml denatured salmon sperm DNA. Under certain conditions, it is also possible to use organic solvents, eg formamide at a concentration of about 35-50% v / v, for the hybridization of eg RNA and DNA.
Useful variations of wash conditions will be apparent to those of skill in the art. Hybridization can suggest evolutionary similarities between nucleotides, especially under conditions of high stringency. Such similarities strongly suggest a similar role for nucleotides and nucleotide-encoded polypeptides.

The term “hybridization complex” refers to a complex formed between two nucleic acid sequences by virtue of the ability to form hydrogen bonds between complementary base pairs. A hybridization complex may be formed in solution (C ₀ t or R ₀ t analysis, etc.). Alternatively, one nucleic acid sequence is present in solution and the other nucleic acid sequence is present on a solid support (eg paper, membrane,
It may be formed between two nucleic acid sequences such as filters, chips, pins or glass slides, or other suitable substrate that is immobilized on a substrate on which cells or their nucleic acids are immobilized).

The terms “insertion” and “addition” refer to a change in an amino acid or nucleotide sequence that adds one or several amino acid residues or nucleotide sequences, respectively.

“Immune response” can refer to a condition associated with inflammation, trauma, immune disorders, contagious diseases or genetic disorders. These conditions can be characterized by the expression of various factors, such as cytokines, chemokines, and other signaling molecules that can act on the cellular and systemic defense system.

An “immunogenic fragment” is a polypeptide or oligopeptide fragment of GBAP capable of eliciting an immune response when introduced into an organism such as a mammal. The term "immunogenic fragment" also includes any polypeptide or oligopeptide fragment of GBAP useful in any method of raising antibodies as disclosed herein or as known in the art. ..

The term “microarray” refers to the composition of multiple polynucleotides, polypeptides or other compounds on a substrate.

The term “element” or “array element” refers to a hybridizable polynucleotide located on the surface of a substrate in a microarray environment.

The term “modulate” refers to a change in the activity of GBAP. Modulation can, for example, increase or decrease protein activity, binding properties and other biological, functional or immunological properties of GBAP.

The terms “nucleic acid” and “nucleic acid sequence” refer to nucleotides, oligonucleotides, polynucleotides or fragments thereof. The terms "nucleic acid" and "nucleic acid sequence" refer to DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded, or represent the sense or antisense strand, or It may also refer to peptide nucleic acid (PNA) or any DNA-like or RNA-like substance.

“Operably linked” refers to the condition in which a first nucleic acid sequence is placed into a functional relationship with a second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences may be in close proximity or contiguous if it is necessary to join the two protein coding regions in the same reading frame.

“Peptide nucleic acid” (PNA) is an antisense molecule or anti-genetic substance, consisting of an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of lysine-terminated amino acid residues. Refers to something. The terminal lysine confers solubility to the components. PNAs preferentially bind complementary single-stranded DNA or RNA to stop the expansion of transcription, and can be polyethylene glycolated to prolong PNA lifespan in cells.

“Post-translational modifications” of GBAP may include lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic resolution and other modifications known in the art. These processes can occur synthetically or biochemically. Biochemical modifications depend on the enzymatic environment of GBAP and can vary by cell type.

“Probe” refers to a nucleic acid sequence encoding GBAP, a complementary sequence of GBAP, or a fragment thereof, and is used for the detection of identical nucleic acid sequences, allelic nucleic acid sequences or related nucleic acid sequences. A probe is an isolated oligonucleotide or polynucleotide attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent reagents and enzymes. A "primer" is a short nucleic acid, usually a DNA oligonucleotide, which can anneal to a target polynucleotide by forming complementary base pairs. The primer can then extend to the target DNA strand by a DNA polymerase enzyme. Primer pairs are used to amplify (and identify) nucleic acid sequences by, for example, polymerase chain reaction (PCR).
Can be used for.

Probes and primers as used in the present invention usually contain at least 15 contiguous nucleotides of known sequence. Longer probes and primers to increase specificity, eg at least 20, 25, 30, of the disclosed nucleic acid sequences,
Probes and primers that consist of 40, 50, 60, 70, 80, 90, 100 or at least 150 contiguous nucleotides may be used. Some probes and primers are much longer than this. It is understood that any length of nucleotides supported herein, including tables, figures and sequence listings, can be used.

For preparation and use of probes and primers, see Sambrook, J. et al. (1.
989) Molecular Cloning: A Laboratory Manual , 2nd edition, Volume 1-3, Cold Spring H
arbor Press, Plainview NY, Ausubel, FM et al., (1987) Current Protocols in Molecular Biology , Greene Pubi. Assoc. & Wiley-Intersciences, New York.
NY, Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications Ac
See ademic Press, San Diego CA, etc. PCR primer pairs are computer programs for that purpose, such as Primer (Version 0.5, 1991, Whitehe
Ad Institute for Biomedical Research, Cambridge MA).

Oligonucleotides used as primers are selected for such purposes 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,
It is also useful for analyzing oligonucleotides and polynucleotides of up to 5,000 sizes obtained from input polynucleotide sequences of up to 32 kilobases. A similar primer selection program incorporates additional functionality for expanded capacity. For example, the PrimOU primer selection program (available to the public from the Genome Center at the University of Texas Southwestern Medical Center in Dallas, Texas) is capable of selecting a particular primer from a megabase sequence, thus covering the entire genome. Useful for designing primers. Prim
er3 Primer Selection Program (Whitehead I, Cambridge, MA)
(available publicly from the Institute / MIT Genome Research Center)
A "mispriming library" can be input, and the user specifies the sequence to be avoided as a primer binding site. Primer3 is particularly useful for the selection of oligonucleotides for microarrays (the source code of the latter two primer selection programs may be modified from individual sources to meet user-specific needs). The PrimerGen Program (UK Human Genome Mapping Project in Cambridge, UK-Publicly available from the Resource Center) designs primers based on multiple sequence alignments, thereby maximally or minimally conserving the aligned nucleic acid sequences. Allows selection of primers to hybridize with any of the regions. Therefore, this program is useful for identifying unique and conserved fragments of oligonucleotides and polynucleotides. Oligonucleotide and polynucleotide fragments identified by any of the above selection methods can be used to identify fully or partially complementary polynucleotides in hybridization techniques, such as PCR or sequencing primers, as microarray elements, or in nucleic acid samples. It is useful as a specific probe. The method for selecting the oligonucleotide is not limited to the above method.

A “recombinant nucleic acid” is a sequence that is either a non-native sequence or a sequence that has been produced by artificially combining two or more segments of a sequence that would be separated unless artificially combined. . This artificial combination is often accomplished by chemical synthesis, but more commonly by artificial manipulation of isolated segments of nucleic acids, such as by genetic engineering techniques such as those described in Sambrook et al., Supra. To do. The term recombinant nucleic acid also includes mutant nucleic acids in which a portion of the nucleic acid is simply added, substituted or deleted. Frequently, recombinant nucleic acids include a nucleic acid sequence operably linked to a promoter sequence. Such recombinant nucleic acid may be an integral element of the vector, such as that used to transform a cell.

Alternatively, such recombinant nucleic acid may be an integral element of the viral vector, for example based on vaccinia virus. Vaccinia virus is used for vaccination of mammals in which recombinant nucleic acid is expressed and induces a protective immune response in mammals.

A “regulatory element” is a nucleic acid sequence usually derived from the untranslated region of a gene, including enhancers, promoters, introns and 5 ′ and 3 ′ untranslated regions (UTRs).
)including. Regulatory elements interact with host or viral proteins that regulate transcription, translation or RNA stability.

A “reporter molecule” is a chemical or biochemical moiety used to label nucleic acids, amino acids or antibodies. Reporter molecules include radionuclides, enzymes,
Fluorescent agents, chemiluminescent agents, chromogenic agents, substrates, cofactors, inhibitors, magnetic particles and other ingredients known in the art.

An “RNA equivalent” with respect to a DNA sequence is a reference DNA, except that all of the generated nitrogen base thymine has been replaced with uracil and that the sugar backbone is composed of ribose rather than deoxyribose. It is composed of a linear nucleotide sequence identical to the sequence.

The term “sample” is used in its broadest sense. A sample suspected of containing GBAP-encoding nucleic acid or fragments thereof, or GBAP itself, may be extracted from body fluids, cells isolated from cells, chromosomes, organelles or membranes, cells, or lysed. Or may be composed of genomic DNA, RNA or cDNA bound to a substrate, tissue, tissue print, or the like.

The term “specific binding” or “specifically binds” refers to the interaction between a protein or peptide and an agonist, antibody, antagonist, small molecule, any natural or synthetic binding moiety. This interaction is meant to depend on the presence or absence of a particular structure of the protein (eg an antigenic determinant or epitope) that the binding molecule recognizes. For example, if the antibody is specific for epitope "A", the presence of the polynucleotide containing free unlabeled A in a reaction involving epitope A, free unlabeled A, and the antibody will result in a label that binds to the antibody. A
Will be reduced.

The term “substantially purified” is a nucleic acid or amino acid sequence that has been removed or isolated or separated from its natural environment and is at least about 60%, preferably at least 60%, of the other naturally associated components. Indicates at least about 75%, and most preferably at least about 90% free.

“Substitution” means the replacement of one or several amino acids or nucleotides by different amino acids or nucleotides.

“Substrate” refers to any suitable solid or semi-solid support, including membranes, filters, chips, slides, wafers, fibers, magnetic non-magnetic beads, gels, tubes, plates, polymers. , Fine particles, capillaries are included. The substrate can have various surface morphologies such as walls, grooves, pins, channels, pores, etc., on which the polynucleotide or polypeptide binds.

“Transcriptional image” refers to the collective pattern of gene expression by a unique cell type or tissue at a given time and condition.

“Transformation” refers to the process by which exogenous DNA enters a host cell and modifies the host cell. Transformation can occur under natural or artificial conditions according to various methods known in the art and is based on any known method of inserting an exogenous nucleic acid sequence into a prokaryotic or eukaryotic host cell. You can The method of transformation is selected according to the type of host cell to be transformed. Transformation methods include, but are not limited to, methods using viral infection, electroporation, heat shock, lipofection and particulate irradiation. The term "transformed" cell includes not only transiently transformed cells that express the inserted DNA or RNA within a limited time, but also stably transformed cells. There is also included one in which the DNA inserted therein is capable of replicating autonomously as a plasmid or as a part of the host chromosome.

As used herein, a “genetic transformant” is any organism, including but not limited to animals and plants, in which one or several cells of the organism are , Having a heterologous nucleic acid introduced, for example, by transformation techniques well known in the art. The introduction of the nucleic acid into the cells is carried out directly or indirectly by introducing them into the precursors of the cells, by deliberate genetic manipulation, for example by microinjection or by introduction of recombinant virus. The term genetic engineering does not refer to classical cross breeding or in vitro fertilization, but to the introduction of recombinant DNA molecules. Genetic transformants contemplated according to the present invention include bacteria, cyanobacteria, fungi and plants and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, such as infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the invention to such organisms are well known and provided in the references such as Sambrook et al. (1989) supra.

A “variant” of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence homology with the particular nucleic acid sequence over the length of the entire nucleic acid sequence. At that time, "BLAST 2 Sequences" tool Version 2.0 set to default parameters
.9 (May 7, 1999) with blastn. Such nucleic acid pairs may be, for example, at least 50%, 60%, 70%, 80%, 85%, 90%, for a given length.
It may show 95%, 98% or more homology. Certain variants may be described as, for example, "allelic" variants (supra), "splice" variants, "species" variants or "polymorphic" variants. Splice variants may have considerable homology to the reference molecule, but will typically have a large or small number of polynucleotides due to the alternative splicing of exons during mRNA processing. The corresponding polypeptide may have additional functional domains or may lack domains present in the reference molecule. Species variants are polynucleotide sequences that differ from one another. The resulting polypeptides usually have significant amino acid homology to each other. Polymorphic variants differ in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants may also include "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence changes by one nucleotide base. The presence of SNPs may indicate, for example, a particular population, medical condition or propensity.

A “variant” of a particular polypeptide sequence is defined as a polypeptide sequence that has at least 40% homology to the particular polypeptide sequence over the length of one of the polypeptide sequences. Here, "BLAST
2 Sequences ”tool Version 2.0.9 (May 7, 1999) to run blastp. Such a polypeptide pair may have, for example, at least 50% for a given length.
, 60%, 70%, 80%, 90%, 95%, 98% or more homology.

Invention The present invention relates to a novel GTP binding-related protein (GBAP), a polynucleotide encoding GBAP, and a method for diagnosing, treating and preventing an immune system disease, a reproductive disorder, a nervous system disease and a cell signal transduction disorder. It is based on the discovery of the method of utilizing the sequence of.

Table 1 shows the Incyte clones used to construct the full-length nucleotide sequence encoding GBAP. Columns 1 and 2 show the SEQ ID NOs for the polypeptides and polynucleotides, respectively (SEQ ID NO). Column 3 shows the clone ID of the Incyte clone and the nucleic acid encoding each GBAP is the one identified here. Column 4 shows the cDNA library and the clone in column 3 was isolated from it. Column 5 is
Incyte clones and corresponding cDNA libraries are shown. The Incyte clone for which the cDNA library is not shown was obtained from a pooled cDNA library. The Incyte clone in row 5 was used to construct a consensus nucleotide sequence for each GBAP. The Incyte clone in row 5 is useful as a fragment in hybridization techniques.

The columns of Table 2 show various properties of each polypeptide of the invention. Column 1 is the SEQ ID NO, column 2 is the number of amino acid residues in each polypeptide, column 3 is the potential phosphorylation site, column 4 is the potential glycosylation site, column 5 is Sign
ature) amino acid residues having a sequence and a motif, column 6 shows a homologous sequence identified by BLAST analysis, and column 7 shows an analytical method and, in some cases, a searchable database in which the analytical method can be used. The analytical methods in column 7 were used to characterize each polypeptide from sequence homology and protein motifs.

The columns of Table 3 show the tissue specificity and the disease, disorder or condition associated with the nucleotide sequence encoding GBAP. Column 3 of Table 3 shows the nucleotide sequence numbers and column 2 shows the fragments of the nucleotide sequences of column 1. These fragments are useful in, for example, hybridization or amplification techniques to identify SEQ ID NOS: 67-132 and distinguish the polynucleotide sequences associated with SEQ ID NOS: 67-132. Polynucleotides encoded by these fragments are useful, for example, as immunogenic peptides. Column 3 shows the tissue categories expressing GBAP as a percentage of GBAP expression in the whole tissue. Column 4 shows diseases, disorders or conditions associated with GBAP-expressing tissues as a percentage of total GBAP-expressing tissues. Column 5 shows the vector used to subclon each cDNA library. Expression of SEQ ID NO: 84 in lung tissue and SEQ ID NO: 132
Of particular note is the tissue-specific expression of. Over 90% of tissues expressing SEQ ID NO: 132 are derived from the nervous system (particularly the brain).

The columns of Table 4 provide a description of the tissues used to create the cDNA library. GBAP
The clone of the cDNA coding for was isolated from this cDNA library.
Column 1 shows the nucleotide sequence number, column 2 shows the cDNA library that is the clonal isolation source, and column 3 shows the source of tissue and other bibliographic information related to the cDNA library in column 2.

SEQ ID NO: 70 maps to chromosome 7 within the interval 111.6-123.4 centimorgans. This interval contains genes that are reduced in adenomas. SEQ ID NO: 74 maps to chromosome 11 within the interval of 104.8-123.5 centimorgans. This interval includes genes associated with cerebellar degenerative disease, ataxia-telangiectasia. SEQ ID NO: 75 maps to chromosome 17 within the interval of 62.9-65.0 centimorgans. SEQ ID NO: 77 maps to chromosome 3 within the interval of 12.9 to 16.5 centimorgans. SEQ ID NO: 80 is 42.0-57.3
Maps to chromosome 9 within the centimorgan interval. SEQ ID NO: 86 is 159
Maps to chromosome 1 within the interval of 0.6-164.1 centimorgans. SEQ ID NO: 87 maps to chromosome 11 within the interval 147.2-151.6 centimorgans. SEQ ID NO: 90 maps to chromosome 1 within the interval of 219.2-223.0 centimorgans. This interval includes a gene encoding RAB interacting protein. SEQ ID NO: 92 and SEQ ID NO: 106 are both 48.8 to 81.6.
Maps to chromosome 1 within the centimorgan interval. This interval includes familial hypercholesterolemia, glucose transport deficiency, infantile hypoalkaline phosphatase disease, infantile neuronal ceroid lipofuscinosis, Kostmann disease, polyphyseal dysplasia, and late cutaneous porphyria. And genes associated with T cell acute lymphocytic leukemia 1. SEQ ID NO: 93 is chromosome 1 within the interval 76.5-87.6 centimorgans.
Map to 2. This interval also includes genes associated with type IIID mucopolysaccharidosis, pseudovitamin D deficient rickets and renal amyloidosis. SEQ ID NO: 94 and SEQ ID NO: 109 are both on chromosome 1 within the interval of 143.1-146.6 centimorgans, on chromosome 14 within the interval of 46.8-50.9 centimorgans, 88.1-
It maps to chromosome 16 within the 90.2 centimorgan interval and to chromosome 19 within the 58.7-97.5 centimorgan interval. The interval between 46.8 and 50.9 centimorgans on chromosome 14 also contains genes associated with dopa ataxia. Colorectal cancer, DNA ligase I deficiency, glutaric aciduria IIB, myotonic dystrophy, renal amyloidosis, T-cell acute lymphocytic leukemia and Genes associated with xeroderma pigmentosum D are also included. SEQ ID NO: 97 maps to chromosome 2 within the interval 236.2-269.5 centimorgans. This interval also includes genes associated with Crigler-Najjar syndrome, familial hypercholesterolemia, mouth disease and primary oxalic aciduria. SEQ ID NO: 101 maps to chromosome 2 within the interval 225.6 to 233.1 centimorgans and to chromosome 6 within the interval 132.7 to 144.4 centimorgans, 117.9 to 120.8. Maps to chromosome 11 within the centimorgan interval. The 225.6 to 233.1 centimorgan interval on chromosome 2 also contains the gene associated with Wardenburg Syndrome 1. 1 of chromosome 6
The 32.7 to 144.4 centimorgan interval also includes genes associated with familial disseminated atypical mycobacterial infection and tarsal root punctate epiphyseal dysplasia. 1 of chromosome 11
The 17.9 to 120.8 centimorgan interval also includes genes associated with acute intermittent porphyria. SEQ ID NO: 111 is on chromosome 19 within the interval of 35.5-49.4 cmorgan, on chromosome 1 within the interval of 16.4 cmorgan from the p-terminus, and within the interval of 147.2 cmorgan to the q-terminus within the interval of 16.4 cmorgan. Map to chromosome 11. SEQ ID NO: 112 is on chromosome 1 within the interval of 41.7-49.4 centimorgans.
Map to 9. SEQ ID NO: 113 maps to chromosome 9 within the interval 136.2-163.0 centimorgans. SEQ ID NO: 115 is 95.5-103.7
Maps to chromosome 14 within the centimorgan interval and to X chromosome (23) within the 55.5 centimorgan interval from the p-terminus. SEQ ID NO: 117 maps to chromosome 13 at 46.9 centimorgans. SEQ ID NO: 118 is 16.4 to 22.9.
Maps to chromosome 1 within the centimorgan interval. SEQ ID NO: 121 is 11
Maps to chromosome 12 within the interval of 6.6 to 118.9 centimorgans. SEQ ID NO: 128 maps to chromosome 1 within the interval 16.4 centimorgans from the p-terminus.

The present invention also includes variants of GBAP. A preferred GBAP variant amino acid sequence has at least about 80%, about 90%, or even about 95% concordance with the GBAP amino acid sequence, such that it has at least one functional or structural characteristic of GBAP. It is a mutant.

The present invention also includes polynucleotides encoding GBAP. In one embodiment,
The present invention includes a polynucleotide sequence having a sequence selected from the group comprising SEQ ID NOs: 67 to 132 encoding GBAP. The polynucleotide sequences of SEQ ID NOs: 67 to 132 as shown in the sequence listing have the same value as the equivalent RNA sequence as shown in the sequence listing, but the appearance of the nitrogen base thymine is replaced with uracil. And the sugar backbone is composed of siribose rather than deoxyribose.

The present invention also includes mutant sequences of the polynucleotide sequence encoding GBAP.
Specifically, the variant sequences of such polynucleotide sequences have at least about 70%, or at least about 85%, or even at least about 95% identity with the polynucleotide sequence encoding GBAP. In some embodiments of the invention at least about 70% amino acid sequence selected from the group consisting of SEQ ID NOs: 67-132.
Alternatively, a variant sequence of a polynucleotide sequence having a sequence selected from the group consisting of SEQ ID NOs: 67-132 having a consensus of at least about 85%, or even at least about 95%. Variants of any of the above polynucleotides may be GBAP
Can encode an amino acid sequence having at least one functional or structural feature of

Those skilled in the art will appreciate that as a result of the degeneracy of the genetic code, a large number of polynucleotide sequences encoding GBAP (polynucleotide sequences having minimal similarity to those of known or naturally occurring genes) Understand that it can produce Thus, the invention may cover any and all possible polynucleotide sequence variants that may be produced by the selection of combinations based on possible codon choices. These combinations are based on the standard triplet genetic code as applied to the native GBAP polynucleotide sequences, and all such mutations are considered to be explicitly disclosed.

The nucleotide sequence encoding GBAP and variants thereof will generally be hybridizable to the nucleotide sequence of naturally occurring GBAP under suitably selected stringent conditions, although GBAP or a derivative thereof may be substantially It would be beneficial to create nucleotide sequences that code for different codon usages, eg, for inclusion of non-naturally occurring codons. Based on the frequency with which the host utilizes a particular codon, the codon can be selected to increase the expression rate of the peptide occurring in a particular eukaryotic or prokaryotic host. Without changing the encoded amino acid sequence
Another reason for substantially altering the nucleotide sequence encoding GBAP and its derivatives is the production of RNA transcripts with properties that are preferable to those produced from the native sequence, eg having a longer half-life.

The present invention also includes making DNA sequences encoding GBAP, GBAP derivatives and fragments thereof, or fragments thereof, entirely by synthetic chemistry. After construction, the synthetic sequence may be inserted into any of a variety of available expression vectors and cell lines using reagents well known in the art. In addition, synthetic chemistry may be used to induce mutations in the sequence encoding GBAP or any fragment thereof.

Furthermore, the present invention also provides a polynucleotide sequence capable of hybridizing to the polynucleotide sequences according to the claims, particularly the sequences represented by SEQ ID NOs: 67 to 132 and fragments thereof under various stringent conditions. Included (Wahl, GM and SL
. Berger (1987) Methods Enzymol. 152: 399-407, Kimmel. AR (1987) Method
s Enzymol. 152: 507-511. Hybridization conditions, including annealing and washing conditions, are described in the "Definition" section.

Methods for DNA sequencing are well known in the art, and any method of the invention may be practiced with DNA sequencing methods. Enzymes can be used in the DNA sequencing method, such as the Klenow fragment of DNA polymerase I, SEQUEN.
ASE (US Biochemical, Cleveland OH), Taq polymerase (PE Biosystems, Fos
ter City CA), thermostable T7 polymerase (Amersham, Pharmacia Biotech, Pisc
ataway NJ) can be used. Alternatively, for example, the ELONGASE amplification system (Lif
A polymerase and a proofreading exonuclease can be used together, as found in e Technologies, Gaithersburg MD). MICROLAB 2200 liquid transfer system (Hamilton, Reno, NV), PTC200 thermal cycler (MJ Researc)
h, Watertown MA) and ABI CATALYST 800 Thermal Cycler (PE Biosystems)
Etc. to automate sequence preparation. Sequencing is then performed using the ABI 373 or 377 DNA Sequencing System (PE Biosystems), MEGABACE 1000 DNA Sequencing System (Molecular Dynamics, Sunnyvale CA) or other methods well known in the art. The resulting sequences are analyzed using various algorithms well known in the art. (Ausubel, FM (1
997) Short Protocols in Molecular Biology , John Wiley & Sons, New York N
Y, unit 7.7, Meyers, RA (1995) Molecular Biology and Biotechnology , Wi
ley VCH, New York NY, pp. 856-853.

The GBAP-encoding nucleic acid sequence is extended using a variety of methods based on PCR methods utilizing partial nucleotide sequences and well known in the art to provide upstream sequences such as promoters and regulatory elements. Can be detected. For example, the restriction site PCR method, which is one of the methods that can be used, is a method of amplifying an unknown sequence from genomic DNA in a cloning vector using universal primers and nested primers (Sarkar, G. (1993) PCR. See Methods Applic 2: 318-322). Another method is the inverse PCR method, which is a method of amplifying an unknown sequence from a circularized template using primers extended in a wide range of directions. The template is derived from a restriction fragment consisting of a known genomic locus and its surrounding sequences (Triglia, T. et al. (1988) Nuc
leic Acids Res 16: 8186 etc.). The third method is a capture PCR method, which is involved in a method of PCR-amplifying a DNA fragment adjacent to a known sequence of human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods). Applic 1:11
See 1-119 etc.). In this method, it is possible to insert a recombinant double-stranded sequence into an unknown sequence region using digestion with multiple restriction enzymes and a ligation reaction before performing PCR.
Also, there are other methods known in the art that can be used to search for unknown sequences. (See Parker, JD et al. (1991) Nucleic Acids Res. 19: 3055-3060 etc.). In addition, PCR, nested primers and PromoterFinder library (Clontec
h, Palo Alto CA) can be used to walk genomic DNA. This procedure is useful for finding intron / exon junctions without the need to screen the library. For all PCR-based methods, commercially available software such as OLIGO 4.06 primer analysis software (National Bio
sciences, Plymouth MN) or another suitable program,
The primer can be designed to anneal to the template at ~ 30 nucleotides, a GC content of about 50% or greater, and a temperature of about 68 ° C to 72 ° C.

When screening for full-length cDNAs, it is preferable to use libraries that are size-selected to contain larger cDNAs. Furthermore, randomly primed libraries often contain sequences with the 5'regions of genes, which is suitable for situations where oligo d (T) libraries are unable to produce full-length cDNA. Genomic libraries can be useful for extension of sequences into 5'non-transcribed regulatory regions.

Commercially available capillary electrophoresis systems can be used to analyze the size of, or confirm the nucleotide sequence of, sequencing or PCR products. Specifically, capillary sequencing consists of a flowable polymer for electrophoretic separation, a laser-activated fluorescent dye, such as is specific for four different nucleotides, and detection of emitted wavelengths. And a CCD camera used for. Output / light intensity is measured with the appropriate software (PE Biosystems GENOTYP
ER, SEQUENCE NAVIGATOR, etc.) can be used to convert into an electric signal. The entire process from sample loading to computer analysis and electronic data display is computer controllable. Capillary electrophoresis is particularly suitable for sequencing small DNA fragments that may be present in low amounts in a particular sample.

In another embodiment of the present invention, a polynucleotide sequence encoding GBAP or a fragment thereof is used to express a GBAP, a fragment of GBAP or a functional equivalent thereof in a suitable host cell. Can be cloned in. Another DNA sequence encoding a substantially identical or functionally equivalent amino acid sequence due to the unique duplication of the genetic code may be prepared and used for the expression of GBAP.

Using methods commonly known in the art for altering GBAP coding sequences for a variety of purposes including, but not limited to, cloning of gene products, processing, regulation of expression. Thus, the nucleotide sequences of the present invention can be recombined. Nucleotide sequences can be recombined using random fragmentation of gene fragments and synthetic oligonucleotides and DNA shuffling by PCR reassembly. For example, utilizing oligonucleotide-mediated directed mutagenesis to create new restriction sites, alter glycosylation patterns,
Mutations may be introduced that alter codon preferences, generate splice variants, and the like.

Nucleotides of the present invention are described in Molecular Breeding (Maxygen Inc., Santa Clara C
A, U.S. Patent No. 5,837,458, Chang, C.-C. et al. (1999) Nat. Biotechnol. 17: 793-.
797, Christians, FC et al. (1999) Nat. Biotechnol. 17: 259-264, Crameri, A.
(1996) Nat. Biotechnol. 14: 315-319), etc., and the biological properties of GBAP, such as biological activity, enzymatic activity, or binding ability to other molecules or compounds. Can be changed or improved. DNA shuffling is the process of generating a library of gene variants using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening to identify the gene variant with the desired property. Then pool these suitable variants,
Additional iterations of DNA shuffling and selection / screening may be performed. Thus, genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene with random point mutations can be recombined, screened, and then shuffled until the desired property is optimized. Alternatively, a given gene can be recombined with a homologous gene from the same gene family, either homologous or heterologous, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner. Can be transformed.

According to another embodiment, all or part of the GBAP coding sequence may be synthesized using chemical methods well known in the art (Caruthers. MH et al. (1980) Nuc).
leic. Acids Symp. Ser 7: 215-223, Horn, T. et al. (1980) Nucleic. Acids Symp.
Ser. 7: 225-232 etc.). Alternatively, chemical methods may be used to synthesize GBAP itself or a fragment thereof. For example, peptide synthesis can be performed using various liquid or solid phase techniques (Creighton, T. (1984) Proteins, Structures and Molecular Properties , WH Freeman, New York NY, pp. 55-60, Roberge. , JY et al. (19
95) Science 269: 202-204 etc.). Automated synthesis can be accomplished using the ABI 431A Peptide Synthesizer (Perkin Elmer). In addition, the amino acid sequence of GBAP, or any portion thereof, may be altered during direct synthesis and / or binding to sequences obtained from other proteins or any portion thereof to produce variant polypeptides.

Peptides can be substantially purified using preparative high performance liquid chromatography (
Chiez, RMand FZ Regnier (1990) Methods Enzymol. 182: 392-421 etc.). The composition of synthetic peptides can be confirmed by amino acid analysis or sequencing (see Creighton, pp. 28-53, etc., supra).

For expression of biologically active GBAP, the nucleotide sequence encoding GBAP or a derivative thereof can be inserted into a suitable expression vector. A preferred expression vector is a vector containing the elements necessary for the regulation of transcription and translation of the inserted coding sequence in a suitable host. Required elements include regulatory sequences such as enhancers, constitutive and expression-inducible promoters, 5'and 3'untranslated regions in the vector and polynucleotide sequences encoding GBAP. Such elements vary in length and specificity. It is also possible that the unique initiation signal may be used to translate the GBAP coding sequence more effectively. AT for such signals
It includes the G start codon and nearby sequences such as the Kozak sequence. If the GBAP coding sequence and its initiation codon, upstream regulatory sequences are inserted into a suitable expression vector, no additional transcriptional or translational regulatory signals will be required. However, if only the coding sequence or a fragment thereof is inserted, an exogenous translational control signal containing an in-frame ATG initiation codon should be included in the expression vector. Exogenous translational elements and initiation codons can originate from a variety of natural and synthetic sources. The efficiency of expression can be enhanced by the inclusion of enhancers suitable for the particular host cell line used (Scharf, D. et al. (199
4) Results Probl. Cell Differ. 20: 125-162.

Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding GBAP and suitable transcriptional and translational regulatory elements. The method, in vit ro recombinant DNA techniques, synthetic techniques and in vivo genetic recombination technique (Sambrook, J.
Et al. (1989) Molecular Cloning.A Laboratory Manual , Cold Spring Harbor Pr
ess, Plainview NY, chapters 4, 8, 16-17; Ausubel, FM et al. (1995) Current Protocols in Molecular Biology , John Wiley & Sons, New York NY, chapters 9, 13, 16).

A variety of expression vector / host systems may be utilized to retain and express sequences encoding GBAP. Such expression vectors / host systems include, but are not limited to, bacteria transformed with recombinant bacteriophage, plasmids or cosmid DNA expression vectors, yeasts transformed with yeast expression vectors, and viruses. Insect cell lines infected with expression vectors (eg baculovirus) and viral expression vectors (eg cauliflower mosaic virus CaMV or tobacco mosaic virus)
TMV) or bacterial expression vectors (eg Ti or pBR322 plasmids) transformed microorganisms such as plant cell lines, animal cell lines etc. (Sambrook, supra, supra).
Ausubel, Van Heeke, G. and SM Schuster (1989) J. Biol. Chem. 264: 5503-
5509, Bitter, GA et al. (1987) Methods Enzymol. 153: 516-544; Scorer, CA.
(1994) Bio / Technology 12:18 1-184; Engelhard, EK et al. (1994) Proc. Na.
tl. Acad. Sci. USA 91: 3224-3227, Sandig, V. et al. (1996) Hum. Gene Ther. 7:
1937-1945, Takamatsu, N. (1987) EMBOJ. 6: 307-311, Coruzzi, G. et al. (1984) E
MBOJ. 3: 1671-1680, Broglie, R. et al. (1984) Science 224: 838-843, Winter, J.
Et al (1991) Results Probl. Cell Differ. 17: 85-105, The McGraw Hill Yearbook of Science and Technology (1992) McGraw
Hill New York NY, pp.191-196, Logan, J. and T. Shenk (1984) Proc. Natl.
Acad. Sci. USA 81: 3655-3659, Harrington, JJ et al. (1997) Nat. Genet. 15:
See 345-355 etc.). Expression vectors derived from retroviruses, adenoviruses, herpesviruses or vaccinia viruses or expression vectors derived from various bacterial plasmids can be used to deliver polynucleotide sequences to target organs, tissues or cell populations (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5 (6): 3
50-356, Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90 (13): 6340-6344, Bu.
ller, RM et al. (1985) Nature 317 (6040): 813-815; McGregor, DP et al. (1994).
Mol. Immunol. 31 (3): 219-226, Verma, IM and N. Somia (1997) Nature 389:
See 239-242 etc.). The present invention is not limited by the host cell used.

In bacterial systems, a number of cloning and expression vectors may be chosen depending upon the use intended for the polynucleotide sequence encoding GBAP. For example, for routine cloning, subcloning, and propagation of polynucleotide sequences encoding GBAP, PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Li
multi-functional E. coli vectors such as fe Technologies) can be used. By the ligation reaction of the GBAP-encoding sequence to multiple cloning sites of the vector,
The lacZ gene is disrupted, allowing a colorimetric screening method to identify transformed bacteria containing recombinant molecules. In addition, these vectors may also be useful for in vitro transcription of cloned sequences, dideoxy sequencing, single-stranded rescue by helper phage, and generation of nested deletions (Van He
eke, G. and SM Schuster (1989) J. Biol. Chem. 264: 5503-5509.
. When a large amount of GBAP is required, for example, when producing an antibody, a vector which induces GBAP expression at a high level can be used. For example, strong inducible T5 or
A vector containing the T7 bacteriophage promoter may be used.

Yeast expression systems can be used to produce products of GBAP. A large number of vectors containing constitutive or inducible promoters such as α-factor, alcohol oxidase, and PGH promoter can be used for yeast Saccharomyces cerevisiae or Pichia pastoris . In addition, such vectors direct the expressed protein either to be secreted or to be retained intracellularly, allowing integration of foreign sequences into the host genome for stable growth (Ausubel (supra. 1995), Bitter, Scorer, supra.
Etc.).

It is also possible to express GBAP using plant systems. Transcription of the GBAP-encoding sequence is regulated by viral promoters, such as alone or TMV (Takamatsu, N. (1987) E.
C as used in combination with the omega leader sequence from MBO J 6: 307-311)
Promoted by the 35S and 19S promoters from aMV. Alternatively, a plant promoter such as the small subunit of RUBISCO or a heat shock promoter may be used (see Coruzzi, supra, Broglie, supra, Winter, supra). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection ( The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill See New York NY, pp. 191-196).

In mammalian cells, a number of virus-based expression systems are available. When using adenovirus as an expression vector, the GBAP-encoding sequence can be ligated to an adenovirus transcription / translation complex consisting of a late promoter and a triple leader sequence. Insert the viral genome into the essential E1 or E3 region and
Infectious virus expressing GBAP can be obtained (Logan, J. and T. Shenk
(1984) Proc. Natl. Acad. Sci. 81: 3655-3659 etc.). In addition, transcription enhancers such as the Rous Sarcoma Virus (RSV) enhancer can be used to increase expression in mammalian host cells. Proteins can also be expressed at high levels using SV40 or EBV based vectors.

Human artificial chromosomes (HACs) can also be used to transport fragments of DNA larger than those contained in and expressed from the plasmid. HACs of about 6 kb to 10 Mb were prepared for therapeutic purposes and supplied by conventional delivery methods (liposomes, polycationic amino polymers or vesicles) (Harrington. JJ et al. (1997) Nat Gen
et.15: 345-355.).

For long-term production of recombinant proteins in mammalian systems, stable expression of GBAP in cell lines is desirable. For example, a GBAP-encoding sequence is transformed into a cell line using an expression vector containing a viral origin of replication and / or an endogenous expression factor and a selectable marker gene on the same or another vector. It is possible to Approximately 1 in enriched medium after transfer of vector and before transfer to selective medium
Allow the cells to grow for ~ 2 days. The purpose of the selectable marker is to confer resistance to selective medium, and the presence of the selectable marker enables growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate to the cell type.

Transformed cell lines can be recovered using any number of selection systems. Such selection systems include, but are not limited to, the herpesvirus thymidine kinase gene used for tk ^- simple cells and the adenine phosphoribosyl transferase gene used for apr ^- cells (Wigler, M. Et al (1977) Cell 11: 223-2
32, Lowy, I. et al. (1980) Cell 22: 817-823). Also, resistance to antimetabolites, antibiotics or herbicides can be used as the basis for selection. Eg dh
fr confers resistance to methotrexate, neo confers resistance to aminoglycosid neomycin and G-418, als chlorsulfuron and pa
t confers resistance to phosphinothricin acetyltransferase (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77: 3567-3570, Colbere-
Garapin, F. et al. (1981) J. Mol. Biol. 150: 1-14). Other selectable genes, such as trpB and hisD that alter cellular requirements for metabolism, have been described in the literature (Hartman, SC and RC Mulligan (1988) Proc. Natl. Acad. Sci.
USA 85: 8047-8051 etc.). Visible markers such as anitocyanin, green fluorescent protein (GFP; Clontech), β-glucuronidase and its substrate β-glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used to not only identify transformants but also quantify transient or stable protein expression due to a particular vector system (Rhodes, CA (1995) Methods Mol. Biol. . 55: 121-131 etc.).

Although the presence / absence of marker gene expression suggests the presence of the gene of interest, it may be necessary to confirm the presence and expression of that gene. For example, if the GBAP coding sequence is inserted within a marker gene sequence, transformed cells containing the GBAP coding sequence can be identified by the lack of marker gene function. Alternatively, a marker gene can be placed in tandem with the GBAP-encoding sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually also indicates expression of the tandem gene.

[0133] Generally, a variety of methods well known to those of skill in the art can be used to identify host cells that contain a nucleic acid sequence encoding GBAP and that express GBAP. Methods well known to those skilled in the art include, but are not limited to, DNA-DNA or DNA-RNA hybridization, PCR methods, membrane systems for performing detection and / or quantification of nucleic acids or proteins, solution-based or There are protein bioassays or immunoassay technologies, including chip-based technologies.

[0134]   Development of GBAP using specific polyclonal antibody or specific monoclonal antibody
Immunological methods for performing current detection and counting are known in the art. like this
Examples of such techniques include enzyme-linked immunosorbent assay (ELISA),
Examples include geoimmunoassay (RIA) and fluorescence activated cell sorting (FACS). G
Two parts using a monoclonal antibody reactive to two non-interfering epitopes on BAP
Two-site, monoclonal-based immun
oassay) is preferred, but competitive binding assays can also be used. These
Say and other assays are known in the art (Hampton. R. et al. (1990). Serological Methods, a Laboratory Manual.  APS Press. St Paul. MN, Sect.
IV, Coligan, J. E. et al. (1997)Current Protocols in Immunology, Greene Pub
.Associates and Wiley-Interscience, New York NY, Pound, J.D. (1998)Imm unochemical Protocols , Humans Press, Totowa NJ, etc.).

A wide variety of labeling and conjugation methods are known to those of skill in the art and can be used in various nucleic acid and amino acid assays. Methods for producing labeled hybridization or PCR probes for detecting sequences related to GBAP-encoding polynucleotides include oligo-labeling, nick-translation, end-labeling, or labeled nucleotides. There is a PCR method to use. Alternatively, the GBAP coding sequence or any fragment thereof can be cloned into a vector for producing an mRNA probe. Such vectors are known in the art and are commercially available, and in addition to a suitable RNA polymerase such as T7, T3 or SP6 and labeled nucleotides, RNA is expressed in vitro .
It can be used for probe synthesis. Such a method is, for example, Amersham Pha.
It can be carried out using various kits commercially available from rmacia Biotech, Promega (Madison WI), US Biochemical and the like. Suitable reporter molecules or labels that can be used to facilitate detection include substrates, cofactors, inhibitors, magnetic particles, as well as radionuclides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents and the like.

Host cells transformed with a nucleotide sequence encoding GBAP may be cultured under conditions suitable for the recovery and expression of the protein from cell culture. Whether a protein produced by a transformed cell is secreted or remains intracellular depends on the sequence used, the vector, or both. As one of ordinary skill in the art would understand, GB
Expression vectors containing polynucleotides encoding AP can be designed to contain a signal sequence that directs the direct secretion of GBAP across prokaryotic or eukaryotic cell membranes.

Furthermore, the choice of host cell strain may be made by the ability to regulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Modifications of such polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing such as cleaving the "prepro" or "pro" form of the protein can also be utilized to identify protein targeting, folding and / or activity. A variety of host cells (eg, CH 2) with unique cellular machinery and characteristic mechanisms for post-translational activity.
O, HeLa, MDCK, MEK293, WI38, etc.) are American Type Culture Collection (ATC
C, Bethesda, VA) and can be selected to ensure correct modification and processing of foreign proteins.

In another embodiment of the invention, the natural, modified or recombinant nucleic acid sequence encoding GBAP is ligated to a heterologous sequence which results in the translation of the fusion protein in any of the above host systems. You can For example, a chimeric GBAP protein containing a heterologous moiety that can be recognized using commercially available antibodies can facilitate the screening of peptide libraries for inhibitors of GBAP activity. Heterologous protein moieties and heterologous peptide moieties may also facilitate the purification of fusion proteins using commercially available affinity substrates. Such moieties include, but are not limited to, glutathione S transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc,
Has hemagglutinin (HA). GST on immobilized glutathione, MBP on maltose, Trx on phenylarsine oxide, CBP on calmodulin, and 6-
His allows the purification of cognate fusion proteins on metal chelating resins. FLAG
, C-myc and hemagglutinin (HA) allow immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. The fusion protein can also be genetically engineered to include a proteolytic cleavage site between the GBAP coding sequence and the heterologous protein sequence so that GBAP can be cleaved from the heterologous moiety after purification. Methods for expressing and purifying fusion proteins are described in Ausubel (1995), supra, Chapter 10. A variety of commercially available kits can also be used to facilitate expression and purification of the fusion protein.

In yet another embodiment of the invention, radiolabeled GBAP can be synthesized in vitro using TNT rabbit reticulocyte lysate or wheat germ extraction system (Promega). These systems couple transcription and translation of protein coding sequences operably linked to the T7, T3 or SP6 promoters. Translation occurs in the presence of radiolabeled amino acid precursors such as ³⁵ S methionine.

GBAP or fragments thereof of the invention may be used to screen for compounds that specifically bind to GBAP. At least one to more than one test compound can be used to screen for specific binding to GBAP. Examples of test compounds include antibodies, oligonucleotides, proteins (eg receptors) or small molecules.

In one example, the compound thus identified is closely related to a natural ligand of GBAP, such as a ligand or fragment thereof, a natural substrate, a structural or functional mimetic or natural binding partner. (See Chapter 5 of Coligan, JE et al. (1991) Current Protocols in Immunology 1 (2)). Similarly, the compound is GBAP
May be associated with the natural receptor to which it binds, or may be closely associated with at least a fragment of the receptor, eg, the ligand binding site. In any case, the compound may be rationally designed using known techniques. In one example, screening for such compounds involves producing suitable cells that express GBAP either as a secreted protein or on the cell membrane. Suitable cells include cells from mammals, yeast, Drosophila or E. coli. GBAP-expressing cells or GBAP
A cell membrane fragment containing the is contacted with a test compound and analyzed for binding, stimulation or inhibition of either GBAP or the compound.

The assay may simply test bind a test compound to the polypeptide. Here, binding is detected by fluorophores, radioisotopes, enzyme conjugates or other detectable labels. For example, the assay can include either binding at least one test compound in solution to GBAP or immobilizing to a solid support and detecting GBAP binding to the compound. Alternatively, the assay may detect or measure binding of the test compound in the presence of labeled competitor. Furthermore, the assay
It can be carried out with cell free preparations, chemical libraries or natural product mixtures, test compounds can be released in solution or immobilized on a solid support.

The GBAPs or fragments thereof of the present invention may be used to screen for compounds that modulate the activity of GBAP. Such compounds include agonists, antagonists, partial or inverse agonists and the like. In one example, the assay is performed under conditions that allow the activity of GBAP such that GBAP is bound to at least one test compound, and the activity of GBAP in the presence of the test compound is in the absence of the test compound. GBAP at
Compared to the activity of. The change in the activity of GBAP in the presence of the test compound indicates a compound that regulates the activity of GBAP. Alternatively, the test compound is bound under conditions suitable for activity of GBAP to an in vitro or cell free system containing GBAP and the assay is performed. In any of these assays, the test compound that modulates the activity of GBAP can do so indirectly, eliminating the need for direct contact with the test compound. At least one to multiple test compounds can be screened.

In another example, a polynucleotide encoding GBAP or a mammalian homolog thereof is “knocked out” in an animal model system using homologous recombination in embryonic stem (ES) cells. Such techniques are known in the art and are useful in generating animal models of human disease (see, eg, US Pat. Nos. 5,175,383 and 5,767,337). Mouse ES cells, such as the 129 / SvJ cell line, are derived from early mouse fetuses and grow in culture. ES cells are transformed with a vector containing a gene of interest divided by a marker gene such as neomycin phosphotransferase gene (neo: Capecchi, MR (1989) Science 244: 1).
288-1292). The vector integrates into the corresponding region of the host genome by homologous recombination. Alternatively, homologous recombination occurs using the Cre-loxP system, which knocks out the gene of interest in a tissue-specific or developmental stage-specific manner (Marth, JD (1996) C
lin. Invest. 97: 1999-2002; Wagner, KU et al. (1997) Nucleic Acids Res. 25:
4323-43 30). Transformed ES cells are identified and microinjected into, for example, mouse cell blastocysts taken from the C57BL / 6 mouse strain. Blastocysts are surgically introduced into pseudopregnant females and the resulting chimeric progeny are determined and propagated to produce heterozygous or homozygous lines. The transgenic animals thus produced may be tested with potential therapeutic or toxic agents.

Polynucleotides encoding GBAP can also be engineered in vitro in human blastocyst-derived ES cells. Human ES cells have the potential to differentiate into at least eight separate cell lineages, including endoderm, mesoderm and ectoderm cell types. This cell lineage differentiates into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, JA et al. (19
98) Science 282: 1145-1147).

Polynucleotides encoding GBAP may also generate "knock-in" humanized animals (pigs) or transgenic animals (mouse or rat) into model human diseases.
Using knock-in technology, a region of the polynucleotide encoding GBAP
It is injected into ES cells and the injected sequences integrate into the animal cell genome. The transformed cells are injected into the blastula and the blastula is transplanted as described above. In order to obtain information on the treatment of human diseases, transgenic offspring or inbred lines are studied, and powerful drugs are used to treat the transgenic offspring or inbred lines. Alternatively, mammals that have been inbred to overexpress GBAP, for example by secreting GBAP into milk, can also serve as a convenient source of protein (Janne, J. et al. (1998) Biotechnol. Annu.
Rev. 4: 55-74).

There are chemical and structural similarities between regions of therapeutic GBAP and intracellular signaling molecules, such as in the context of sequences and motifs. Furthermore, GBAP expression is closely associated with hematopoietic / inflammatory, nervous, gastrointestinal and reproductive cancers. Therefore GBAP
Are believed to play a role in immune system disorders, reproductive disorders, nervous system disorders and cell signaling disorders. In treating diseases associated with increased expression or activity of GBAP, it is desirable to reduce expression or activity of GBAP. Also, GB
In the treatment of diseases associated with decreased AP expression or activity, it is desirable to increase GBAP expression or activity.

Thus, in certain embodiments, it is possible to administer GBAP or a fragment or derivative thereof to a patient for the treatment or prevention of diseases associated with decreased expression or activity of GBAP. Non-limiting examples of such disorders include immune system disorders, including inflammation, actinic keratoses, acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies. , Ankylosing spondylitis, amyloidosis, anemia,
Arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, Diabetes mellitus, emphysema, erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
Glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, paroxysmal nocturnal hemoglobinuria, hepatitis, hypereosinophilia, irritable bowel syndrome, lymphotoxin-induced temporary lymphopenia, mixed Type connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, myelofibrosis, osteoarthritis, osteoporosis, pancreatitis, Reiter's syndrome, rheumatoid arthritis, scleroderma , Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, primary thrombocytosis, thrombocytopenia, ulcerative colitis, uveitis, Werner syndrome, complications of cancer,
Hemodialysis, extracorporeal circulation, trauma, and hematopoietic cancers including lymphoma, leukemia, myeloma, and reproductive disorders, including abnormal prolactin production, fallopian tube disease, abnormal ovulation and endometriosis. Infertility, including estrus, abnormal menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial or ovarian cancer, uterine fibroids, autoimmune disorders, ectopic pregnancy and malformation, breast cancer, fibrosis Cystic mastopathy and mastasis, including spermatogenesis, abnormal sperm physiology, testicular cancer, prostate cancer, benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, male breast cancer and gynecomastia, and Nervous system diseases are also included, including epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasm, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, muscular atrophic lateral measures. Hard Other motor neuron disorders, progressive neuromuscular atrophy, retinitis pigmentosa, inherited 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 disease and prion diseases including Kuru, Creutzfeldt-Jakob disease and Gastmann-Straussler-Shineker syndrome And fatal familial insomnia, nutritional and metabolic disorders of the nervous system, neurofibromatosis, tuberous sclerosis, cerebellar retinal hemangioblastomatosis.
, Cerebral trigeminal vascular syndrome, mental retardation and other central nervous system development disorders, cerebral palsy, neuroskeletal abnormalities, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nerve diseases, skin Myositis and polymyositis, hereditary, metabolic, endocrine and toxic myopathy, myasthenia gravis, periodic quadriplegia, and psychiatric disorders including mood disorders, anxiety disorders and schizophrenia, and akathisia, It includes amnesia, catatonic disease, diabetic neuropathy, extrapyramidal terminal deficiency syndrome, dystonia, schizophrenia, postherpetic neuralgia and Tourette's disease, and also impaired cell signaling. Is the hypothalamus and inferior hypothalamus resulting from lesions such as primary brain tumors and adenomas, gestational infarction, hypophysectomy, aneurysms, vascular malformations, thrombosis, infections, immune disorders, and complications of head trauma Disorders of the pituitary gland, inappropriate antidiuretic hormone (ADH) secretion syndrome (SIADH) that is more likely to occur due to benign adenoma, and disorders related to hyperpituitary gland such as acromegaly and macromegaly, and goiter and myxedema, bacterial infection Disorders related to hypothyroidism including acute acute thyroiditis, hyperparathyroidism including Conn disease (chronic hypercalemia), pancreatic diseases such as type I and type II diabetes and complications, and hyperplasia and adrenal gland Cortical carcinomas and adenomas, adrenal-related disorders such as hypertension associated with alkalosis, and abnormal prolactin production and infertility in women, endometriosis, menstrual cycle perturbation, polycystic ovary disease, hyperprolactinemia, Selective gonadotropin deficiency (is
olate gonadotropin deficiency), amenorrhea, milk leak, hermaphroditism, hirsutism and virilization, breast cancer, postmenopausal osteoporosis, male Leydig cell hyperplasia, male menopause, germ cell aplasia, Leydig cell tumor Endocrine disorders, including androgen resistance associated with lack of androgen receptor, 5α-reductase syndrome, and diseases related to gonadal steroid hormones such as female mammary disorders,
It also includes abnormal cell proliferation, including actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis. , Paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia and cancer including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, 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, Includes thyroid and uterine cancer.

In another example, a vector capable of expressing GBAP or a fragment or derivative thereof is administered to a patient to treat diseases associated with decreased expression or activity of GBAP, including but not limited to the above mentioned diseases. Treatment or prevention is also possible.

In yet another embodiment, a pharmaceutical composition comprising substantially purified GBAP is administered to a patient with a suitable pharmaceutical carrier, including but not limited to the GBAPs described above.
It is also possible to treat or prevent diseases associated with decreased expression or activity of.

In yet another embodiment, an agonist that regulates the activity of GBAP is administered to a patient to treat or prevent diseases associated with decreased expression or activity of GBAP, including but not limited to those mentioned above. It is also possible to do so.

In yet another example, a patient can be administered an antagonist of GBAP to treat or prevent a disorder associated with increased expression or activity of GBAP. Non-limiting examples of such disorders include the immune system disorders, reproductive disorders, nervous system disorders and cell signaling disorders described above. In one embodiment, an antibody that specifically binds to GBAP can be used directly as an antagonist or indirectly as a targeting or transport mechanism that transports the drug to cells or tissues that express GBAP.

In another example, a vector expressing a complement of a polynucleotide encoding GBAP was administered to a patient and was associated with increased expression or activity of GBAP, including, but not limited to, the disorders described above. It is also possible to treat or prevent the disease.

In another example, any protein, antagonist, antibody, agonist, complementary sequence or vector of the invention can be administered in combination with another suitable therapeutic agent. Suitable therapeutic agents for use in combination therapy may be selected by those of ordinary skill in the art according to conventional pharmaceutical principles. When combined with a therapeutic agent, a synergistic effect can be brought about in the treatment or prevention of various diseases mentioned above. By using this method, it is possible to obtain a medicinal effect with a small amount of each drug, thereby reducing the possibility of side effects.

GBAP antagonists may be prepared using methods generally known in the art. Specifically, purified GBAP can be used to make antibodies or to screen libraries of therapeutics to identify those that specifically bind to GBAP.
Antibodies to GBAP can also be produced using methods generally known in the art. Such antibodies may include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments and fragments produced by Fab expression libraries. Neutralizing antibodies (ie, antibodies that inhibit dimer formation) are usually suitable for treatment.

To produce antibodies, goat, rabbit, rat, mouse, human, etc. are injected by injecting GBAP, or any fragment or oligopeptide of GBAP having immunogenic properties. A variety of hosts can be immunized, including.
Depending on the host species, various adjuvants may be used to enhance the immune response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gel adjuvants such as aluminum hydroxide, lysolecithin, pluronic polyol, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol. There is a cleaning agent such as. Among the adjuvants used in humans, BCG (bacillus Calmette-Guerin) and Corynebacterium parvum are particularly preferred.

Oligopeptides, peptides or fragments used to induce antibodies to GBAP are preferably those having an amino acid sequence of at least about 5 amino acids, generally at least about 10 amino acids. Desirably, these oligopeptides, peptides or fragments are identical to a portion of the amino acid sequence of the native protein and include the entire amino acid sequence of a small naturally occurring molecule. Short stretches of GBAP amino acids can be fused with sequences of another protein (eg KLH) to produce antibodies to the chimeric molecule.

Monoclonal antibodies against GBAP can be produced by continuous cell lines in culture, using any technique that produces antibody molecules. Such techniques include, but are not limited to, hybridoma technology, human B cell hybridoma technology and EBV-hybridoma technology (Kohler, G. et al. (1975) Nature 256: 495-497, Kozbor.
, D. et al. (1985) .J. Immunol. Methods 81: 31-42, Cote, RJ et al. (1983) Proc.
Natl. Acad. Sci. USA 80: 2026-2030, Cole, SP et al. (1984) Mol. Cell Biol.
62: 109-120 etc.).

In addition, it is possible to use techniques developed to produce “chimeric antibodies”, such as splicing of a mouse antibody gene into a human antibody gene to obtain a molecule with suitable antigen specificity and biological activity. Possible (Morrison, SL et al. (1984) P
Roc. Natl. Acad. Sci. USA 81: 6851-6855, Neuberger, MS et al. (1984) Nature.
312: 604-608, Takeda, S. et al. (1985) Nature 314: 452-454 etc.). Alternatively,
The techniques described for producing single chain antibodies can be applied to produce GBAP-specific single chain antibodies using methods known in the art. Antibodies with related specificities but different idiotypic composition can also be produced by chain shuffling from random combinatorial immunoglobulin libraries (Burton DR
(1991) Proc. Natl. Acad. Sci. USA 88: 10134-10137).

The production of antibodies may also be carried out by inducing in vivo production in a lymphocyte population, or by screening immunoglobulin libraries or panels of high specific binding reagents as disclosed in the literature (Orlandi, R . Et (
1989) Proc. Natl. Acad. Sci. USA 86: 3833-3837, Winter, G. et al. (1991) Nat.
ure 349: 293-299 etc.).

It is also possible to produce antibody fragments which have specific binding sites for GBAP. For example, without limitation, such fragments are made by reducing the disulfide bridges of the F (ab ') ₂ fragment and the F (ab') ₂ fragment produced by pepsin digestion of the antibody molecule. There are Fab fragments. Alternatively, it is possible to rapidly and easily identify a monoclonal Fab fragment with a desired specificity by preparing a Fab expression library (see Huse, WD et al. (1989) Science 256: 1275-1281 etc.).

A variety of immunoassays can be used to screen to identify antibodies with the desired specificity. Numerous protocols for immunoradiometric or competitive binding assays using either polyclonal or monoclonal antibodies with established specificity are known in the art. Such immunoassays typically involve measuring complex formation between GBAP and its specific antibody.
Using a monoclonal antibody reactive to two non-interfering GBAP epitopes,
Two-site monoclonal-based immunoassays are commonly used, but competitive binding assays may be used (Pound, supra).

Various methods can be used in conjunction with radioimmunoassay techniques, such as Scatchard analysis, to assess the affinity of the antibody for GBAP. Affinity is represented by the binding constant Ka.
Ka is defined as the molar concentration of GBAP antibody complex divided by the molar concentration of free antibody and free antigen at equilibrium. Polyclonal antibodies have heterogeneous affinities for various GBAP epitopes, and the Ka determined for polyclonal antibody reagents represents the average affinity or avidity of GBAP antibodies. Monoclonal antibody is specific GB
The Ka, which is monospecific for the AP epitope and determined for the monoclonal antibody reagent, represents a true measure of affinity. High affinity antibody reagents with Ka values in the range of about 10 ⁹ to 10 ¹² L / mol are preferably used in immunoassays in which the GBAP antibody complex must withstand vigorous manipulation. Low affinity antibody reagents with Ka values in the range of about 10 ⁶ to 10 ⁷ L / mol are used for immunopurification and similar procedures where GBAP must ultimately dissociate from the antibody in its activated state. Preferred (Catty, D. (198
8) Antibodies, Volume I: A Practical Approach , IRL Press, Washington, DC
, Liddell, JEand Cryer, A. (1991) A Practical Guide to Monoclonal Ant ibodies , John Wiley & Sons, New York NY).

The titer and avidity of polyclonal antibody reagents can be further evaluated to determine the quality and suitability of such reagents for certain downstream applications. For example,
Polyclonal antibody reagents containing at least 1-2 mg / ml, preferably 5-10 mg / ml of specific antibody are commonly used in processes requiring precipitation of GBAP antibody complexes. Guidance on antibody specificity, antibody titer, avidity, antibody quality and use in various applications is generally available (Catty, Coligan, supra).
See the literature, etc.).

In another embodiment of the invention, the polynucleotide encoding GBAP, any fragment of GBAP or the complementary sequence may be used for therapeutic purposes. In some embodiments, GB
It may be used if the complementary sequence of the polynucleotide encoding the AP is suitable for blocking the transcription of mRNA. Specifically, cells can be transformed with a sequence complementary to a polynucleotide encoding GBAP. Therefore, a complementary molecule or fragment is
It can be used to regulate AP activity or to regulate gene function. Such techniques are already well known in the art and allow sense or antisense oligonucleotides or large fragments to be designed from various positions extending into the coding or control region of the GBAP coding sequence. (Agrawal, S., ed.
(1996) Antisense Therapeutics , Humana Press Inc., Totawa NJ, etc.).

When used in therapy, any gene delivery system suitable for introducing antisense sequences into suitable target cells can be used. Antisense sequences can be transported intracellularly in the form of expression plasmids that express sequences complementary to at least a portion of the cellular sequence encoding the target protein during transcription (Slater, JE et al. (199
8) J. Allergy Clin. Immunol. 102 (3): 469-475, Scanlon, KJ et al. (1995) 9 (13).
): 1288-1296.). Antisense sequences can also be introduced into cells using viral vectors such as retroviruses and adeno-associated viral vectors (Miller, AD (1990) Blood 76: 271, Ausubel, Uckert, W., supra). an
d W. Walther (1994) Pharmacol. Ther. 63 (3): 323-347 etc.). Other gene transfer mechanisms include liposome systems, artificial viral envelopes and other systems known in the art (Rossi, JJ (1995) Br. Med. Bull. 51 (1): 217-22.
5; Boado, RJ et al. (1998) J. Pharm. Sci. 87 (11): 1308-1315, Morris, MC et al.
(1997) Nucleic Acids Res. 25 (14): 2730-2736.

In another embodiment of the invention, polynucleotides encoding GBAP can be used for somatic or germ cell gene therapy. By carrying out gene therapy, (i) gene deficiency (for example, X chromosome chain inheritance (Cavazzana-Calvo, M. et al.
0) Severe combined immunodeficiency (SCID) -X characterized by Science 288: 669-672)
1), severe combined immunodeficiency associated with congenital adenosine deaminase (ADA) deficiency (Blaese, RM et al. (1995) Science 270: 475-480, Bordignon, C. et al.
(1995) Science 270: 470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 7).
5: 207-216: Crystal, RG et al. (1995) Hum. Gene Therapy 6: 643-666, Crystal
, RG et al. (1995) Hum. Gene Therapy 6: 667-703), thalassamia.
), Familial hypercholesterolemia, hemophilia caused by factor VIII or IX deficiency (Crystal, 35 RG (1995) Science 270: 404-410, Verma, IM and So
mia. N. (1997) Nature 389: 239-242), (ii) expressing a conditionally lethal gene product (eg in the 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: 1139.
5-11399), hepatitis B or C virus (HBV, HCV), fungal parasites such as Candida albicans and P aracoccidioides brasiliensis , and Plasmodium falciparum
And a protein having a protective function against protozoan parasites such as Trypanosoma cruzi can be expressed. When a gene deficiency required for the expression or regulation of GBAP causes a disease, expressing GBAP from a suitable population of transduced cells may alleviate the onset of symptoms due to the gene deficiency.

In a further embodiment of the present invention, mammalian expression vectors encoding GBAP are prepared and introduced into GBAP-deficient cells by mechanical means, whereby diseases or abnormalities caused by GBAP deficiency are treated. treat. Mechanical transfer techniques used for cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (
ii) ballistic gold particle delivery, (iii
) Liposome-mediated transfection, (iv) Receptor-mediated gene transfer, and (v) Use of DNA transposons (Morgan, RA and WF Anderson (1993) Annu. Rev. Biochem
.62: 191-217, Ivics, Z. (1997) Cell 91: 501-510; Boulay, JL. And H. Reci
pon (1998) Curr. Opin. Biotechnol. 9: 445-450).

Expression vectors that can influence the expression of GBAP include, but are not limited to, PC
DNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vector (Invitrogen, Carlsbad CA)
, PCMV-SCRIPT, PCMV-TAG, PEGSH / PERV (Stratagene, La Jolla CA) and PTET-O
There are FF, PTET-ON, PTRE2, PTRE2-LUC and PTK-HYG (Clontech, Palo Alto CA).
GBAP is (i) a constitutively active promoter (eg cytomegalovirus (CMV
), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK) or β-actin gene), (ii) an inducible promoter (eg, tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl . Acad. Sci. U
.SA 89: 5547-5551, Gossen, M. et al. (1995) Science 268: 1766-1769, Rossi, F.
.MV and HM Blau (1998) Curr. Opin. Biotechnol. 9: 451-456), commercially available In
Vitrogen's T-REX plasmid), ecdysone inducible promoter (I
nvitrogen plasmids PVGRXR and PIND), FK506 / rapamycin inducible promoter or RU486 / mifepristone inducible promoter (Rossi, FMV and HM Blau, supra), or (iii) GBAP from normal individuals. It can be expressed using the natural promoter or the tissue-specific promoter of the endogenous gene encoding the gene.

Commercially available liposome transformation kits (eg PerFec available from Invitrogen)
t Lipid Transfection Kit) allows one of skill in the art to introduce polynucleotides into target cells in culture without resorting to much experience. In another embodiment, the calcium phosphate method (Graham. FL and AJ Eb (1973) Virology 52:45.
6-467) or electroporation (Neumann, B. et al. (1982) EMBO J. 1: 841-845). Introducing DNA into primary cells requires modification of standardized mammalian transfection protocols.

In another embodiment of the invention, the disease or disorder caused by a gene deficiency associated with GBAP expression encodes GBAP under the control of (i) a retroviral terminal repeat (LTR) promoter or an independent promoter. (Ii) suitable polynucleotide
Preparation and treatment of retroviral vector consisting of RNA packaging signal and (iii) Rev responsive element (RRE) with additional retroviral cis-acting RNA sequence and coding sequence required for efficient vector growth can do.
Retroviral vectors (eg PFB and PBF NEO) are commercially available from Stratagene and published data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. US
A. 92: 6733-6737). Reference to the above data is made a part of this specification. The vector is propagated in a suitable vector-producing cell line (VPCL), which expresses an envelope gene with tropism for the receptor on the target cell or a promiscuous envelope protein such as VSVg (Armentano, D. et al. 19
87) J. Virol. 61: 1647-1650, Bender, MA et al. (1987) J. Virol. 61: 1639-164
6, Adam, MA and AD 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). U.S. Pat. No. 5,910,434 ("Method for obtaining regi
trovirus packaging cell lines producing high transducing efficiency retr
oviral cells ") disclose methods for obtaining retroviral packaging cell lines, which are incorporated herein by reference. Propagation of retroviral vectors, cell populations (eg CD4 ⁺ Transduction of (T cells) and returning the transduced cells to the patient is a method known to those skilled in the art of gene therapy and has been described in numerous references (Ranga, U. et al. (1997). ) J. Virol.
71: 7020-7029, Bauer, G. et al. (1997) Blood 89: 2259-2267, Bonyhadi, ML (1
997) J. Virol. 71: 4707-4716, Ranga, U. et al. (1998) Proc. Natl. Acad. Sci.
USA 95: 1201-1206, Su, L. (1997) Blood 89: 2283-2290).

In another example, an adenovirus-based gene therapy delivery system is used to deliver a polynucleotide encoding GBAP to cells having one or more genetic abnormalities associated with the expression of GBAP. The construction and packaging of adenovirus-based vectors is known to those of skill in the art. Replication-defective adenovirus vector
It has been shown to be variable for the introduction of genes encoding immunomodulatory proteins into intact pancreatic islets (Csete, ME et al. (1995) Transplantation 2
7: 263-268). A possible adenovirus vector is US Pat. No. 5,707,618 (“Adenovirus vectors for gene therapy”) to Armentano.
, Which is incorporated herein by reference. Regarding the adenovirus vector, Antinozi, PA et al. (1999) Annu. Rev. Nutr. 19: 511-
See also 544 and Verma, IM and N. Somia (1997) Nature 18: 389: 239-242. Both documents are incorporated herein by reference.

In yet another embodiment, a herpes gene therapy delivery system is used to deliver a polynucleotide encoding GBAP to a target cell having one or more genetic abnormalities associated with GBAP expression. The use of herpes simplex virus (HSV) based vectors is particularly useful in introducing GBAP into cells of the central nervous system where HSV is tropic. Construction and packaging of herpes-based vectors is known to those of skill in the art. Vectors of the replication competent herpes simplex virus (HSV) type I system have been used to deliver reporter genes to the primate eye (Liu, X. et al. (1999) Exp. Eye Res.
.169: 385-395). For the production of HSV-1 viral vectors, US Pat. No. 5,804,413 (“Herpes simplex virus swains for gene transfer”) was granted to DeLuca.
), Which is incorporated herein by reference. US Patent No.
No. 5,804,413 describes recombinant HSV d92 containing a genome with at least one endogenous gene that is introduced into cells under the control of a promoter suitable for purposes including human gene therapy. The above patents also include ICP4, ICP27 and IC
Disclosed is the generation and use of a recombinant HSV line that is eliminated for P22. HS
For V vectors, Goins, WF et al. (1999) J. Virol. 73: 519-532 and Xu,
See also H. et al. (1994) Dev. Biol. 163: 152-161. Both documents are incorporated herein by reference. Manipulation of cloned herpesvirus sequences, passage of recombinant virus after transfection of multiple plasmids containing different parts of the giant herpesvirus genome, growth and multiplication of herpesvirus, and infection of cells with herpesvirus. Is a technique known to those skilled in the art.

In another example, an alphavirus (positive single-stranded RNA virus) vector is used to deliver a polynucleotide encoding GBAP to target cells. Extensive biological studies of the prototype alphavirus Semliki Forest Fever virus (SFV) have shown that the gene transfer vector is based on the SFV genome (Garoff, H. and K.-J. . Li (1998) Cun. Opin. Biotech. 9: 464-469). During replication of alphavirus RNA, subgenomic RNA that normally encodes the viral capsid protein is produced. This subgenomic RNA replicates to higher levels than full-length genomic RNA, resulting in overproduction of capsid proteins relative to viral proteins with enzymatic activity (eg, proteases and polymerases).
Similarly, by introducing the coding sequence for GBAP into the alphavirus genome of the capsid coding region, a large number of GBAP coding RNAs are produced in the vector-transfected cells and high levels of GBAP are synthesized. The ability to establish a persistent infection of hamster normal kidney cells (BHK-21) with mutants of Sindbis virus (SIN), while alphavirus infection is usually associated with cell lysis within days, is not Have suggested that the lytic replication of alphavirus can be suitably modified so that it can be applied to gene therapy (Dryga, SA et al. (1997) Virology 228: 74-83). The wide host range of alphaviruses allows the introduction of GBAP into a variety of cell types. Specific transduction of a subset of cells in a population may require selection of cells prior to transduction. Methods of treating alphavirus infectious cDNA clones, transfection of alphavirus cDNA and RNA, and methods of infection with alphavirus are known to those of skill in the art.

It is also possible to inhibit gene expression using, for example, an oligonucleotide derived from a transcription start site that is between about −10 and about +10 counted from the start site. Similarly, inhibition is possible using the triple helix base pair formation method. Triple helix base pairing is useful because it inhibits the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors or regulatory molecules. Recent advances in treatment with triple helix DNA have been described in the literature (Gee, JE
(1994) in: Huber, BEand BI Carr, Molecular and Immunologic Approaches , Futura Publishing Co., Mt. Kisco, NY, pp. 163-177 , etc.). Complementary sequences or antisense molecules can also be designed to block translation of mRNA by blocking the binding of transcripts to ribosomes.

Ribozymes are enzymatic RNA molecules that can be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action is a complementary target that precedes nucleotide breaks.
It is involved in the sequence-specific hybridization of ribozyme molecules to RNA.
For example, recombinant hammerhead ribozyme molecules specifically and effectively catalyze nucleotide breaks in the GBAP-encoding sequence.

Specific ribozyme cleavage sites within any potential RNA target are GUA, GUU, GUC
It is first identified by scanning the target molecule for ribozyme cleavage sites, including sequences. Once identified, 15-2 corresponding to the region of the target gene containing the cleavage site for secondary structural features that render the oligonucleotide dysfunctional
It allows the evaluation of short RNA sequences of 0 ribonucleotides. The suitability of candidate targets can also be evaluated by testing the operability of hybridization with complementary oligonucleotides using a ribonuclease protection assay.

Complementary ribonucleic acid molecules and ribozymes of the invention can be made using any method well known in the art for nucleic acid molecule synthesis. An optional method is to chemically synthesize an oligonucleotide such as a solid phase phosphoramidite compound. Alternatively, RNA molecules may be produced by in vitro and in vivo transcription of the DNA sequence encoding HRIP. Such DNA sequences can be incorporated into a wide variety of vectors using suitable RNA polymerase promoters such as T7 and SP6. Alternatively, those cDNA products that synthesize complementary RNA constitutively or inducibly can be introduced into cell lines, cells or tissues.

RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, flanking sequences at the 5'and / or 3'ends of the molecule, or phosphorothionate or 2'O rather than phosphodiesterase linkages within the backbone of the molecule. Use of methyl is included. This concept is unique to the production of PNA and can be extended to all these molecules. It includes acetyl-, methyl-, thio-, and similar modifications of adenine, cytidine, guanine, thymine, and uridine, which are not readily recognized by endogenous endonucleases, as well as non-conventional bases such as inosine. , By including queosin (queosine), wybutosine (wybutosine) and the like.

Additional embodiments of the invention include methods of screening for compounds effective in mutating expression of a polynucleotide encoding GBAP. Compounds effective for, but not limited to, variant expression of specific polynucleotides include oligonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, transcription factors and other polypeptide transcription control factors, and specific polynucleotide sequences that interact with them. There are non-polymeric chemical entities that can act. Effective compounds may mutate polynucleotide expression by acting as either inhibitors or enhancers of polynucleotide expression. Therefore, in the treatment of diseases associated with increased expression or activity of GBAP, compounds that specifically inhibit the expression of polynucleotides encoding GBAP would be of therapeutic benefit and would be associated with decreased expression or activity of GBAP. In treating diseases, compounds that specifically enhance the expression of polynucleotides encoding GBAP may be of therapeutic benefit.

At least one to a plurality of test compounds may be screened for effectiveness in mutating the expression of a specific polynucleotide. The test compound is obtained by any method commonly known in the art. Such methods include mutating the expression of the polynucleotide, selecting from existing, commercially available or proprietary, natural or unnatural compound libraries, and chemical and / or structural analysis of the target polynucleotide. There are chemical modifications of compounds that are known to be effective in the rational design of compounds based on properties and in the selection of combinatorially or randomly generated libraries of compounds. A sample containing a polynucleotide encoding GBAP is exposed to at least one test compound and is thus obtained. The sample can have, for example, intact cells, permeabilized cells, in vitro cell release or reconstituted biochemical systems. Changes in expression of a polynucleotide encoding GBAP are assayed by any method commonly known in the art. Normal,
Expression of specific nucleotides is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding GBAP. The amount of hybridization can be quantified and thereby form the basis for comparison of the expression of polynucleotides exposed and unexposed to one or more test compounds. Detection of changes in expression of the polynucleotide exposed to the test compound indicates that the test compound is effective in mutating expression of the polynucleotide. For compounds effective for mutating specific polynucleotides, for example, Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435, Arndt, GM et al. (2000) Nucleic Acids Res .28: E15) or He
Human cell lines such as La cells (Clarke, ML et al. (2000) Biochem. Biophys. Res. Comm.
un. 268: 8-13). A particular embodiment of the invention is
Involved in screening combinatorial libraries of oligonucleotides (deoxyribonucleotides, ribonucleotides, peptide nucleic acids, modified oligonucleotides) for antisense activity against specific polynucleotide sequences (Bruice, TW et al. (1997) US Patent 5,686,242, Bruice, TW et al. (2000)
U.S. Pat. No. 6,022,691).

A number of methods are available for introducing vectors into cells or tissues, in vivo
Equally suitable for in vitro and ex vivo use. For ex vivo therapy, the vector can be introduced into hepatocytes taken from a patient, cloned and propagated and autologously transplanted back into the same patient. Transfection, ribosome injection or transport by polycationic aminopolymers can be performed using methods well known in the art (Goldman, CK et al. (1997) Nat. Biotechnol. 15:
462-466. Etc.).

Any of the above treatment methods can be applied to all subjects in need of treatment including mammals such as humans, dogs, cats, cows, horses, rabbits and monkeys.

An additional embodiment of the present invention relates to the administration of pharmaceutical ingredients having the active ingredient usually formulated with pharmaceutically acceptable excipients. Excipients include, for example, cellulose, gums and proteins. Various formulations are commonly known and details are given in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such pharmaceutical ingredients include GBAP, antibodies against GBAP, mimetics, agonists,
It may consist of an antagonist, or a GBAP inhibitor.

The pharmaceutical ingredients used in the present invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, arachnoid. Intracavitary, intraventricular, pulmonary, percutaneous, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal.

Pulmonary administration pharmaceutical ingredients may be prepared in liquid or dry powder form. Such pharmaceutical ingredients are typically aerosolized shortly before inhalation by the patient. In the case of small molecules (eg traditional low molecular weight organic drugs), aerosol delivery of fast-acting formulations is known in the art. In the case of macromolecules (eg larger peptides and proteins)
Recent improvements in pulmonary transport through the alveolar region of the lung in the field have made it possible to substantially transport drugs such as insulin into the blood circulation (Patton,
See JS et al., US Pat. No. 5,997,848). Pulmonary transport is superior in that it is administered without needle injection, obviating the need for potentially toxic penetration enhancers.

Pharmaceutical ingredients suitable for use in the present invention include those ingredients that contain the active ingredient in an amount sufficient to achieve the intended purpose. Determination of the effective dose is within the ability of those skilled in the art.

A special form of pharmaceutical ingredient is prepared for direct intracellular delivery of macromolecules, including GBAP or fragments thereof. For example, a liposome preparation containing a cell-impermeable polymer is
It can promote cell fusion and intracellular transport of macromolecules. Or GBAP or a fragment thereof
It is also possible to bind from the HIV Tat-1 protein to the cationic N-terminal part. The fusion protein thus produced is known to transduce cells of all tissues including the mouse model system brain (Schwarze, SR et al. (1999) Science 285: 1.
569-1572).

For any compound, in a cell culture assay, such as a neoplastic cell culture assay, or in an animal model, such as a mouse, rabbit, dog or pig, one first estimates the effective dose of the treatment. be able to. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

The therapeutically effective dose refers to the amount of active ingredient that ameliorates symptoms and conditions. Examples of such active ingredients are GBAP or fragments thereof, antibodies to GBAP, GBAP.
There are agonists, antagonists or inhibitors of. Medicinal efficacy and toxicity are determined by standard drug techniques in cell culture or animal studies, eg, ED ₅₀ (
It can be determined, for example, by measuring the pharmaceutically effective amount of 50% of the population) or LD ₅₀ (lethal dose of 50% of the population). The dose ratio relative to the therapeutic effect of toxic effects is the therapeutic index and it can be expressed as the ratio LD ₅₀ / ED _50. Pharmaceutical ingredients that exhibit a high therapeutic index are desirable. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for use in humans. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dosage will vary within this range depending upon the dosage form employed, the sensitivity of the patient and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide effective levels of the active ingredient or to maintain the desired effect. Factors relating to the subject include the severity of the disease, the normal health condition of the patient, the age, weight and sex of the patient, time and frequency of administration, drug combination, reaction sensitivity, response to treatment and the like. Long-acting pharmaceutical ingredients may be administered at intervals of once every 3 to 4 days, once every week, or once every two weeks, depending on the half-life and clearance rate of the particular formulation.

A usual dose is about 0.1 to 100,000 μg, depending on the route of administration,
The total is up to about 1g. Guidance regarding specific dosages and delivery methods is found in the literature and is usually available to the onsite physician. Those skilled in the art
A recipe for nucleotides that is different than the recipe for proteins or inhibitors will be utilized. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

Diagnosis In another embodiment, for the diagnosis of a disease characterized by the expression of GBAP, or in an assay for monitoring patients treated with GBAP or an agonist, antagonist or inhibitor of GBAP, Antibodies that specifically bind GBAP may be used. Antibodies useful for diagnostic purposes are formulated in the same manner as described in the treatment section above. Diagnostic assays for GBAP include methods that utilize antibodies and labels to detect GBAP in human body fluids or in extracts of cells and tissues. The antibody may be used with or without modification and may be labeled by covalent or non-covalent attachment of the reporter molecule. A wide variety of reporter molecules are known in the art and can be used. Some reporter molecules have been described above.

Various protocols for measuring GBAP, such as ELISA, RIA, FACS, etc., are known in the art and provide the basis for diagnosing corrected or abnormal levels of GBAP expression. GBAP is obtained by binding an antibody against GBAP to a body fluid or cells collected from a normal mammalian subject such as a human subject under conditions suitable for complex formation.
Normal or standard values of expression are determined. The amount of standard complex formed can be quantified by various methods such as photometry. The amount of GBAP expressed in the subject, control, and lesion samples from the specimen are compared to standard values. The deviation between the standard value and the subject is the parameter for diagnosing the disease.

According to another embodiment, the polynucleotide encoding GBAP can be used for diagnostic purposes. Polynucleotides that can be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNA. The polynucleotides can be used to detect and quantify gene expression in a sample such that GBAP expression in the sample can be correlated with disease. Diagnostic assays can be used to measure the absence, presence and overexpression of GBAP, and to monitor the preparation of GBAP levels during therapeutic intervention.

In one embodiment, to identify a nucleic acid sequence encoding GBAP, hybridization with a PCR probe capable of detecting a polynucleotide sequence, including genomic sequence, encoding GBAP or a closely related molecule. Can be used. Whether the probe has a high specific region such as a 5'regulatory region or a low specific region such as a conserved motif, it identifies only the native sequence encoding GBAP, mutant or related sequences It depends on the specificity of the probe and the stringency of hybridization or amplification.

The probe can also be used to detect related sequences, which sequences may also have at least 50% homology to any sequence encoding GBAP. The hybridization probe of the present invention can be DNA or RNA, and can be derived from the sequences of SEQ ID NOs: 67 to 132, or the genomic sequence including the promoter, enhancer and intron of the GBAP gene.

A means for generating specific hybridization probes for GBAP-encoding DNA is to include a polynucleotide sequence encoding GBAP or a GBAP derivative in the mRNA sequence.
Included is a method of cloning into a vector for making NA probes. mRNA
Vectors for making probes are known to those of skill in the art, are commercially available, and can be used to synthesize RNA probes in vitro by adding a suitable RNA polymerase and suitable labeled nucleotides. . Hybridization probes can be labeled with different populations of reporters. Examples of reporter populations include radionuclides such as ³² P or ³⁵ S, or enzyme labels such as alkaline phosphatase attached to the probe via an avidin / biotin binding system.

Polynucleotide sequences encoding GBAP can be used for the diagnosis of diseases associated with the expression of GBAP. Non-limiting examples of such disorders include immune system disorders, including inflammation, actinic keratoses, acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies. , Ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, emphysema, fetal erythroblastosis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture syndrome, gout, Graves' disease, Hashimoto thyroiditis, paroxysmal nocturnal hemoglobin. Urine, hepatitis, hypereosinophilia, irritable bowel syndrome, lymphotoxin-induced temporary lymphopenia, mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, myocardium or heart Membrane inflammation Myelofibrosis, osteoarthritis, osteoporosis, pancreatitis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, primary thrombocytosis, Includes thrombocytopenia, ulcerative colitis, uveitis, Werner syndrome, cancer complications, hemodialysis, extracorporeal circulation, trauma, and hematopoietic cancers including lymphoma, leukemia, myeloma, and reproductive disorders. , Among them, abnormal prolactin production, fallopian tube disease,
Infertility including abnormal ovulation and endometriosis, and abnormal estrus, abnormal menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial or ovarian cancer, uterine fibroids, autoimmune disorders, ectopic pregnancy And teratogenesis, breast cancer, fibrocystic mastopathy and milk leak, and spermatogenesis abnormalities, abnormal sperm physiology, testicular cancer, prostate cancer, benign prostatic hyperplasia, prostatitis,
Peyronie's disease, impotence, male breast cancer and gynecomastia, as well as nervous system diseases, including epilepsy, ischemic cerebrovascular disease, 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, retinitis pigmentosa, hereditary ataxia, multiple sclerosis and other demyelination Disease, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, purulent intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, kuru, Prion diseases including Creutzfeldt-Jakob disease and Gastmann-Straussler-Scheinker syndrome, fatal familial insomnia, trophic and metabolic disorders of the nervous system, neurofibromatosis, tuberous sclerosis Cerebellum retinal vascular neuroblastoma disease (cerebelloretinal hemangioblast
cerebral trigeminal vascular syndrome, mental retardation and other central nervous system development disorders, cerebral palsy, neuroskeletal abnormalities, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nerve disorders, skin Myositis and polymyositis, hereditary, metabolic, endocrine and toxic myopathy, myasthenia gravis, periodic quadriplegia, and psychiatric disorders including mood disorders, anxiety disorders and schizophrenia, and akathisia, It includes amnesia, catatonic disease, diabetic neuropathy, extrapyramidal terminal deficiency syndrome, dystonia, schizophrenia, postherpetic neuralgia and Tourette's disease, and also impaired cell signaling. Indicates hypothalamus and lesions resulting from lesions such as primary brain tumors and adenomas, gestational infarction, pituitary resection, aneurysm, vascular malformation, thrombosis, infection, immune abnormality, and complications due to head injury. Pituitary disorders, inappropriate antidiuretic hormone (ADH) secretion syndrome (SIADH) that is more likely to occur due to benign adenoma, and disorders associated with pituitary hypertrophy, including acromegaly and macromegaly, and goiter and myxedema, bacteria Disorders related to hypothyroidism including infectious acute thyroiditis, hyperparathyroidism including Conn's disease (chronic hypercalemia), pancreatic diseases such as type I and type II diabetes and complications, hyperplasia and Adrenal related disorders such as adrenal cortex carcinoma and adenoma, hypertension associated with alkalosis, and abnormal prolactin production and infertility in women, endometriosis, menstrual cycle perturbation, polycystic ovarian disease, hyperprolactinemia , Selective gonadotropin deficiency, amenorrhea, galactorrhea, hermaphroditism, hirsutism and virilization, breast cancer, postmenopausal osteoporosis, Leydig cell hypermorphism in men Gonadal steroid hormone-related diseases such as adult, male climacteric, germ cell aplasia, hypersexuality associated with Leydig cell tumor, androgen resistance associated with androgen receptor deficiency, 5α-reductase syndrome, female mastopathy Endocrine disorders including and are also included, and cell proliferation abnormalities are also included, among which actinic keratoses, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed bonds Tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia,
Cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, specifically adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglia, gastrointestinal tract, heart, kidney ,
Liver, lung, muscle, ovary, pancreas, parathyroid gland, penis, prostate gland, salivary gland, skin, spleen,
Includes testicular, thymic, thyroid, and uterine cancers. Polynucleotide sequences encoding GBAP are derived from Southern, Northern, dot blot and other membrane-based techniques, PCR, dipstick, pin and multi-format ELISA-like assays, and mutant GBAP. It can be used in microarrays that utilize bodily fluids or tissues collected from patients to detect expression. Such qualitative or quantitative methods are known in the art.

In some forms, the nucleotide sequences encoding GBAP may be useful in assays that detect related disorders, particularly the disorders described above. The nucleotide sequence encoding GBAP is labeled by standard methods and can be added to a sample of bodily fluid or tissue taken from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the samples are washed and the signal is quantified and compared to standard values. If the amount of signal in the patient sample is significantly altered compared to the control sample, the level of mutation of the nucleotide sequence encoding GBAP in the sample is indicative of the presence of the associated disease. Such assays can also be used to predict the effects of particular treatments in animal studies, clinical trials, or to monitor the treatment of individual patients.

In order to provide diagnostic criteria for diseases associated with GBAP expression, a normal or standard profile for expression is established. This is because the body fluid or cells extracted from a normal animal or human subject is treated with GBAP under conditions suitable for hybridization or amplification.
Can be achieved by ligating with a sequence coding for or a fragment thereof. Standard hybridizations can be quantitated by comparing the values obtained from experiments performed with known amounts of substantially purified polynucleotide to those obtained from normal subjects. The standard value thus obtained can be compared with the value obtained from a sample obtained from a patient who shows signs of disease. Deviation from standard values is used to verify the presence of disease.

Once the presence of the disease has been demonstrated and the treatment protocol has begun, hybridization assays are routinely used to determine whether the patient's expression levels begin to approach the levels observed in normal subjects. Get repeat. The results obtained from the serial assays can be used to show the effect of treatment over a period of days to months.

For cancer, the presence of abnormal amounts of transcripts (under- or over-expression) in living tissue from an individual indicates the predisposition to the disease or detects the disease before actual clinical symptoms appear. May provide a method of doing so. With a clearer diagnosis of this kind,
It allows medical professionals to use preventative or aggressive treatment early to prevent the development or further progression of cancer.

The additional diagnostic utility of oligonucleotides designed from GBAP coding sequences may involve the use of PCR. These oligomers can be chemically synthesized, enzymatically produced, or produced in vitro . The oligomer is
Preferably, it contains a fragment of a polynucleotide encoding GBAP or a fragment of a polynucleotide complementary to the polynucleotide encoding GBAP, and is used to identify a specific gene or condition under optimal conditions. Also, the oligomers can be used for the detection and / or quantification of closely related DNA or RNA sequences under mildly stringent conditions.

In certain embodiments, oligonucleotide primers derived from a polynucleotide sequence encoding GBAP can be used to detect single nucleotide polymorphisms (SNPs). SNPs are substitutions, insertions and deletions that often cause congenital or acquired genetic diseases in humans. Without limitation, SNP detection methods include restriction enzyme digestion (SSCP) and fluorescent SSCP (fSSCP). In SSCP, polymerase chain reaction (PC) was performed using an oligonucleotide primer derived from a polynucleotide sequence encoding GBAP.
R) is used to amplify the DNA. DNA can be derived, for example, from diseased or normal tissue, biopsy samples, body fluids and the like. The SNPs in the DNA are two single-stranded PCR products.
It makes a difference in the secondary and tertiary structure. Differences can be detected using gel electrophoresis in non-denaturing gels. In fSCCP, oligonucleotide primers are fluorescently labeled. This enables detection of amplimers with high-throughput equipment such as DNA sequencing machines. Furthermore, in silico SNP (in silico SNP, is
A sequence database analysis method called SNP) can identify polymorphisms by comparing the sequences of individual overlapping DNA fragments as aligned to a common consensus sequence. These computer-based methods filter out sequence variations due to laboratory adjustments of DNA and sequencing errors using statistical models and automated analysis of DNA sequence chromatograms. In another embodiment, SNPs are detected and characterized by mass spectrometry, for example using the High Throughput MASSARRAY system (Sequenom, Inc., San Diego CA).

Methods that can be used to quantify the expression of GBAP also include radiolabelling or biotin labeling of nucleotides, coamplification of regulatory nucleic acids and interpolating the results obtained from standard curves (Melby, PC et al. 1993) J. Immunol. Methods, 159: 235-24.
4, Duplaa, C. et al. (1993) Anal. Biochem. 212: 229-236). High-throughput format assays in which the oligomer of interest is present in different dilutions and quantified quickly by spectrophotometric or non-color response can accelerate quantitation rates for multiple samples .

In yet another example, oligonucleotides or longer fragments from any of the polynucleotide sequences described herein can be used as targets in microarrays. Microarrays can be used in transcription imaging techniques that simultaneously monitor the associated expression levels of multiple genes. In this regard, Seilhamer, JJ et al., "Comparative Gene Transcrip," of US Pat. No. 5,840,484.
T Analysis "and incorporated herein by reference. Microarrays can also be used to identify genetic variants, mutations and polymorphisms. Use this information To determine gene function, understand the genetic basis of the disease,
Diseases can be diagnosed, the progression / regression of the disease as a function of gene expression can be monitored, and the activity of the drug in treating the disease can be developed and monitored. In particular, this information can be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment for the patient. For example, based on the patient's pharmacogenomic profile, therapeutic agents can be selected that are highly effective and show few side effects to the patient.

In another example, an antibody specific for GBAP, GBAP or a fragment thereof may be used as an element on the microarray. Microarrays can be used to monitor or measure protein-protein interactions, drug-target interactions and gene expression profiles as described above.

One example pertains to the use of the polynucleotides of the invention to generate a tissue or cell type transcriptional image. Transcriptional images represent the overall pattern of gene expression by a particular tissue or cell type. The overall gene expression pattern is
Analysis by quantifying multiple expressed genes and their relative abundance under given conditions and time (Seilliamer et al., US Pat. No. 5,840,484, "Comparative Gene").
Transcript Analysis ", incorporated herein by reference.
. Thus, a transcriptional image may be produced by hybridizing a polynucleotide or a complement thereof of the invention to the entire transcription or reverse transcription of a particular tissue or cell type. In one example, a polynucleotide of the invention or its complement has a subset of elements on a plurality of microarrays and hybridisation occurs in a high throughput format. The resulting transcriptional image will provide a profile of gene activity.

Transcriptional images can be generated using transcripts isolated from tissues, cell lines, biopsies or biological samples. Transcriptional images may reflect gene expression in vivo for tissue or biopsy samples and in vitro for cell lines.

Transcriptional images to generate expression profiles of the polynucleotides of the invention can be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals as well as toxicity studies of industrial and natural environmental compounds. All compounds induce characteristic gene expression patterns that represent mechanisms of action and toxicity, often termed molecular fingerprints or toxic signatures (Nuwaysir, EF et al. (1999) Mol.
. Carcinog. 24: 153-159; Steiner, S. and NL Anderson (2000) Toxicol. Le
tt. 112-113: 467-471, specifically incorporated herein by reference). If a test compound has a signature similar to that of a compound with known toxicity, it may share toxic properties. These fingerprints or signatures are most informative and sophisticated when they contain expression information from multiple genes and gene families. Ideally, measuring expression across the genome gives the highest quality signature. Equally important are genes whose expression is mutated by any test compound, since the expression levels of these genes can be used to normalize the rest of the expression data. The normalization procedure serves to compare expression data after treatment with different compounds. Although assigning gene function to elements of the toxic signature helps to interpret toxicity mechanisms, knowledge of gene function is not necessary to statistically match signatures that lead to prediction of toxicity (eg, Institute of Environmental Health Sciences, USA). (National Institute of Environmental
Health Sciences) published on February 29, 2000, http://www.niehs.nih.gov/o
See Press Release 00-02 available at c / news / toxchip.htm). Therefore, in toxicological screening using the toxic signature, it is important and desirable to include all expressed gene sequences.

In one example, the toxicity of a test compound is calculated by treating a biological sample containing nucleic acid within the test compound. The expressed in treated biological sample can be hybridized to one or several probes specific for a polynucleotide of the invention, thereby quantifying the transcription level corresponding to the polynucleotide of the invention. Transcript levels in treated biological samples are compared to levels in untreated biological samples. The difference in the transcription level of both samples is indicative of the toxic response caused by the test compound in the treated samples.

Another example relates to the use of the polypeptide sequences of the invention to analyze a tissue or cell type proteome. The term proteome refers to the overall pattern of protein expression in a particular tissue or cell type. Each protein component of the proteome can be individually targeted for further analysis. Proteome expression patterns or profiles are analyzed by quantifying the number of expressed proteins and their relative abundance at a given time under given conditions. Thus, by isolating and analyzing polypeptides of a particular tissue or cell type, a proteomic profile of cells can be generated. In one example, the proteins obtained from the sample in one dimension are separated by isoelectric focusing and then by two-dimensional gel electrophoresis such that they are separated by molecular weight in two dimensions by sodium dodecyl sulfate slab gel electrophoresis. Can be achieved (Steiner and Anderson, supra). Proteins are usually visualized as discrete and uniquely located spots in the gel by staining the gel with substances such as Coomassie blue or silver or fluorescent stains. The optical density of each protein spot is usually proportional to the level of protein in the sample. Compare the optical densities of equally located protein spots from different samples (eg, biological samples treated with test compounds or therapeutics or untreated biological samples) to determine the protein spots associated with the treatment Identify any changes in density. The proteins in the spots are partially aligned and mass spectrometrically analyzed, eg, using standard methods utilizing chemical or enzymatic cleavage. Identification of a protein in a spot can be determined by comparing its subsequence (preferably at least 5 contiguous amino acid residues) to a polypeptide sequence of the invention. In some cases, additional sequence data can be obtained for final protein identification.

Antibodies specific for GBAP can also be used to generate proteome profiles to quantify the level of GBAP expression. In one example, protein expression levels are identified by using antibodies as elements on the microarray and exposing the microarray to a sample to detect the level of protein binding to each array element (Lueking, A. et al. (1999). Anal. Biochem. 270: 103-111; Mendoze, LG
et al. (1999) Biotechniques 27: 778-788). Detection can be performed by various methods known in the art. For example, proteins in the sample can be reacted with thiol-reactive or amino-reactive fluorescent compounds to detect the amount of fluorescent binding at each array element.

The toxicity signature at the proteome level is also valuable for toxicological screening and should be analyzed in parallel with the toxicity signature at the transcription level. Some proteins in tissues have poor interrelationships between transcription and protein abundance (An
derson, NL and J. Seilhamer (1997) Electrophoresis 18: 533-537), the proteome toxicity signature is useful when analyzing compounds that do not significantly affect the transcriptional image but alter the proteome profile. possible. Furthermore, analysis of transcripts in body fluids is difficult due to the rapid degradation of mRNA, so the proteome profile is more reliable and informative in such cases.

In another example, the toxicity of a test compound is calculated by treating a biological sample containing the protein with the test compound. The proteins expressed in the treated biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the corresponding protein in the untreated biological sample. The difference in the amount of protein in both samples indicates a toxic response to the test compound in the treated samples. Identification of individual proteins is performed by sequencing the amino acid residues of the individual proteins and comparing these subsequences to the polypeptides of the invention.

In another example, the toxicity of a test compound is calculated by treating a biological sample containing the protein with the test compound. Proteins obtained from biological samples are incubated with antibodies specific for the polypeptides of the invention. The amount of protein recognized by the antibody is quantified. The amount of protein in the treated biological sample is compared to the amount of untreated biological sample. The difference in the amount of protein in both samples indicates a toxic response to the test compound in the treated samples.

Microarrays were prepared and used using methods well known in the art,
And analyze (Brennan, TM et al. (1995) U.S. Pat. No. 5,474,796, Schena,
M. et al. (1996) Proc. Natl. Acad. Sci. USA 93: 10614-10619, Baldeschweiler.
(1995) PCT application No. WO95 / 251116, Shalon, D. et al. (1995) PCT application No. WO95 /
35505, Heller, RA et al. (1997) Proc. Natl. Acad. Sci. USA 94: 2150-2155.
, Heller, MJ et al. (1997) US Pat. No. 5,605,662). Various types of microarrays are known and are described in detail in DNA Microarrays: A Practical Approach , M. Schena, ed. (1999) Oxford University Press, London. The document is incorporated herein by reference in its entirety.

In another embodiment of the invention, a nucleic acid sequence encoding GBAP can be used to produce a hybridization probe that is effective in mapping the native genomic sequence. Either coding or non-coding sequences can be used, and in some instances non-coding sequences are preferred over coding sequences. For example, conservation of coding sequences within members of a multigene family can result in unwanted cross-hybridization during chromosome mapping. The nucleic acid sequence may be a specific chromosome, a specific region of a chromosome or an artificially formed chromosome, for example, human artificial chromosome (HAC), yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), bacterial P1 product, or single chromosome cDNA. Maps to the library (Harrington,
JJ et al. (1997) Nat Genet. 15: 345-355, Price, CM (1993) Blood Rev. 7:12.
7-134, Trask, BJ (1991) Trends Genet. 7: 149-154 etc.). Once mapped, the nucleic acid sequences of the present invention can be used, for example, to generate a genetic linkage map that correlates the inheritance of a pathology with the inheritance of a particular chromosomal region or restriction fragment length polymorphism (RFLP).

Fluorescence in situ hybridization (FISH) can be correlated with other physical and genetic map data (see Heinz-Ulrich, et al. (1995) in Meyers, pp. 965-968., Supra). ). 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. The correlation between the position of the gene encoding GBAP on the physical chromosomal map and the predisposition to a particular disease may serve to define the region of DNA associated with that disease, and thus It can be an attempt to position clone.

Physical mapping techniques such as binding analysis using validated chromosomal markers and chromosomal in situ hybridization methods can be used to extend the genetic map. Placing a gene on the chromosome of another mammal, such as a mouse, may reveal related markers even when the number or arm of a particular human chromosome is unknown. This information is valuable to researchers investigating genetic disorders using positional cloning and other gene discovery techniques. Once the disease or syndrome is loosely located by genetic linkage to a particular gene region, such as the 11q22-23 region of ataxia telangiectasia, any mapping to that region will result in relevant genes for further investigation. Alternatively, it can represent a regulatory gene (Gatti, RA et al. (198
8) Nature 336: 577-580, etc.). The nucleotide sequences of the present invention may be used for discovering differences in chromosomal position among healthy persons, carriers, and infected persons due to translocation, inversion, and the like.

In another embodiment of the invention, GBAP, catalytic fragments of GBAP, immunogenic fragments, or oligopeptides thereof may be used to screen libraries of compounds using a variety of drug screening techniques. it can. The fragments used for drug screening may be free in solution, immobilized on a solid support, retained on the cell surface, or located intracellularly. The formation of a binding complex between GBAP and the drug under test is measurable.

Another drug screening method is used for high throughput screening of compounds that have a suitable binding affinity for a protein of interest (Geysen
, Et al. (1984) PCT application No. WO84 / 03564, etc.). In this method, a large number of different small test compounds are synthesized on a solid substrate. The test compound is reacted with GBAP or a fragment thereof and washed. The bound GBAP is then detected by methods well known in the art. Purified GBAP can also be coated directly on plates used in the drug screening techniques described above. In another example, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

In another example, a competitive drug screening assay can be used. In this assay, a neutralizing antibody capable of binding GBAP specifically competes with a test compound for binding GBAP. In this method, the antibody detects the presence of peptides that share one or several antigenic determinants with GBAP.

In another embodiment, if the new technology relies on currently known nucleotide sequence characteristics, including, but not limited to, the triplet genetic code and specific base pair interactions, The nucleotide sequence encoding GBAP can be used in any molecular biology technique that is still to be developed.

Without further elaboration, one of ordinary skill in the art will be able to utilize the invention to the full extent from the foregoing description. Therefore, the examples described below are for illustrative purposes only and are not intended to limit the invention in any way.

All patents, patent applications and publications disclosed herein, especially US Pat.
60 / 144,595, 60 / 150,460 and 60 / 159,849 are incorporated herein by reference.

Example 1 Preparation of cDNA Library RNA was purchased from Clontech or isolated from the tissues listed in Table 4. Some tissues were homogenized and dissolved in guanidinium isothiocyanate solution,
There are also tissues that have been homogenized and dissolved in a mixture of phenol or a suitable denaturant. The mixture of modifiers is, for example, TRIZOL (Life Technologies), which is a single-phase solution of phenol and guanidinium isothiocyanate. The resulting lysate was either centrifuged in cesium chloride or extracted with chloroform.
RNA was precipitated from the lysates using either isopropanol, sodium acetate and ethanol, or another method.

To increase the RNA purity, RNA extraction with phenol and precipitation was repeated as many times as necessary. In some cases RNA was treated with DNase. Most libraries have oligo d (T) -linked paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, V
alencia CA) or OLIGOTEX mRNA purification kit (QIAGEN).
A was isolated. Alternatively, RNA was isolated directly from tissue lysates using another RNA isolation kit, such as the POLY (A) PURE mRNA purification kit (Ambion, Austin TX).

[0230] In some cases, RNA was provided to Stratagene and the corresponding cDNA library was produced by the same. If not, use UNIZAP vector system (Stratagene) or SUPERSCRIPT using recommended methods or similar methods known in the art.
CDNA was synthesized using a plasmid system (Life Technologies) to prepare a cDNA library (see Ausubel, 1997, unit 5.1-6.6, etc., described above). Reverse transcription is
Starting with oligo d (T) or random primers. The synthetic oligonucleotide adapter was ligated to the double-stranded cDNA, and the cDNA was digested with a suitable restriction enzyme.
For most libraries, cDNA size (300-1000 bp) selection is SEPH
ACRYL S1000, SEPHAROSE CL2B or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis was used. The cDNA was ligated to the compatible restriction enzyme sites of the polylinker of the appropriate plasmid. Suitable plasmids include, for example, the PBLUESCRIPT plasmid (Stratag
ene), pSPORT1 plasmid (Life Technologies) or plNCY (Incyte Pharmac
euticals, Palo Alto CA) etc. The recombinant plasmid is XL1- from Stratagene.
The cells were transformed into competent E. coli cells containing Blue, XL1-BIueMRF or SOLR, or Life Technologies DH5α, DH10B or ELECTROMAX DH10B.

2 Isolation of cDNA Clone The plasmid obtained as described in Example 1 was UNIZAP vector system (
Harvested from host cells by in vivo excision with Stratagene) or by cell lysis. Magic or WIZARD miniprep DNA purification system (Promega), A
GTC miniprep purification kit (Edge Biosystems, Gaithersburg MD), QIAGEN's QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid and QIAWELL 8 Ultra Plasmid
Plasmids were purified using at least one of the purification systems, the REAL Prep 96 plasmid kit. After settling, resuspending in 0.1 ml distilled water,
Stored at 4 ° C with or without lyophilization.

In another example, plasmid DNA was amplified from host cell lysates using a direct ligation PCR method in a high throughput format (Rao, VB (1994) Anal. Biochem. 216).
: 1-14). Host cell lysis and thermal cycling processes were performed in a single reaction mixture. Samples were processed and stored in 384-well plates and the concentration of amplified plasmid DNA was adjusted to PICOGREEN dye (Molecular Probes, Eugene OR) and Fluoroskan.
Fluorimetric quantification using a II fluorescence scanner (Labsystems Oy, Helsinki, Finland).

3 Sequencing and Analysis The Incyte cDNA recovered as described in Example 2 was sequenced as follows. The cDNA sequencing reaction can be performed using standard methods or high throughput equipment such as ABI.
CATALYST 800 Thermal Cycler (PE Biosystems) or PTC-200 Thermal Cycler (MJ Research) with HYDRA Microdispenser (Robbins Scientific)
) Or MICROLAB 2200 (Hamilton) liquid transfer system. cDN
A sequencing reaction is performed using reagents provided by Amersham Pharmacia Biotech or A
BI sequencing kit, eg ABI PRISM BIGDYE Terminator cycle sequen
Prepared using the reagents provided in the cing ready reaction kit (PE Biosystems). MEGABACE 1000 DNA Sequencing System (Molecular Dynamics) for electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides
Or ABI PRISM 373 using standard ABI protocol and base pair calling software
Alternatively, the 377 Sequencing System (PE Biosystems) or other sequence analysis system well known in the art was used. Reading frames within the cDNA sequences were determined using standard methods (outlined in Ausubel, 1997, unit 7.7, supra). Several cDNA sequences were selected and the sequences extended using the method disclosed in Example 6.

A polynucleotide sequence derived from the cDNA sequence was constructed and analyzed using a combination of software utilizing algorithms well known to those of skill in the art. Table 5 shows the outline of tools, programs and algorithms used, applicable explanations, references, and threshold parameters. The tools, programs and algorithms used are shown in column 1 of table 5 and a brief description thereof in column 2. Column 3 is the preferred citation, and all references are incorporated by reference in their entirety. Where applicable,
Column 4 shows the score, probability value and other parameters used to evaluate the strength of the match between the two sequences (the higher the score, the higher the homology between the two sequences).
. Sequence analysis was performed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence alignments are generated using the default parameters specified by the clustal algorithm when incorporated into the MEGALIGN multi-sequence alignment program (DNASTAR), which also calculates the percent match between aligned sequences. did.

The polynucleotide sequences were validated by removing the vector, linker and polyA sequences and by masking ambiguous base pairs. In doing so, algorithms and programs based on BLAST, dynamic programming, and flanking dinucleotide frequency analysis were used. Then, using BLAST, FASTA and BLIMPS based programs, to obtain annotations in the programs, public databases such as GenBank primates and rodents, mammals, vertebrates, eukaryotic databases, and The array for the choice of BLOCKS, PRINTS, DOMO, PRODOM and PFAM was queried. The sequences were assembled into full-length polynucleotide sequences using programs based on Phred, Phrap and Consed and screened against open reading frames using programs based on GenMark, BLAST and FASTA. The full-length polynucleotide sequence is translated to derive the corresponding full-length amino acid sequence, and then the GenBank database (above), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM and Prosite databases, PFAM and other Hidden Markov Model (HMM ) Full-length sequences were analyzed by querying the base protein family database. HMM is a probabilistic approach for analyzing the consensus primary structure of gene families (see Eddy, SR (1996) Curr. Opin. Struct. Biol. 6: 361-365).

The programs described above for the construction and analysis of full length polynucleotide and amino acid sequences can also be used to identify fragments of the polynucleotide sequence from SEQ ID NOs: 67-132. About 20 to about 4 useful for hybridization and amplification
Fragments of 000 nucleotides were described in the "Invention" section above.

4 Analysis of Polynucleotide Expression Northern analysis is an experimental technique used to detect the presence of transcribed genetic information, in which the RN of a labeled nucleotide sequence from a particular cell type or tissue.
Is involved in hybridization to the membrane to which A is bound (Sambrook, supra.
(See Chapter 7, Ausubel. FM et al., Chapters 4 and 16).

Using similar computer technology applied to BLAST, GenBank and LifeSeq (Incyt
searching for identical or related molecules in nucleotide databases such as e Pharmaceuticals). Northern analysis is by far faster than many membrane-based hybridizations. In addition, the sensitivity of computer searches can be modified to determine whether or not a particular identity should be classified as exact or homologous. The search criterion is the product score, which is defined by the following formula.

[0239]

[Equation 1]

The product score takes into account both the degree of similarity between the two sequences and the length with which the sequences 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. The two sequences may share more than one HSP (which may be 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.

The results of the Northern analysis are reported as the percentage distribution of libraries in which transcripts encoding GBAP were produced. The analysis is involved in the categorization of cDNA libraries by organ / tissue and disease. Organ / tissue categories include cardiovascular, skin, development, endocrine, gastrointestinal, hematopoietic / immune, musculoskeletal, neural, reproductive and urological. Disease / condition categories include cancer, inflammation, trauma, cell proliferation, nerves, retention (
pooled) is included. The number of libraries expressing the target sequence was counted for each category and divided by the number of libraries in all categories. Percentages of tissue-specific and disease / condition-specific expression are shown in Table 3.

5 The cDNA sequence used to sequence the chromosome mapping SEQ ID NOs: 67-132 of GBAP, which encodes the polynucleotide , using BLAST and other implementations of the Smith-Waterman algorithm,
The sequences were compared to sequences obtained from the Incyte LIFE SEQ database and public domain databases. Sequences from the databases that match SEQ ID NOs: 67-132 were sequenced into clusters of flanking and overlapping sequences using an assembly algorithm such as Phrap (Table 5). Was the clustered sequence pre-mapped using radiation hybrid and genetic map data available from public sources such as the Stanford Human Genome Center (SHGC), Whitehead Genome Institute (WIGR) and Genethon? Was measured. As a result of including the mapped sequence in the cluster, the entire sequence of the cluster including the individual sequence number was assigned to the map position.

SEQ ID NOs: 70, 74, 75, 77, 80, 86, 87, 90, 92, 93, 9
4, 97, 101, 106, 109, 111, 112, 113, 115, 118
, 121 and 128 on the genetic map are described in the "Invention" section as the range or interval of human chromosomes. SEQ ID NOs: 94, 101, 109, 11
1 and 115 have reported one or more genetic map locations, SEQ ID NO: 94,
It shows that the previously mapped sequences that do not exactly match 101, 109, 111 and 115 but have similarities are assembled into each cluster. The map position of the centimorgan interval is measured relative to the end of the p-arm of the chromosome (centimorgan (cM) is a unit of measure based on the recombination frequency between chromosomal markers. On average, 1 cM is human. 1 megabase of DNA (M
It is almost equal to b). However, this value varies widely due to recombination hotspots and coldspots). The cM distance is based on the genetic markers mapped by Genethon so as to provide a border for radiation hybrid markers such that the sequences are contained within each cluster.

6 Extension of GBAP Encoding Polynucleotides The full length nucleic acid sequences of SEQ ID NOS: 67-132 were generated by extending the fragments using oligonucleotide primers designed from the appropriate fragment of the full length molecule.
One primer was synthesized to initiate 5'extension of the known fragment and the other primer was synthesized to initiate 3'extension of the known fragment. The starting primer is designed to have a length of about 22 to 30 nucleotides, a GC content of 50% or more, and a length of about 68 to 7 nucleotides.
The OLIGO 4.06 software (Na
designed from cDNA using National Biosciences) or another suitable program. Hairpin structures and primer extension of the nucleotides that resulted in primer-primer dimers were all avoided.

Sequences were extended with selected human cDNA libraries. 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 using 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 contains DNA template and 200 nm of each primer.
l, a reaction buffer containing Mg ²⁺ , (NH ₄ ) ₂ SO ₄ and β-mercaptoethanol, and Taq
DNA polymerase (Amersham Pharmacia Biotech) and ELONGASE enzyme (Life Tech
nologies) and Pfu DNA polymerase (Stratagene). The parameters used for the primer pair PCI A and PCI B are as follows. Step 1: 94 ° C. for 3 minutes Step 2: 94 ° C. for 15 seconds Step 3: 60 ° C. for 1 minute Step 4: 68 ° C. for 2 minutes Step 5: Steps 2, 3, and 4 are repeated 20 times Step 6: 68 ° C. 5 minutes Step 7: Stored at 4 ° C. For the primer pair T7, SK +, the following parameters were used instead of the above parameters. Step 1: 94 ° C. for 3 minutes Step 2: 94 ° C. for 15 seconds Step 3: 57 ° C. for 1 minute Step 4: 68 ° C. for 2 minutes Step 5: Steps 2, 3, 4 repeated 20 times Step 6: 68 ° C. Step 5: Store at 4 ℃ for 5 minutes. PICOGREEN quantitative reagent dissolved in 1X TE (0.25% (v / v) PICOGREEN, Molecular
100 μl of Probes, Eugene OR) and 0.5 μl of undiluted PCR product are distributed into each well of an opaque fluorometer plate (Coming Costar, Acton MA) to allow the DNA to bind to the reagents. The DNA concentration in each well was measured by. Fluoroskan II (Lab
systems Oy, Helsinki, Finland). Aliquot 5 of reaction mixture
-10 μl were analyzed by electrophoresis on a 1% agarose minigel to determine which reaction was successful in extending the sequence.

The extended nucleotides were desalted and concentrated, transferred to a 384-well plate and subjected to CviJ
Cholera virus endonuclease (Molecular Biology Research, Madison W
Digested with I) and sonicated or sheared prior to 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 (Stratag).
ene) to fill the restriction site overhang and transfect competent E. coli cells. Transfected cells were selected on antibiotic containing medium and individual colonies were selected and grown overnight at 37 ° C in 384 well plates in LB / 2x carb liquid medium.

Cells are lysed and Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu
DNA was amplified by PCR using DNA polymerase (Stratagene). The parameters used at that time are as follows. Step 1: 94 ° C. for 3 minutes Step 2: 94 ° C. for 15 seconds Step 3: 60 ° C. for 1 minute Step 4: 72 ° C. for 2 minutes Step 5: Steps 2, 3 and 4 are repeated 29 times Step 6: 72 ° C. 5 minutes Step 7: DNA stored at 4 ° C. was quantified by the PICOGREEN reagent (Molecular Probes) described above. Samples with low DNA recovery were reamplified using the same conditions as above. 20 samples
Dilute with% dimethylsulfoxide (1: 2, v / v) and use DYENAMIC energy transfer
sequencing primer and DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (PE
Biosystems) was used for sequencing.

Similarly, to obtain 5'regulatory sequences using the above procedure with oligonucleotides designed for extension and an appropriate genomic library, SEQ ID NOs: 67-13 were used.
Two nucleotide sequences are used.

7 Labeling and use of individual hybridization probes Utilizing the hybridization probes from SEQ ID NOs: 67-132,
Screen cDNA, genomic DNA or mRNA. 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 the latest software such as OLIGO 4.06 software (National Biosciences) and
0 pmol of each oligomer and 250 μCi of [γ- ³² P] adenosine triphosphate (Amersha
m Pharmacia Biotech) and T4 polynucleotide kinase (DuPont NEN, Boston
MA) to label. The labeled oligonucleotide is SEPHAD
EX G-25 Ultrafine molecular size exclusion dextran bead column (Amersham Pharmacia
Substantially purified using Biotech). 10 ⁷ min -1 in a typical membrane-based hybridization analysis of human genomic DNA digested with any one of Ase I, Bgl II, Eco RI, Pst I, Xba I or Pvu II (DuPont NEN) endonuclease. An aliquot containing a count of labeled probes is used.

[0251] The DNA obtained from each digest was fractionated on a 0.7% agarose gel and subjected to nylon membrane (Ny.
tran 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. Visualize the hybridization pattern using autoradiography or alternative imaging means,
Compare.

8 Microarrays Chaining or synthesis of array elements on the surface of microarrays may be performed by photolithography, piezoelectric printing (inkjet printing; see Baldeschweiler et al., Supra),
It can be achieved using mechanical microspotting techniques and their derivatives. In each of the above techniques, the substrate should be a solid, non-porous solid (Schena (1999). Supra). Recommended substrates include silicon, silica,
There are slide glasses, glass chips, and silicon wafers. Alternatively, an array similar to dot blotting or slot blotting can be used to
Elements may be placed and attached to the surface of the substrate using chemical or mechanical attachment procedures. Regular arrays can be produced manually or using available methods and machines and can have any suitable number of elements (Schena, M. et al. (1995) Scien.
ce 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 gene sequence fragment (EST), or fragments or oligomers thereof, may constitute an element of a microarray. Fragments or oligomers suitable for hybridization can be selected using software known in the art such as LASERGENE 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 strand buffer, 0.03 unit / μl RNase inhibitor, 500 μM dATP, 500
Reverse-transcribe using μM dGTP, 500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The reverse transcription reaction is carried out in 25 volume ml containing poly (A) ⁺ RNA using the GEMBRIGHT kit (Incyte). Specific control poly (A) ⁺ RNA is synthesized from non-coding yeast genomic DNA by in vitro transcription after incubation at 370 ° C. for 2 hours. 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 2 hours.
Incubate for 0 minutes to stop the reaction and degrade RNA. Samples were prepared from two consecutive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after conjugation both reaction samples are ethanol precipitated with 1 ml glycogen (1 mg / ml), 60 ml sodium acetate and 300 ml 100% ethanol. Next, SpeedVAC (Savant I
nstruments Inc., Holbrook NY) to dry and finish the sample, 14 μl
Resuspend in 5X SSC / 0.2% SDS.

Microarray Preparation The arrays of the invention are used to make array elements. Each array element is amplified 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 in 30 cycles of PCR from an initial amount of 1-2 ng to a final amount greater than 5 μg. The amplified array element is SEPHACRYL-400 (A
mersham Pharmacia Biotech).

Purified array elements are immobilized on polymer-coated glass slides. During and after the treatment, a large amount of distilled water washing solution is used to ultrasonically wash the microscope slide glass (Corning) in 0.1% SDS and acetone. 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR),
West Chester PA), washed extensively in distilled water,
Coat with 0.05% aminopropylsilane (Sigma) in 5% ethanol.

Array elements are added to a coated glass substrate using the method described in US Pat. No. 5,807,522. The patent is incorporated herein by reference. 1 μl of the array element DNA having an average concentration of 100 ng / μl is filled in the open capillary print element by a high speed robot device. The instrument now deposits approximately 5 nl of array element sample per slide.

For the microarray, STRATALINKER UV crosslinking agent (Stratagene) was used for UV.
Crosslink. The microarray is washed once with 0.2% SDS at room temperature and three times with distilled water. After incubating the microarray in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60 ° C., 0.2% SDS and Nonspecific binding sites are blocked by washing with distilled water.

Hybridization For the hybridization reaction, 9 μl of a sample mixture containing 0.2 μg each of the Cy3 and Cy5-labeled cDNA synthesis products in 5 × SSC, 0.2% SDS hybridization buffer is used. The sample mixture is heated to 65 ° C. for 5 minutes, aliquoted on the surface of the microarray 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 interior of the chamber is kept at 100% humidity by adding 140 μl of 5 × SSC to the corners 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 then in the second wash buffer (0.1 × SSC) at 45 ° C. for 1 min.
Wash 3 times for 0 minutes each and dry.

The detection reporter labeled hybridization complex is 48 for excitation of Cy3.
Detection with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of producing spectral lines at 8 nm and 632 nm for excitation of Cy3. A 20 × microscope objective (Nikon, Inc., Melville NY) is used to focus the pump laser light on the array. Computer-controlled microscope with slide containing array
Place it on the XY stage and pass the objective lens for raster scanning. 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 phosphors. The emitted light is based on the wavelength depending on the two phosphors and two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater
NJ). The signal is filtered using a suitable filter placed between the array and the photomultiplier tube. The maximum emission of the phosphor used is 56 for Cy3.
It is 5 nm and 650 nm for Cy5. The device can record spectra from both phosphors simultaneously, but typically scans each array twice with a suitable filter in the laser source, once for each phosphor.

Scan sensitivity is usually calibrated using the signal intensity emitted by a cDNA control species added to a sample mixture of known concentration. A specific position on the array contains a complementary DNA sequence, and the intensity of the signal at that position is determined by the weight ratio of the hybridizing species.
1: Correlate to 100,000. Two samples from different sources (eg representative test cells and control cells), each labeled with a different fluorophore, are hybridized to a single array to identify differentially expressed genes. In some cases, a labeled sample of calibrated cDNA with two fluorophores is labeled and an equal volume is added to the hybridization mixture to perform the calibration.

The output of the photomultiplier tube is a 12-bit RTI-835H analog-digital (A / D) conversion board (Analog Device) installed in an IBM compatible PC computer.
s, Inc., Norwood MA). The digitized data is displayed as an image and the signal intensities are mapped using a linear 20 color transform to a pseudo color range that extends from blue (low signal) to red (high signal). The data are also analyzed quantitatively. When simultaneously exciting and measuring two different phosphors, the emission spectrum of each phosphor is first used to correct the data for optical crosstalk between the phosphors (due to the overlapping emission spectrum).

The 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).

9 Complementary Polynucleotides A sequence complementary to the sequence encoding GBAP, or any portion thereof, may be a natural GB
Used to detect, reduce or inhibit the expression of AP. About 15-30
Although the use of oligonucleotides containing base pairs is described, essentially the same method can be used for smaller or larger sequence fragments. Oligo4.0
6 Software (National Biosciences) and sequences encoding GBAP are used to design appropriate oligonucleotides. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5'sequence and used to prevent the promoter from binding to the coding sequence. To inhibit translation, complementary oligonucleotides are designed so that the ribosome does not bind to the GBAP-encoding transcript.

10 GBAP Expression GBAP expression and purification can be performed using bacterial or virus-based expression systems. For expression of GBAP in bacteria, the cDNA is subcloned into a suitable vector containing an antibiotic resistance and an inducible promoter that enhances 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 GBAP when induced with isopropyl β-D thiogalactopyranoside (IPTG). Expression of GBAP in eukaryotic cells is performed by infecting insect cell lines or mammalian cell lines with Autographica californica nuclear polyhedrosis virus (AcMNPV), which is generally known as baculovirus. The baculovirus indispensable polyhedrin gene is replaced with the cDNA encoding GBAP either by homologous recombination or by bacterial-mediated gene transfer involving the transfer plasmid transfer. Viral infectivity is maintained and high levels of cDNA transcription are driven by the strong polyhedrin promoter. 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, GBAP as a fusion protein is synthesized using, for example, glutathione S transferase (GST) or peptide epitope tags such as FLAG or
Use 6-His. By using these, affinity-based purification of recombinant fusion proteins from crude cell lysates can be performed rapidly in one step. GST
Is a 26kDa enzyme from Schistosoma japonicum, which allows purification of the fusion protein on immobilized glutathione while maintaining protein activity and antigenicity (Amer
sham Pharmacia Biotech). After purification, the GST portion was
Can be proteolytically cleaved from. FLAG is an 8-amino acid peptide that is commercially available monoclonal and polyclonal anti-FLAG antibody (Eastma
n Kodak) to allow immunoaffinity purification. 6-His, which is a continuous extension of 6 histidine residues, allows purification on a metal chelate resin (QIAGEN). Methods for protein expression and purification are described in Ausubel (1995), chapters 10 and 16 above. GBAP purified by these methods can be used directly to perform the assays of Examples 11 and 15.

11 Demonstration of GBAP Activity GBTP's GTP activity is determined in an assay that measures GBAP binding to α-P ³² labeled GTP. First, the purified GBAP is blotted onto a filter and rinsed in the appropriate buffer. The filters are then incubated in a buffer containing radiolabeled α-P ³² . The filters are washed in buffer to remove unbound GTP and counted in a radioisotope counter. Non-specific binding is determined in an assay containing a 100-fold excess of unlabeled GTP. The amount of specific binding is proportional to the activity of GTP.

GBAP GTPase activity is determined in an assay that measures the conversion of α-P ³² GTP to α-P ³² GDP. GBAP is incubated with α-P ³² GTP in buffer for a suitable period of time and after centrifugation the reaction is terminated by heat or acid precipitation. Aliquots of the supernatant are subjected to polyacrylamide gel electrophoresis (PAGE) and unlabeled standards are used to separate both GDP and GTP. Remove the GDP spot and count with a radioisotope counter. The amount of radioactivity recovered in GDP is proportional to the GTPase activity of GBAP.

[0270]   12 Functional assays   GBAP function is a function of GBAP at physiologically elevated levels in mammalian cell culture systems.
Evaluation is by expression of the AP coding sequence. High level expression of cDNA and cDNA
Subcloning into a mammalian expression vector containing a strong promoter. Election
For the vector to be excised, pCMV SPORT plasmid (Life Technologies) and pCR 3
.1 plasmid (Invitrogen), both of which are cytomegalovirus promoted
Have a tar. 5-10 μg recombinant using liposome preparation or electroporation
Of the E. coli vector into a human cell line, such as an endothelium-derived or hematopoietic-derived cell line.
Pour in. Furthermore, 1 to 2 μg of plus containing the sequence encoding the labeled protein
Mido is co-transfected. Expression of the labeled protein allows it to
Means are provided to distinguish the transfected cells. Also, depending on the expression of the labeled protein
, The expression of the cDNA from the recombinant vector can be accurately predicted. Labeled proteins are examples
For example, green fluorescent protein (GFP; Clontech), CD64 or CD64-GFP fusion protein
You can choose from quality. Flow site, an automated, laser-optics-based technology
Transformed cells expressing GFP or CD64-GFP using metric (FCM)
Identify and assess the apoptotic state of the cell and other cellular characteristics. FCM is a cell
Detecting the uptake of fluorescent molecules to diagnose phenomena that precede or coincide with death
Weigh. Such a phenomenon is caused by propidium iodide.
Changes in nuclear DNA content measured by DNA staining, incorporation of bromodeoxyuridine
Down-regulation of DNA synthesis, measured by decreased amount, by reactivity with specific antibodies
Changes in measured protein expression on the surface and inside the cell, and fluorescence
Plasma membrane composition measured by binding of combined annexin V protein to cell surface
There is a change of. For flow cytometry methods, Ormerod, M. G. (1994). Flow Cytometry There is a description in Oxford, New York, NY.

The effect of GBAP on gene expression depends on the sequence encoding GBAP and CD64 or CD64-G.
Highly purified cell populations transfected with either FP 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 cells and non-transformed cells are
Effective separation can be achieved using magnetic beads coated with human IgG or antibodies against CD64 (DYNAL, Lake Success. NY). mRNA can be purified from cells by methods known in the art. The expression of mRNA encoding GBAP or other gene of interest can be analyzed by Northern analysis or microarray technology.

Production of 13 GBAP-specific antibody Polyacrylamide gel electrophoresis (PAGE; Harrington, MG (1990) Method
S. Enzymol. 182: 488-495 et al.) or GBAP substantially purified using other purification techniques to immunize rabbits and produce antibodies using standard protocols.

Alternatively, the GBAP amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine highly immunogenic regions. Then, a corresponding oligopeptide is synthesized, and this oligopeptide is used to generate an antibody by a method well known to those skilled in the art. The selection of appropriate epitopes, for example those near the C-terminus or in adjacent hydrophilic regions, is known in the art (see Ausubel, 1995, supra, chapter 11, etc.).

Normally, oligopeptides of about 15 residues in length are labeled with ABI 43 using Fmoc chemistry.
Synthesized using a 1A Peptide Synthesizer (PE Biosystems) and reacted with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to produce KLH
(Sigma-Aldrich, St. Louis MO) to enhance immunogenicity (see A above).
See usubel, 1995, etc.). Rabbits are immunized with the oligopeptide-KLM complex in complete Freund's adjuvant. To test the anti-peptide activity and anti-GBAP activity of the obtained antiserum, the peptide or GBAP was bound to a substrate, blocked with 1% BSA, reacted with rabbit antiserum and washed, and then radioactive iodine was added. React with goat anti-rabbit IgG labeled with.

Purification of native GBAP with 14-specific antibodies Native or recombinant GBAP is substantially purified by immunoaffinity chromatography with GBAP-specific antibodies. The immunoaffinity column uses an anti-GBAP antibody as a resin for activation chromatography, such as CNBr-activated Sepharose (Amersh).
am Pharmacia Biotech). After binding, the resin is blocked and washed according to the manufacturer's instructions.

The culture solution containing GBAP is passed through an immunoaffinity column, and the column is washed under conditions that preferentially adsorb GBAP (eg, high ionic strength buffer in the presence of detergent). Elute the column under conditions that destroy the binding between the antibody and GBAP (eg, buffer solution of pH 2-3 or high-concentration chaotropic agent such as urea or thiocyanate ion).
Collect AP.

Identification of 15 GBAP-Interacting Molecules GBAP or biologically active GBAP fragments are labeled with ¹²⁵ I Bolton-Hunter reagent (Bolton AE and WM Hunter (1973) Biochem. J. 133: 529-539 et al. See). Pre-arranged candidate molecules labeled in the wells of a multi-well plate
Incubate with GBAP, wash and assay any wells with labeled GBAP complex. Using the data obtained by changing the GBAP concentration, the number of GBAP with the candidate molecule,
Calculate affinity and association values.

Alternatively, molecules that interact with GBAP are described in Fields, S. and O. Song (1989, Natur.
e 340: 245-246) or using a commercially available kit based on a two-hybrid system such as the MATCHMAKER system (Clontech).

PATHCALLING that utilizes the yeast two-hybrid system in a high-throughput manner to determine all interactions between proteins encoded by two large libraries of genes.
GBAP can also be used for the process (CuraGen Corp., New Haven CT) (Nandabalan, K
(2000) U.S. Patent No. 6,057,101).

Those 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 THE TABLES Table 1 lists the polypeptide and nucleotide sequences used to assemble the full length sequence encoding GBAP (SEQ ID NO), clone identification number (clone identification number). ID), cDNA library and cDNA fragment.

Table 2 shows the characteristics of each polypeptide sequence, including latent motifs, homologous sequences, the methods, algorithms and searchable databases used to analyze GBAP.

Table 3 lists selected fragments of each nucleic acid sequence, tissue-specific expression patterns of each nucleic acid sequence determined by Northern analysis, diseases, disorders or conditions associated with these tissues, and each DNA. And the vector to which it is cloned.

Table 4 shows the tissues used for the construction of the cDNA library. A cDNA clone encoding GBAP was isolated from here.

Table 5 shows the tools, programs and algorithms used for GBAP analysis, 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]

[Table 31]

[Table 32]

[Table 33]

[Table 34]

[Table 35]

[Table 36]

[Table 37]

[Table 38]

[Table 39]

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 13/00 A61P 37/00 4C084 25/00 43/00 105 4C085 37/00 C07K 14/47 4C086 43 / 00 105 16/18 4C087 C07K 14/47 C12N 1/15 4H045 16/18 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 C12Q 1/02 5/10 1/68 A C12P 21/02 G01N 33/15 Z C12Q 1/02 33/50 Z 1/68 33/53 M G01N 33/15 33/566 33/50 37/00 102 33/53 A61K 31/7088 33/566 35 / 76 37/00 102 48/00 // A61K 31/7088 C12N 15/00 A 35/76 5/00 A 48/00 A61K 37/02 (31) Priority claim number 60/159, 849 (32) Priority date October 15, 1999 (October 15, 1999) (33) Country claiming priority US US) (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), 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, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY , CA, CH, CN, CR, CU, CZ, DE, DK, DM, 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, G, MK, MN, MW, MX, 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 Bandman, Olga 94043, California, USA Mountain View Anna Avenue 366 (72) Inventor Hillman, Jennifer El 94040, Mountain View, USA # 12 Monrode Live 230 (72) Inventor Lar, Pretty 95054 Santa Clara Russ Drive, California, USA 2382 (72) Inventor Ou-Young, Janice 94005, Brisbane Golden Eagle Lane 233 (72) Inventor, Lady , Loopa California, USA 94086 Sunny Ill # 3 West McKinley Avenue 1233 (72) Inventor Young, Junming California 95129 San Jose Barcrane 7125 (72) Inventor Bogun, Maria Earl USA California 94577 San Leandro Santiago Road 14244 (72) Inventor Ryu, Dung Aina Em USA 95136 San Jose Park Belmont Place 55 (72) Inventor Azimzai, Yalda 94545 Hayward Rock Springs Drive 2045 (72) Inventor Patterson, Chandra United States California 94025 Menlo Park # 1 Sherwood Way 490 F Term (reference) 2G045 AA25 AA40 CA26 CB01 CB03 DA12 DA13 DA14 DA36 DA77 FA37 FB02 FB06 FB07 4B024 AA01 AA11 BA43 BA80 CA04 CA07 C A09 CA11 DA01 DA02 DA05 DA11 EA01 EA02 EA03 EA04 FA02 FA06 GA11 HA01 HA12 4B063 QA01 QA08 QA18 QA19 QQ01 QQ05 QQ13 QQ42 QQ52 QR08 QR33 QR42 QR55 QR59 QR62 QR74 QR80 QR82 QS05 QS25 QS34 QS36 QX02 4B064 AG01 CA01 CA19 CC24 DA01 DA13 4B065 AA01X AA57X AA87X AA90Y AB01 BA01 CA24 CA25 CA44 CA46 4C084 AA02 AA03 AA06 AA07 AA13 AA17 BA01 BA02 BA20 BA22 CA53 NA14 ZA012 ZA812 ZB012 ZB212 4C085 AA13 AA14 NA01 A01 A01 A01 A01 A01 B01 A01 A01 B01 A01 B01 A01 B01 A01 B01 A01 B01 A01 B01 A01 B01 A01 B01 A01 B01 A01 B01 A01 B01 A01 B01 A01 B01 A01 B01 A01 B01 A01 B01 A01 B01 A01 B01 B01 B01 B01 A01 B01 A01 B01 B01 B01 B01 B01 B01 B01 A01 B01 B01 B01 B01 ZB21 4H045 AA10 AA11 AA20 AA30 BA10 BA41 CA40 DA00 DA76 EA20 EA50 FA72 FA74

Claims (28)

[Claims] 1. A substantially isolated polypeptide selected from the group comprising the following (a) to (d): (A) Sequence number 1, sequence number 2, sequence number 3, sequence number 4, sequence number 5, sequence number 6, sequence number 7, sequence number 8, sequence number 9, sequence number 10, sequence number 11,
Sequence number 12, sequence number 13, sequence number 14, sequence number 15, sequence number 16, sequence number 17, sequence number 18, sequence number 19, sequence number 20, sequence number 21, sequence number 22, sequence number 24, sequence number 25, sequence number 26, sequence number 27, sequence number 29, sequence number 30, sequence number 31, sequence number 32, sequence number 33, sequence number 34, sequence number 36, sequence number 37, sequence number 38, sequence number 39, Sequence number 4
0, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46
, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52,
Sequence number 53, sequence number 54, sequence number 55, sequence number 56, sequence number 57, sequence number 58, sequence number 59, sequence number 60, sequence number 61, sequence number 62, sequence number 63, sequence number 64, sequence number 65 and the amino acid sequence selected from the group having SEQ ID NO: 66 (b) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,
Sequence number 12, sequence number 13, sequence number 14, sequence number 15, sequence number 16, sequence number 17, sequence number 18, sequence number 19, sequence number 20, sequence number 21, sequence number 22, sequence number 24, sequence number 25, sequence number 26, sequence number 27, sequence number 29, sequence number 30, sequence number 31, sequence number 32, sequence number 33, sequence number 34, sequence number 36, sequence number 37, sequence number 38, sequence number 39, Sequence number 4
0, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46
, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52,
Sequence number 53, sequence number 54, sequence number 55, sequence number 56, sequence number 57, sequence number 58, sequence number 59, sequence number 60, sequence number 61, sequence number 62, sequence number 63, sequence number 64, sequence number 65 and a natural amino acid sequence having an amino acid sequence that is at least 90% identical to the amino acid sequence selected from the group having SEQ ID NO: 66. (c) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, Sequence number 5, sequence number 6, sequence number 7, sequence number 8, sequence number 9, sequence number 10, sequence number 11,
Sequence number 12, sequence number 13, sequence number 14, sequence number 15, sequence number 16, sequence number 17, sequence number 18, sequence number 19, sequence number 20, sequence number 21, sequence number 22, sequence number 24, sequence number 25, sequence number 26, sequence number 27, sequence number 29, sequence number 30, sequence number 31, sequence number 32, sequence number 33, sequence number 34, sequence number 36, sequence number 37, sequence number 38, sequence number 39, Sequence number 4
0, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46
, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52,
Sequence number 53, sequence number 54, sequence number 55, sequence number 56, sequence number 57, sequence number 58, sequence number 59, sequence number 60, sequence number 61, sequence number 62, sequence number 63, sequence number 64, sequence number 65 and a biologically active fragment of an amino acid sequence having an amino acid sequence selected from the group having SEQ ID NO: 66 (d) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,
Sequence number 12, sequence number 13, sequence number 14, sequence number 15, sequence number 16, sequence number 17, sequence number 18, sequence number 19, sequence number 20, sequence number 21, sequence number 22, sequence number 24, sequence number 25, sequence number 26, sequence number 27, sequence number 29, sequence number 30, sequence number 31, sequence number 32, sequence number 33, sequence number 34, sequence number 36, sequence number 37, sequence number 38, sequence number 39, Sequence number 4
0, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46
, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52,
Sequence number 53, sequence number 54, sequence number 55, sequence number 56, sequence number 57, sequence number 58, sequence number 59, sequence number 60, sequence number 61, sequence number 62, sequence number 63, sequence number 64, sequence number 65 and an immunogenic fragment having an amino acid sequence selected from the group having SEQ ID NO: 66. 2. SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, sequence No. 12, SEQ ID No. 13, Sequence No. 14, Sequence No. 15, Sequence No. 16, Sequence No. 17, Sequence No. 18, Sequence No. 19, Sequence No. 20, Sequence No. 21, Sequence No. 22, Sequence No. 24, Sequence No. 25 , SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 3
3, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39
, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45,
Sequence number 46, sequence number 47, sequence number 48, sequence number 49, sequence number 50, sequence number 52, sequence number 53, sequence number 54, sequence number 55, sequence number 56, sequence number 57, sequence number 58, sequence number 59. The isolated polypeptide of claim 1 selected from the group having 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66. 3. An isolated polynucleotide encoding the polypeptide of claim 1. 4. An isolated polynucleotide encoding the polypeptide of claim 2. 5. SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 7
0, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75
, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80,
Sequence number 81, sequence number 82, sequence number 83, sequence number 84, sequence number 85, sequence number 86, sequence number 87, sequence number 88, sequence number 90, sequence number 91, sequence number 92, sequence number 93, sequence number 95, sequence number 96, sequence number 97, sequence number 98, sequence number 99, sequence number 100, sequence number 102, sequence number 103, sequence number 104, sequence number 105, sequence number 106, sequence number 107, sequence number 1
09, sequence number 110, sequence number 111, sequence number 112, sequence number 113, sequence number 114, sequence number 115, sequence number 116, sequence number 118, sequence number 1
19, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 1
The isolated polynucleotide of claim 4, selected from the group having 28, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131 and SEQ ID NO: 132. 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 (e): (A) Sequence number 67, sequence number 68, sequence number 69, sequence number 70, sequence number 71, sequence number 72, sequence number 73, sequence number 74, sequence number 75, sequence number 7
6, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81
, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86,
Sequence number 87, sequence number 88, sequence number 90, sequence number 91, sequence number 92, sequence number 93, sequence number 95, sequence number 96, sequence number 97, sequence number 98, sequence number 99, sequence number 100, sequence number 102, SEQ ID NO: 103, SEQ ID NO: 104
, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114
, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124
, A polynucleotide sequence selected from the group having SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131 and 132 (b) SEQ ID NO: 67, SEQ ID NO: 68, sequence number 69, sequence number 70, sequence number 71, sequence number 72, sequence number 73, sequence number 74, sequence number 75, sequence number 7
6, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81
, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86,
Sequence number 87, sequence number 88, sequence number 90, sequence number 91, sequence number 92, sequence number 93, sequence number 95, sequence number 96, sequence number 97, sequence number 98, sequence number 99, sequence number 100, sequence number 102, SEQ ID NO: 103, SEQ ID NO: 104
, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114
, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124
, At least 70% identical to the polynucleotide sequence selected from the group having SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131 and 132. Natural polynucleotide sequence (c) Polynucleotide complementary to the polynucleotide of (a) (d) Polynucleotide complementary to the polynucleotide of (b) (e) RNA equivalents of (a) to (d) 12. At least 60 of the polynucleotide of claim 11.
An isolated polynucleotide comprising a contiguous nucleotide of. 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 or absence of the hybridization complex, and optionally the amount of the complex, if any. And the probe specifically hybridizes to the target polynucleotide under conditions such that a hybridization complex is formed between the probe and the target polynucleotide. Method. 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 in 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, which comprises an effective amount of the polypeptide of claim 1 and a pharmaceutically acceptable excipient. 17. The polypeptide comprises SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, Sequence number 77, sequence number 78, sequence number 79, sequence number 80, sequence number 81, sequence number 82, sequence number 83, sequence number 84, sequence number 85, sequence number 86, sequence number 87, sequence number 88, sequence number 9
0, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 96
, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 10
2, sequence number 103, sequence number 104, sequence number 105, sequence number 106, sequence number 107, sequence number 109, sequence number 110, sequence number 111, sequence number 11
2, sequence number 113, sequence number 114, sequence number 115, sequence number 116, sequence number 118, sequence number 119, sequence number 120, sequence number 121, sequence number 12
2, sequence number 123, sequence number 124, sequence number 125, sequence number 126, sequence number 127, sequence number 128, sequence number 129, sequence number 130, sequence number 13
17. The component of claim 16, comprising an amino acid sequence selected from the group having 1 and SEQ ID NO: 132. 18. A method of treating a disease or medical condition associated with reduced expression of functional GBAP, comprising the step of administering the component of claim 16 to a patient in need of such treatment. A method characterized by the following. 19. A method for screening a compound for confirming its effectiveness as an agonist of the polypeptide according to claim 1, comprising: (a) exposing a sample containing the polypeptide according to claim 1 to the compound. A method comprising the steps of: (b) detecting an agonist activity in the sample. 20. A component comprising an agonist compound identified by the method of claim 19 and a pharmaceutically acceptable excipient. 21. A method of treating a disease or medical condition associated with reduced expression of functional GBAP, comprising the step of administering a component of claim 20 to a patient in need of such treatment. A method characterized by the following. 22. A method of screening a compound for confirming 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. A method comprising the steps of: (b) detecting an 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 overexpression of functional GBAP, comprising the step of administering the component of claim 23 to a patient in need of such treatment. A method characterized by. 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, which comprises (a) a condition under which the activity of the polypeptide according to claim 1 is allowed. 1
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) in the presence of the test compound. Comparing the activity of the polypeptide of claim 1 with the activity of the polypeptide of claim 1 in the absence of the test compound in the presence of the test compound. 9. 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. A method comprising the following: exposing a sample containing the target polynucleotide to a compound, and (b) detecting a mutant expression of the target polynucleotide. 28. A method for calculating the toxicity of a test compound, comprising: (a) treating a biological sample containing nucleic acid with the test compound, and (b) at least the polynucleotide of claim 11. A specific hybridization complex is formed between a probe containing 20 contiguous nucleotides and a target polynucleotide of the biological sample having the polynucleotide sequence of the polynucleotide of claim 11 or a fragment thereof. Under the conditions as described above, the step of hybridizing the nucleic acid of the treated sample with the probe, (c) the step of quantifying the amount of the hybridization complex, and (d) the treated organism. The amount of the hybridization complex in the biological sample is determined by the amount of the hybridization complex in the untreated biological sample. Comparing the amount of the Retantiation Complex with the difference in the amount of the Hybridization Complex in the treated biological sample is indicative of toxicity of the test compound. .