JP2002542782A - Human membrane-related protein - Google Patents

Abstract

  (57) [Summary] The present invention provides human membrane associated proteins (HUMAPs) and polynucleotides that identify and encode HUMAPs. The invention also provides expression vectors and host cells, antibodies, agonists, antagonists. Further, the present invention provides a method for diagnosing, treating, and preventing a disease associated with HUMAP expression.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a nucleic acid sequence and an amino acid sequence of a human membrane-associated protein (carbohydrate-modifying enzyme), and to the use of these sequences to diagnose cell signaling, abnormal cell differentiation, and abnormal cell proliferation. And treatment, prevention.

BACKGROUND OF THE INVENTION Eukaryotic cells surround a cell and maintain an environment within the cell that is different from the outside environment.
Surrounded by cell membranes. Furthermore, eukaryotes, unlike prokaryotes, are rich in organelles and vesicle structures. Many of the metabolic reactions that distinguish eukaryotic and prokaryotic biochemistry occur within these structures. Cell membranes and membranes surrounding organelles and vesicles contain phosphoglycerides and fatty acids, cholesterol, phospholipids,
It consists of glycolipids, proteoglycans, and proteins. These components impart individuality and functionality to the membranes to which they relate.

[0003] Most of integral membrane proteins known integral membrane protein is a transmembrane protein characterized extracellular and transmembrane domains, the intracellular domain (TM). TM domains usually consist of 15-20 hydrophobic amino acids presumed to adopt an α-helix structure. TM proteins are classified into bitopic (type I and type II) and polytopic (polytopic: type III and IV) (Singer, SJ (1990) Annu. Re.
v. Cell Biol. 6: 247-96). Bitopic proteins penetrate the curtain once and polytopic proteins contain multiple transmembrane segments. TM proteins function as cell surface receptors and receptor interacting proteins, transporters of ions and metabolites, ion channels, and cell type-specific surface antigens.

[0004] Many membrane proteins (MPs) contain amino acid sequence motifs that target these proteins to specific subcellular sites. Examples of these motifs include PD
Z domain, KDEL, RGD, NGR and GSL sequence motifs, von Willebrand factor
A (vWFA) domain and EGF-like domain. RGD, NGR and GSL motif-containing peptides have been used as drug delivery carriers in cancer treatments targeting tumor blood vessels (Arap, W. et al. (1998) Science, 279: 377-380). In addition, MPs also contain amino acid sequence motifs, such as the carbohydrate recognition domain (CRD), that mediate interactions with extracellular and intracellular molecules.

[0005] TM proteins may also function as mediators of cell-cell adhesion. For example,
Emp (erythroblast macrophage protein), which mediates the attachment of erythroblasts to macrophages, has a putative TM domain near its N-terminus. Emp-mediated contact between erythroblasts and macrophages is presumed to promote terminal differentiation of erythroid cells and suppress the apoptotic process (Hanspal, M. et al. (1998) Blood 92: 2940-
2950).

One of the functions of TM proteins is to facilitate cell-to-cell communication. Neulexin is a family of neuronal cell surface receptor proteins with one TM region, which aids axonal guidance and synapse formation. A variety of alternative splicing produces hundreds of different neulexins in the brain (Ushkaryov, YA
(1992) Science 257: 50-56).

[0007] Some TM proteins function as transporters and channels in cell membranes. Rh
(Rhesus monkey) The erythrocyte blood group protein family thus functions in the erythrocyte membrane. This family includes Rh50 glycoprotein and Rh30 polypeptide that together form a complex. This complex is essential for the expression of the Rh antigen and for the completion of the erythrocyte membrane. The Rh deficiency syndrome, an autosomal recessive disorder associated with chronic hemolytic anemia and spherocytosis, is caused by a mutation in Rh (Huang,
CH (1998) J. Biol. Chem. 273: 2207-2213).

[0008] Tumor antigens are cell surface molecules that are expressed on tumor cells differently from normal cells.
This tumor antigen makes it possible to immunologically distinguish tumor cells from normal cells. This tumor antigen can also be a target for the diagnosis and treatment of human cancer (Takagi, S. et al.
(1995) Int. J. Cancer 61: 706-715; Liu, E. et al. (1992) Oncogene 7: 1027-103.
2).

[0009] Other types of cell surface antigens include those identified on white blood cells of the immune system. These antigens were identified by "shotgun" technology using a systematic monoclonal antibody (mAb). Such techniques produce hundreds of mAbs against known cell surface leukocyte antigens. These antigens are classified into a "cluster of differentiation" (CD) based on a common immunocytochemical localization pattern in various differentiated and undifferentiated leukocyte cell types. Each antigen in a cluster identifies one type of cell surface protein and either a "CD" or a "
It is called a “differentiated cluster”. Several genes encoding proteins identified by the CD antigen were cloned and verified by standard molecular biology techniques.
CD antigens are characterized by transmembrane proteins and cell surface proteins both for the fixed covalently linked to the cell membrane fatty acid-containing glycolipid such as glycosylphosphatidylinositol (GPI) (Barclay, AN other. (1995) The Leucocyte Anti gen Facts Book , Academic Press, San Diego, CA, pp. 17-20).

[0010] The TM cell surface glycoprotein CD69 is an early activation antigen of T lymphocytes. CD69
Is similar to a member of the supergene family of type II integral membrane proteins with a type C lectin domain. Although the exact function of the CD69 antigen is not clear, studies show that these proteins transduce cell division signals across cell membranes and are upregulated by lymphocyte activation (Hammann, J. et al.). (1993) J.
Immunol. 150: 4920-35 4927).

[0011] Some surface membrane proteins and anchor-type membrane proteins are not transmembrane proteins but are attached to cell membranes by interaction with membrane anchors or integral membrane proteins. Membrane anchors are covalently linked to proteins after translation and include such moieties as prenyl, myristyl and glycosylphosphatidylinositol (GPI). Membrane localization of superficial and anchored proteins is important in processes such as receptor-mediated signaling. For example, prenylation of Ras is required for its localization to the cell membrane and its normal and carcinogenic effects on signal transduction.

The Ly-6 antigen is a family of membrane-bound proteins, expressed primarily on the surface of T lymphocytes and in small amounts on the surface of other leukocytes and their precursors (Barclay A. et al.
N. et al. (1995) The Leucocyte Antigen Facts Book , Academic Press, San Dieg
o, CA, pp. 352-354; see Friedman, S. et al. (1990) Immunogenetics 31: 104-111). Ly-6 antigen is attached to the cell surface by a GPI anchor. Each Ly-6 antigen is about 135 amino acids long and has about 50% homology to each other. Although the exact function of the Ly-6 antigen is not clear, the results of the study suggest that these proteins penetrate cell membranes, transmit cell division signals, and are upregulated by leukocyte activation.

Examples of superficial membrane proteins include the mucin glycoprotein ASGP-1 (ascites sialoglycoprotein-1) from the cell line 13762 rat mammary adenocarcinoma.
1) There is. ASGP-1 associates with a transmembrane partner to form a heterodimer on the cell surface. It has been proposed that this complex alters the activity of growth factors and affects the process of tumor progression (Wu, K. et al. (1994) J. Biol. Chem. 269: 11950-11955).
.

By discovering each novel human membrane-associated protein and a polynucleotide encoding the same, it is possible to provide a novel composition useful for diagnosing, treating, and preventing cell signal transduction and abnormal cell differentiation and abnormal cell proliferation. Can answer the needs of this field.

SUMMARY OF THE INVENTION The present invention is generally referred to as "HUMAP", and individually referred to as "HUMAP-1" and "HUMAP-2", respectively.
"," HUMAP-3 "," HUMAP-4 "," HUMAP-5 "," HUMAP-6 "," HUMAP-7 "," H
UMAP-8, HUMAP-9, HUMAP-10, HUMAP-11, HUMAP-12, HUMAP
-13 "," HUMAP-14 "," HUMAP-15 "," HUMAP-16 ", and" HUMAP-17 ", which are purified polypeptides that are human membrane-associated proteins. In one embodiment of the present invention, a) an amino acid sequence selected from the group consisting of: SEQ ID NO: 1-17, b) SEQ ID NO:
A) a naturally occurring amino acid sequence having 90% or more sequence identity with an amino acid sequence selected from the group consisting of ID NO: 1-17; c) the amino acid sequence selected from the group consisting of SEQ ID NO: 1-17; An isolated polypeptide comprising a biologically active fragment, or d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17. Alternatively, provided is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1-17.

Further, the present invention relates to a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, and b) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17 of 90% or more. A naturally occurring amino acid sequence having sequence identity, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-17, or d) SEQ ID NO: 1-
An isolated polynucleotide encoding a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of: Alternatively, the polynucleotide is selected from the group consisting of SEQ ID NOs: 18-34.

Further, the present invention relates to a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-17, and b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-17 by 90% or more. A) a naturally occurring amino acid sequence having the sequence identity of: c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, or d) SEQ ID NO: 1.
A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of -17. Alternatively, the invention provides a cell transformed with the recombinant polynucleotide. In yet another alternative, the invention provides a transgenic organism comprising the recombinant polynucleotide.

Also, the present invention relates to a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-17, and b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-17 by 90% or more. A) a naturally occurring amino acid sequence having the sequence identity of: c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, or d) SEQ ID NO: 1.
The present invention provides a method for producing a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of -17. The method comprises the steps of: a) culturing cells transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide under conditions suitable for expression of the polypeptide. And b) recovering the polypeptide thus expressed.

Furthermore, the present invention relates to a) an amino acid sequence selected from a group consisting of SEQ ID NO: 1-17, and b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-17 by 90% or more. A) a naturally occurring amino acid sequence having the sequence identity of: c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, or d) SEQ ID NO: 1.
An isolated antibody that specifically binds to a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of -17.

Further, the present invention relates to a) a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 18-34; b) a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 18-34; A naturally occurring polynucleotide sequence having at least% sequence identity,
c) An isolated polynucleotide comprising a polynucleotide sequence complementary to a) or d) a polynucleotide sequence complementary to b). Alternatively, the polynucleotide comprises at least 60 contiguous nucleotides.

Further, the present invention relates to a method comprising: a) a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 18-34; b) a polynucleotide selected from the group consisting of SEQ ID NOs: 18-34 and 90% A naturally occurring polynucleotide sequence having the above sequence identity, c)
A method is provided for detecting a target polynucleotide in a sample having a polynucleotide sequence complementary to a) or d) a polynucleotide sequence comprising a polynucleotide sequence complementary to b). The method comprises the steps of: a) hybridizing the sample with a probe comprising at least 16 contiguous nucleotides comprising a sequence complementary to a target polynucleotide in the sample, wherein the probe and the target polynucleotide And b) detecting the presence or absence of the hybridization complex, if the probe is present, under the conditions that a hybridization complex is formed in Optionally measuring the amount.
Alternatively, the probe comprises at least 30 contiguous nucleotides.
In yet another alternative, the probe comprises at least 60 contiguous nucleotides.

Furthermore, the present invention relates to the present invention, wherein a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-17, and b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-17 by 90% or more. A) a naturally occurring amino acid sequence having the sequence identity of: c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, or d) SEQ ID NO: 1.
A pharmaceutical composition comprising an effective amount of a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of -17 and a suitable pharmaceutical excipient. Further, the present invention provides a method for treating a disease or a symptom thereof associated with reduced expression of functional HUMAP, which comprises administering the pharmaceutical composition to a patient.

Furthermore, the present invention relates to a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-17, and b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-17 by 90% or more. A) a naturally occurring amino acid sequence having the sequence identity of: c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, or d) SEQ ID NO: 1.
A method for screening a compound effective as an agonist of a polypeptide containing an immunogenic fragment of an amino acid sequence selected from the group consisting of -17. The method includes the steps of: a) exposing a sample containing the polypeptide to a compound; and b) detecting agonist activity of the sample. Alternatively,
The present invention provides a pharmaceutical composition comprising an agonist compound identified by the above method and a suitable pharmaceutical excipient. In yet another alternative, the invention provides a method of treating a disease or condition associated with reduced expression of functional HUMAP, comprising administering the pharmaceutical composition to a patient.

Furthermore, the present invention relates to the present invention, wherein a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-17, and b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-17 by 90% or more. A) a naturally occurring amino acid sequence having the sequence identity of: c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17, or d) SEQ ID NO: 1.
A method for screening a compound effective as an antagonist of a polypeptide containing an immunogenic fragment of an amino acid sequence selected from the group consisting of -17.
The method includes the steps of: a) exposing a sample comprising the polypeptide to a compound; and b) detecting antagonist activity of the sample. Alternatively, the present invention provides a pharmaceutical composition comprising an antagonist compound identified by the above method and a suitable pharmaceutical excipient. In a further alternative, the invention provides a method of treating a disease or condition associated with over-expression of functional HUMAP, comprising administering the pharmaceutical composition to a patient.

The invention further provides a method of screening for a compound effective to alter the expression of a target polynucleotide comprising a sequence selected from the group consisting of SEQ ID NOs: 18-34, comprising: The screening method is provided, comprising: exposing a sample containing a polynucleotide to a compound; and b) detecting a change in expression of the target polynucleotide.

(Description of the Present Invention) Before describing the protein and nucleic acid sequences and methods of the present invention, the present invention is not limited to the specific devices, materials and methods disclosed herein, but rather modifies its embodiments. Please understand what you can do. Further, the terms used herein are used only for the purpose of describing a specific embodiment, and are limited only by the claims described below.
It should also be understood that they are not intended to limit the scope of the present invention.

In the present specification and claims, “a”, “the” and the like refer to a singular form.
Note that the plural includes the plural unless explicitly stated in the context. Thus, for example, "a host cell" includes a plurality of host cells, and the "antibody" includes a plurality of antibodies, including equivalents well known to those skilled in the art.

All technical and scientific terms used herein are, unless otherwise defined,
This has the same meaning as commonly interpreted by a general engineer in the technical field to which the present invention belongs.
Although all devices and materials and methods similar or equivalent to those described herein can be used in the practice and testing of the present invention, the preferred devices, materials and methods are described here.
All references mentioned herein are cited to describe and disclose the cell lines, protocols, reagents, and vectors described in the literature that may be used in connection with the present invention. Citing a conventional invention does not, however, be construed as impairing the novelty of the present invention.

Definitions The term "HUMAP" refers to substantially any species obtained from all species, including natural, synthetic, semi-synthetic or recombinant, particularly mammals including cows, sheep, pigs, mice, horses and humans. Refers to the amino acid sequence of purified HUMAP.

The term “agonist” refers to a molecule that enhances or mimics the biological activity of HUMAP. The agonists may interact directly with HUMAP or interact with components of the biological pathways involving HUMAP to regulate the activity of HUMAP, such as proteins, nucleic acids, carbohydrates, small molecules, any other compounds, A composition may be included.

The term “allelic variant” refers to another form of the gene encoding HUMAP. An allelic variant is caused by at least one mutation in the nucleic acid sequence, and
Alternatively, it becomes a mutant polypeptide, and its structure or function may or may not change. Some genes have no naturally occurring allelic variant, one or a large number thereof. In general, mutations that result in allelic variants are due to natural deletions, additions, or substitutions of nucleotides. Each of these mutations
It may occur alone or simultaneously with other mutations, and may occur once or more in a given sequence.

[0032] "Mutant" nucleic acid sequences encoding HUMAP may comprise various nucleotide deletions, insertions,
Alternatively, it refers to a polypeptide having the same polypeptide as HUMAP or having at least one of the functional characteristics of HUMAP even when substitution occurs. This definition includes improper or unexpected hybridization of the polynucleotide sequence encoding HUMAP with an allelic variant at a position that is not a normal chromosomal locus, as well as specific oligonucleotide probes of the polynucleotide encoding HUMAP. And polymorphisms that are easily detectable or difficult to detect using The encoded protein can also be mutated and contain deletions, insertions, or substitutions of amino acid residues that produce a silent change and are functionally equivalent to HUMAP. Intentional amino acid substitutions can be biologically or immunologically
To the extent that the activity of HUMAP is retained, it can be based on similarities in residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity. For example, negatively charged amino acids can include aspartic acid and glutamic acid, and positively charged amino acids can include lysine and arginine. Amino acids with similar hydrophilicity values and polar uncharged side chains may include asparagine, glutamine, serine, threonine. Amino acids with similar hydrophilicity values and uncharged side chains include:
Leucine, isoleucine, valine, glycine, alanine, phenylalanine and tyrosine may be included.

The terms “amino acid” and “amino acid sequence” refer to oligopeptides, peptides, polypeptides, protein sequences, or any fragment thereof, and include natural and synthetic molecules. If the "amino acid sequence" is a naturally occurring protein molecule,
"Amino acid sequence" and like terms do not limit the amino acid sequence to the complete, intact amino acid sequence associated with the described protein molecule.

The term “amplification” relates to making a copy of a nucleic acid sequence. Generally, amplification is performed by the polymerase chain reaction (PCR) technique well known in the art.

The term “antagonist” is a molecule that inhibits or attenuates the biological activity of HUMAP. Antagonists directly interact with HUMAP or interact with components of the biological pathways involving HUMAP to regulate antibodies, nucleic acids, carbohydrates,
It can include proteins such as small molecules, any other compounds or compositions.

The term “antibody” refers to intact molecules, such as Fab and F (ab ′) ₂ , and their fragments, Fv fragments, that are capable of binding an antigenic determinant. Antibodies that bind HUMAP polypeptides can be produced using intact molecules or fragments thereof, including small peptides that immunize the antibody. The polypeptide or oligopeptide used to immunize an animal (eg, a mouse, rat, or rabbit) can be synthesized from the translation of RNA or chemically and optionally linked to a carrier protein. It is also possible. Commonly used carriers that are chemically linked to the peptide include bovine serum albumin, thyroglobulin, and keyhole limpet haemonian (KLH). The animal is then immunized with the conjugated peptide.

The term “antigenic determinant” refers to a region of a molecule (ie, an epitope) that makes contact with a particular antibody
Point to. When a protein or a fragment of a protein is used to immunize a host animal, various regions of the protein may contain antibodies that specifically bind to an antigenic determinant (a specific region or three-dimensional structure on the protein). Can be induced. Antigenic determinants can compete with the intact antigen (ie, the immunogen used to elicit the immune response) to bind the antibody.

As used herein, “antisense” refers to any composition capable of forming base pairs with the sense strand of a particular nucleic acid sequence. Antisense components include DNA, RNA, peptide nucleic acid (PNA), and modified backbone linkages such as phosphorothioate, methylphosphonate, and benzylphosphonate.
And an oligonucleotide having an altered sugar such as 2'-methoxyethyl sugar or 2'-methoxyethoxy sugar, and 5-methylcytosine or 2'-deoxyuracil, 7-deaza-2'-deoxyguanosine and the like Oligonucleotides having an altered base of Antisense molecules can be created by any method, including chemical synthesis and transcription. Once introduced into a cell, a complementary antisense molecule base pairs with a naturally occurring nucleic acid sequence created by the cell to form a duplex and inhibit transcription and translation. The expression “negative” or “minus” is the antisense strand, and the expression “positive” or “plus” is the sense strand.

The term “biologically active” refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Similarly, the term "immunologically active" refers to natural or recombinant HUMAP, synthetic HUMAP, or any of their oligopeptides, which elicits a specific immune response in a suitable animal or cell to a specific antibody. Refers to the ability to combine with

The terms “complementary” and “complementarity” refer to the spontaneous binding of polynucleotides to form base pairs. For example, the sequence "5'AGT3 '" binds to the complementary sequence "3'TCA5'". The complementarity between two single-stranded molecules can be partial, where only some nucleic acids bind, or there can be complete complementarity between the single strands, resulting in complete complementarity. The degree of complementarity between nucleic acid strands greatly affects the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions that depend on binding between nucleic acid strands, as well as in the design or use of peptide nucleic acid (PNA) molecules.

“Composition comprising a given polynucleotide sequence” or “composition comprising a given amino acid sequence” refers in a broad sense to any composition comprising a given nucleotide or amino acid sequence. The composition may include a dry formulation or an aqueous solution.
A composition comprising a polynucleotide sequence encoding HUMAP or a fragment of HUMAP,
It can be used as a hybridization probe. This probe can be stored in a lyophilized state, and can be bound to a stabilizer such as a carbohydrate.
In hybridization, the probe is developed in an aqueous solution containing a salt (eg, NaCl), a surfactant (eg, SDS: sodium dodecyl sulfate), and other substances (eg, Denhardt's solution, dried milk, salmon sperm DNA, etc.). obtain.

A “consensus sequence” is a nucleic acid sequence that has been sequenced to separate unwanted bases and is 5 ′ and / or 3 ′ using XL-PCR ^™ (Perkin Elmer, Norwalk, Conn.). Sequenced nucleic acid sequence that has been extended in the direction or GELVI
One or more Insight clones using a computer program for the construction of fragments, such as the EW fragment construction system (GCG, Madison, WI);
And, in some cases, nucleic acid sequences constructed by duplication of one or more public domain ESTs. Some consensus sequences are constructed by both extension and duplication.

The term “conservative amino acid substitution” refers to a substitution that does not significantly alter the properties of the original protein. That is, the substitution does not significantly change the structure or function of the protein,
The structure of the protein, especially its function, is preserved. The following shows conservative amino acid substitutions in which the original amino acid of one protein is replaced by another amino acid. 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 In general, for conserved amino acid substitutions, a) the backbone structure of the polypeptide in the substituted region, eg, β-sheet or α-helix conformation, b) the charge of the molecule at the substituted site or Hydrophobicity and / or c) the majority of the side chains are preserved.

The term “deletion” refers to a change in the amino acid sequence that lacks one or more amino acid residues, or a change in the nucleic acid sequence that lacks one or more nucleotides.

The term “derivative” refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide sequence include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide has at least one of the biological or immunological functions of a natural molecule (unmodified molecule).
Encodes a polypeptide that maintains its identity. Derivative polypeptides are polypeptides that have been modified by glycosylation, polyethyleneglycolation, or any similar process that maintain at least one of the biological or immunological functions of the original polypeptide. That is.

The term “fragment” refers to a unique portion of HUMAP or a polynucleotide encoding HUMAP that is identical to, but shorter in length than, its parent sequence. The maximum length of a “fragment” is one nucleotide /
This is the length obtained by subtracting amino acid residues. For example, some fragments contain from 5 to 1000 contiguous nucleotide or amino acid residues. Probes, primers, antigens, therapeutic molecules or fragments used for other purposes are at least 5, 10, 15
, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250
Or 500 consecutive nucleotides or amino acid residues in length. Fragments may be preferentially selected from particular regions of the molecule. For example, a polypeptide fragment can include a predetermined length of contiguous amino acids selected from the first 250 or 500 amino acids set forth in a predetermined sequence (or the first 25% or 50% of the polypeptide). . These lengths are examples, and any length described in the specification including the sequence listing and the tables and drawings can be included in the examples of the present invention.

Certain fragments of SEQ ID NO: 18-34 include, for example, regions of the unique polynucleotide sequence that uniquely identify SEQ ID NO: 18-34, which are different from other sequences in the same genome. Certain fragments of SEQ ID NO: 18-34 are useful, for example, in hybridization and amplification techniques, or similar methods that distinguish SEQ ID NO: 18-34 from related polynucleotide sequences. The exact fragment length or region of SEQ ID NO: 18-34 that corresponds to a fragment can be routinely determined by techniques common in the art based on the purpose of the fragment.

Certain fragments of SEQ ID NOs: 1-17 are encoded by certain fragments of SEQ ID NOs: 18-34. Certain fragments of SEQ ID NO: 1-17 include regions of the unique amino acid sequence that specifically identify SEQ ID NO: 1-17. For example, certain fragments of SEQ ID NO: 1-17 are useful as immunogenic peptides for the production of antibodies that specifically recognize SEQ ID NO: 1-17. The exact fragment length or region of SEQ ID NO: 1-17 that corresponds to a fragment can be routinely determined by techniques common in the art based on the purpose of the fragment.

The term “similarity” refers to a degree of complementarity. This includes partial similarities and complete similarities. The term "identity" can also be referred to as "similarity". Partially complementary sequences that at least partially block hybridization of the same sequence to a target nucleic acid are termed "substantially similar." The inhibition of hybridization between the perfectly complementary sequence and the target sequence is tested using a hybridization assay (Southern or Northern blotting, solution hybridization, etc.) under mild stringent conditions. Substantially similar sequences or hybridization probes compete under mild stringent conditions for binding of a completely similar (identical) sequence to the target sequence. This does not mean that non-specific binding is tolerated under mild stringent conditions, but under mild stringent conditions the binding of the two sequences to each other is specific (ie, selective). You have to interact. Using a second target sequence that is not even partially complementary (eg, less than 30% similarity or identity), it can be tested for the absence of non-specific binding. In the absence of non-specific binding, substantially similar sequences or probes do not hybridize to the second non-complementary target sequence.

The terms “percent identity” or “percent identity” for a polynucleotide sequence refer to the identity of matching residues between two or more polynucleotide sequences that are aligned using a standardized algorithm. Percentage. Such an algorithm can insert gaps in the sequences to make a more meaningful comparison between the two sequences in a standardized and reproducible manner to optimize the alignment between the two sequences.

The percent identity between polynucleotide sequences can be determined using the default parameters of the CLUSTAL V algorithm incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package and includes a suite of molecular biology analysis programs (DNASTAR, Madison W
I). This CLUSTAL V is compatible with Higgins, DG and PM Sharp (1989) CABIOS
5: 151-153, Higgins, DG et al. (1992) CABIOS 8: 189-191. For alignment of pairs of polynucleotide sequences, the default parameter is Kt
Set uple = 2, gap penalty = 5, window = 4, “diagonals saved” = 4. A "weighted" residue weight table was selected as the default. The percent identity is reported by CLUSTAL V as the "percent similarity" of the paired aligned polynucleotide sequences.

Alternatively, a set of commonly used and freely available sequence comparison algorithms is
NCBI, Bethesda, MD, and the Internet (http://WWW.ncbi.nlm.nih.gov/BLAS
National Center for Biotechnology Information (NCB)
I) Basic Local Alignment Search Tool (BLAST) (Altschul, SF et al. (1990) J
Mol. Biol. 215: 403-410). The BLAST software suite includes a variety of sequence analysis programs, including "blastn," used to align known polynucleotide sequences with other polynucleotide sequences in various databases. A tool called "BLAST 2 Sequences" is available,
Used to compare two nucleotide sequence pairs directly. "BLAST 2 Sequen
"ces" can be used interactively by accessing http://WWW.ncbi.nlm.nih.gov/gorf/b12.html. The "BLAST 2 Sequences" tool can be used for both blastn and blastp (described below). The BLAST program is commonly used with default setting gaps and other parameters. For example,
When comparing two nucleotide sequences, one may use the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) with default parameters set to blast
would use n. Such default parameters are, for example, as follows. 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 May be measured with respect to the entire length of the predetermined sequence, or for a shorter length, for example, a fragment obtained from a certain large predetermined sequence, , 20 or 30
, 40, 50, 70, 100, 200 nucleotides. Such lengths are merely examples, and any length fragment of a sequence set forth in the specification, including the sequence listing and the tables and drawings, may be used to indicate the length at which percent identity is measured. it can.

[0053] Nucleic acid sequences that do not show a high degree of identity can still encode similar amino acid sequences due to the degeneracy of the genetic code. It should be understood that degeneracy can be used to alter a nucleic acid sequence to produce a variety of nucleic acid sequences, each encoding substantially the same protein.

The terms “percent identity” or “% identity” as used in a polypeptide sequence refer to the identity of matching residues between two or more polypeptide sequences that are aligned using a standardized algorithm. Percentage. Methods for polypeptide sequence alignment are well known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, as described in detail above, generally preserve the charge and hydrophobicity of the substitution site and maintain the structure (and therefore function) of the polypeptide.
Is saved.

The percent identity between polypeptide sequences can be determined using the default parameters of the CLUSTAL V algorithm incorporated in the MEGALIGN version 3.12e sequence alignment program (supra). For pairwise polypeptide sequence alignments using CLUSTAL V, the default parameters are Ktuple = 1, gap
Set penalty = 3, window = 5, and “diagonals saved” = 5. The PAM250 matrix is selected as the default residue weight table. As with polynucleotide alignments, the percent identity of a pair of aligned polypeptide sequences is reported by CLUSTAL V as "percent similarity".

Alternatively, the NCBI BLAST software suite is used. For example, when comparing two polypeptide sequences in pairs, one would use blastp with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000), set with default parameters. . Such default parameters are, for example, as follows. Matrix: BLOSUM62 Open Gap: 11 and Extension Gap: 1 penalties Gap x drop-off: 50 Expect: 10 Word Size: 3 Filter: on The percentage of identity can be determined by, for example, It may be measured relative to the full length of the peptide sequence, or for shorter lengths, for example, fragments obtained from a given large polypeptide sequence, for example, at least 15, 20, or 30, 40, contiguous. , 50, 70, 150 residues. Such lengths are merely examples, and any length fragment of a sequence set forth in the specification, including the sequence listing and the tables and drawings, may be used to indicate the length at which percent identity is measured. it can.

“Human artificial chromosome (HAC)” is a DNA of about 6 kb (kilobase) to 10 Mb in size.
A linear small chromosome that can contain a sequence and contains all elements necessary for the separation and maintenance of stable mitotic chromosomes.

The term “humanized antibody” refers to an antibody molecule in which the amino acid sequence of the non-antigen binding region has been changed in order to more closely resemble a human antibody while retaining its original binding ability.

“Hybridization” is the process of annealing in which a single-stranded polynucleotide forms a base pair with a complementary single-stranded strand under predetermined hybridization conditions. Specific hybridization means that two nucleic acid sequences have high identity. Under conditions that permit annealing, a specific hybridization complex is formed and remains hybridized after the washing step. The washing step is particularly important in determining the stringency or stringency of the hybridization process; under more stringent conditions, nonspecific binding, ie, binding of pairs between nucleic acid strands that are not perfectly matched. Decrease. The conditions under which annealing between nucleic acid sequences is permissible are routinely determined by those skilled in the art, and are constant during hybridization, but the washing process is altered during the process to achieve the desired stringency. And hybridization specificity is obtained. Conditions that allow annealing include, for example, about 6 × SSC, about 1% (w / v) SDS at about 68 ° C., and about 100%.
Contains μg / ml sheared denatured salmon sperm DNA.

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

High stringency hybridization between polynucleotides of the present invention involves a washing step at about 68 ° C. for 1 hour in the presence of about 0.2 × SSC and about 1% SDS. Alternatively, it is performed at a temperature of 65 ° C, 60 ° C, 55 ° C, 42 ° C. The concentration of SSC ranges from about 0.1 to 2x SSC in the presence of about 0.1% SDS. Normally,
Non-specific hybridization is blocked using a blocking agent. Such blocking agents include, for example, denatured salmon sperm DNA at about 100-200 μg / ml. An organic solvent such as formamide at a concentration of about 35-50% v / v
It can be used in certain cases, such as DNA hybridization. Useful modifications of these washing conditions are well known to those skilled in the art. Hybridization under particularly high stringency conditions can indicate evolutionary similarities between nucleotides. Such similarities strongly suggest that their nucleotides and encoded polypeptides play a similar role.

The term “hybridization complex” refers to a complex of two nucleic acid sequences formed by hydrogen bonding between complementary bases. Hybridization complex in solution (e.g., C 0 _t or R _{0 t} analysis) or formed by, or one nucleic acid sequence and the solid support material in the solution (e.g., paper, membranes, filters, chips, pins ,
Or a glass slide or any suitable substrate for immobilizing cells and their nucleic acids)
And another nucleic acid sequence fixed to the

The term “insertion” or “addition” refers to a change in the amino acid or nucleic acid sequence to which one or more amino acid residues or nucleotides are added, respectively.

“Immune response” refers to conditions associated with inflammatory diseases and trauma, immune disorders, infections, genetic diseases, and the like. These conditions are characterized by the expression of various factors that affect the cell line and the systemic defense system, such as cytokines and chemokines and other signaling molecules.

The term “microarray” refers to a sequence of different polynucleotides arranged on a substrate.

The term “element” or “array element” as used in the context of a microarray refers to a hybridizable polynucleotide arranged on the surface of a substrate.

The term “modulation” refers to a change in the activity of HUMAP. For example, the modulation results in a change in the protein activity or binding properties of HUMAP, or other biological, functional or immunological properties.

The terms “nucleic acid” and “nucleic acid sequence” refer to nucleotides, oligonucleotides, polynucleotides, or fragments thereof. In addition, it refers to single-stranded or double-stranded, sense or antisense strand DNA or RNA of genomic or synthetic origin, peptide nucleic acid (PNA), any DNA-like substance, and RNA-like substance.

“Operably linked” refers to the state in which a first nucleic acid sequence and a second nucleic acid sequence are in a functional relationship. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. Generally, operably linked DNA sequences are very close or contiguous if, in the same reading frame, the regions encoding the two proteins need to be joined.

“Peptide nucleic acid (PNA)” refers to an antisense molecule or an antigenic agent comprising an oligonucleotide at least about 5 nucleotides in length attached to a peptide backbone of amino acid residues terminating in lysine. This terminal lysine renders the composition soluble. PNAs can preferentially bind to complementary single-stranded DNA or RNA, stop transcript elongation, and become polyethylene glycolated to extend their lifespan in cells.

The term “probe” refers to a nucleic acid sequence encoding HUMAP, its complementary sequence, or a fragment thereof, which is used to detect the same or allele nucleic acid sequence or a related nucleic acid sequence. A probe is an isolated oligonucleotide or polynucleotide to which a detectable label or reporter molecule has been attached. Typical signs include
There are radioisotopes and ligands, chemiluminescent reagents and enzymes. "Primer"
A short nucleic acid, usually a DNA oligonucleotide, that can form complementary base pairs and anneal to a target polynucleotide. After the primers anneal to the polynucleotide, the DNA of the target is
It extends along a single strand. The primer set can be used, for example, for amplification (and identification) of a nucleic acid sequence in a PCR method.

The probes and primers used in the present invention have at least 15
Of consecutive nucleotides. Longer probes and primers can be used to increase specificity. For example, at least 20 or 25, 30, 40, 50, 60, 70, 80, 90, 100 consecutive of the disclosed nucleic acid sequences.
, 150 nucleotides. It should be understood that probes and primers that are considerably longer than the examples described above can be used, and that nucleotides of any length shown in the tables, drawings and sequence listings herein can be used.

For preparation and use of probes and primers, see, for example, Sambrook,
J. et al., 1989, Molecular Cloning: A Laboratory Manual, Second
Volume 1-3 of the edition (Cold Spring Harbor Press, Plainview NY) or Ausubel, FM
1987, by the name `` Current Protocols in Molecular Biology '' (Greene
Pubi. Assoc. & Wiley-Intersciences, New York NY), and Innis et al.
1990, `` PCR Protocols, A Guide to Methods and Applications '' (Academic
mic Press, San Diego CA.). A primer set for PCR is, for example, Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research)
, Cambridge MA), can be used to derive from certain known sequences.

The oligonucleotide used as a primer is selected by a computer program for selecting primers well known in the art. For example, OLIGO 4.06 software allows the selection of primer pairs for PCR up to 100 nucleotides each,
And analysis of large polynucleotides and oligonucleotides up to 5,000 nucleotides from an input polynucleotide sequence of up to 32,000 bases. Similar primer selection programs include additional features that enhance capacity. For example, PrimOU primer selection program (Genome Center at Uni
versity of Texas South West Medical Center, Dallas TX)
Since a specific primer can be selected from the megabase sequence, it is useful for designing primers in a genome-wide scope. Primer3 primer selection program (Whitehead Institute / MIT Center for Genome Research,
Cambridge MA1) allows users to specify sequences they want to avoid as primer binding sites.
) "Can be entered. Primer3 is also particularly useful for selecting oligonucleotides for microarrays (the source code of the latter two primer selection programs can be obtained from each source and modified to meet user needs). You can also). PrimerGen program (UK Human Genome Mapping Pro
ject Resource Center, available from Cambridge UK), which designs primers based on multiple sequence alignments, primers that hybridize to either the most conserved or the least conserved regions of the aligned nucleic acid sequences Can be selected. Thus, the program is useful for identifying unique and conserved oligonucleotide and polynucleotide fragments. Oligonucleotide or polynucleotide fragments identified by any of the selection methods described above can be used, for example, to identify polynucleotides or sequencing primers, microarray elements, or polynucleotides that completely or partially complement the nucleic acids of the sample. Useful for hybridization techniques, such as a probe. The method for selecting the oligonucleotide is not limited to the method described above.

As used herein, a “recombinant nucleic acid” is not a natural sequence, but a sequence that is an artificial combination of two or more separate segments of a sequence. Although this artificial combination is often made by chemical synthesis, it is more common to artificially manipulate discrete segments of nucleic acids using genetic engineering techniques such as those described in Sambrook, supra. is there. The “recombinant nucleic acid” also includes a nucleic acid that has been changed simply by adding, replacing, or deleting a part of the nucleic acid. A recombinant nucleic acid can include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid can be, for example, part of a vector used to transform a cell.

Alternatively, such a recombinant nucleic acid is a vaccinia virus-based virus that, when used in vaccination of a mammal expressing the recombinant nucleic acid, elicits a protective immune response in the mammal. It can be part of a vector.

As used herein, “RNA equivalent” to a DNA sequence is composed of the same linear nucleic acid sequence as a reference DNA sequence, except that thymine, a nitrogenous base, is substituted with uracil, Spine consists of ribose instead of deoxyribose.

The term “sample” is used in its broadest sense. Samples presumed to contain HUMAP-encoding nucleic acids or fragments thereof, and HUMAP itself, include body fluids, extracts from cells and chromosomes and organelles isolated from cells, organelles, membranes, cells, and solutions. Genomic DNA, RNA, cDNA present in or immobilized on a substrate, as well as tissues or tissue prints, may also be included.

The terms “specific binding” and “specifically binds” refer to the interaction between a protein or peptide and an agonist, antibody, antagonist, small molecule, or any natural or synthetic binding composition. Point. This interaction is dependent on the presence of a particular structure of the protein, such as an antigenic determinant or epitope, recognized by the binding molecule. For example, if the antibody is specific for epitope "A", the unbound labeled "A" and the reaction solution containing the antibody may contain a polypeptide comprising epitope A or unlabeled "A". ”Reduces the amount of label A that binds to the antibody.

The term “substantially purified” refers to a nucleic acid or amino acid sequence that has been removed from its natural environment and that has been isolated or separated, wherein the naturally associated composition has at least about 60 % Or more, preferably about 75% or more removed, most preferably 90% or more removed.

“Substitution” refers to the replacement of one or more amino acids or nucleotides with another amino acid or nucleotide, respectively.

The term “substrate” refers to any suitable solid or semi-solid support, including membranes and filters, chips, slides, wafers, fibers, magnetic or non-magnetic beads, gels, tubes, plates, polymers, microscopic Particles and capillaries are included. The substrate has various surface features, such as walls or trenches, pins, channels, pores, etc., to which polynucleotides or polypeptides bind.

“Transformation” is the process by which foreign DNA enters and changes a recipient cell. Transformation can occur under natural or artificial conditions by a variety of methods known in the art, and is performed by any known method for inserting foreign nucleic acid sequences into prokaryotic or eukaryotic host cells. be able to. The method of transformation
The choice depends on the type of host cell to be transformed. The methods include, but are not limited to, viral infection, electroporation, lipofection, and microparticle irradiation. A "transformed" cell includes a stably transformed cell in which the introduced DNA is capable of replicating autonomously as a plasmid or as part of the host chromosome. Furthermore, cells that express the introduced DNA or introduced RNA for a limited period of time are also included.

As used herein, the term “genetically modified organism” refers to any organism in which a human has introduced a heterologous nucleic acid into one or more cells of the organism using a gene recombination technique well known in the art. And include, but are not limited to, animals and plants. Heterologous nucleic acids are introduced into cells directly or indirectly into their precursors by careful genetic manipulations such as microinjection or infection with recombinant viruses. "Genetic manipulation" refers to the introduction of a recombinant DNA molecule rather than the typical cross breeding and in vitro fertilization. Genetically modified organisms according to the present invention include bacteria and cyanobacteria, fungi, plants and animals. The isolated DNA of the present invention can be introduced into a host by methods well known in the art, such as infection, transfection, transformation, transconjugation, and the like. Techniques for introducing the DNA of the present invention into such organisms are well known and are described in Sambrook et al. (1989), supra.

The “variant sequence” of a specific nucleic acid sequence is referred to as “BLAST” in the default parameter settings.
By blastn using the "2 Sequences" tool Version 2.0.9 (May-07-1999), the identity of a particular nucleic acid sequence to a length of the nucleic acid sequence is at least 40%
Is the nucleic acid sequence determined as follows. Such pairs of nucleic acids may be, for example, at least 50% or 60%, 70%, 80%, 85%, 90%,
It can show 95%, 98% or more identity. Certain variant sequences can be referred to, for example, as "allelic" variant sequences (described above) or "splice" variant sequences, "species" variant sequences, "polymorphic" variant sequences. A splice variant may have very high identity to a reference molecule, but will have more or less polynucleotides due to alternative splicing of exons during mRNA processing. The corresponding polypeptide may have additional functional domains present in the reference molecule, or may lack domains present in the reference molecule. Species variant sequences are polynucleotide sequences that vary from species to species. The resulting polypeptides have high amino acid identity with each other. Polymorphic variant sequences differ in the polynucleotide sequence of a particular gene in a given species. A polymorphic variant sequence can also include one of the polynucleotide sequences.
It may also include "single nucleotide polymorphisms" (SNPs) in which two nucleotides differ. The presence of a SNP may, for example, represent a certain population, condition, or characteristic of the condition.

A “variant” of a particular polypeptide sequence is referred to as the default parameter setting “
A polypeptide sequence in which the identity of the particular polypeptide sequence to a length of a nucleic acid sequence has been determined to be at least 40% by blastp using the "BLAST 2 Sequences" tool Version 2.0.9 (May-07-1999). That is. Such pairs of polypeptides may be, for example, at least 50% or 60%,
It may show 0%, 80%, 85%, 90%, 95%, 98% or more identity.

(Invention) The present invention is based on the discovery of a novel human membrane-associated protein (HUMAP) and a polynucleotide encoding HUMAP. The use of these compositions in

Table 1 shows the Incyte clones used to construct the full length nucleotide sequence encoding HUMAP. Columns 1 and 2 show the SEQ ID NO of the polypeptide and nucleotide sequences, respectively. Column 3 shows the clone ID of the Incyte clone from which the nucleic acid encoding each HUMAP was identified, and column 4 shows the cDNA library from which those clones were isolated. Column 5 shows Incyte clones and their corresponding cDNA libraries. Incyte clones for which no cDNA library is shown are from the pooled cDNA library. The Incyte clones in row 5 are used to construct a consensus nucleotide sequence for each HUMAP and are useful as fragments in hybridization techniques.

Each column of Table 2 shows various properties of each polypeptide of the invention. Column 1 is SEQ ID NO, column 2 is the number of amino acid residues in each polypeptide, column 3 is a potential phosphorylation site, column 4 is a potential glycosylation site, and column 5 is the signature (sign).
ature) Amino acid residues with sequence and motif, column 6 shows homologous sequences identified by BLAST analysis. Column 7 indicates the analysis method, and possibly a searchable database to which the analysis method can be applied. The analysis method in column 7 was used to characterize each polypeptide by sequence homology and protein motifs.

The columns of Table 3 show the tissue specificities and diseases, disorders, and conditions associated with the nucleotide sequence encoding HUMAP. Column 1 of Table 3 contains the nucleotide sequence number (SE
Q ID NO). Column 2 shows a fragment of the nucleotide sequence of column 1. These fragments are useful, for example, in hybridization or amplification techniques to identify SEQ ID NOs: 18-34 and distinguish SEQ ID NOs: 18-34 from related polynucleotide sequences. The polypeptides encoded by these fragments are useful, for example, as immunogenic peptides. Column 3 shows the name of the tissue expressing HUMAP and its percentage in all tissues expressing HUMAP. Column 4 shows the diseases or abnormalities, symptoms associated with tissues that express HUMAP, and their percentage in all tissues that express HUMAP. Column 5 shows the vector used for subcloning each cDNA library.

Each column in Table 4 describes a tissue used for preparing a cDNA library from which a clone of cDNA encoding HUMAP was isolated. Column 1 contains the nucleotide SEQ
Column 2 indicates the cDNA library from which the clones were isolated, and column 3 indicates the origin and details of the tissue associated with the cDNA library in column 2.

[0092] SEQ ID NO: 31 maps to the chromosome 4 within the interval 77.30-88.85 centiMorgan.

[0093] The present invention also includes variants of HUMAP. Preferred variants of HUMAP have at least one of the functional and / or structural characteristics of HUMAP and have at least about 80% amino acid sequence identity to the HUMAP amino acid sequence, or at least about 90%.
And even at least about 95% amino acid sequence identity.

[0094] The present invention also provides polynucleotides encoding HUMAP. In certain embodiments, the invention provides a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 18-34 encoding HUMAP. SEQ ID shown in Sequence Listing
The polynucleotide sequence of NO: 18-34 contains an RNA sequence equivalent in which the nitrogenous base thymine is replaced by uracil and the backbone of the sugar chain is ribose instead of deoxyribose.

[0095] The invention also includes mutant sequences of the polynucleotide sequence encoding HUMAP.
In particular, a variant sequence of such a polynucleotide sequence will have at least 80% polynucleotide sequence identity to the polynucleotide sequence encoding HUMAP, or at least 85% polynucleotide sequence identity, or even at least 95%. Has polynucleotide sequence identity. Certain embodiments of the present invention are directed to SEQ ID N
O: SEQ ID No .: at least 80% polynucleotide sequence identity, or at least 85% polynucleotide sequence identity, or even at least 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of 18-34 ID NO: 18−
34 provides a mutant sequence of the polynucleotide sequence comprising a sequence selected from the group consisting of: Each of the above-described polynucleotide variants encodes an amino acid sequence having at least one of the functional or structural characteristics of HUMAP.

[0096] The various polynucleotide sequences encoding HUMAP that can be created by the degeneracy of the genetic code include those that have minimal similarity to the polynucleotide sequences of any known naturally occurring genes. , One skilled in the art will understand. Thus, the present invention may include all possible polynucleotide sequence variations that may be made by selection of combinations based on possible codon choices. These combinations are made based on the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring HUMAP, and consider that all mutations are explicitly disclosed.

[0097] The nucleotide sequence encoding HUMAP and its variant sequences are generally capable of hybridizing to the nucleotide sequence of a naturally occurring HUMAP under suitably selected stringent conditions, but have non-naturally occurring codons. It may be advantageous to create a nucleotide sequence encoding HUMAP or a derivative thereof having codons of substantially different uses, such as inclusion. Codons can be selected based on the frequency with which particular codons are utilized by the host to increase the rate at which expression of the peptide occurs in a particular eukaryotic or prokaryotic host. Another reason to substantially alter the nucleotide sequence encoding HUMAP and its derivatives without altering the encoded amino acid sequence is that RNA with favorable properties, such as a longer half-life, than transcripts made from naturally occurring sequences. To make a transcript.

The present invention also includes the generation of DNA sequences encoding HUMAP and its derivatives, or fragments thereof, entirely by synthetic chemistry. After construction, the synthetic sequence can 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 can be used to introduce mutations in the sequence encoding HUMAP or any fragment thereof.

The present invention further provides a polynucleotide sequence capable of hybridizing with the claimed polynucleotide sequences, in particular, SEQ ID NO: 18-34 and fragments thereof, under various stringent conditions. Included (e.g., Wahl, GM and SL Berger
(1987) Methods Enzymol. 152: 399-407; and Kimmel.AR (1987) Methods En
zymol. 152: 507-511). Hybridization conditions, including annealing and washing conditions, are described in Definitions.

[0100] Any of the embodiments of the invention can be performed using DNA sequencing methods well known in the art. This method includes, for example, the Klenow fragment of DNA polymerase I, SE
QUENASE (US Biochemical, Cleveland OH), Taq polymerase (Perkin Elmer)
, Thermostable T7 polymerase (Amersham, Pharmacia Biotech Piscataway NJ),
Alternatively, an enzyme such as a combination of a proofreading exonuclease and a polymerase as found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD) is used. Preferably, a MICROLAB2200 liquid transfer system (Hamilton, Reno, NV),
PTC200 Thermal Cycler200 (MJ Research, Watertown MA) and ABI CATALYST 80
Automate sequence preparation using an instrument such as 0 (Perkin-Elmer). Next, ABI 373
Alternatively, sequencing is performed using the 377 DNA sequencing system (Perkin-Elmer), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics. Sunnyvale CA) or other methods well known in the art. The resulting sequence is analyzed using various algorithms well known in the art (e.g., Ausubel, FM (1997) Short Protocols in Molecular Biology , John Wiley & Sons, New York NY, unit 7.7;
Meyers, RA (1995) Molecular Biology and Biotechnology , Wiley VCH, New
York NY, pp. 856-853.).

In various methods based on the PCR method well known in the art, partial nucleotide sequences are used to extend a nucleic acid sequence encoding HUMAP so that sequences upstream of promoters and regulatory elements can be removed. To detect. For example, one method utilizing restriction site PCR involves amplifying an unknown sequence from genomic DNA in a cloning vector using common primers and nested primers (eg, Sarkar, G. (1993) PCR M).
ethods Applic 2: 318-322). Another method using the inverse PCR method uses primers that amplify an unknown sequence from a circularized template that has been extended in a wide range of directions. This template is derived from a restriction fragment containing the known genomic locus and surrounding sequences (see, for example, Triglia, T. et al. (1988) Nucleic Acids Res 16: 8186). Capture pc
A third method using the R method involves PCR amplification of DNA fragments adjacent to known sequences of human and yeast artificial chromosomal DNA (see, eg, Lagerstrom, M. et al. (1991) PCR Methods Ap.
plic 1: 111-119). In this method, it is possible to insert a recombinant double-stranded sequence into a region of unknown sequence before performing PCR using digestion and ligation with a number of restriction enzymes. It is also possible to obtain unknown sequences using other methods well known in the art. (For example, Parker, JD et al. (1991) Nucleic Acids Res
19: 3055-3060). Furthermore, the use of PCR, nested primers, and a PROMOTERFINDER library (Clontech, Palo Alto CA) enables walking in genomic DNA. This method eliminates the need to screen libraries and is useful for finding intron / exon junctions. For all PCR-based methods, the primers are commercially available OLIGO 4.06 Primer Analysis software (National Bioscien
ces, Plymouth MN) or another suitable program.
It is designed to anneal to a template at a temperature of about 68 ° C to 72 ° C with 0 nucleotides, a GC content of 50% or more.

When screening for full-length cDNAs, it is preferable to use libraries whose size has been selected to include large cDNAs. Furthermore, if the oligo d (T) library cannot produce full-length cDNA, a randomly primed library, often containing a sequence having the 5 'region of the gene, is useful. Genomic libraries may be useful for extension of sequence into 5 'non-transcribed regulatory regions.

Sequencing or PCR using a commercially available capillary electrophoresis system
Analysis or confirmation of the size of the nucleotide sequence of the product is possible. For more information,
Capillary sequencing uses a flowable polymer for electrophoretic separation, a laser-activated fluorescent dye specific to four different nucleotides, and a CCD camera used to detect the emitted wavelength It is possible.
Output / light intensity is determined by appropriate software (eg GENOTYPER and SEQUENCE NAVIGA
TOR, Perkin-Elmer), and the process from sample loading to computer analysis and electronic data display can be controlled by computer. Capillary electrophoresis is particularly suitable for sequencing small pieces of DNA that may be present in small amounts in a particular sample.

In another embodiment of the present invention, a polynucleotide sequence encoding HUMAP, or a fragment thereof, is cloned into a recombinant DNA molecule to express HUMAP, a fragment, or a functional equivalent, in a suitable host cell. It is possible. Due to the inherent degeneracy of the genetic code, alternative DNA sequences encoding substantially the same or functionally equivalent amino acid sequences can be made, and these sequences can be used for cloning and expression of HUMAP.

The nucleotide sequence of the present invention can be recombined using methods generally known in the art to change the sequence encoding HUMAP for various purposes. This purpose includes, but is not limited to, cloning, processing and / or regulating expression of the gene product. Recombination of nucleotide sequences is possible using mixing of DNA with random fragments or PCR reassembly of gene fragments and synthetic oligonucleotides. For example, oligonucleotide-mediated directed mutagenesis can be used to introduce mutations that create new restriction sites, alter glycosylation patterns, alter codon preferences, create splice variants, and the like.

The nucleotides of the present invention were prepared using MOLECULARBREEDING (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
Et al. (1996) Nat. Biotechnol. 14: 315-319) and the ability to bind HUMAP to biological or enzymatic activities or other molecules or compounds by shuffling using DNA shuffling techniques. The biological properties of HUMAP such as can be changed or improved. DNA shuffling is a process for preparing a library of gene variants by recombination of gene fragments by PCR. Next, the library is selected or screened to identify gene variants having the desired properties. These preferred variants are pooled and the DNA shuffling and selection / screening is repeated. Thus, diverse genes are created by artificial breeding and rapid molecular evolution. For example, recombination, screening and shuffling can be performed on a fragment of one gene having a mutation at a random position until the desired property is optimized. Alternatively, a fragment of a given gene is recombined with a fragment of a homologous gene of the same gene family from the same or a different species, thereby regulating the gene diversity of a number of naturally occurring genes in a regulatable manner according to the protocol. Sex can be maximized.

According to another embodiment, the sequence encoding HUMAP can be synthesized, in whole or in part, using chemical methods well known in the art (eg, Caruthers. MH, et al. (198).
0) Nucl. Acids Res. Symp. Ser 7: 215-223; and Horn, T. et al. (1980) Nucl. Aci.
ds Res. Symp. Ser. 225-232). Alternatively, HUMAP itself or a fragment thereof can be synthesized using chemical methods. For example, peptide synthesis can be performed using various solid-phase techniques (eg, Roberge, JY et al. (1995) Science 26).
9: 202-204). Automation of synthesis can also be achieved using, for example, an ABI 431A peptide synthesizer (Perkin Elmer). Further, the amino acid sequence of HUMAP, or any portion thereof, may be altered by alterations during direct synthesis and / or in combination with sequences from other proteins or any portion thereof using chemical methods. It is possible to make a polypeptide.

The peptide was purified by high performance liquid chromatography for separation (eg, Chiez, RM).
And FZ Regnier (1990) Methods Enzymol. 182: 392-421). The composition of the synthesized peptide can be confirmed by amino acid analysis or sequencing (see, for example, Creighton. T. (1983) Proteins , Structures and Molecular Properties , WH Freeman, New York, NY).
.

To express biologically active HUMAP, the nucleotide sequence encoding HUMAP or a derivative thereof is inserted into a suitable expression vector. This expression vector is
Contains elements necessary for the regulation of transcription and translation of the inserted coding sequence into a suitable host. These elements include regulatory sequences such as enhancers, constitutive and inducible promoters, 5 'and 3' untranslated regions in the vector and polynucleotide sequences encoding HUMAP. Such elements vary in their length and specificities. With certain initiation signals, it is possible to achieve more efficient translation of the sequence encoding HUMAP. Such signals include the ATG start codon and nearby sequences such as the Kozak sequence. If the sequence encoding HUMAP, its initiation codon, and 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 including an in-frame ATG initiation codon must be included in the expression vector. Exogenous translational elements and initiation codons can be obtained from a variety of sources, both natural and synthetic. Increasing the efficiency of expression can be achieved by including enhancers suitable for the particular host cell line used. (For example, Scharf, D
Et al. (1994) Results Probl. Cell Differ. 201-18-162).

The sequences encoding HUMAP, suitable transcription and translation, using methods well known to those skilled in the art.
It is possible to create an expression vector containing regulatory elements. These methods include: in vitro Recombinant DNA technology, synthetic technology, andin vivoGenetic recombination techniques are included.
(For example, Sambrook, J. et al. (1989)Molecular Cloning. A Laboratory Manual , Cold Spring Harbor Press, Plainview NY, Chapters 4 and 8, and 16-17; and
 Ausubel, F.M. et al. (1995)Current Protocols in Molecular Biology, John W
iley & Sons, New York NY, ch. Chapters 9 and 13 Chapters 1-4).

[0111] A variety of expression vector / host systems can be utilized to retain and express sequences encoding HUMAP. These include, but are not limited to, microorganisms such as bacteria transformed with a recombinant bacteriophage, plasmid, or cosmid DNA expression vector, yeast transformed with a yeast expression vector, and viral expression vectors. (E.g., baculovirus) -infected insect cell lines or plants transformed with a viral expression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or a bacterial expression vector (e.g., Ti or pBR322 plasmid). Cell lines and animal cell lines are included. The present invention is not limited by the host cell used.

[0112] In bacterial systems, a number of cloning and expression vectors are available depending upon the use for which the polynucleotide sequence encoding HUMAP will be used. For example, HUMA
For routine cloning, subcloning, and propagation of a polynucleotide sequence encoding P, a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or pSPORT1 plasmid (GIBCO BRL) can be used. Ligation of sequences encoding HUMAP into multiple cloning sites of the vector results in lac
The Z gene is disrupted, allowing a colorimetric screening method to identify transformed bacteria containing the recombinant molecule. Furthermore, using these vectors, it is possible to transcribe cloned sequences in vitro , dideoxin screening, rescue single stranded by helper phage, and create nested deletions. (E.g., Van Heeke, G. and SM Schuster (1989) J. Biol. Chem. 264: 5503-5509
See.). For example, if a large amount of HUMAP is required for antibody production, use HUMAP
A vector that induces high-level expression of can be used. For example, vectors containing a T5 or T7 bacteriophage promoter that strongly induces expression can be used.

A yeast expression system can be used for the expression of HUMAP. A variety of vectors containing constitutive or inducible promoters such as α-factor, alcohol oxidase, and PGH promoters can be used in yeast Saccharomyces cerevisiae or Pichia pastoris . In addition, such vectors induce either the secretion or retention of the expressed protein in cells, incorporating foreign sequences into the host genome for stable growth. (For example, Ausubel .; and Bitter, GA et al. (1987) Method
s Enzymol. 153: 51-794: Scorer.CA et al. (1994) Bio / Technology 121-181-18.
(See 4.) Plant systems can also be used for HUMAP expression. Transcription of the sequence encoding HUMAP is carried out, for example, by using a viral promoter such as CaMV-derived 35S and 19S promoters alone or an omega leader sequence derived from TMV (Takamatsu, N. et al. (1987) EMBO J 6: 307-311). Is promoted in combination with. These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. (For example, The McGraw Hill Yearbook of Science and Technology (1992)
McGraw Hill NY, pp. 191-196).

[0114] In mammalian cells, a variety of virus-based expression systems may be utilized. When an adenovirus is used as an expression vector, a sequence encoding HUMAP may be ligated to an adenovirus transcript / translation complex consisting of the late promoter and tripartite leader sequence. By insertion into the non-essential E1 or E3 region of the viral genome,
It is possible to obtain a live virus expressing HUMAP in infected host cells (Logan
, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81: 3655-3659). In addition, transcription enhancers such as the Rous sarcoma virus (RSV) enhancer can be used to increase expression in mammalian host cells. To express proteins at high levels, vectors based on SV40 or EBV can be used.

[0115] Human artificial chromosomes (HACs) can be used to supply fragments of DNA that are expressed in a plasmid and larger than those contained therein. About 6 kb to 10 Mb of HACs for treatment
Is prepared and supplied by conventional transport methods (liposomes, polycation amino polymers, or vesicles). (For example, Harrington. JJ et al. (1997) Nat Genet. 15: 3
45-355.).

For long-term production of mammalian-based recombinant proteins, stable expression of HUMAP in cell lines is desirable. For example, a HUMAP-encoding sequence can be transformed into a cell line using an expression vector. Such expression vectors include replication and / or endogenous expression elements of viral origin and a selectable marker gene on the same or another vector. After the introduction of the vector, the cells are allowed to grow for about 1-2 days in an enriched medium before being transferred to a selective medium. The purpose of the selectable marker is to confer resistance to the selective mediator, and its presence allows the growth and recovery of cells that successfully express the introduced sequence. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate for the cell type.

[0117] Any number of selection systems can be used to recover transformed cell lines. The selection systems include, but are not limited to, include herpes simplex virus thymidine kinase gene and adenine phosphoribosyltransferase genes, respectively tk ^- or apr ^- used in the cell. (For example, Wigler, M. et al. (1
977) Cell 11: 223-232; and Lowy, I. et al. (1980) Cell 22: 817-823). Also, resistance to antimetabolites, antibiotics or herbicides can be used as a basis for selection. For example, dhfr confers resistance to methotrexate, neo confers resistance to aminoglycoside neomycin and G-418, als or pat confers resistance to chlorsulfuron (cHUMAPsulfuron), phosphinotricin acetyltransferase (eg, , Wigl
er, M. et al. (1980) Proc. Natl. Acad. Sci. 77: 3567-3570; Colbere-Garapin,
F. et al. (1981) J. Mol. Biol. 150: 1-14). In addition, genes available for selection, such as trpB and hisD, which alter what cells need for metabolism, have been described in the literature (eg, Hartman, SC and RC Mulligan (1988) Proc. Natl. Acad.
Sci. 85: 8047-51). Anitocyanin, green fluorescent protein (GFP; Clon
tech), β-glucuronidase and its substrate GUS, luciferase and its substrate luciferin and other visible markers are used. Green fluorescent protein (GFP) (Clon
tech, Palo Alto, CA) can also 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 (eg, Rhodes, CA et al. (19)
95) Methods Mol. Biol. 55: 121-131).

Even if the presence / absence of marker gene expression indicates the presence of the gene of interest, it may be necessary to confirm the presence and expression of that gene. For example, if a sequence encoding HUMAP is inserted into a marker gene sequence, transformed cells containing the sequence encoding HUMAP can be identified by a lack of marker gene function. Alternatively, the marker gene can be aligned with the sequence encoding HUMAP under the control of one promoter. Expression of the marker gene in response to induction or selection usually also indicates expression of the tandem gene.

In general, host cells that contain a nucleic acid sequence encoding HUMAP and that express HUMAP can be identified using various methods well known to those skilled in the art. These methods include DNA-DNA or DNA-RNA hybridization, PCR, and protein biology, including membrane-based, solution-based, or chip-based techniques for detection and / or quantification of nucleic acids or proteins. Test methods or immunological assays include, but are not limited to.

HU using either specific polyclonal or monoclonal antibodies
Immunological methods for detecting and measuring MAP expression are well known in the art. Such techniques include immunosorbent assays (ELISA), radioimmunoassays (RIAs), and fluorescence activated cell sorters (FACS) linked to enzymes. Two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on HUMAP
) Is preferred, but competitive binding assays can also be used. These and other assays are well known in the art. (For example, Hampton.
R. et al. (1990) Serological Methods, a Laboratory Manual. APS Press. St Pa.
ul.MN, Sect.IV; Coligan, JE et al. Current Protocols in Immunology , Gree
ne Pub. Associates and Wiley-Interscience, New York. NY; and Pound, JD
(1990) Immunochemical Protocols , Humans Press, Totowa NJ).

Various labeling and conjugation techniques are well known to those skilled in the art and can be used in various nucleic acid and amino acid assays. Methods for generating labeled hybridization probes or PCR probes for detecting sequences related to a polynucleotide encoding HUMAP include oligo-labeling, nick translation, end-labeling, or labeled nucleotides. The PCR amplification used is included.
Alternatively, the sequence encoding HUMAP, or any fragment thereof, can be cloned into a vector for generating an mRNA probe. Such vectors, which are well known in the art and are commercially available, can be converted to a suitable R7, such as T7, T3, or SP6.
RNA in vitro with the addition of NA polymerase and labeled nucleotides
It can be used for probe synthesis. These methods are described, for example, in Amersham Pha
rmacia Biotech and Promega (Madison WI), US Biochemical Corp (Clevelan
dOH) can be carried out using various commercially available kits. Suitable reporter molecules or labels that can be used for easy detection include substrates, cofactors, inhibitors, magnetic particles, and radionuclides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents, and the like.

[0122] Host cells transformed with the nucleotide sequence encoding HUMAP are cultured under conditions suitable for the expression and recovery of the protein from cell culture. Whether a protein produced from a transformed cell is secreted or remains in the cell depends on the sequence used and / or the vector. One of skill in the art will appreciate that expression vectors containing a polynucleotide encoding HUMAP can be designed to include a signal sequence that directs secretion of HUMAP across prokaryotic and eukaryotic cell membranes.

In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion. Such polypeptide modifications include acetylation, carboxylation, glycosylation, phosphorylation, lipidation (
lipidation), and acylation.
Post-translational processing that cleaves the "prepro" or "pro" form of the protein can be used to identify the target protein, folding and / or activity. A variety of host cells (eg, CHO, HeLa, MDCK, MEK293, WI38) with specific cellular devices and characteristic mechanisms for post-translational activity can be obtained from American Type Culture C
Available from ollection (ATCC; Bethesda, MD) and selected to ensure correct modification and processing of the foreign protein.

In another embodiment of the present invention, a natural or altered or recombinant nucleic acid sequence encoding HUMAP is linked to a heterologous sequence which results in translation of the fusion protein of any of the host systems described above. For example, a chimera containing a heterologous moiety that can be recognized by a commercially available antibody
HUMAP proteins may facilitate the screening of peptide libraries for inhibitors of HUMAP activity. Also, the heterologous protein portion and the heterologous peptide portion can facilitate purification of the fusion protein using commercially available affinity substrates. These include glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6
-His, FLAG, c-mc, hemagglutinin (HA), but are not limited thereto. GST and MBP, Trx, CBP, 6-His allow purification of homologous fusion proteins with each of immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal chelating resin.
FLAG, c-mc, and hemagglutinin (HA) allow immunoaffinity purification of the fusion protein using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. Also, if the fusion protein is engineered so that the fusion protein contains a proteolytic cleavage site between the sequence encoding HUMAP and the heterologous protein sequence, HUMAP can be cleaved from the heterologous portion after purification. Methods for expression and purification of the fusion protein are described in Ausubel. (1995, supra ch 10). Various commercially available kits can be used to facilitate expression and purification of the fusion protein.

In another embodiment of the invention, the synthesis of radiolabeled HUMAP in vitro using a TNT rabbit reticulocyte lysate or a wheat germ extract system (Promega) is possible. These systems link transcription and translation of sequences encoding proteins operably linked to the T7 or T3, SP6 promoter. Translation occurs in the presence of a radiolabeled amino acid precursor, for example, ³⁵ S methionine.

Fragments of HUMAP can be made by direct peptide synthesis using solid phase technology as well as recombinant products (see, eg, Creighton, pp. 55-60, supra). Protein synthesis can be performed manually or automatically. Automated synthesis, for example, ABI 431A
It can be performed using a peptide synthesizer (Perkin Elmer). HUMAP
Are synthesized separately and then ligated to produce the full-length molecule.

Therapeutics There are chemical and structural similarities between certain regions of HUMAP and certain regions of the human membrane-associated protein, for example, in the context of sequence and motifs. In addition, HUMAP expression is closely linked to cell proliferation and cancer, inflammation. Therefore, HUMAP is thought to play a role in cell signaling, cell differentiation abnormalities, and cell proliferation abnormalities.
In the treatment of a disease associated with an increase in HUMAP expression or activity, it is desirable to reduce HUMAP expression or activity. In the treatment of a disease associated with a decrease in the expression or activity of HUMAP, it is desirable to increase the expression or activity of HUMAP.

Accordingly, in one embodiment, it is possible to administer HUMAP or a fragment or derivative thereof to a patient for the treatment or prevention of a disease associated with reduced HUMAP expression or activity. Examples of such diseases include, but are not limited to, abnormal cell proliferation, including actinic keratosis and atherosclerosis, bursitis, cirrhosis, hepatitis, mixed binding Tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and adenocarcinoma and leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, Specifically, adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary, pancreas,
Includes cancers of the parathyroid gland, penis, prostate, salivary gland, skin, spleen, testis, thymus, uterus, and also includes abnormal cell differentiation, including tubular acidosis, anemia, Cushing's syndrome, and chondrodysplasia Dwarfism, Duchenne-Becker muscular dystrophy, epilepsy, gonadal dysplasia, WAGR syndrome (Wilms tumor, aniridia, genitourinary disorders, mental retardation), Smith-Magenis syndrome, myelodysplasia Syndrome, hereditary mucosal epithelial dysplasia, hereditary keratoderma, Charcot-Marie
Hereditary neuropathies such as tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as chorea (Syndenham's chorea) and cerebral palsy, spinal cord fission, anencephaly, cranial spondylolysis, congenital Includes developmental disorders, including glaucoma, cataracts, sensory nerve hearing loss, and abnormalities in immune cell activity, and also includes any tissue, organ, or system of the patient, such as the brain or adrenal gland, kidney, skeletal or reproductive system Cell proliferation and differentiation, embryogenic form,
Any disease associated with metaplasia and also includes abnormal cell signaling, including primary brain tumors and adenomas, gestational infarction, pituitary resection, aneurysms, vascular malformations, thrombosis, and infection Disorders of the hypothalamus and pituitary gland resulting from lesions such as infectious diseases, immune abnormalities, and complications due to head trauma, and inappropriate antidiuretic hormones (AD
H) Disorders related to hyperpituitary gland including secretory syndrome (SIADH) and acromegaly, giantosis, and disorders related to goiter and myxedema, hypothyroidism including bacterial infectious acute thyroiditis, Conn. Hyperparathyroidism including chronic hypercalemia, pancreatic diseases such as type I and type II diabetes and complications, and adrenal glands such as hyperplasia and adenoma carcinomas and adenomas, and hypertension associated with alkalosis Disorders and abnormal prolactin production and infertility in women, endometriosis, menstrual cycle perturbation, polycystic ovarian disease, hyperprolactinemia, selective gonadotropin deficiency (isolated gonadot
ropin deficiency), amenorrhea, milk leakage, semi-Yin Yang, hirsutism and virilization, breast cancer,
Postmenopausal osteoporosis, male Leydig cell hyperplasia, male menopause, germ cell aplasia, hypersexuality associated with Leydig cell tumors, androgen resistance associated with lack of androgen receptor, 5α-reductase syndrome, female Endocrine disorders, including those associated with gonadal steroid hormones such as mastosis.

In another embodiment, HUMAP or a fragment or derivative thereof is expressed for the treatment or prevention of a disease associated with reduced HUMAP expression or activity, including but not limited to the diseases listed above. The resulting vector can be administered to a patient.

In yet another embodiment, HUMAPs, including but not limited to the diseases listed above,
Pharmaceutical compositions comprising substantially purified HUMAP can be administered to a patient together with a suitable pharmaceutical carrier for the treatment or prevention of a disease associated with reduced expression or activity of the compound.

In yet another embodiment, HUMAPs include, but are not limited to, the diseases listed above.
For the treatment or prevention of a disease associated with a decrease in the expression or activity of HUMAP, an agonist that modulates the activity of HUMAP can be administered to a patient.

In a further embodiment, an HUMAP antagonist can be administered to a patient for the treatment or prevention of a disease associated with increased HUMAP expression or activity.
Examples of such diseases include, but are not limited to, the above-described abnormal cell signaling and cell differentiation, and abnormal cell proliferation. In one embodiment, an antibody that specifically binds to HUMAP can be used directly as an antagonist or indirectly as a targeting or delivery mechanism to deliver an agent to cells or tissues that express HUMAP.

In another example, the complementary sequence of a polynucleotide encoding HUMAP for the treatment or prevention of a disease associated with increased expression or activity of HUMAP, including but not limited to the diseases listed above. Can be administered to a patient.

In another embodiment, any of the proteins, antagonists, antibodies, agonists, complementary sequences, vectors of the invention may be administered in combination with another suitable therapeutic agent. One skilled in the art can select a suitable therapeutic agent to use in the combination therapy according to conventional pharmaceutical principles. Combinations with therapeutic agents may have synergistic effects in the treatment or prevention of the various diseases listed above. Using this method, it is possible to achieve a medicinal effect with a small amount of each drug, and to reduce the possibility of a wide range of side effects.

[0135] Antagonists of HUMAP can be produced using methods common in the art. Specifically, antibodies can be produced using purified HUMAP, or libraries of therapeutic agents can be screened to identify those that specifically bind to HUMAP. HUMAP antibodies can also be produced using methods common in the art.
Such antibodies include polyclonal, monoclonal, chimeric,
Includes single strands, Fab fragments, and fragments created by Fab expression libraries. However, it is not limited to these. For therapeutic use, neutralizing antibodies (ie, those that inhibit dimer formation) are particularly preferred.

For the production of antibodies, various hosts, including goats, rabbits, rats, mice, humans and others, may be injected with HUMAP or any fragment, or its oligopeptides with immunogenic properties. Can be immunized with. Depending on the host species, various adjuvants can be used to enhance the immune response. Such adjuvants include surfactants such as Freund's adjuvant, mineral gel adjuvants such as aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, peptides, oily emulsions, keyhole limpet hemocyanin, and dinitrophenol. However, the present invention is not limited to these. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parv um it is particularly preferred.

Oligopeptides, peptides used to elicit antibodies against HUMAP,
Alternatively, the fragment is composed of at least about 5 amino acids, and generally is preferably composed of about 10 or more amino acids. These oligopeptides or peptides,
Or their fragments desirably are identical to a portion of the amino acid sequence of the native protein, and also include the entire amino acid sequence of a small naturally occurring molecule. Short stretches of HUMAP amino acids can be fused to sequences of another protein, such as KLH, to produce antibodies against the chimeric molecule.

[0138] Monoclonal antibodies to HUMAP can be produced by continuous cell lines in culture using any technique that produces antibody molecules. These techniques include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and EBV-hybridoma technology (eg, Kohler
(1975) Nature 256: 495-497; Kozbor, D. et al. (1985) .J. Immunol. M.
ethods 81-8-42; Cote, RJ et al. (1983) Proc. Natl. Acad. Sci. 80: 2026-20
30; Cole, SP et al. (1984) Mol. Cell Biol. 62: 109-120).

In addition, techniques such as splicing a mouse antibody gene with a human antibody gene developed to produce a “chimeric antibody” are used to obtain molecules with suitable antigen specificity and biological activity ( For example, Morrison, SL et al. (1984) Proc. N
atl. Acad. Sci. 81-4851-4855; Neuberger, MS et al. (1984) Nature 312: 604.
-608; Takeda, S. et al. (1985) Nature 314: 452,454). Alternatively, applying the described techniques for the production of single chain antibodies using methods well known in the art,
Generate HUMAP specific single chain antibodies. Antibodies of related idiotype composition with related specificity can also be generated by chain mixing from random combinations of immunoglobulin libraries (see, eg, Burton DR (1991) Proc. Natl.
Acad. Sci. 88: 11120-3).

Antibodies can be used to trigger in vivo production in lymphocyte populations, or to screen immunoglobulin libraries or to screen a panel of highly specific binding reagents as indicated in the literature. (For example, Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833).
-3837; Winter, G. et al. (1991) Nature 349: 293-299).

[0141] Antibodies containing specific binding sites for HUMAP can also be produced. For example, such fragments include F (ab ') generated by pepsin digestion of an antibody molecule and F (ab') generated by reducing the disulfide bridges of the fragment.
b fragments, including but not limited to. Alternatively, creating a Fab expression library allows for rapid and easy identification of monoclonal Fab fragments with the desired specificity (eg, Huse, WD et al. (1989) Science 254: 12).
75-1281).

Screening is performed using various immunoassays to identify antibodies with the desired specificity. Numerous protocols for competitive binding, using either polyclonal or monoclonal antibodies with sequestered specificity, or immunoradioactivity, are well known in the art. Usually such immunoassays include HUMA
Includes measurement of complex modulation between P and its specific antibody. Two incoherent HUMAPs
Two-site, monoclonal-based immunoassays using monoclonal antibodies reactive to the epitope are commonly used, but competitive binding assays can also be used (Pound, supra).

Using various methods such as Scatchard analysis with radioimmunoassay technology, H
Evaluate the affinity of the antibody for UMAP. Affinity is represented by the binding constant Ka, which is the value obtained by dividing the molar concentration of the HUMAP antibody complex by the molar concentration of free antibody and free antigen under equilibrium conditions. The Ka of a polyclonal antibody drug with heterogeneous affinity for a number of HUMAP epitopes represents the average affinity or avidity of the antibody for HUMAP. The Ka of a monoclonal antibody drug monospecific for a particular HUMAP epitope represents a true measure of affinity. High-affinity antibody drugs with a Ka value of 10 ⁹ to 10 ¹² L / mol are preferably used for immunoassays in which the HUMAP antibody complex must withstand severe manipulations. Low affinity antibody drugs with a Ka value of 10 < ⁶ > to 10 < ⁷ > L / mol are preferably used for immunopurification and similar treatments where HUMAP needs to be finally dissociated from the antibody in an activated state. (Catty, D. (19
88) Antibodies, Volume I: A Practical Approach .IRL Press, Washington, D
C; Liddell, JE and Cryer, A. (1991) A Practical Guide to Monoclonal Antibodies , John Wiley & Sons, New York NY).

To determine the quality and suitability of such pharmaceuticals in certain downstream applications,
The antibody titer and binding activity of the polyclonal antibody drug are further evaluated. For example, polyclonal antibody medicaments containing at least 1-2 mg / ml of specific antibody, preferably 5-10 mg / ml of specific antibody, are generally used in processes where the HUMAP antibody complex must be precipitated. Guidance on antibody specificity and titer, avidity, antibody quality and usage in various applications is generally available. (For example, C
atty, supra, and Coligan et al., supra).

In another embodiment of the invention, a polynucleotide encoding HUMAP, or any fragment or complement thereof, can be used for therapeutic purposes. In certain embodiments, if the complementary sequence of the polynucleotide encoding HUMAP is suitable for blocking transcription of mRNA, it can be used. In particular, cells can be transformed with sequences complementary to a polynucleotide encoding HUMAP. Thus, complementary molecules or fragments can be used to modulate the activity of HUMAP, or to regulate gene function. Such techniques are well known in the art, and sense or antisense oligonucleotides or large fragments can be designed from the control region of the sequence encoding HUMAP, or from various locations along the coding region.

[0146] Expression vectors derived from retroviruses, adenoviruses, herpes or vaccinia, or various bacterial plasmids can also be used to carry the nucleotide sequence to the target organ, tissue or cell population. Vectors expressing nucleic acid sequences that are complementary to HUMAP encoding polynucleotides can be made using methods well known to those of skill in the art (see, for example, Sambrook et al., Supra, and Ausubel et al., Supra). .

[0147] The gene encoding HUMAP can be stopped by transforming a cell or tissue with an expression vector that expresses polynucleotides encoding HUMAP or fragments thereof at high levels. Such constructs can be used to introduce sense or antisense sequences that cannot be translated into cells. Such vectors, even when not incorporated into DNA, continue to transcribe mRNA molecules until their function is impaired by endogenous nucleases. Non-replicating vectors also maintain transient expression for over a month, and may last longer if suitable replication elements are part of the vector system.

As described above, gene expression is determined by the sequence complementary to the regulatory 5 'or regulatory region of the gene encoding HUMAP or an antisense molecule (DNA or RNA, PNA)
Can be adjusted by designing the For example, oligonucleotides derived from the transcription start site from about -10 to about +10 from the start site can be used. Similarly, it can be blocked using "triple helix" and base pairing. Triple helix structures are useful because they prevent the double helix from spreading sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (eg, Gee, JE et al. (1994) In: H
uber, BE and BI Carr, Molecular and ImmunologiHUMAPproaches , Futura
Publishing Co., Mt. Kisco, NY). Complementary sequences or antisense molecules can also be designed to prevent translation of the mRNA by preventing the transcript from binding to the ribosome.

[0149] Ribozymes, which are enzymatic RNA molecules, can be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by nucleotide strand breaks.
For example, it includes a recombinant hammerhead ribozyme molecule that specifically and effectively catalyzes nucleotide chain cleavage of a sequence encoding HUMAP.

A specific ribozyme cleavage site within any potential RNA target is identified by the following sequence GUA
, GUU, and GUC and are identified initially by scanning the target molecule for ribozyme cleavage sites. Once identified, evaluate short RNA sequences of 15-20 ribonucleotides corresponding to the region of the target gene containing the cleavage site for secondary structural features that impair oligonucleotide function Is possible. The suitability of a candidate target can also be assessed by using a ribonuclease protection assay to test the ease of hybridization with a complementary oligonucleotide.

[0151] Complementary ribonucleic acid molecules and ribozymes of the invention can be made for synthesis of nucleic acid molecules using methods well known in the art. These methods include those for chemically synthesizing oligonucleotides such as solid phase phosphoramidite compounds. Alternatively, an RNA molecule can be generated in vitro and in vivo by transcription of a DNA sequence encoding HUMAP.Such DNA sequences can be produced from various vectors using a suitable RNA polymerase promoter, such as T7 or SP6. It is possible to incorporate it inside. Alternatively, these cDNA constructs that synthesize complementary RNA constitutively or inducibly can be introduced into a cell line, cell, or tissue.

[0152] Modification of an RNA molecule can increase intracellular stability and increase half-life. Possible modifications include the addition of flanking sequences at the 5 'and / or 3' ends of the molecule, or the use of phosphorothioate or 2'O methyl rather than phosphodiesterase linkages in the backbone of the molecule. Are not limited to these. This concept inherent in the production of PNAs is based on adenine, cytidine, guanine, thymine, which are not easily recognized by endogenous endonucleases.
And the inclusion of non-conventional bases such as inosine, queosine and wybutosine, as well as acetyl-, methyl-, thio-, and similar modified forms of uridine. Can be expanded to

[0153] vectors available are a number of ways of introducing into a cell or tissue, in vivo, are equally suitable for use in in vitr o, and ex vivo. In the case of ex vivo treatment, the vector is introduced into hepatocytes collected from a patient and cloned and propagated to return the same patient by autologous transplantation. Transfection, ribosome injection or delivery by polycation amino polymer can be performed using methods well known in the art (eg, Goldman, CK et al. (1997) Nature Biotechnology 15: 462).
-66 :).

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

Another embodiment of the present invention relates to the administration of a medicament or sterile composition with a pharmaceutically acceptable carrier for all of the above-mentioned therapeutic effects. Such a pharmaceutical composition comprises HUMAP, an antibody of HUMAP, a mimetic, an agonist, an antagonist, an inhibitor of HUMAP, or the like. The composition can be administered alone or in combination with one or more other agents, such as stabilizers, to a sterile, biocompatible pharmaceutical carrier. Such pharmaceutical carriers include, but are not limited to, saline, buffered saline, glucose, water, and the like. The composition can be administered alone or with another drug, such as a drug or hormone.

The pharmaceutical compositions used in the present invention can be administered using various routes. This route includes oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal Is not limited to these.

In addition to the active ingredients, these pharmaceutical compositions contain suitable pharmaceutically acceptable additives, including excipients and adjuvants, which make the active compound a pharmaceutically acceptable drug. Carrier can be included. Detailed techniques for formulation and administration are described in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, PA).

Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art at dosages suitable for oral administration. Such carriers enable the pharmaceutical composition to be consumed by the patient in tablets, pills, dragees, capsules,
It is formulated as a liquid, gel, syrup, mud, suspension.

Pharmaceutical preparations for oral use are prepared by mixing the active compound with solid pharmaceutical additives, treating the resulting granule mixture (after grinding if desired) into tablets or dragee cores. cores). Suitable excipients include sugars including lactose, sucrose, mannitol, or sorbitol, starch from corn, wheat, rice, potato, or other plants, cellulose such as methylcellulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose, Carbohydrate or protein excipients such as gums, including gum arabic and tragacanth, and proteins such as gelatin and collagen.
If desired, disintegrating or solubilizing agents may be added, such as, for example, cross-linked polyvinyl pyrrolidone, tangerine, alginic acid, or a salt thereof, sodium alginate.

Dragee cores are used with suitable coatings, such as concentrated sugar solvents. Such concentrated sugar solvents can include gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solvents, and suitable organic or mixed solvents. Dyestuffs or pigments are added to the tablets or dragees for product identification or to indicate the amount of active compound, ie, dosage.

Pharmaceutical preparations for oral use include push-fit capsules made of gelatin, as well as encapsulated capsules made of gelatin with a coating, such as glycerol or sorbitol. Push-fit capsules contain the active ingredient in admixture with excipients and binders such as lactose or starch, lubricants such as talc or magnesium stearate, and stabilizers if desired. In soft capsules, the active compound is treated with or without a stabilizer.
It can be dissolved or suspended in a suitable solution such as a fatty oil, a solution, or a polyethylene glycol solution.

Pharmaceutical agents suitable for parenteral administration are formulated in aqueous solutions, with physiologically compatible buffers such as Hanks's solution, Ringer's solution, physiological saline buffer being preferred. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be formulated as suitable oily injection suspensions. Suitable hydrophilic solutions or vehicles include fatty oils such as sesame oil, and synthetic fatty acids such as ethyl oleate, triglycerides or ribosomes. Non-lipid polycationic amino polymers are used for delivery purposes. Optionally, the suspension may contain suitable stabilizers or agents that increase the solubility of the compounds to allow for concentrated solutions.

For topical or nasal administration, penetrants appropriate to the particular barrier to be used are used in the formulation. Such penetrants are well-known to those skilled in the art.

The pharmaceutical compositions of the present invention can be prepared using methods well known in the art, for example, conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, encapsulating, or lyophilizing processes. Can be manufactured.

The pharmaceutical compositions are formulated as salts, and can be formed with a number of acids, including but not limited to hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. Salts are more soluble in aqueous or other protic solvents than the corresponding free base systems. Another drug form includes 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2%
A lyophilized powder that contains some or all of ７7% mannitol, has a pH range of 4.5-5.5, and is combined with a buffer prior to use can be used.

After the pharmaceutical compositions have been formulated, they can be packaged in a suitable box and labeled as medication for the indicated condition. For administration of HUMAP, such labels include the quantity,
Will include frequency, and method of administration.

Suitable pharmaceutical compositions for use in the present invention include compositions that contain an effective amount of the active ingredient to achieve its intended purpose. One of skill in the art can determine a dose that is sufficiently effective on his own.

For any composition, a therapeutically effective dose can be estimated initially either from tumor cell assays, for example on tumor cells, or in animal models. Usually, a mouse, rabbit, dog, pig, or the like is used as an animal model. Animal models can also be used to determine suitable enrichment ranges and routes of administration. Based on such treatment, beneficial dosages and routes for administration in humans can be determined.

A medically effective dose is related to the amount of active ingredient that ameliorates the symptoms or condition, eg, HUMAP or a fragment thereof, an antibody to HUMAP, an agonist or antagonist of HUMAP, an inhibitor, and the like. Medication efficacy and toxicity may be calculated, for example, by calculating the ED ₅₀ (50% of the population has a medicinal effect on taking) or the LD ₅₀ (50% of the population is fatal on taking) statistics. Can be determined by standard pharmaceutical techniques in cell culture or animal studies. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD ₅₀ / ED ₅₀ . Pharmaceutical compositions that exhibit high therapeutic indices are desirable. The data obtained from the cell culture assays and animal studies is used in formulating a range of dosage for human application. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. 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 patient requiring treatment. Dosage and administration are adjusted to provide effective levels of the active ingredient or to maintain the desired effect. Factors that may be considered as dose components include disease severity, general health of the patient, age, weight, and gender of the patient, time and frequency of administration, concomitant medications, response sensitivities, and Response is included. Long-acting pharmaceutical compositions can be administered once every three or four days, once a week, once every two weeks, depending on the half-life and clearance rate of the particular formulation.

[0171] Normal dosage amounts will vary depending on the route of administration, but will range from about 0.1 to 100,000 μg, up to about 1 gram. Guidance on specific dosages and modes of delivery is provided in the literature and is generally available to practitioners in the art. One of skill in the art will utilize formulations of nucleotides that are different from the protein or inhibitor. Similarly, delivery of a polynucleotide or polypeptide will be specific to a particular cell, condition, location, etc.

Diagnosis In another example, an antibody that specifically binds to HUMAP is diagnosed with a disease characterized by HUMAP expression, or is treated with HUMAP, an agonist or antagonist of HUMAP, or an inhibitor. Used in assays to monitor Antibodies useful for diagnosis are formulated in the same manner as described for therapy. HUMAP diagnostic assays include methods for detecting HUMAP from human body fluids or from cells or tissues using antibodies and labels. These antibodies can be used with or without modification and can be labeled by covalent or non-covalent attachment of a reporter molecule. Various reporter molecules known in the art may be used, some of which are described above.

A variety of protocols for measuring HUMAP, including ELISA, RIA, and FACS, are well known in the art and provide a basis for diagnosing abnormal or abnormal levels of HUMAP expression. Normal or standard HUMAP expression values are determined by combining an antibody to HUMAP with a body fluid or cells collected from a subject, such as a normal mammal, e.g., a human, under conditions suitable for complex formation. I do. The amount of standard complex formation can be quantified by various methods, such as photometric. The amount of HUMAP expression in the subject, control and disease, samples from biopsy tissue are compared to baseline values. The deviation between the reference value and the subject is a diagnostic indicator.

According to another embodiment, a polynucleotide encoding HUMAP can be used for diagnosis. Polynucleotides used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. The polynucleotide is used to detect and quantify the expression of a gene in a biopsy tissue that expresses HUMAP, which can be correlated with disease. This diagnostic assay is used to determine the presence, absence, and overexpression of HUMAP, and to monitor the modulation of HUMAP levels during treatment.

In one embodiment, the nucleic acid sequence encoding HUMAP can be identified by hybridization using a PCR probe capable of detecting a polynucleotide sequence comprising the gene sequence encoding HUMAP or a closely related molecule. is there. For example, 5 '
The specificity of a probe, whether it is made from a highly specific region that is a regulatory region, for example, a conserved motif, or a region with a lower degree of specificity, and the stringency of hybridization or amplification depends on whether the probe uses HUMAP. It will depend on whether only the encoding natural sequence is identified, or whether only the natural sequence encoding the allele or related sequence is identified.

Probes may also be used for the detection of related sequences and have at least 50% sequence identity with any sequence encoding HUMAP. The target hybridization probe of the present invention can be DNA or RNA, and can be derived from the sequence of SEQ ID NO: 18-34 or a genomic sequence including the promoter, enhancer, and intron of the HUMAP gene.

As a method for producing a hybridization probe specific to DNA encoding HUMAP, there is a method of cloning a polynucleotide sequence encoding HUMAP and a HUMAP derivative into a vector for producing an mRNA probe. Such vectors are commercially available and well known to those skilled in the art and are used to synthesize RNA probes in vitro by adding a suitable RNA polymerase and suitable labeled nucleotides. The hybridization probe is, for example, ³² P
Alternatively, it may be labeled with a population of various reporters, such as a radionuclide such as ³⁵ S, or an enzyme label such as alkaline phosphatase bound to the probe by an avidin / biotin (biotin) binding system.

[0178] A polynucleotide sequence encoding HUMAP can be used to diagnose a disease associated with HUMAP expression. Examples of such diseases include, but are not limited to, cell proliferation abnormalities, including actinic keratosis and atherosclerosis, bursitis, cirrhosis, hepatitis, mixed binding Tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and adenocarcinoma and leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, Specifically, the adrenal gland, bladder,
Bone, bone marrow, brain, breast, cervix, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary gland, skin, spleen, testis, thymus,
Includes uterine cancer, and also includes abnormal cell differentiation, including tubular acidosis, anemia, Cushing's syndrome, achondroplasia dwarfism, Duchenne-Becker muscular dystrophy, epilepsy, and gonad dysplasia , WAGR syndrome (Wilms tumor, aniridia, genitourinary disorders, mental retardation), Smith-Magenis syndrome
syndrome), myelodysplastic syndrome, hereditary mucosal epithelial dysplasia, hereditary keratosis, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, chorea ( Seizure disorders such as Syndenham's chorea) and cerebral palsy,
Includes developmental disorders, including spinal cord fission, anencephaly, cranial spina bifida, congenital glaucoma, cataracts, sensorineural hearing loss, and abnormalities in immune cell activity, and also include, for example, brain and adrenal glands, kidney, skeletal or reproductive systems Includes any disease related to cell proliferation and differentiation, including embryonic morphogenesis and metaplasia, including any tissues and organs and systems of the patient, including abnormalities in cell signaling, including primary Hypothalamic and pituitary disorders resulting from lesions such as brain tumors and adenomas, gestational infarction, pituitary resection, aneurysms, vascular malformations, thrombosis, infections, immune abnormalities, complications from head trauma, and benign adenomas Inappropriate inappropriate antidiuretic hormone (ADH) secretion syndrome (SIADH) and disorders associated with hyperpituitary gland including acromegaly, giantosis, and thyroid function including goiter and myxedema, bacterial infectious acute thyroiditis About hypotension Disorders, hyperparathyroidism including Conn's disease (chronic hypercalemia), pancreatic diseases such as type I and type II diabetes and complications, carcinomas and adenomas of hyperplasia and adrenal cortex, hypertension associated with alkalosis, etc. Disorders of the adrenal gland and abnormal prolactin production and infertility in women, endometriosis, menstrual cycle perturbation, polycystic ovarian disease, hyperprolactinemia, selective gonadotropin deficiency (
Isolated gonadotropin deficiency), associated with amenorrhea, lactation, semi-yin yang, hirsutism and virilization, breast cancer, postmenopausal osteoporosis, male Leydig cell hyperplasia, male menopause, germ cell aplasia, Leydig cell tumor Endocrine disorders, including sexual hyperactivity, androgen resistance associated with androgen receptor deficiency, and diseases associated with gonadal steroid hormones such as 5α-reductase syndrome and female mastosis. The polynucleotide sequence encoding HUMAP can be obtained by Southern, Northern, dot blot, or other membrane-based techniques, PCR, dipstick (dipstick).
It can be used for microarrays using body fluids or tissues collected from patients to detect the expression of mutant HUMAP, as well as stick, pin, ELISA assays. Such qualitative or quantitative methods are well known in the art.

[0179] In one embodiment, the nucleotide sequence encoding HUMAP comprises a related disease,
It will be particularly useful in assays to detect the above-mentioned diseases. The nucleotide sequence encoding HUMAP may be labeled by standard techniques and added to a sample of body fluid or tissue taken from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared to a reference value. If the amount of signal in the patient's sample varies significantly relative to the control sample, the level of mutation in the nucleotide sequence encoding HUMAP in the sample will reveal the presence of the associated disease. Such assays can be used to estimate the efficacy of a particular treatment in monitoring animal studies, clinical trials, or treatment of an individual patient.

An outline of normal or standard expression is established to provide a basis for the diagnosis of diseases associated with HUMAP expression. This can be achieved by combining a HUMAP-encoding sequence or a fragment thereof with a body fluid or cell extracted from a normal subject, either animal or human, under conditions suitable for hybridization or amplification. . Standard hybridization can be quantified by comparing values obtained from normal subjects to values from experiments in which known amounts of substantially purified polynucleotides are used. Standard values obtained from normal samples can be compared to values obtained from subjects exhibiting symptoms of the disease. The deviation between the reference value and the subject's value is used to determine whether the subject is affected.

Once the presence of the disease has been determined and the treatment protocol has begun, the hybridization assay is repeated on a regular basis to estimate if the level of expression in the subject has begun to approach the value shown in a normal patient Is possible. The results of the repeated assays can be used to see the effect of the treatment over a period of days to months.

In cancer, abnormal amounts of transcripts in living tissue from an individual are predisposed to the development of the disease, and can provide a method of detecting the disease before actual clinical symptoms occur. is there. This type of more definitive diagnosis allows medical professionals to use preventative or aggressive treatments early to prevent the development or progression of cancer.

Additional diagnostic uses for oligonucleotides designed from the sequence encoding HUMAP may include the use of PCR. Such oligomers can be produced chemically, enzymatically, or in vitro . The oligomer preferably includes a fragment of a polynucleotide encoding HUMAP, or a fragment of a polynucleotide complementary to the polynucleotide encoding HUMAP, and is used to identify a specific gene or condition under optimal conditions. You. Oligomers can also be used for the detection and / or quantification of closely related DNA or RNA sequences under slightly stringent conditions.

Methods that can be used to quantify HUMAP expression include radiolabeling or biotin labeling of nucleotides, coamplification of regulatory nucleic acids, and standard curves with results added. (For example, Melby, PC, etc. (1993) J. I.
mmunol. Methods, 159: 235-44; see Duplaa, C. et al. (1993) Anal. Biochem. 229-236). The rate of quantification of many samples was accelerated by using a high-throughput assay in which the oligomer of interest was included in various dilutions and quantification was rapid by spectrophotometry or non-color response.

In another example, oligonucleotides or longer fragments from any of the polynucleotide sequences described herein are used as targets in a microarray. Microarrays are used to simultaneously monitor the expression levels of a very large number of genes and identify gene mutations, mutations and polymorphisms. This information is useful for determining gene function, interpreting the genetic basis of disease, diagnosing disease, and monitoring and developing the activity of therapeutic agents.

Microarrays are prepared, used, and analyzed by methods well known in the art. (E.g., Brennan, TM et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Pro
c. Natl. Acad. Sci. 93: 10614-10619; Baldeschweiler et al. (1995) PCT Application No.W
O95 / 251116; Shalon, D. et al. (1995) PCT Application No.WO95 / 35505; Heller, RA et al. (1
Natl.Acad.Sci. 94: 2150-2155; and Heller, MJ et al. (1997) U.S. Pat.No. 5,605,662) Another embodiment of the invention also employs a nucleic acid sequence encoding HUMAP. Thus, it is possible to generate hybridization probes useful for mapping naturally occurring genomic sequences. This array maps to: Specific chromosomes, specific regions of chromosomes or artificially generated chromosomes, such as human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), bacterial P1 products or single chromosomal cDNA libraries. (Eg Harrington, 1.3. Other
(1997) Nat Genet. 15: 345-355; Price, CM (1993) Blood Rev. 5-87-134,
And Trask, BJ (1991) Trends Genet. 7: 149-154) In situ fluorescence hybridization (FISH) will correlate with other physical chromosome mapping techniques and genetic map data (eg, Heinz- (See Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.). Examples of genetic map data can be found in various scientific journals or on the World Wide Web site of Online Mendelian Inheritance in Man (OMIM). On the physical chromosome map
The correlation between the location of the gene encoding HUMAP and a particular disease, or a predisposition to a particular disease, can help determine the region of DNA associated with such a disease. The nucleotide sequences of the present invention may also be used to detect differences in gene sequences between normal, carrier, and infected individuals.

The genetic map can also be extended using physical mapping techniques, such as in situ hybridization of chromosomal preparations and binding analysis using defined chromosomal markers. By placing the gene on the chromosome of another mammal, such as a mouse,
Even if the number or arm of a particular human chromosome is not known, relevant markers are often revealed. New arrays are created by physical mapping
It can also be assigned to chromosome arms. This is valuable information for researchers studying genetic diseases using positional cloning or other gene discovery techniques. The location of the disease or syndrome is, for example, 11q22-
Once roughly determined by gene binding to a particular gene region, such as 23, any sequence mapping for that region represents a relevant or regulatory gene for further investigation (eg, by Gatti, RA et al. (1988) Nature 336: 577-580
See). In addition, using the nucleotide sequence of the present invention of interest, normal, holder,
That is, a difference in chromosome position due to translocation or inversion between infected persons may be detected.

In another embodiment of the present invention, HUMAP, a catalytic or immunogenic fragment thereof, or an oligopeptide thereof can be used to screen a library of compounds in any of a variety of drug screening techniques. Fragments used for such screening are free in solution, fixed to a solid support, retained on the surface of cells, or present intracellularly. Complex formation due to binding of HUMAP to the agent to be tested may be measured.

Another method used for drug screening is used to increase the throughput of screening compounds with suitable binding affinity for the protein of interest (eg, Geysen, et al. (1984) PCT Application No. See WO84 / 03564). In this method, a significant number of different small test compounds are synthesized on plastic pins or other substrates. The test compound is washed after reacting with HUMAP or a fragment thereof. Next, the bound HUMAP is detected by methods well known in the art.
Purified HUMAP can also be coated directly on plates used in the drug screening techniques described above. Alternatively, 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 which neutralizing antibodies capable of binding to HUMAP specifically compete with the test compound for binding to HUMAP. In this method, the antibody detects the presence of any peptide that shares one or more antigenic determinants with HUMAP.

In another embodiment, using the nucleotide sequence encoding HUMAP in developing molecular biology techniques, nucleotides such as, but not limited to, the currently known triplet code and specific base pairing interactions New techniques that depend on the properties of the sequence can be provided.

Those skilled in the art will be able to utilize the present invention to the fullest extent only with the foregoing description without further explanation. Accordingly, the specific preferred embodiments described below are for purposes of illustration and not limitation.

All patent applications, patents, and publications mentioned above and below are specifically incorporated herein by reference, including US Patent Application Serial Nos. 60 / 130,694 and 60 / 140,580.

Example 1 Preparation of cDNA Library RNA was purchased from Clontech or isolated from the tissues listed in Table 4. First, a part of this tissue is homogenized and dissolved in a guanidinium isothiocyanate solution, while another part of this tissue is homogenized and dissolved in phenol, or TRIZOL (Life Technologies), guanidinium isothiocyanate. Dissolved in a mixture of suitable modifiers, such as a single phase solution of thiocyanate and phenol. The lysate was centrifuged on cesium chloride or extracted with chloroform. RNA was precipitated from this lysate with either isopropanol or sodium acetate and ethanol, or otherwise.

Extraction and precipitation of RNA with phenol were repeated as necessary to increase RNA purity. In some cases, the RNA is treated with a DNase. For most libraries, oligo d (T) linked paramagnetic particles (Promega) or OLIGOTEX latex particles (QIAGEN
Valencia CA) and OLIGOTEX mRNA purification kit (QIAGEN) to isolate poly (A +) RNA. Alternatively, isolation was performed directly from tissue lysates using another RNA isolation kit, such as the POLY (A) PURE mRNA purification kit (Ambion, Austin TX).

In some cases, RNA was provided to Stratagene and Stratagene generated a corresponding cDNA library. Otherwise, UNIZAP vector system (Stratagene)
Alternatively, cDNA was synthesized using the SUPERSCRIPT plasmid system (Life Technologies) by a recommended method or a similar method well known in the art to prepare a cDNA library.
(See, for example, Ausubel, 1997, supra, units 5.1-6.6). Reverse transcription is oligo d (T)
Or started with random primers. After binding the synthetic oligonucleotide adapter to the double-stranded cDNA, the cDNA was digested with one or more suitable restriction enzymes. Most libraries use SEPHACRYL S 1000 or SEPHAROSE
CL2B, SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech
), CDNA size (300-1000 bp) was selected by agarose gel electrophoresis. PBLUESCRIPT plasmid (Stratagene) or pSPORT1 plasmid (Life Technolo
gies), pcDNA2.1 plasmid (Invitrogen Carlsbad CA), plNCY plasmid (Incyt
e Pharmaceuticals, Palo Alto CA). The cDNA was ligated to compatible restriction enzyme sites on a polylinker of a suitable plasmid such as e.g. This recombinant plasmid was transformed into XL1-Blue, XL1-BIueMRF, SOLR from Stratagene, or DH5α or DH from Life Technologies.
The cells were introduced into competent E. coli cells containing 10B and ELECTROMAX DH 10B and incorporated.

2 Isolation of cDNA clones Plasmids were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or cell lysis. Magic or WIZARD Minipreps
DNA purification system (Promega) and AGTC Miniprep purification kit (Edge Biosystems, G
aithersburg MD), QIAGEN's QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QI
Plasmids were purified using at least one of the AWELL 8 Ultra Plasmid purification system, REAL Prep 96 plasmid kit. After precipitation, they were resuspended in 0.1 ml of distilled water and stored at 4 ° C. with or without lyophilization.

[0198] Alternatively, plasmid DNA was amplified from host cell lysates by high-throughput direct binding PCR. (Rao, VB (1994) Anal. Biochem. 216: 1-14). The lysis and thermal cycling process of the host cells was performed in a single reaction mixture. Samples are processed, transferred to a 384-well plate and stored, and the concentration of the amplified plasmid DNA is quantified using a PICOGREEN dye (Molecular Probes, Eugene OR) and a Fluoroskan II fluorescence scanner (Labsystems Oy, Helsinki, Finland). Was measured.

3 Sequencing and Analysis The sequencing reaction of the cDNA was performed using standard methods or ABI CATALYST 800 (
Perkin-Elmer) thermal cycler or PTC-200 thermal cycler (MJ Research)
HYDRA micro dispenser (Robbins Scientific) or MICROLAB 2200 (Ham
ilton) High throughput equipment such as in combination with a liquid transfer system. For preparation of the cDNA sequencing reaction, a reagent contained in an ABI sequencing kit such as Amersham Pharmacia Biotech's reagent or ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer) was used. For electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides, the MEGABACE 1000 DNA Sequencing System (Molecular Dynami
cs), ABI PRISM 37 using standard ABI protocol and base pairing software
A 3 or 377 sequencing system (Perkin-Elmer), other sequence analysis systems known in the art, were used. Reading frames for cDNA sequences are read using standard methods (Ausubel, 1997).
, Supra, unit 7.7). Some of the cDNA sequences were selected and extended in the manner described in Example 6 above.

The construction and analysis of polynucleotide sequences obtained from cDNA sequencing was performed in combination with software utilizing algorithms well known to those skilled in the art.
Table 5 shows an overview of the tools, software and algorithms used, their descriptions, references and threshold parameters. Column 1 in Table 5 is the tools and programs and algorithms used, column 2 is a brief description of them, column 3 is a cited reference that is incorporated herein by reference, column 4 is a two-part sequence. Shows the score, probability value, and other parameters used for the evaluation of the degree of coincidence (the higher the probability value, the higher the homology between the sequences). For sequence analysis, use MACDNASIS PRO software (Hitachi Softwa
re Engineering, S. San Francisco CA) and LASERGENE software (DNASTAR)
Was used.

Confirmation of polynucleotide sequences can be accomplished by using BLAST and dynamic programming, programs and algorithms based on dinucleotide nearest neighbor analysis to remove vector and linker, polyA sequences and mask ambiguous base pairs. I went in. next,
Using programs based on BLAST, FASTA, and BLIMPS, the public databases of primates and rodents, mammals, vertebrates, and eukaryotes in the public database, and databases such as BLOCKS, PRINTS, DOMO, PRODOM, and PFAM These sequences were queried against sequences selected from to obtain annotations. These sequences were assembled into full-length polynucleotide sequences using programs based on Phred and Phrap, Consed and screened for open reading frames with programs based on BLAST and FASTA, BLIMPS. Translate the full-length polynucleotide sequence to derive the corresponding full-length amino acid sequence, GenBank database (above) and databases such as SwissProt, BLOCKS, PRINTS, DOMO, PRODOM and Prosite,
Alternatively, these full-length sequences were analyzed by querying a protein family database based on the Hidden Markov Model (HMM) such as PFAM. HMMs use probabilities to analyze the consensus primary structure of gene families (eg, Ed
dy, 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: 18-34. Fragments of up to about 20-4000 nucleotides are useful for hybridization and amplification and have been described in the above invention.

4 Northern Analysis Northern analysis is an experimental technique used to detect the presence of gene transcripts, in which labeled nucleotides on a membrane to which RNA from a particular cell type or tissue is bound. With sequence hybridization (see, eg, Sambrook, supra,
Chapter 7; and Ausubel. FM et al., Supra, see Chapters 4 and 16).

Using similar computer techniques used for BLAST, GenBank or LIFESEQ (Inc.
Search for identical or related molecules in nucleotide databases such as yte Pharmaceuticals). This analysis is much faster than many membrane-based hybridizations. Further change the sensitivity of computer search, any specific matches,
It can be determined whether the classification is an exact match or a homologous match. The criterion for the search is the product score defined as (% sequence identity x% maximum BLAST score) / 100. The product score takes into account both the similarity between the two sequences and the length of the sequence match. For example, for a product score of 40, the match is 1-2
It is accurate within% error, and at 70 the match will be accurate. Similar molecules are typically identified by selecting a molecule that exhibits a product score in the range of 15 to 40, although lower scores may identify related molecules.

The results of the Northern analysis are reported as the distribution percentage of the library in which transcripts encoding HUMAP have occurred. The analysis also includes the classification of cDNA libraries by organ / tissue and disease. Organ / tissue categories include cardiovascular, skin, developmental, endocrine, gastrointestinal, hematopoietic / immune, musculoskeletal, neural, reproductive, urinary. Disease categories include cancer, inflammation, trauma, cell proliferation, nerves, pooled. For each category, the number of libraries expressing the sequence of interest was counted and divided by the number of libraries in the entire range. Table 3 shows the ratio (percent) specifically expressed in each tissue and the ratio expressed in each disease.

5 Chromosomal Mapping of the Polynucleotide Encoding HUMAP The cDNA sequence used to construct SEQ ID NOs: 27-34 was compared to BLAST and Smith-Water
Using the implementation of the man algorithm, the sequences were compared to sequences from the Insight LIFESEQ database and the public domain database. Sequences from these databases that match SEQ ID NOs: 27-34 were incorporated into clusters of contiguous and overlapping sequences using construction algorithms such as Phrap (Table 5). Stanford Human
Radiation hybrids available from public sources such as the Genonse Center (SHGC), Whitehead Institute for Genome Research (WIGR) and Genethon
hybrid) and gene mapping data to determine if the clustered sequence has already been mapped. If the cluster contained a sequence that was mapped, all sequences in that cluster (including the specific SEQ ID NO) were assigned to that mapped location.

The genetic map location of SEQ ID NO: 31 is described in the section (invention) herein as a section or range of human chromosomes. The range of map locations in centiMorgans is
Measured from the end of the short arm (p) of the chromosome (Centimorgan (cM) is a unit that represents the distance based on the crossover rate between genes on the same chromosome. On average, 1 cM is 1 megabase of the human chromosome , But can vary greatly due to high and low recombination rates). The distance cM is based on Genethon-mapped gene markers that can detect the boundaries of radiation hybrid markers whose sequences are included in each cluster.

6 Extension of the Polynucleotide Encoding HUMAP The full-length nucleic acid sequence of SEQ ID NO: 18-34 is synthesized using the oligonucleotide primers designed from the appropriate fragments of the full-length molecule. The fragments were made by extension. One primer was synthesized to initiate 5 'extension of a known fragment, and the other primer was synthesized to initiate 3' extension of a known fragment. The starting primer is about 22 to about 30 nucleotides in length, has a GC content of about 50% or more, using OLIGO 4.06 software (National Biosciences) or other suitable program, and has a GC content of about 68%. It was designed to anneal to the target sequence at a temperature of ７２72 ° C. Nucleotides were extended to avoid hairpin structures and primer-primer dimers.

The sequence was extended using a selected human cDNA library. If more than one step extension is necessary or desired, additional or nested primer sets are designed.

Amplification was performed with high fidelity by a PCR method using a method known in the art. PCR is PTC-200
Performed in a 96-well block plate using a thermal cycler (MJ Research, Inc.). The reaction mixture was composed of template DNA and 200 nmol of each primer, Mg ²⁺ , (NH ₄ ) ₂ SO ₄ and β
-Buffer containing mercaptoethanol, Taq DNA polymerase (Amersham Pha
rmacia Biotech), ELONGASE enzyme (Life Technologies), Pfu DNA polymerase (St
ratagene). Amplification was performed on the primer set, PCI A and PCI B, with the following parameters. 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 Repeat Steps 2, 3, and 4 20 times Step 6 68 ° C. for 5 minutes Step 7 Stored at 4 ° C. Alternatively, amplification was performed on the primer set, T7 and SK +, with the following 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 Repeat Steps 2, 3, and 4 20 times Step 6 68 ° C. for 5 minutes Step 7 Store at 4 ° C.

The DNA concentration in each well was dissolved in 1 × TE and 0.5 μl of undiluted PCR product
100 μl PICOGREEN quantitative reagent (0.25% (v / v) PICOGREEN; Molecular Probes, Eugen
eOR) is distributed to each well of an opaque fluorometer plate (Coming Costar, Acton MA) to measure the DNA so that it can bind to the reagent. Fluoroskan this plate
Scan with II (Labsystems Oy, Helsinki, Finland) and measure the fluorescence of the sample to quantify the concentration of DNA. A 5-10 μl aliquot of the reaction mixture is analyzed by electrophoresis on a 1% agarose minigel to determine which reaction has successfully extended the sequence.

The extended nucleotides were desalted and concentrated before being transferred to a 384-well plate.
JI cholera virus endonuclease (Molecular Biology Research, Madison W
I) digested and sonicated or sheared before religating to pUC18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on a low concentration (0.6-0.8%) agarose gel to cut the fragments, and the agar was digested with Agar ACE (Promega). T4 ligase (New England B
clones expanded using iolabs, Beverly MA) with a pUC 18 vector (Amersham Pha
rmacia Biotech) and treated with Pfu DNA polymerase (Stratagene) for extension of the restriction site to transfect competent E. coli cells. The transfected cells were selected and transferred to a medium containing an antibiotic, and each colony was cut out and cultured in a 384/2 plate of LB / 2X carbenicillin culture solution at 37 ° C. overnight.

The cells were lysed and Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu
DNA was amplified by PCR using a DNA polymerase (Stratagene) according to the following procedure. 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 Repeat steps 2, 3 and 4 29 times Step 6 72 ° C for 5 minutes Step 7 Store at 4 ° C. DNA was quantified with PICOGREEN reagent (Molecular Probes) as described above. Samples with poor DNA recovery were amplified again under the conditions described above. The sample was diluted with 20% dimethylsulphoxide (1: 2, v / v) and DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or ABI PRISM BIGDYE Terminator cycle se
Sequencing was performed using a quencing ready reaction kit (Perkin-Elmer).

Similarly, the procedure described above utilizes the nucleotide sequence of SEQ ID NO: 18-34, and uses the oligonucleotide designed for this extension and a suitable gene library to provide 5 ′
The regulatory sequence was obtained.

7 Labeling and Use of Individual Hybridization Probes cDNA, mRNA, or genomic DNA is screened using hybridization probes derived from SEQ ID NOs: 18-34. Particular mention is made of the labeling of oligonucleotides of about 20 base pairs, but essentially the same procedure is used for larger cDNA fragments. Oligonucleotides were designed using state-of-the-art software such as OLIGO 4.06 software (National Bioscience), and 50 pmol of each oligomer and 250 μCi of [γ- ³² P] adenosine triphosphate (A
mersham, Chicago, IL) and T4 polynucleotide kinase (DuPont NEN, Bost)
on MA). The labeled oligonucleotide is applied to a SEPHADEX G-25 ultra-fine exclusion dextran bead column (Amersham Phar
(Macia Biotech). An aliquot containing per minute 10 ⁷ counts of labeled probe, following endonuclease, Ase I, Bgl II, Eco R
Human genome cut with one of I, Pst I, Xba1 or Pvu II (DuPont NEN)
Used in typical membrane-based hybridization analysis of DNA.

DNA from each cut is fractionated on a 0.7% agarose gel and transferred to a nylon membrane (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is performed at 40 ° C. for 16 hours. To remove non-specific signals, the blots are washed sequentially at room temperature, for example, under conditions of up to 0.1 × saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visualized and compared by autoradiography or another imaging means.

8 It is possible to synthesize array elements on the surface of a substrate using microarray chemical bonding methods and inkjet devices (see, eg, Baldeschweiler, supra). Using an array similar to dot or slot blots, the elements are exposed to heat, UV,
It is arranged and bonded to the surface of the substrate using a mechanical or chemical bonding method. Typical arrays can be made manually or using available methods and machines, and can include any suitable number of elements. After hybridization, unhybridized probe is removed and the level and pattern of fluorescence is determined using a scanner. The scanned images can be analyzed to determine the degree of complementarity and the relative amount / expression level of each probe that hybridizes to the element on the microarray.

[0218] Full-length cDNAs, expressed gene sequence fragments (ESTs), or fragments thereof, can be elements of a microarray. Fragments suitable for hybridization can be obtained from LASERGEN
The selection can be made using software well known in the art such as E software (DNASTAR). A full-length cDNA, EST, or a fragment thereof corresponding to one of the nucleic acid sequences of the present invention, or a cDNA arbitrarily selected from a cDNA library related to the present invention is aligned on a suitable substrate such as a glass slide. . The cDNA is fixed to the slide using, for example, UV cross-linking, subjected to heat treatment and chemical treatment, and finally dried (for example, Schena, M. et al. (1995) Science 27).
0: 467-470; and Shalon, D. et al. (1996) Genome Res. 6: 639-645). A fluorescent probe is provided and used to hybridize to elements on the substrate. The substrate is analyzed in the manner described above.

9 Complementary Polynucleotides The sequence encoding HUMAP, or a sequence complementary to any portion thereof, is used to reduce or inhibit the expression of naturally occurring HUMAP. About 15 ~
Although the use of oligonucleotides containing about 30 base pairs is described, essentially the same method can be used for smaller or larger sequence fragments. The appropriate oligonucleotides are designed using Oligo 4.06 software (National Biosciences) and the coding sequence of HUMAP. 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 to prevent ribosomes from binding to transcripts encoding HUMAP.

10 HUMAP Expression HUMAP expression and purification are performed using bacterial or viral expression systems. To express HUAMP in bacteria, the cDNA is cloned into a suitable vector containing an inducible promoter that expresses the antibiotic resistance gene and cDNA transcript at high levels. Such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter associated with the lac operator regulatory element and the T5 or T7 bacteriophage promoter. The transgenic vector is introduced into a suitable bacterial host, for example, BL21 (DE3). Antibiotic-resistant bacteria express HUMAP when induced with isopropyl β-D thiogalactopyranoside (IPTG).
Expression of HUMAP in eukaryotic cells, usually carried out by infecting an insect cell systems or mammalian cell lines recombinant Autog raphica californica nuclear polyhedrosis virus called a baculovirus. The non-essential polyhedrin gene of baculovirus
The cDNA encoding HUMAP is replaced by either bacterial-mediated gene transfer using a transfer plasmid intermediate or by homologous recombination. The virus remains infectious and the strong polyhedrin promoter causes high levels of cDNA transcript expression. Recombinant baculovirus is most often Spodoptera frugiperda (S
f9) Used to infect insect cells, but sometimes to infect human hepatocytes (Engelhard, EK et al. (1994) Proc. Natl. Acad. Sci. USA
91: 3224-3227: Sandig, V. et al. (1996) Hum. Gene Ther. 7: 1937-1945)
.

[0221] In most expression systems, HUMAP is, for example, glutathione S-transferase (GST
) Or as a fusion protein with a peptide epitope tag such as FLAG or 6-His, allowing rapid one-step affinity purification of the recombinant fusion protein from unpurified cell lysates. The 26 kilodalton enzyme GST from Schistosoma japonicum allows purification of the fusion protein on immobilized glutathione while maintaining protein activity and antigenicity (Amersham Pharmac
ia Biotech). After this purification, the GST moiety is proteolytically cleaved from HUMAP at the specifically engineered site. FLAG, a peptide consisting of 8 amino acids, commercially available monoclonal or polyclonal anti-FLAG antibody (Eastman Kodak)
Enables immunoaffinity purification. 6-His, a stretch of six consecutive histidine residues, allows for purification on metal chelating resins (QIAGEN). Methods for protein expression and purification are described in Ausbel (1995, supra, ch. 10
And 16). HUMAPs purified by this method can be used directly in the following assays.

11 Demonstration of HUMAP activity HUMAP activity is demonstrated by general immunoblotting techniques or HUMAP-specific activity assays described below. In a general method, cell lines or tissues transformed with a vector containing the HUMAP coding sequence are assayed by immunoblotting to determine HUMAP activity. Transformation 12 Functional Assays The function of HUMAP is assessed by expression of sequences encoding HUMAP at physiologically elevated levels in mammalian cell culture systems. The cDNA is subcloned into a mammalian expression vector containing a strong promoter that expresses the cDNA at high levels. Such vectors include pCMV SPORT ^™ (Life Technologies.) And pCR 3.1
(Invitrogen, Carlsbad, CA), both of which contain the cytomegalovirus promoter. 5-10 μg of the recombinant vector is transiently transfected into human cell lines, for example endothelial or hematopoietic, by liposome preparation or electroporation. In addition, 1-2 μg of plasmid containing the sequence encoding the labeled protein is co-transfected. Expression of the labeled protein allows one to distinguish between transfected and non-transfected cells. Also, depending on the expression of the labeled protein,
The expression of the cDNA from the recombinant vector can be accurately predicted. Such labeled proteins include green fluorescent protein (GFP; Clontech), and CD64 or CD64-GFP fusion proteins. Identify transfected cells expressing GFP or CD64-GFP using automated flow cytometry (FCM) using laser optics based technology,
Evaluate the apoptotic state and other cellular properties of the cell. In addition, the FCM detects and measures the incorporation of a fluorescent molecule that diagnoses the phenomenon of preceding or simultaneous cell death. These phenomena include changes in nuclear DNA content as measured by staining DNA with propidium iodide, down-regulation of DNA synthesis as measured by reduced uptake of bromodeoxyuridine, and specific antibodies. And changes in plasma membrane composition as measured by the binding of the fluorescent complex annexin V protein to the cell surface, as measured by the reactivity of E. coli.
Flow cytometry was performed by Ormerod, MG (1994) Flow Cytometry Oxford, New
York, NY.

The effect of HUMAP on gene expression was determined by the sequence encoding HUMAP and CD64 or CD64.
Can be assessed using highly purified cell populations transfected with either -GFP. CD64 or CD64-GFP is expressed on the transformed cell surface and binds to a conserved region of human immunoglobulin G (IgG). Transformed and non-transformed cells can be separated using magnetic beads coated with either human IgG or antibodies to CD64 (DYNAL. Lake Success. NY). mRNA can be purified from cells by methods well known in the art. Expression of mRNA encoding HUMAP and other genes of interest can be analyzed by Northern analysis or microarray technology.

13 Preparation of Antibodies Specific for HUMAP Polyacrylamide Gel Electrophoresis (PAGE; eg, Harrington, MG (1990)
Rabbits are immunized using standard protocols with HUMAP, substantially purified by Methods Enzymol. 1816-3088-495) or other purification techniques, to produce antibodies.

Alternatively, the HUMAP amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine the region of high immunogenicity, and the corresponding oligopeptide is synthesized and used in a manner well known to those skilled in the art. Produces antibodies. The selection of suitable epitopes, such as those near the C-terminus or in adjacent hydrophilic regions, is well known in the art (see, eg, Ausubel, 1995, supra, Chapter 11).

Typically, oligopeptides about 15 residues in length are prepared using the ABI 431A from Applied Biosystems.
It was synthesized by fmoc method chemistry using a peptide synthesizer (Perkin-Elmer), and N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS
) To enhance immunogenicity by binding to KLH (Sigma-Aldrich, St. Louis MO) (see, eg, Ausubel, 1995, supra). Rabbits are immunized with the oligopeptide-KLH conjugate in Freund's complete adjuvant. To test the anti-peptide activity and anti-HUMAP activity of the obtained antiserum, peptide or H
UMAP is bound to the substrate, blocked with 1% BSA, reacted with rabbit antiserum, washed, and further reacted with radioactive iodine-labeled goat anti-rabbit IgG.

14 Purification of Spontaneous HUMAP Using Specific Antibodies Spontaneous HUMAP or recombinant HUMAP is substantially purified by immunoaffinity chromatography using an antibody specific for HUMAP. An immunoaffinity column is formed by covalently binding an anti-HUMAP antibody to an activated chromatography resin such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After binding, the resin is blocked and washed according to the manufacturer's instructions.

The culture medium containing HUMAP is passed through an immunoaffinity column, and the column is washed under conditions that allow preferential adsorption of HUMAP (for example, with a buffer having a high ionic strength in the presence of a surfactant). The column is eluted under conditions that disrupt the binding of the antibody to HUMAP (eg, with a buffer of pH 2-3 or a high concentration of chaotropic ions such as urea or thiocyanate ions) to recover HUMAP.

15 Identification of molecules that interact with HUMAP HUMAP, or a biologically active fragment thereof, was prepared using the ¹²⁵ I Bolton Hunter reagent (see, eg, Bolton AE and WM Hunter (1973) Biochem. J. 133: 529). Label with. Candidate molecules pre-arranged in a multiwell plate are labeled with labeled HU
Incubate with MAP, wash and assay all wells with labeled HUMAP complex. Using the data obtained at different HUMAP concentrations, values for the quantity and affinity of HUMAP bound to the candidate molecule and for association are calculated.

[0230] Alternatively, molecules that interact with HUMAP are identified in Fields, S. and O. Song (1989, Natur
e 340: 245-246) and the MATCHMAKER system (Clo
Analyze using a commercially available kit based on a two-hybrid system (e.g., ntech).

[0231] Those skilled in the art will be able to make various modifications of the described methods and systems of the invention without departing from the scope and spirit of the invention. Although the invention has been described with reference to certain preferred embodiments, it is to be understood that the scope of the invention should not be unduly limited to such specific 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 within the scope of the following claims.

BRIEF DESCRIPTION OF THE TABLES Table 1 shows the polypeptide and nucleotide sequence SEQ ID NOs, clone identification numbers, cDNs used to generate the full length sequence encoding HUMAP.
1 shows an A library and a cDNA fragment.

Table 2 summarizes the characteristics of each polypeptide sequence, including latent motifs and homologous sequences, and
The method, algorithm, and searchable database used for HUMAP analysis are shown.

Table 3 shows selected fragments of each nucleic acid sequence, the tissue-specific expression patterns of each nucleic acid sequence determined by Northern analysis, the diseases, disorders and conditions associated with these tissues, and the DNA Indicates the cloned vector.

Table 4 shows the tissues used to construct a cDNA library from which cDNA clones encoding HUMAP were isolated.

Table 5 shows the tools, programs, and algorithms used in the analysis of HUMAP, along with 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]

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 1/16 A61P 3/10 4C086 1/18 5/02 4H045 3/10 5/06 5/02 5 / 14 5/06 5/26 5/14 5/30 5/26 7/00 5/30 7/02 7/00 7/06 7/02 9/00 7/06 9/10 101 9/00 9/12 9/10 101 13/00 9/12 13/02 13/00 13/08 13/02 13/12 13/08 15/00 13/12 15/08 15/00 15/12 15/08 15/14 15 / 12 17/00 15/14 17/06 17/00 19/00 17/06 19/08 19/00 19/10 19/08 21/04 19/10 25/08 21/04 25/18 25/08 27/00 25/18 27/06 27/00 27/12 27/06 31/00 27/12 35/00 31/00 35/02 35/00 37/02 35/02 43/00 105 37/02 111 43/00 105 C07K 14/47 111 16/18 C07K 14/47 C12N 1/15 16/18 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 C12Q 1/68 A5 / 10 C12N 15/00 ZNAA C12P 21/02 5 / 00 A C12Q 1/68 A61K 37/02 (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, 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, CU, CZ, DE, DK, 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, MD, MG , MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Tang, Wy Tom 95118, California, United States of America San Jose Runwick Court 4230 (72) Inventor Lal, Pleity 95054, California, United States of America Santa Clara Las Drive 2382 (72) Inventor Yue, Henry 94087 Sunnyvale Louis Avenue, California, United States 826 (72) Inventor Lady, Loopa 94086, Sunnyvale West, McKinley Drive, California 1233 (72) Inventor Azimsay, Yalda 94545, Hayward Hayward, California, United States of America Rock Springs Drive 2045 (7 2) Inventor Bogun, Mariah Earl San Diego, CA 94577 San Leandro Santiago Road 14244 F Term (Reference) 4B024 AA01 AA11 CA04 CA09 FA02 GA11 HA03 HA12 HA17 4B063 QA01 QA18 QQ43 QR56 QS34 QX07 4B064 AG01 CA19 CC24 DA01 DA13 4 BA02 CA24 CA44 CA46 4C084 AA02 AA06 AA13 AA17 BA02 BA44 CA18 CA53 DC50 NA14 ZA062 ZA182 ZA322 ZA332 ZA362 ZA422 ZA452 ZA512 ZA552 ZA662 ZA752 ZA812 ZA892 ZA942 ZA962 ZB072 ZB12 ZC12 ZA06 ZB02 ZC026 ZA36 ZA42 ZA45 ZA51 ZA55 ZA66 ZA75 ZA81 ZA89 ZA94 ZA96 ZB07 ZB26 ZB27 ZB35 ZC04 ZC06 ZC10 ZC11 ZC35 4H045 AA10 AA20 AA30 CA40 DA50 DA75 EA28 FA74

Claims (23)

[Claims] 1. An isolated polypeptide, comprising: a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 17 (SEQ ID NO: 1-17); b) A naturally occurring amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17; c) an amino acid selected from the group consisting of SEQ ID NOs: 1-17 Comprising a biologically active fragment of the sequence and d) an amino acid sequence selected from the group consisting of an immunogenic fragment of the amino acid sequence selected from the group consisting of SEQ ID NOs: 1-17. An isolated polypeptide which is characterized. 2. The isolated polypeptide of claim 1 selected from the group consisting of SEQ ID NOs: 1-17. 3. An isolated polynucleotide encoding the polypeptide of claim 1. 4. The isolated polynucleotide of claim 3, selected from the group consisting of SEQ ID NOs: 18-34. 5. A recombinant polynucleotide comprising a promoter sequence operably linked to the polynucleotide of claim 3. 6. A cell transformed with the recombinant polynucleotide of claim 5. 7. A transgenic organism comprising the recombinant polynucleotide of claim 5. 8. A method for producing the polypeptide of claim 1, comprising: a) operably binding to a polynucleotide encoding the polypeptide of claim 1 under conditions suitable for expression of said polypeptide. Culturing cells transformed with the recombinant polynucleotide comprising the promoter sequence thus obtained, and b) recovering the polypeptide so expressed. Production method. 9. An isolated antibody that specifically binds to the polypeptide of claim 1. 10. An isolated polynucleotide comprising: a) a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 18-34; and b) a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 18-34. A) a naturally occurring polynucleotide sequence having at least 90% sequence identity with the polynucleotide sequence obtained, c) a polynucleotide sequence complementary to a), and d) a polynucleotide sequence complementary to b). E) an isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of: RNA equivalents of a) -d) above. 11. An isolated polynucleotide comprising at least 60 contiguous nucleotides of the polynucleotide of claim 10. 12. A method for detecting a target polynucleotide in a sample having the polynucleotide sequence of claim 10, comprising: a) at least 16 contiguous sequences comprising a sequence complementary to said target polynucleotide in said sample. Hybridizing a probe containing nucleotides to be sampled with the sample, wherein the probe specifically binds to the target polynucleotide under conditions where a hybridization complex is formed by the probe and the target polynucleotide. And b) detecting the presence or absence of the hybridization complex, and optionally measuring the yield, if present, of the target polynucleotide. Detection method. 13. The method of claim 12, wherein said probe comprises at least 30 contiguous nucleotides. 14. The method of claim 12, wherein said probe comprises at least 60 contiguous nucleotides. 15. A pharmaceutical composition comprising an effective amount of the polypeptide of claim 1 and a pharmaceutically acceptable excipient. 16. A method for treating a disease or a symptom thereof associated with reduced expression of a functional HUMAP (purified polypeptide which is a human membrane-associated protein), wherein the pharmaceutical composition according to claim 15 is used for such a method. A method of treatment, comprising administering to a patient in need of treatment. 17. A method of screening for a compound that is effective as an agonist of the polypeptide of claim 1, comprising: a) exposing a sample containing the polypeptide of claim 1 to the compound; Detecting the agonist activity. 18. A pharmaceutical composition comprising an agonist compound identified by the screening method of claim 17, and a pharmaceutically acceptable excipient. 19. A method for treating a disease or a symptom thereof associated with decreased expression of functional HUMAP, comprising administering the pharmaceutical composition of claim 18 to a patient in need of such treatment. A characteristic treatment method. 20. A method of screening a compound that is effective as an antagonist of the polypeptide of claim 1, comprising: a) exposing a sample containing the polypeptide of claim 1 to the compound; Detecting the antagonist activity. 21. A pharmaceutical composition comprising an antagonist compound identified by the screening method of claim 20, and a pharmaceutically acceptable excipient. 22. A method for treating a disease or a symptom thereof associated with overexpression of functional HUMAP, comprising administering the pharmaceutical composition of claim 21 to a patient in need of such treatment. A characteristic treatment method. 23. A method for screening a compound that effectively alters the expression level of a target polynucleotide comprising the sequence of claim 4, comprising: a) exposing a sample containing the target polynucleotide to the compound; b) detecting a change in the expression of the target polynucleotide.