JP2002525032A - DNA replication and repair related proteins - Google Patents

Abstract

  (57) [Summary] The present invention provides human DNA replication and repair associated proteins (DRASP) and polynucleotides that identify and encode DRASP. The present invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. Furthermore, the present invention provides a method for diagnosing, treating, or preventing a disease associated with the expression of DRASP.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Technical Field The present invention, DNA replication and the nucleic acid sequence and amino acid sequence of the repair-related proteins,
The invention also relates to the use of these sequences in the diagnosis, prevention and treatment of developmental disorders, proliferative disorders including cancer, and autoimmune disorders / inflammatory diseases.

[0002] The copy of the background of the individual cells of the invention are those essential to the survival of multicellular organisms. Cells are replicated as the organism grows and are replaced in response to normal aging as well as destruction by trauma and infection. Cells preserve genetic material by replicating DNA. Somatic cells are replicated by mitosis. In this process,
The DNA is unwound and the two strands of each double-stranded molecule are replicated. The same pair of DNA strands migrate to different sites and integrate into the two nuclei of the two daughter cells. Meiosis produces eggs and sperm cells, which divide and migrate to give daughter cells with half the chromosome number of the parent cell. Synthesis, migration, division, replication, unwinding (u
Despite the physical and chemical complexity of ncoiling, various cellular structures, regulatory mechanisms, and reaction pathways allow rapid and accurate cell replication. In addition, other mechanisms have been developed to correct the damage and errors incurred during replication and division. Cells can also undergo metastasis (cancerous growth) or apoptosis (regulatory destruction).

[0003] Annexin I (also called lipocortin I) is a calcium / phospholipid binding protein that promotes membrane fusion and is involved in exocytosis. Annexin I has been shown to play a role in cell proliferation, differentiation, and apoptosis (Kim et al. (1997) Biochem Mol. Biol. Int. 43: 521-528: Sol).
ito et al. (1998) Cell Growth Differ. 9: 327-336). This protein binds 2-4 calcium ions with high affinity and is common to all annexin proteins.
It is thought to contain four homologous repeated amino acid sequences. These paired repeats have been suggested to form one binding site for calcium and phospholipids (SWISS-PROT: P04083). Annexin I regulates the activity of the enzyme phospholipase A2, which plays a key role in inducing mitosis (Kim et al. (1998) Mol. Cells 8:90
-95). Annexin I levels increase during recovery from renal ischemia, a process involved in hyperplasia and hypertrophy (MeKanna et al. (1992) J. Cell. Physiol. 153: 467-
476). Annexin I levels are increased in proliferating hepatocytes, and it has been proposed that annexin is a useful marker for identifying hepatocytes with active, pre- and post-proliferation (Masaki et al.). (1994) Hepatology 20: 425-435). Furthermore, annexin I levels may be correlated with the development of malignant breast cancer (A
hn et al. (1997) Clin. Exp. Metastasis 15: 151-156). Treatment with okadaic acid, which increases levels of annexin I in promyelocytic leukocyte cell lines, differentiates leukemic cells into macrophage-like cells rather than undergoing mitosis (Sato et al. (
1995) Biochim. Biophys. Acta 1266: 23-30).

[0004] DNA can be damaged by growth. Double-strand breaks and single-stranded gaps in damaged DNA are repaired by mechanisms involved in a complex series of biochemical reactions known as recombination. Various genes involved in recombination have been identified in prokaryotes, and these genes are highly conserved. Genes homologous to recombinant genes in bacteria and yeast have been identified in higher eukaryotes (Sh
inohara et al. (1995) Trends Biochem. Sd. 20: 387-391).

[0005] The prokaryotic protein RecF is a single-stranded DNA binding protein that is thought to bind to ATP. RecF is required for recombinant DNA repair (Howard-Flanders,
F. (1975) Basic Life Sc 5A: 265-274). For example, E. coli performs homologous genetic recombination in multiple pathways, and RecF can function as a switch between replication and recombination by facilitating the conversion of replication to an intermediate for recombination ( Clark et al. (1994) Crit.
Rev. Microbiol. 20: 125-142). The gene shown to encode the RecF protein shows relatively low sequence homology across species (Roberts et al. (1997) J
Bacteriol. 179: 2319-2330; Gorbalenys et al. (1990) J. Mol. Biol. 213: 583-59.
1). Therefore, even some homology to a known RecF is considered important.

[0006] Cells are constantly facing replication errors and environmental attacks (eg, ultraviolet radiation) that can damage DNA. DNA damage consists of any changes that alter the structure of the molecule. DNA changes can generally be classified into two types: single base changes and structural distortions. Single base changes affect the sequence but not the overall structure of the DNA. Single base changes do not affect transcription or repair, and thus affect later generations. Structural distortion affects the structure of DNA. Removal of a single-stranded nick or base prevents the strand from functioning as a viable template for DNA or RNA synthesis. Coupling between bases within or within the strand, or the addition of bulky adducts to the bases, can distort the double helix structure and inhibit transcription and replication. Any DNA damage can cause mutations and cause diseases such as cancer.

[0007] Changes in DNA are recognized by repair systems within the cell. These repair systems function to correct the damage and counteract any deleterious effects of mutational phenomena. These repair systems are generally classified into three types: direct repair, excisional repair, and recovery. If the repair system is eliminated, the cells become very sensitive to environmental mutagens such as ultraviolet light. Diseases associated with loss of the DNA repair system often show signs of being sensitive to environmental mutagens. Examples of such diseases include xeroderma pigmentosum (XP), Bloom syndrome (BS), and Werner syndrome (WS) (Yamagata, K. et al. (1998) Proc. Natl. Acad. Sci. USA 95: 8733-873
8) Direct repair involves inversion or simple removal of damaged areas of DNA.
Mismatches for normal bases are repaired in the repair system based on certain trends. For example, inappropriate combinations of GT base pairs are frequently caused by deamination of 5-methylcytosine to form thymine. Thus, the repair system converts improperly combined GT base pairs to GC instead of AT. Repair favors unmethylated strands in semi-methylated DNA, as the strands correspond to newly synthesized daughter strands. Recognition of hemi-methylated DNA and repair of mismatches in the unmethylated strand are encoded by the products of the genes mutH, mutL, mutS, which specifically recognize mismatched base pairs, the uvrD gene Helicase and dam
Related to the methylase encoded by the gene.

[0008] Excision repair is a system in which mismatched or damaged bases are removed from DNA, and a new stretch of DNA is synthesized instead. During the dissection process, the damaged structures are recognized by endonucleases that cut the DNA strands on both sides of the lesion. 5'-3 'endonuclease damaged during resection
Remove DNA strand stretch. During the synthesis process, the resulting single-stranded region serves as a template for DNA polymerase to synthesize a replacement for the excised sequence. Finally, DNA ligase covalently links the old material with the 3 'end of the new material.

[0009] Although eukaryotic DNA repair systems have been described, they are based primarily on homology to the elaborated prokaryotic DNA repair systems. In prokaryotes, there are two types of excision repair. Short-patch repair
repair: SPR) removes an average of 20 nucleotides. SPR for resection repair
It accounts for 99% and performs the constitutive function of the cell. Long-patch repai
r: LPR) removes an average of 1,500 nucleotides, but can remove more than 9,000 bases. LPR accounts for the remaining 1% of excision repairs and is triggered by DNA damage. Short patch repair and long patch repair involve the uvr genes uvrA, uvrB, uvrC, which encode components of the repair endonuclease. Endonucleases recognize bulky lesions such as pyridine dimers and cut 7 nucleotides from the 5 'and 3 nucleotides from the 3' of the site of injury. Some E. coli enzymes can excise bulk lesions from DNA, where single-strand-specific endonucleases, and those containing the 5'-3 'endonuclease activity of VII DNA PolI and PolII. Resection is performed. DNA PolI is thought to perform most excision repairs in vivo. Also, the helicase activity of uvrD is required.

If replication occurs before repair of the bulky lesion in the DNA template, the DNA polymerase terminates synthesis of the daughter strand near the lesion and resumes replication at some distance along the template. This process leaves substantial gaps in the newly synthesized strand. Other parental strands (complementary strands) that do not contain damage give rise to normal daughter strands. Gap repair involves the "stealing" of homologous single-stranded DNA from a normal double-stranded strand. After this single-strand exchange, the recipient's duplex has a (damaged) parent strand facing the wild-type strand. The donor duplex has a normal parent strand facing the gap. This gap is filled by the normal replication machinery, resulting in a normal duplex. This system does not repair the damage, but limits the damage to the original strain. If the lesion is not actually removed by the excision repair system, a similar recombination repair must be repeated after every replication cycle. Genes involved in retrieval repair include recB, recC, recF, recQ, and recA. These genes are 2
Divided into two recombination repair pathways, recA is a component of both pathways, recBC is a component of one pathway, and recFQ is another. The recBC gene encodes two subunits of exonuclease V. In the recA mutant, exonuclease V is uncontrolled and causes extensive DNA digestion. The recQ gene encodes a helicase capable of unwinding a wide variety of DNA substrates and includes a conjugation molecule formed by the RecA protein (Harmon. FG and Kowalczydowski, SC (19).
98) Gene Dev. 15 12: 1134-1144). The function of the recF gene product is unknown.

[0011] The RecA protein has two different functions. The first function is the double-stranded D of single-stranded DNA.
Involved in invading NA. This function requires that RecA have a site responsible for DNA binding, and this activity is directly involved in the normal recombination as well as the recovery system. The second function involves activation of gene expression. RecA is a treatment that prevents DNA repair (
Activated by ultraviolet irradiation or the like). Once activated, RecA triggers the potential proteolytic activity of the target protein, and the resulting proteolysis targets regulatory proteins of genes that repress transcription in specific operons. Activation of RecA is responsible for inducing the expression of many genes, the products of which have repair functions including, for example, the long patch repair mode.

[0012] Eukaryotic homologs to prokaryotic DNA repair enzymes have been described. The Rad51 family of proteins is a homologue of RecA (Baumann, P. and We
st, SC (1998) Trends Biochem. Sci. 23: 247-251). Yeast Sgs1 and human B
LN and WRN are homologues of RecQ helicase (Yamagata, supra ). Signs of the importance and presence of the mammalian repair system are recognized by certain human genetic disorders, including the recessive disorder xeroderma pigmentosum (XP). The disease results in hypersensitivity to sunlight (especially ultraviolet light), resulting in skin defects. Bloom's syndrome, a human recessive disease, results in increased frequency of chromosomal abnormalities, including sister chromosome exchanges. Because the BLN gene corresponds to the RecQ helicase homolog, Bloom syndrome suggests that mammalian cells may have a recombinant repair system (Yamagata, supra ).

[0013] The disclosure of novel DNA replication and repair-related proteins and polynucleotides encoding them will provide new diagnostic, therapeutic, and prophylactic approaches for developmental disorders, proliferative disorders, including cancer, and autoimmune disorders / inflammatory diseases. To meet the needs of those skilled in the art.

SUMMARY OF THE INVENTION The present invention features a DNA replication and repair related protein that is a substantially purified polypeptide, collectively referred to as "DRASP." In one embodiment, the present invention relates to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 (S
EQ ID NO: 1-5) as well as a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of fragments thereof.

The present invention also provides a substantially purified amino acid sequence having at least 90% or more amino acid identity to at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5 and fragments thereof. Provide mutants. The present invention further provides an isolated and purified polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-5 and fragments thereof. In addition, the present invention
QID NO: 1-5 and isolated and purified having at least 90% or greater polynucleotide sequence identity to a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: Mutated sequence of the polynucleotide.

Further, the present invention provides an isolated, hybridizable under stringent conditions to a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5 and fragments thereof. Provide a purified polynucleotide. The present invention also provides an isolated and purified polynucleotide having a sequence complementary to a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5 and fragments thereof. I will provide a.

Further, the present invention relates to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and
Provided is an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 10 (SEQ ID NO: 6-10) and fragments thereof. The present invention also relates to an isolated and purified polynucleotide having at least 90% or more polynucleotide sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 6-10 and fragments thereof. Mutant sequences are provided.
The present invention further provides an isolated and purified polynucleotide having a sequence complementary to a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 6-10 and fragments thereof.

The present invention also provides a method for detecting a polynucleotide in a sample containing a nucleic acid, the method comprising the steps of: (a) hybridizing at least one polynucleotide of a sample with a complementary sequence of the polynucleotide; Forming a hybridization complex, and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding a polypeptide in the biological sample. And a detection process having a characteristic. In one embodiment of the invention, the method further comprises amplifying the polynucleotide prior to hybridization.

Further, the present invention provides an expression vector comprising at least a fragment of a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5 and fragments thereof. In another embodiment, the expression vector is contained in a host cell.

The present invention also provides a method for producing a polypeptide, the method comprising: (a) a host cell comprising an expression vector containing at least a fragment of a polynucleotide under conditions suitable for expression of the polypeptide. And (b) recovering the polypeptide from the medium of the host cell.

Further, the present invention provides a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-5 and fragments thereof together with a suitable pharmaceutical carrier. provide.

The invention further includes a purified antibody that binds to a polypeptide selected from the group consisting of SEQ ID NOs: 1-5 and fragments thereof. The present invention also provides a purified agonist and a purified antagonist of the polypeptide.

Further, the present invention provides a method of treating or preventing a disease associated with reduced activity or expression of DRASP, the method comprising:
Administering an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of NO: 1-5 and fragments thereof together with a suitable pharmaceutical carrier.

Further, the present invention provides a method of treating or preventing a disease associated with increased activity or expression of DRASP, the method comprising:
NO: 1-5 and a process of administering an effective amount of an antagonist of a polypeptide having an amino acid sequence selected from the group consisting of fragments thereof.

[0025] Proteins form the present invention for carrying out the invention, nucleic acid sequences, and before describing how the present invention,
It should be understood that the invention is not limited to the particular devices, materials, and methods disclosed herein, but that embodiments thereof can be varied. The terminology used herein is used for the purpose of describing specific embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Please also understand.

As used herein, including the claims, the singular forms “a” and “the”
Note that the notation also has multiple meanings unless the context clearly indicates otherwise. Thus, for example, reference to "a host cell" includes a plurality of such host cells, and reference to "antibody" includes reference to one or more antibodies and their equivalents well known to those of skill in the art. Represents.

Unless defined otherwise, all technical and specific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are described herein. All references mentioned in the present invention are cited to describe and disclose cell lines, protocols, reagents, and vectors that can be used in connection with the present invention as reported in the literature. Nothing in this disclosure should be construed as an admission that the present invention cannot precede such disclosure by the prior invention.

The definition term "DRASP" is derived from any species, particularly bovine, ovine, porcine, murine, equine, and preferably specific mammalian species, including humans, Substantially purified DRASP, obtained from semi-synthetic or recombinant sources
Refers to the amino acid sequence of

The term “agonist” refers to a molecule that, when bound to DRASP, enhances the effect of DRASP or extends the duration of the effect. Agonists can include proteins, nucleic acids, carbohydrates, or any other molecules that bind to and modulate the action of DRASP.

The term “allelic variant” is an allelic variant of the gene encoding DRASP. The allelic variant has been mutated due to at least one mutation in the nucleic acid sequence.
mRNA or polypeptide is produced, but their structure or function may or may not change. Any given natural or recombinant gene may be free of allelic forms, one present, or multiple.
Common mutations that generally result in an allelic variant are due to natural deletions, additions or substitutions of nucleotides. Each of these types of changes, alone or in combination with other changes, can occur one or more times in a given sequence.

As used herein, an “altered” nucleic acid sequence encoding DRASP includes various nucleotides that result in a polynucleotide encoding a polypeptide having the same DRASP or at least one of the functional features of DRASP. And those sequences with deletions, insertions, or substitutions of This definition includes polymorphisms that are easily detectable or difficult to detect using specific oligonucleotide probes of DRASP,
Also included are improper or unexpected hybridizations to allelic variant sequences having a predetermined position other than the normal chromosomal location of the polynucleotide sequence encoding DRASP. The encoded protein can also be "mutated", including substitutions, deletions, or insertions of amino acid residues that produce a silent change, resulting in a functionally equivalent DRASP. It is. Intentional amino acid substitutions may involve similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues, as long as the biological or immunological activity of DRASP is retained. It can be done based on gender. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids having an uncharged polar head group with similar hydrophilicity values include: Includes leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

As used herein, “amino acid” or “amino acid sequence” refers to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment thereof,
Refers to naturally occurring or synthetic molecules. Fragments of DRASP preferably have a length of about 5 to about 15 amino acids and retain the biological or immunological activity of DRASP. In this context, a "fragment", "immunogenic fragment", or "antigenic fragment" is preferably about 5-15 amino acids long (most preferably 14 amino acids long)
Refers to fragments of DRASP that retain the biological or immunological activity of DRASP. Although "amino acid sequence" is referred to herein as referring to the amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and similar terms refer to the complete, intact amino acid sequence associated with the listed protein molecule. Is not used to limit the amino acid sequence.

As used herein, “amplification” is the generation of additional copies of a nucleic acid sequence, usually performed using the polymerase chain reaction (PCR) technique well known to those skilled in the art.

The term “antagonist” refers to a molecule that, when bound to DRASP, reduces the extent of the effect of the biological or immunological activity of DRASP or reduces the duration of the effect.
Antagonists may include proteins, nucleic acids, carbohydrates, or any other molecule that reduces the effect of DRASP.

The term “antibody” also refers to intact molecules and fragments thereof, such as Fa, F (ab) ₂ , and Fv fragments, that can bind antigenic determinants. Antibodies that bind DRASP polypeptides can be prepared using intact polypeptides or using fragments thereof containing small peptides involved as antigens to immunize. Animals (for example,
The polypeptide or oligopeptide used to immunize a mouse, rat, or rabbit) is obtained from translation or chemically synthesized of RNA and can be coupled to a carrier protein if necessary. Commonly used carriers that bind chemically to the peptide include bovine serum albumin and thyroglobulin, keyhole limpet hemocyanin (KLH). The animal is then immunized with the conjugated peptide.

The term “antigenic determinant” refers to a fragment of a molecule (ie, an epitope) that makes contact with a particular antibody. When a host animal is immunized with a protein or protein fragment, many regions of the protein trigger the production of antibodies that specifically bind to antigenic determinants (defined regions or three-dimensional structures of the protein). I can do it. Antigenic determinants can compete with the original antigen (ie, the immunogen used to elicit the immune response) for binding to the antibody.

The term “antisense” refers to any component that contains a nucleic acid sequence that is complementary to the “sense” strand of a particular nucleic acid sequence. Antisense molecules can be created by any method, including synthesis and transcription. This complementary nucleotide, once introduced into the cell,
Combines with natural sequences produced by cells to form duplexes, preventing transcription and translation. The expression “minus (−)” may refer to the antisense strand, and the expression “plus (+)” may refer to the sense strand.

The term “biologically active” refers to a protein that has a structural, regulatory, or biochemical function of a naturally occurring molecule. Similarly, "immunologically active" refers to natural,
Refers to the ability of recombinant or synthetic DRASP, or any of its oligopeptides, to elicit a specific immune response in appropriate animals or cells and to bind to specific antibodies.

The terms “complementary” or “complementarity” are the natural associations of polynucleotides by base pairing under any salt and temperature conditions. For example, the sequence "5'AG-T3 '"
Binds to the complementary sequence "3'TC-A5 '". Complementarity between two single-stranded molecules can be either "partial", such as when only some nucleic acids are bound, or "complete", such as when there is complete complementarity between the single-stranded molecules. Some are. The degree of complementarity between nucleic acid strands greatly affects the efficiency and strength of hybridization between nucleic acid strands.
This can be attributed to amplification reactions that depend on the binding of nucleic acid strands, peptide nucleic acids (
PNA) is of particular importance in the design and use of molecules.

As used herein, “composition comprising a given polynucleotide sequence” or “composition comprising a given amino acid sequence” generally refers to any composition comprising a given polynucleotide or amino acid sequence. The composition may include a dry formulation or an aqueous solution. A composition comprising a polynucleotide encoding DRASP or a fragment of DRASP can be used as a hybridization probe. The probe can be stored in lyophilized form and conjugated to a stabilizing agent such as a carbohydrate. In hybridization, the probe is attached to a salt (eg,
NaCl), surfactants (eg, sodium dodecyl sulfate (SDS)) and other substances (
For example, it can be dispersed in an aqueous solution containing denhardt solution, dried milk, salmon sperm DNA, and the like.

The term “consensus sequence” refers to a nucleic acid sequence from which unnecessary bases have been separated by resequencing, or using a XL-PCR kit (The Perkin Elmer Corp., Norwalk, Conn.) In the 5 ′ direction and / or A nucleic acid sequence that has been extended and resequenced in the 3 ′ direction, or a computer program (eg, GELVIEW ^™ Fragment A)
ssembly system, GCG, Madison Wis.) is a nucleic acid sequence created by combining duplicate sequences of two or more Incyte clones. Some sequences are extended and combined to create a consensus sequence.

The term “correlated with the expression of a polynucleotide” is defined by Northern analysis
Detection of the presence of a nucleic acid identical or related to the nucleic acid sequence encoding DRASP indicates the presence of the nucleic acid encoding DRASP in the sample, and thus correlates with expression of a transcript from the polynucleotide encoding DRASP. Has been expressed.

As used herein, a “deletion” is a change in the nucleotide or amino acid sequence that results in the deletion of one or more nucleotide or amino acid residues.

The term “derivative” refers to a chemical modification of a polypeptide or polynucleotide sequence. For chemical modification of a polynucleotide sequence, for example, an alkyl group from hydrogen,
Substitution to an acyl group or an amino group is included. A polynucleotide derivative encodes a polypeptide that retains the biological or immunological functions of the naturally occurring molecule. Derivative polypeptides are glycosylated, polyethylene glycolated (pegylat
ion) or any other process that retains the biological or immunological function of the original polypeptide.

The term “similarity” refers to a degree of complementarity. There may be partial similarities or complete similarities. "Identity" can be used in place of the term "similarity".
A partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid refers to "substantially similar." Inhibition of hybridization between the completely complementary sequence and the target sequence can be assayed under mild stringency using a hybridization assay (Southern or Northern method, solution hybridization, etc.). A substantially similar sequence or hybridization probe will compete with and inhibit the target sequence for binding to a completely similar (identical) sequence under mild stringency. This does not mean that loose stringency conditions are such that they allow non-specific binding; under loose stringency conditions, the binding of two sequences to each other is specific. It is necessary that the interaction be selective (ie, selective). The absence of non-specific binding can be tested by using a second target sequence that does not have even a partial degree of complementarity (ie, less than about 30% similarity or identity). . In the absence of non-specific binding, a generally similar sequence or probe will not hybridize to a second non-complementary target sequence.

The term “percent identity” or “% identity” refers to the percentage of sequence similarity found in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, for example, by using the MEGALIGN program (DNASTAR, Inc., Madison WI). The MegAlign program aligns two or more sequences according to a variety of user-selected methods, such as, for example, the clustal method (see, eg, Higgins, DG and Sharp, PM (1988) Gene 73: 237-244). Can be created. The clustal algorithm classifies sequences into clusters by examining the distance between all pairs. Clusters are aligned in pairs,
Then they are aligned in groups. Two amino acid sequences (eg, sequence A
And the percentage of similarity between (SEQ ID NO: B and SEQ ID NO: B) is (total number of matching residues between sequence A and sequence B) / (length of sequence A-number of gap residues in sequence A) -Number of gap residues in sequence B) x 100. Low similarity or dissimilarity gaps between two amino acids are not included in determining similarity percentages. Also, the percent identity between nucleic acid sequences can be determined, for example, by Jotun Hein
It can be calculated or counted by other methods well known to those skilled in the art, such as the Method (see, eg, Hein, J. (1990) Methods Enzymol. 183: 626-645). Furthermore, identity between sequences can be determined by other methods well known to those skilled in the art, for example, by changing hybridization conditions.

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

The term “humanized antibody” refers to an antibody molecule in which the amino acid sequence in the non-antibody binding region has been replaced such that the antibody is closer to a human antibody while retaining its original binding ability.

The term “hybridization” refers to any process by which a strand of nucleic acid joins with a complementary strand through base pairing.

The term “hybridization complex” refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A hybridization complex may either be formed in solution (e.g., C ₀ t or R ₀ t analysis), or nucleic acid one of the nucleic acids present in the solution and the solid support (e.g., paper, membranes, filters, A pin, or another glass substrate, a glass slide, a polymer, or another suitable substrate to which the cells or their nucleic acids are fixed.

As used herein, “insertion” or “addition” refers to a nucleotide sequence or amino acid sequence in which one or more nucleotides or amino acid residues are added to a sequence found in a naturally occurring molecule, respectively. Refers to the change.

“Immune response” refers to a condition associated with inflammation, trauma, immune disorders, or infectious or hereditary diseases. These conditions are characterized by the expression of various factors that act on cellular and systemic defense systems, such as cytokines, chemokines, and other signaling molecules.

The term “microarray” refers to an individual array arranged on a substrate such as, for example, a paper, nylon, or other type of membrane, filter, chip, glass slide, or any other suitable solid support. Refers to an array of polynucleotides or oligonucleotides.

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

The term “modulate” refers to a change in the activity of DRASP. For example, modulation results in an increase or decrease in protein activity, an alteration in binding properties, or other alterations in DRASP biological, functional, immunological properties, etc.

As used herein, “nucleic acid” or “nucleic acid sequence” refers to nucleotides, oligonucleotides, polynucleotides, or any fragment thereof. These terms also refer to single-stranded or double-stranded, sense or antisense strands of genomic or synthetic DNA or RNA, peptide nucleic acids (PNA), and any DNA-like or RNA-like material. Point. The term "fragment" refers to a nucleic acid sequence that includes a region of a unique polynucleotide sequence that specifically identifies SEQ ID NOs: 6-10 (eg, different from any other sequence in the same genome). . For example, fragments of SEQ ID NO: 6-10 are useful for hybridization and amplification techniques, and also for determining similarity that distinguishes SEQ ID NO: 6-10 from related polynucleotide sequences. . SEQ ID NO: 6
A -10 fragment is at least about 15-20 nucleotides in length. SEQ ID NO: 6
The exact length of -10 and the region of SEQ ID NO: 6-10 corresponding to this fragment is routinely determined using one of the general techniques well known to those skilled in the art based on the intended purpose of the fragment. Can be determined. Alternatively, when the fragment is translated, the functional characteristics (eg, antigenicity of the full-length polypeptide, etc.) and the structural characteristics (eg, ATP of the full-length polypeptide, etc.)
A binding site, etc.).

The term “functionally related”, ie, “operably linked”, refers to a nucleic acid sequence that is functionally related. When the promoter controls the transcription of the encoded polypeptide,
A promoter is operably associated with, ie, operably linked to, a coding sequence. When a functionally related, ie, operably linked, nucleic acid sequence is present at or near the reading frame, a given genetic element (eg, a repressor gene) is continuously bound to the encoded polypeptide. Instead, it binds to an operator sequence that still controls expression of the polypeptide.

As used herein, an “oligonucleotide” is a nucleic acid sequence that can be used in a PCR amplification or hybridization assay or a microarray and is at least about 6 nucleotides to about 60 nucleotides in length, and is preferably Is 1
5 to 30 nucleotides, more preferably 20 to 25 nucleotides. As used herein, “oligonucleotide” is generally synonymous with the terms “amplimer”, “primer”, “oligomer”, and “probe” as commonly defined in the art.

As used herein, “peptide nucleic acid” (PNA) is an antisense molecule or anti-gene agent (anti-genetic agent) containing an oligonucleotide having a length of 5 nucleotides or more linked to a peptide backbone of lysine-terminated amino acid residues. gene agent). Terminal lysine confers solubility to its composition. PNAs preferentially bind to complementary single-stranded DNA or RNA and stop transcript elongation, and can also PNA-polyethylene glycol extend the life of PNAs in cells.

The term “sample” is used in its broadest sense. Samples suspected of containing the nucleic acid encoding DRASP, or fragments thereof, or DRASP itself,
Chromosomes isolated from cells, organelles, or extracts from cell membranes, cells,
Genomic DNA, RNA or cDNA (in solution or attached to a solid support),
It may include an organization, an organization print, and the like.

The term “specific binding” or “specifically binds” refers to antibodies and the interaction of proteins or peptides, antibodies and antagonists. This interaction means that it depends on the presence of a particular structure of the protein (eg, an antigenic determinant or epitope recognized by the binding molecule). For example, if the antibody is specific for epitope "A", a polynucleotide comprising epitope A (ie, free, unlabeled A) in a reaction involving labeled free "A" and the antibody The presence of a reduces the amount of labeled A bound to the antibody.

The term “stringent conditions” refers to conditions that permit hybridization of a polynucleotide to the polynucleotides set forth in the claims. The exact conditions are dictated by the salt concentration, the concentration of the organic solvent (eg, formamide), the temperature, and other conditions well known to those skilled in the art. In particular, lowering the salt concentration, increasing the formamide concentration, and increasing the hybridization temperature increase the stringency.

The term “substantially purified” is a nucleic acid or amino acid sequence that has been removed from its natural environment, isolated or separated from other components with which it is naturally associated, It is a nucleic acid sequence or an amino acid sequence whose components have been removed by 60% or more, preferably 75% or more, and most preferably 90% or more.

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

As used herein, “substrate” refers to any suitable solid or semi-conductive material, including membranes, filters, chips, slides, wafers, fibers, magnetic or non-magnetic beads, gels, tubes, plates, polymers, microparticles, capillaries. It is a solid support. Substrates can take a variety of surface forms, such as walls, grooves, pins, channels, and holes to which polynucleotides and polypeptides bind.

The definition of the term “transformation” refers to a process by which exogenous DNA enters and changes a recipient cell. Transformation can occur under natural or artificial conditions using a variety of methods well known to those of skill in the art, and any known method for introducing foreign nucleic acid sequences into prokaryotic or eukaryotic host cells. May be based on the method. Transformation methods are selected according to the type of host cell to be transformed and include, but are not limited to, viral infection, heat shock, electroporation, lipofection, and methods using microprojectile bombardment. It is. Such `` transformed '' cells include stably transformed cells capable of replicating the inserted DNA autonomously, or as part of the host chromosome, It also refers to cells that transiently express DNA or RNA introduced for a limited time.

A “variant” of a DRASP polypeptide as used herein is an amino acid sequence in which one or more amino acid residues have been mutated. The variant may contain "conservative" changes, in which the substituted amino acid has similar structural and chemical properties, for example, replacing leucine with isoleucine. In rare cases, variants may be changed "non-conservatively", for example, glycine is replaced with tryptophan. Similar small changes include amino acid deletions or insertions, or both. Using well-known computer programs, such as, for example, LASERGENE software (DNASTAR), determine that any amino acid residue can be substituted, inserted, or removed without compromising biological or immunological activity. Guidance can be provided.

A “mutant sequence,” as used in the context of a polynucleotide sequence, may include a polynucleotide sequence associated with DRASP. This definition includes, for example, "allele" (
Supra), "splice", "species", and "polymorphic" mutant sequences. A splice variant can have significant identity to a reference molecule, but generally has an increased or decreased number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide has an additional functional domain or no domain. Species variant sequences are polynucleotide sequences that vary from species to species.
The resulting polypeptides generally have significant amino acid identity with each other. Polymorphic variant sequences are variations in the polynucleotide sequence of a particular gene between a given individual species. Also, polymorphic variant sequences can include "single nucleotide polymorphisms" (SNPs) in which one base of the polynucleotide sequence changes. The existence of SNP
For example, it may represent a population, a medical condition, or a propensity for a medical condition.

[0069] This invention is the prevention of a new human DNA replication and repair-related protein (DRASP), polynucleotides encoding DRASP, and generating disorders, proliferative disorders, including cancer, and autoimmune disorders / inflammatory diseases , Diagnostics, or therapeutics.

Table 1 is a table of the Incyte clones used to obtain the full length nucleotide sequence encoding DRASP. The first and second columns contain the amino acid and nucleic acid sequence IDs.
Indicates the number (SEQ ID NO). The third column shows the clone ID of the Incyte clone from which the nucleic acid encoding each DRASP was identified, and the fourth column shows the cDNA library from which these clones were isolated. Column 5 shows Insights clones, their corresponding cDNA libraries, and shotgun sequences, which are useful as fragments in hybridization techniques and are part of the consensus nucleotide sequence of each DRASP.

Each column of Table 2 shows various properties of each polypeptide of the present invention, and column 1 shows SEQ ID NO:
ID NO, the second row is the number of amino acid residues of each polypeptide, the third row is a potential phosphorylation site, the fourth row is a potential glycosylation site, and the fifth row is a signature sequence (signature sequences).
) And the amino acid residues containing the motif, column 6 is the identification of each protein, column 7 is the analysis method used to identify each protein by sequence homology and protein motif.

Each column in Table 3 shows tissue specificity, as well as diseases related to the nucleotide sequence encoding DRASP. The first column of Table 3 shows the polynucleotide SEQ ID NO. The second column shows the tissue classification representing the percentage of the total tissue classification that expresses DRASP. The third column shows the classification of the disease associated with those tissues expressing DRASP. The fourth column shows the vector used for subcloning of the cDNA library.

The fragments of the nucleotide sequence encoding DRASP shown below may be used to identify SEQ ID NOs: 6-10, as well as hybridization or amplification techniques to distinguish SEQ ID NOs: 6-10 from similar polynucleotide sequences. It is useful in Useful fragments include:
A fragment of SEQ ID NO: 6 of nucleotides from about 90 to about 120 (encoding a fragment of DRASP-1 of about 30 to about 40 amino acids) and a fragment of about 165 to about 19;
A fragment of SEQ ID NO: 7 of the fifth nucleotide (encoding a fragment of DRASP-2 of about 55 to about 65 amino acids) and a SEQ ID NO of about 833 to about 889 nucleotides : 8, a fragment of SEQ ID NO: 9 from about 848 to about 892 nucleotides, and a SEQ ID NO: 9 of about 101 to about 130 nucleotides
NO: 10 fragments are included.

The present invention also includes mutants of DRASP. A DRASP variant preferably has at least 80%, more preferably at least 90%, most preferably at least 95% or more amino acid sequence identity to the DRASP amino acid sequence, and has functional or structural characteristics of DRASP. Including at least one of

[0075] The invention further includes polynucleotides encoding DRASP. In certain embodiments, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 6-10 encoding DRASP.

Further, the present invention includes a mutant sequence of a polynucleotide encoding DRASP. In particular, such polynucleotide variant sequences will have at least 80%, more preferably at least 90%, most preferably at least 95% or more polynucleotide sequence identity to the polynucleotide sequence encoding DRASP. Certain embodiments of the present invention include a variant sequence of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 6-10, which is selected from the group consisting of SEQ ID NOs: 6-10. At least 80%, more preferably at least 90%, most preferably at least 95% or more polynucleotide sequence identity to the nucleic acid sequence. Any of the above mutant sequences of the polynucleotide can encode an amino acid sequence containing at least one functional or structural characteristic of DRASP.

The degeneracy of the genetic code results in the generation of a large number of DRASP-encoding polynucleotide sequences, including those with minimal similarity to known and naturally occurring gene polynucleotide sequences. Will be understood by those skilled in the art. Thus, the present invention contemplates all possible variations in the polynucleotide sequence that can be created by selecting combinations based on possible codon choices. These combinations are to be created according to the standard triplet genetic code as applied to the nucleotide sequence of naturally occurring DRASP, and all such variations are to be considered specifically disclosed herein.

Preferably, the nucleotide sequence encoding DRASP and its mutant sequences are capable of hybridizing, under appropriately selected stringency conditions, to the nucleotide sequence of naturally occurring DRASP. It is advantageous to create a nucleotide sequence encoding DRASP or a derivative thereof having substantially different codon usage, such as including non-naturally occurring codons. Depending on the frequency with which particular codons are utilized by the host, codons can be selected to increase the rate at which expression of the peptide occurs in eukaryotic or prokaryotic hosts. Another reason for substantially altering the nucleotide sequence encoding DRASP and its derivatives without altering the encoded amino acid sequence is that it has a longer half-life than transcripts generated from naturally occurring sequences. This is to create an RNA transcript having the desired properties.

The present invention also includes the production of DNA sequences encoding DRASP and derivatives of DRASP, or fragments thereof, exclusively by synthetic chemistry. After construction, the synthetic sequences can be inserted into any of a number of available expression vectors and cell lines using reagents well known to those of skill in the art. In addition, synthetic chemistry can be used to introduce mutations into the DRASP-encoding sequence or any fragment thereof.

Also included in the invention are hybrids, under various stringent conditions, to the claimed polynucleotide sequences, and especially to SEQ ID NOs: 6-10 and fragments thereof. Soyable polynucleotide sequences are included (see, eg, Wahl, G.
M. and SL Berger (1987) Methods Enzymol. 152: 399-407; Kimmel, AR (1987).
Methods Enzymol. 152: 507-511). For example, the exact salt concentration is usually around 750
less than about mM mM sodium chloride and less than about 75 mM sodium chloride and preferably less than about 500 mM sodium chloride and less than about 50 mM trisodium citrate, most preferably less than about 250 mM sodium chloride and less than about 25 mM. Is trisodium citrate. For example, in the absence of an organic solvent such as formamide, less stringent hybridization occurs, while in the presence of about 35% or more formamide, most preferably about 50% or more formamide, more stringent hybridization. Strict temperature conditions are usually about 30 ° C. or higher, more preferably about 37 ° C.
° C or higher, most preferably about 42 ° C or higher. Variable additional parameters such as, for example, hybridization time, drug concentration such as sodium dodecyl sulfate (SDS), inclusion or exclusion of carrier DNA, are well known to those skilled in the art. By combining these various requirements, various levels of stringency are achieved. In a preferred embodiment, the hybridization is performed at a temperature of 30 ° C., 750 mM sodium chloride, 75 mM trisodium citrate, and 1% SDS.
In a more preferred embodiment, a temperature of 37 ° C., 500 mM sodium chloride, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ss
DNA). In the most preferred embodiment,
Temperature 42 ° C, 250 mM sodium chloride, 25 mM trisodium citrate, 1% SDS, 50%
The hybridization is carried out on formamide and 200 μg / ml of ssDNA. Useful alterations of these conditions will be readily apparent to those skilled in the art.

The stringency can also vary during the washing process after hybridization. The exact conditions for washing can be defined by salt concentration and temperature. As noted above, decreasing the salt concentration or increasing the temperature can increase the stringency of the wash. For example, the exact salt concentration conditions for the washing process are preferably less than about 30 mM sodium chloride and less than about 3 mM trisodium citrate, most preferably less than about 15 mM sodium chloride and less than about 1.5 mM citric acid. 3 sodium. Strict temperature conditions for the washing process are usually about 25 ° C. or more, more preferably about 42 ° C. or more,
Most preferably it is above about 68 ° C. In a preferred embodiment, the washing step is performed at a temperature of 25 ° C., 30 ° C.
Performed in mM sodium chloride, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing process comprises a temperature of 42 ° C., 15 mM sodium chloride,
Performed in 1.5 mM trisodium citrate, and 0.1% SDS. In the most preferred embodiment, the washing step is performed at a temperature of 68 ° C., 15 mM sodium chloride, 1.5 mM trisodium citrate, and 0.1% SDS. Further modifications of these conditions will be readily apparent to those skilled in the art.

[0082] DNA sequencing methods are well known and can be used in any of the embodiments of the present invention. The method is based on the Klenow fragment of DNA polymerase I, SEQUEN
ASE (US Biochemical, Cleveland, OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Pharmacia Biotech, Piscataway NJ), or
Use enzymes such as a combination of proofreading exonuclease and polymerase as found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD). For sequence preparation, use Hamilton MICROLAB 2200 (Hamilton
, Reno, NV), Peltier Thermal Cycler 200 (PTC200; MJ Research, Watertown, MA)
) And ABI CATALYST 800 (Perkin Elmer). Next, use the ABI 373 or 377 DNA sequencing system (Perkin
-Elmer), MEGABACE Capillary Electrophoresis System (Molecular Dynamics, Sunn)
Perform sequencing with yvale CA). The resulting sequence is analyzed using various algorithms well known to those skilled in the art (eg, Ausubei, FM (1997)
Short Protocols in Molecular Biology , John Wiley & Sons, New York NY, u
nit 7.7; Meyers, RA (1995) Molecular Biology and Biotechnology , Wiley
VCH, New York NY, pp. 856-853).

The nucleic acid sequence encoding DRASP may be extended using partial nucleotide sequences in a variety of PCR-based methods well known in the art to cleave upstream sequences such as promoters and regulatory elements. Can be detected. For example, one of the methods used, restriction site PCR, amplifies an unknown sequence from genomic DNA in a cloning vector using common primers and nested primers (eg, Sarkar, G. (1993)). See PCR Methods Applic. 2: 318-322). Another method that uses the inverse PCR method uses primers that extend widely and amplify the unknown sequence from the circularized template. This template is derived from a restriction fragment containing a known genomic locus and sequences around it. (Triglia, T. et al. (1988) Nucleic Acids Res. 16: 8186)
. As a third method, the capture PCR method includes PCR amplification of a DNA fragment adjacent to a known sequence of human and yeast artificial chromosomal DNA (for example, L
agerstrom, M. et al. (1991) PCR Methods Applic 1: 111-119). In this method, the digestion and ligation of a number of restriction enzymes can be used to insert the recombinant double-stranded sequence into a region of unknown sequence before performing PCR. Also, other methods that can be used to recover unknown sequences are well known to those of skill in the art (eg, Parker, JD
(1991) Nucleic Acids Res. 19: 3055-306). Furthermore, walking within genomic DNA is possible using PCR, nested primers, and a PROMOTERFINDER library (Clonetech, Palo Alto, CA). This method eliminates the need to screen the library and is useful for finding intron / exon transitions. All PCs
The method R method based, primer, OLIGO ^TM 4.06 Primer Analy
Using commercially available software such as sis software (National Biosciences Inc., Plymouth, MN) or another suitable program, the GC is 22-30 nucleotides in length.
The content can be designed to anneal to the mold at a temperature of about 50% or more and at a temperature of about 68C to 72C.

When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include large cDNAs. In addition, a random-primed library that often contains the sequence of the 5 ′ region of a gene is an oligo d (T)
This is suitable when a full-length cDNA cannot be obtained from the library. Genomic libraries can also serve for extension of sequence in the 5 'non-transcribed region.

For size analysis or confirmation of the nucleotide sequence of the product of sequencing or PCR,
A commercially available capillary electrophoresis system can be used. In particular, capillary sequencing may use a flowable polymer for electrophoretic separation, a laser-activated fluorescent dye specific for four different nucleotides, and a CCD camera that detects the emitted wavelength. The output / light intensity is determined by the appropriate software (
For example, it is converted into an electric signal using GENOTYPER and SEQUENCE NAVIGATOR (Perkin Elmer), and the whole process from sample loading to computer analysis and electronic data display can be computer controlled. Capillary electrophoresis is particularly suitable for sequencing small pieces of DNA that may be present in limited amounts in a particular sample.

In another embodiment of the invention, a polynucleotide sequence encoding DRASP, or a fragment thereof, is used in a recombinant DNA molecule to express DRASP, a fragment thereof, or a functional equivalent thereof, in a suitable host cell. Can be oriented. Due to the inherent degeneracy of the genetic code, other DNAs that encode substantially identical, ie, functionally equivalent, amino acid sequences
A sequence is created and can be used for expression of DRASP.

For altering the sequence encoding DRASP for various reasons, including but not limited to altering the cloning, processing and / or expression of the gene product,
The nucleotide sequences of the present invention can be manipulated using methods well known to those skilled in the art. The nucleotide sequence can be manipulated using DNA mixing by random fragmentation or PCR reconstitution of gene fragments and synthetic oligonucleotides. For example, oligonucleotide-mediated site-directed mutagenesis allows for the insertion of mutations that create new restriction sites, alter glycosylation patterns, alter codon preferences, generate splicing variants, and the like.

In another embodiment of the present invention, the entire DRASP-encoding sequence, or a portion thereof, can be synthesized using chemical techniques well known to those skilled in the art (eg, Caruther
sMH et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223; Horn, T. et al. (1980) Nucl.
Res.Symp.Ser.225-232). Alternatively, DRASP itself or a fragment thereof can be synthesized using chemical techniques. For example, peptide synthesis can be performed using various solid phase techniques (eg, Roberge, JY et al. (1995) Science 269: 202-20).
4). In addition, the synthesis can be automated using ABI 431A peptide synthesizer (Perkin Elmer). In addition, variant polypeptides can be produced by modifying the amino acid sequence of DRASP or any portion thereof in direct synthesis and / or binding to sequences derived from other proteins or any portion thereof. It is.

The peptides can be substantially purified by preparative high performance liquid chromatography (eg, Chiez, RM and Regnier. FZ (1990) Methods Enzy.
mol. 182: 392-421). The composition of the synthesized peptide can be confirmed by amino acid analysis or sequencing (Creighton, T. (1983) Proteins. Structured Molecular Properties , WH Freeman and Co., New York, NY).

In order to express a biologically active DRASP, the nucleotide sequence encoding DRASP, or a derivative thereof, is required to have the appropriate expression vector (ie, necessary for the transcription and translation of the coding sequence inserted into the appropriate host). Vector containing the element). These elements include regulatory sequences such as enhancers, constitutive and inducible promoters, 5 'and 3' untranslated regions in the vector and the polynucleotide sequence encoding DRASP. The length and specificity of such elements can vary. Specific initiation signals can be used to effect more efficient translation of the DRASP-encoding sequence. Such signals include ATG
Includes start codons and adjacent sequences such as, for example, Kozak sequences. When the sequence encoding DRASP, its initiation codon, and upstream regulatory sequences are inserted into the appropriate expression vector, additional transcriptional and translational regulatory signals may be unnecessary. However, if only the coding sequence or a fragment thereof is inserted, exogenous translational control signals, including the ATG start codon in the reading frame, must be provided by the expression vector. Exogenous translational elements and initiation codons can consist of various natural and synthetic forms. By including an appropriate enhancer in the particular host cell strain used, it is possible to increase the efficiency of expression (eg, Sc
harf, D. et al. (1994) Results Probl. Cell Differ. 20: 125-162.).

To prepare an expression vector containing a DRASP-encoding sequence and appropriate transcriptional and translational regulatory regions, methods well known to those skilled in the art are used. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination techniques (see, eg, Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual , Cold S.
pring Harbor Press, Plainview, NY, ch. 4, 8, and 16-17; Ausubel, FM et al. (1995
) Current Protocols in Molecular Biology , John Wiley & Sons, New York, N
Y, see ch. 9, 13, and 16).

[0092] A variety of expression vector / host systems are available for containing and expressing the sequence encoding DRASP. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors, yeast transformed with yeast expression vectors, and viral expression vectors ( For example, an insect cell line infected with a baculovirus), transformed with a viral expression vector (eg, cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or a bacterial expression vector (eg, Ti or pBR322 plasmid) Plant cell lines or animal cell lines are included. The present invention is not limited by the host cell utilized.

[0093] In bacterial systems, a number of cloning and expression vectors may be selected depending upon the purpose for which the polynucleotide sequence encoding DRASP is to be used. For example, DRASP
Routine cloning, subcloning, and propagation of a polynucleotide sequence encoding a PBLUESCRIPT (Stratagene, La Jolla CA) or pSportl plasmid
(Life Technologies) or other multifunctional E. coli vectors. Ligation of the sequence encoding DRASP into multiple cloning sites of the vector disrupts the lacZ gene, allowing a colorimetric screening method to identify transformed bacteria containing the recombinant molecule. In addition, these vectors provide for in vitro transcription, dideoxy sequencing,
Useful for single-stranded rescue by helper phage and generation of nested deletions (
For example, Van Heeke. G. and SM Schuster (1989) J. Biol. Chem. 264: 5503-55.
09). For example, when a large amount of DRASP is required for the purpose of antibody production or the like, a vector that provides high-level DRASP expression can be used. For example, a strong triggering T
Vectors containing the 5 or T7 bacteriophage promoter can be used.

A yeast expression system can be used to generate DRASP. Yeast Saccharomyces cerevisiae
Alternatively, in Pichia pastoris , various vectors containing a constitutive or inducible promoter such as α-factor, alcohol oxidase, and PGH can be used. In addition, such vectors direct either secretion or intracellular retention of the expressed protein and allow for the integration of exogenous sequences into the host genome for stable growth (eg, Ausubel as described above). , supra; and Grant et al. (1987) Methods Enzymo
l. 153: 516-54; see Scorer, CA et al. (1994) Bio / Technology 12: 181-184).

[0095] Plant systems can also be used for expression of DRASP. Transcription of the DRASP-encoding sequence can be promoted by a viral promoter, such as the CaMV 35S and 19S promoters alone or in combination with an omega leader sequence from TMV (Takamatsu, N. et al. (1987)).
EMBO J. 6: 307-311). 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, New York; pp.191
-196).

[0096] In mammalian cells, a number of viral-based expression systems are available. When an adenovirus is used as an expression vector, the sequence encoding DRASP can be ligated into an adenovirus transcription / translation complex consisting of the late promoter and tripartite leader sequence. Insertion at a non-essential E1 or E3 region of the viral genome results in an infectious virus that expresses DRASP in host cells (eg, Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. . 81:36
55-3659). In addition, transcription enhancers such as the Rous sarcoma virus (RSV) enhancer can be used to increase expression in mammalian host cells. Also, SV40 or EBV based vectors can be used for high level expression of the protein.

The use of a human artificial chromosome (HAC) can also supply a larger DNA fragment than can be contained and expressed in a plasmid. For therapeutic purposes, 6 kb to 10 Mb of HAC can be constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) (eg,
Harrington, JJ et al. (1997) Nat Genet. 15: 345-355).

To ensure long-term production of recombinant mammalian proteins, stable expression of DRASP in cell lines is preferred. For example, using an expression vector
The RASP-encoding sequence can be transformed into a cell line, which expression vector contains replication and / or endogenous expression elements of viral origin and a selectable marker gene on the same or a separate vector. It is. After the introduction of the vector, the cells are allowed to grow for 1-2 days in a concentrated medium before switching to a selective medium. The purpose of a selectable marker is to confer resistance to a selective mediator, and to allow the growth and recovery of cells that successfully express the introduced sequence due to their presence. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate for the cell type.

[0099] Any number of selection systems can be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes,
tk ^- and apr ^-. As used in the cell (e.g., Wigler, M et al (1977) Cell 11:22
3-32; Lowy, I. et al. (1980) Cell 22: 817-23). 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 the aminoglycoside neomycin and G-418, and als or pat Chlorsulfuron, phosphinotricin acetyltransferase
(Eg, Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.
77: 3567-70; see Colberre-Garapin, F. et al. (1981) J. Mol. Biol. 150: 1-14). Additional selectable genes are described in the literature, for example, trpB and hisD, which alter the cell's need for metabolites (eg, Hartman, SC and RC).
Mulligan (1988) Proc. Natl. Acad. Sci. 85: 8047-8051). For example, visible markers such as anthocyanin, green fluorescent protein (GFP; Clontech), β-glucuronidase and its substrate β-glucuronide, or luciferase and its substrate luciferin can be used. These markers are widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression by a particular vector system (eg, Rhodes, CA et al. (1995) Methods Mol. Biol.
55: 121-131).

The presence / absence of marker gene expression also indicates the presence of the gene of interest, but may require confirmation of the presence and expression of that gene. For example, if a sequence encoding DRASP is inserted within a marker gene sequence, transformed cells containing the sequence encoding DRASP can be identified by the absence of marker gene function. Alternatively, the sequence encoding DRASP and the marker gene can be placed in tandem under the control of a single promoter. Expression of the marker gene in response to selection or induction usually indicates expression of the tandemly arranged sequence as well.

In general, various methods well known to those skilled in the art can be used to identify host cells that contain a nucleic acid sequence encoding DRASP and express DRASP. Such methods include, but are not limited to, DNA-DNA or DNA-RNA hybridization, PCR amplification, and membranes, solutions, or chips based on techniques for detecting and / or quantifying nucleic acid and protein sequences. Including protein bioassays or immunoassays.

Using either specific polyclonal or monoclonal antibodies
Immunological techniques for detecting and measuring the expression of DRASP are well known to those skilled in the art. Examples of such techniques include enzyme linked immunoassays (ELISA), radioimmunoassays (RIAs) and fluorescent cell analysis separations (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on DRASP
ay) is preferred, but competitive binding experiments can also be used. These and other assays are disclosed by those skilled in the art (e.g., Hampton, R. et al. (1990); Se rological Methods, a Laboratory Manual, APS Press, St Paul, MN Section IV
Coligan, JE et al. (1997) Current Protocols in Immunology , Greene Pub. As
sociates and Wiley-lnterscience, New York , NY; and Pound, JD (1998) Imm unochemical Protocols, Humana Press, see Totowa NJ).

A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic and amino acid assays. In order to detect a sequence related to the polynucleotide encoding DRASP, means for producing a labeled hybridization probe or PCR probe include oligo labeling (oligolabeling),
This includes nick translation, end-labeling, or PCR amplification using labeled nucleotides. Alternatively, the sequence encoding DRASP, or any fragment thereof, can be cloned into a vector for the production of an mRNA probe. Such vectors are well known to those of skill in the art and are commercially available, and can be used, for example,
RNA probes can be synthesized in vitro by adding a suitable RNA polymerase such as T7, T3 or SP6 and labeled nucleotides. These methods are described in various commercially available kits (Amersham Pharmacia Biotech, Promega (Madison W
I), and US Biochemical). Suitable reporter molecules or labels used to facilitate detection include radionuclides, enzymes, fluorescent agents, chemiluminescent or dye-producing agents, substrates, cofactors, inhibitors, magnetic particles, and the like.

Under conditions suitable for the recovery and expression of proteins from cell culture, DRASP
The host cell transformed with the nucleotide sequence encoding can be cultured. The protein produced by the transformed cells depends on the sequence used and / or
Or it can be secreted or retained intracellularly depending on the vector. As will be appreciated by those skilled in the art, an expression vector comprising a polynucleotide encoding DRASP is
It can be designed to include a signal sequence that directs DRASP secretion through prokaryotic or eukaryotic cell membranes.

In addition, host cell strains are chosen for their ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing that cleaves the "prepro" form of the protein can be used to identify the target, folding and / or activity of the protein. Various host cells (e.g., CHO, HeLa) that have specific characteristic mechanisms and cellular machinery for post-translational activity
, MDCK, MEK293, and WI38) are available from the American Type Culture Collection (ATCC, M
anassas, VA) and can be selected to ensure correct modification and processing of the foreign protein.

In another embodiment of the present invention, a naturally occurring nucleic acid encoding DRASP, a modified nucleic acid,
Alternatively, the recombinant nucleic acid sequence can be linked to a heterologous sequence which translates into the fusion protein of any of the aforementioned host systems. For example, a chimeric DRASP protein containing a heterologous moiety that can be identified by commercially available antibodies can facilitate screening of peptide libraries for inhibitors of DRASP activity. Also, using commercially available affinity substrates, heterologous proteins and peptide moieties can facilitate purification of the fusion protein. Such moieties include, but are not limited to, glutathione S transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c -myc, hemagglutinin (HA)
Is included. GST, MBP, Trx, CBP, and 6-His allow purification of homologous fusion proteins with each of immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal chelating resins. FLAG, c-mc, and hemagglutinin (HA) allow for immunoaffinity purification of the fusion protein using commercially available monoclonal and polyclonal antibodies that specifically identify these epitope tags. Also, the fusion protein can be engineered such that the fusion protein contains a protein cleavage site located between the sequence encoding DRASP and the heterologous protein sequence, and the DRASP can be cleaved from the heterologous portion after purification. Methods for expression and purification of the fusion protein are described in Ausubel's literature (1995, supra , ch 10). In addition, various commercially available kits can be used to promote expression and purification of the fusion protein.

In yet another embodiment of the present invention, radiolabeled DRASP can be synthesized in vitro using a TNT rabbit reticulocyte lysate or a wheat germ extract system (Promega). These systems couple the transcription and translation of a protein-encoding sequence operably linked to a T7, T3, or SP6 promoter. Transcription takes place in the presence of a radiolabeled amino acid precursor, preferably ³⁵ S-methionine.

Not only by recombinant production, but also by direct peptide synthesis using solid phase technology,
DRASP fragments can be created (see, for example, Creighton, supra, pp. 55-60). Protein synthesis can be performed manually or automatically. Automated synthesis can be performed, for example, using an ABI 431A peptide synthesizer (Perkin Elmer). Various fragments of DRASP can be synthesized separately and then combined to create the full length molecule.

There are chemical and structural similarities (eg, in sequence and motif context) between therapeutic DRASP and DNA replication and repair proteins. Furthermore, DRASP expression is closely associated with cell proliferation, cancer, and inflammation. Thus, DRASP is thought to be associated with developmental disorders, proliferative disorders, including cancer, and autoimmune disorders / inflammatory diseases. In a disease in which the activity or expression of DRASP is reduced, it is desirable to increase the activity or expression of DRASP. In a disease in which the activity or expression of DRASP is increased, it is desirable to decrease the activity or expression of DRASP.

Thus, in one embodiment, DRASP or a fragment or derivative thereof can be administered to a subject for the treatment or prevention of a disease in which DRASP activity or expression is reduced. Such diseases include, but are not limited to, tubular acidosis, anemia, achondroplasia dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysplasia, Smith-Magenis syndrome, myelodysplasia Syndrome, hereditary mucosal epithelial dysplasia, hereditary keratoderma, hypothyroidism, hydrocephalus, seizures (eg,
Chorea and cerebral palsy), spina bifida, encephalopathy, cranial spondylolysis, congenital glaucoma, cataract and other disorders, as well as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, Cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and adenocarcinoma, leukemia (including Bloom syndrome) , Lymphoma, melanoma, myeloma, multiple endocrine tumors (Werner syndrome), sarcomas, teratocarcinomas, and especially the adrenal gland, bladder, bone, bone marrow,
Brain, breast, neck, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary gland, skin, spleen, testis, thymus, thyroid, and uterus Proliferative disorders such as cancer, and acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergy, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmunity Hemolytic anemia, autoimmune thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, emphysema, lymphocotoxin temporary lymphopenia, fetal erythroblasts Bulbar disease, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, inflammation of the myocardium or pericardium, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, joints Rheumatism, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic thrombotic purpura, ulcerative colitis, uveitis, Werner syndrome, and complications of cancer, hemodialysis, and extracorporeal circulation And autoimmune abnormalities / inflammatory diseases such as viruses, bacteria, fungi, parasites, protozoa, and parasite-like infections, and trauma.

In another example, a vector capable of expressing DRASP or a fragment or derivative thereof may be administered to a subject for the treatment or prevention of a disease, including but not limited to those described above.

In another embodiment, a purified DRASP-containing pharmaceutical composition is administered to a subject, along with a suitable pharmaceutical carrier, for the treatment or prevention of a disease, including but not limited to those described above. Is also good.

In another embodiment, an agonist that modulates the activity of DRASP may be administered to a subject for the treatment or prevention of a disease, including but not limited to those described above.

In yet another embodiment, an antagonist of DRASP may be administered to a subject for the treatment or prevention of a disease in which the activity or expression of DRASP is increased. Examples of such diseases include, but are not limited to, those mentioned above. In one embodiment, an antibody that specifically binds DRASP is used directly as an antagonist, or
It can be used indirectly as a delivery mechanism or targeting to bring the drug to cells or tissues that express RASP.

In another example, a vector capable of expressing the complement of a polynucleotide encoding DRASP may be administered to a subject for the treatment or prevention of a disease, including but not limited to those described above. .

In another embodiment, any of the proteins, antagonists, antibodies, agonists, complementary sequences or vectors of the invention may be administered in combination with other suitable therapeutic agents. One of skill in the art would be able to select appropriate agents for use in combination therapy based on conventional pharmaceutical principles. The combination of therapeutic agents can function synergistically to effect treatment or prevention of the various reactions described above. By using this method, the therapeutic effect can be increased with a smaller dose of each drug, and thus the possibility of side effects can be reduced. DRASP antagonists can be prepared using methods well known to those skilled in the art. In particular, a library of drugs can be screened to produce antibodies using purified DRASP or to identify those that specifically bind to DRASP. DRASP antibodies can be produced using methods well known to those skilled in the art. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries. Neutralizing antibodies (ie, those that inhibit dimer formation) are particularly suitable for therapeutic use.

To produce antibodies, various hosts, including goats, rabbits, rats, mice, humans, and the like, are immunized by injecting DRASP or any fragment thereof having immunological properties or oligopeptides thereof. Can be Rats and mice are preferred as hosts for downstream applications involving monoclonal antibody production. Depending on the species of the host, various adjuvants can be used to enhance the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH And dinitrophenol.
Among adjuvants used in humans, BCG (bacilli Calmette - Guerin) and Cor ynebacterium parvum are especially preferable.

The oligopeptide, peptide, or fragment thereof used to elicit DRASP antibodies is preferably at least 5 amino acids, more preferably at least 1 amino acid.
It has an amino acid sequence consisting of four amino acids. Also these oligopeptides,
Preferably, the peptide or fragment is identical to a portion of the amino acid sequence of the native protein and comprises the entire amino acid sequence of a small, naturally occurring molecule. A short stretch of DRASP amino acids can be fused with another protein stretch, such as KLH, to produce chimeric molecule antibodies.

[0119] Monoclonal antibodies to DRASP can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. Such techniques include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and EBV-hybridoma technology (eg, Kohler, G. et al. (1975) Nature 256: 495-49).
7; Kozbor, D. et al. (1985) Immunol. Methods 81: 31-42; Cote, RJ et al. (1983) P
roc. Natl. Acad. Sci. 80: 2026-2030; Cole, SP et al. (1984) Mol. Cell Biol.
62: 109-120).

In addition, techniques developed for the production of “chimeric antibodies”, such as the technique of splicing a mouse antibody gene to a human antibody gene, in order to obtain molecules with appropriate antigen specificity and biological activity (For example, Morrison, SL
Natl.Acad.Sci. 81: 6851-6855; Neuberger, MS et al. (1984) Nat.
ure 312: 604-608; Takeda, S. et al. (1985) Nature 314: 452-454). Alternatively, techniques for the production of single-chain antibodies can be applied using methods well known to those skilled in the art to produce single-chain antibodies specific for DRASP. Antibodies with related specificities but differing idiotypic compositions can be produced by chain shuffling from random combinations of immunoglobulin libraries (eg, Bur).
ton DR (1991) Proc. Natl. Acad. Sci. 88: 10134-10137).

Antibodies can be produced by screening an immunoglobulin library or panel of highly specific binding reagents, or by inducing production in a lymphocyte population in vivo (eg, Orlandi). , R. et al. (1989), Proc.
Natl. Acad. Sci. 86: 3833-3837; Winter, G. et al. (1991) Nature 349: 293-299).

[0122] Antibody fragments which contain specific binding sites for DRASP can also be generated. For example, such fragments include, but are not limited to, F (ab ') ₂ fragments that can be generated by pepsin digestion of antibody molecules, and F (ab') ₂ fragments that are generated by reducing disulfide bridges. Fab fragments that can be used. Alternatively, Fab expression libraries can be constructed so that monoclonal Fab fragments with the desired specificity can be identified quickly and easily (see, for example, Huse, WD et al. (1989) Science 246: 1275-1281).

Various immunoassays can be used for screening to identify antibodies with the desired specificity. Numerous protocols for competitive binding assays or immunoradiometric assays using either monoclonal or polyclonal antibodies with established specificity are well known in the art. Such immunoassays generally involve the measurement of the formation of a complex between DRASP and its specific antibody. Two sites monoclona-based immunoassay using monoclonal antibodies reactive to two non-interfering DRASP epitopes
l based immunoassay) is preferred, but competitive binding assays can also be used (P
sound, supra ).

Using various methods such as Scatchard analysis with radioimmunoassay techniques, DRA
Evaluate the affinity of the SP antibody. Affinity is expressed as the binding constant, Ka, defined as the molar concentration of the OP antibody complex at equilibrium divided by the molar concentrations of free antibody and free antigen. The Ka measured for polyclonal antibody reagents with heterogeneous affinities for multiple DRASP epitopes represents the average affinity or avidity of the DRASP antibody. The Ka measured against a monoclonal antibody reagent monospecific for a particular DRASP epitope represents a true measure of affinity. High affinity antibody reagents with Ka values of 10 ⁹ to 10 ¹² L / mol are preferably used in immunoassays in which the DRASP antibody complex must withstand rigorous manipulations. Low-affinity antibody reagents with Ka values of 10 ⁶ to 10 ⁷ L / mol can be used for immunopurification and similar treatments where DRASP must eventually dissociate from the antibody in the activated state. preferable. (Catty, D. (1
988) Antibodies, Volume I: A Practical Approach .IRL Press, Washington,
DC; Liddell, JE and Cryer, A. (1991) A Practical Guide to Monocional Antibodies , John Wiley & Sons, New York NY).

[0125] The titer and binding activity of the polyclonal antibody reagents are further evaluated to determine the quality and suitability of such reagents for certain downstream applications. For example, a polyclonal antibody reagent containing at least 1-2 mg / ml of a specific antibody, preferably 5-10 mg / ml of a specific antibody, is preferably used in a method requiring precipitation of a DRASP antibody complex. . Treatment for various specificities of antibodies in the application, titer, and avidity, and guidelines for quality and usage of antibodies are generally available (see, for example, Catty, supra, and Coligan et al., Supra).

In another embodiment of the invention, a polynucleotide encoding DRASP, or any fragment or complement thereof, can be used for therapeutic purposes. In one aspect,
In situations where it is desirable to inhibit mRNA transcription, a sequence complementary to the polynucleotide encoding DRASP can be used. In particular, cells can be transformed with sequences complementary to the polynucleotide encoding DRASP. Thus, complementary molecules or fragments can be used to modulate DRASP activity or regulate gene function. At present such techniques are well known and sense or antisense oligonucleotides or larger fragments can be designed from various locations according to the coding and regulatory regions of the DRASP-encoding sequence.

Expression vectors derived from retroviruses, adenoviruses, herpes or vaccinia viruses, or from various bacterial plasmids, can be used to target organs,
It can be used for delivery of a nucleotide sequence to a tissue, ie, a group of cells. Vectors for expressing a nucleic acid sequence complementary to a polynucleotide encoding DRASP can be produced using methods well known to those skilled in the art (see, eg, Sambrook, supra .
And Ausubel, 1995, supra ).

A gene encoding DRASP can be created by transforming a cell or tissue with an expression vector that expresses high levels of a polynucleotide encoding DRASP or a fragment thereof. Such constructs can be used to introduce non-translatable sense or antisense sequences into cells. Even in the absence of integration into DNA, such vectors can continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression can last for a month or more with non-replicating vectors, and even longer if appropriate replication elements are part of the vector system.

As described above, altering gene expression by designing a complementary sequence to the regulatory, 5 ', or regulatory regions of the gene encoding DRASP, ie, an antisense molecule (DNA, RNA or PNA), can be performed. it can. Transcription start point (for example, from the start site
+10 to -10) are preferred. Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it inhibits the ability of the duplex to unwind sufficiently for binding of polymerases, transcription factors, or regulatory molecules (eg, Gee, JE et al. (1994) I.
n: Huber, BE and BI Carr, Molecular and Immunologic Approaches , Futur
a Publishing Co., Mt. Kisco, NY, pp. 163-177). Complementary sequences, ie, antisense molecules, can be designed to inhibit mRNA transcription by blocking transcript binding to ribosomes.

A ribozyme, an enzymatic RNA molecule, can be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves specific hybridization of the sequence of the ribozyme molecule to complementary target RNA, followed by nucleotide strand breaks. For example, engineered hammerhead motif ribozyme molecules can specifically and effectively catalyze nucleotide strand breaks in a DRASP-encoding sequence.

[0131] Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites that include the following sequences GUA, GUU and GUC. Once identified, a short RNA sequence between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site will have secondary structural features that render the oligonucleotide inoperable. Can be evaluated. The suitability of the candidate target is determined by a ribonuclease protection assay (ribonase
uclease protection assay) and can be evaluated by inspection of the accessibility to hybridization with complementary oligonucleotides.

[0132] Complementary ribonucleic acid molecules and ribozymes of the invention can be produced by methods well known in the art for synthesizing nucleic acid molecules. These include techniques for the chemical synthesis of oligonucleotides, such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules can be created by in vitro and in vivo transcription of a DNA sequence encoding DRASP. Such a DNA sequence can be incorporated into a variety of vectors with an appropriate RNA polymerase promoter, such as T7 or SP6. Alternatively, these cDNA constructs that synthesize constitutively or inducibly complementary RNA can be introduced into cell lines, cells or tissues.

[0133] RNA molecules can be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5 'and / or 3' end of the molecule, phosphorothioate or 2'O instead of phosphodiesterase linkages in the backbone of the molecule. -Including using methyl. This concept is unique in PNA production, as well as acetyl-, methyl-, thio-, and similar modified forms of adenine, cytidine, guanine, thymine, and uridine that are not readily recognized by endogenous endonucleases. It can be extended to all these molecules by including less commonly used bases such as, inosine, queosine, and wybutosine.

A number of methods are available for introducing vectors into cells or tissues, and are also suitable for use in vivo , in vitro , and ex vivo . For ex vivo therapy, the vector can be introduced into stem cells taken from a patient and propagated as a clone for autologous transplantation and return to the same patient. Delivery by transfection, or liposome injection or delivery by polycationic amino polymer can be performed using methods well known in the art (eg, Goldman, CK et al. (1997) Nature Biotechnology 15: 462-). 466).

Any of the foregoing treatments can be applied to any subject in need of treatment, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably humans.

In a further embodiment of the invention, the pharmaceutical ingredient is administered with a pharmaceutically acceptable carrier, for any of the therapeutic effects described above. Such a pharmaceutical ingredient is DRASP, DRASP
Antibodies, mimics, agonists, antagonists or inhibitors of DRASP. The component is administered alone or in combination with one or more other agents, such as a stabilizing formulation, to any sterile, biocompatible pharmaceutical carrier, which includes, but is not limited to, saline , Buffered saline, glucose or water. Such compositions may be administered to the patient alone or in combination with other drugs, drugs or hormones.

The route of administration of the pharmaceutical composition used in the present invention is not limited to the following, but is oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal,
Intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration can be included.

In addition to the active ingredient, these pharmaceutical ingredients include suitable pharmaceutically acceptable carriers, including excipients and adjuvants that facilitate the processing of the active ingredient into pharmaceutically usable formulations. sell. Further details on techniques for formulation or administration can be found in the latest edition of Remingtons Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).

Pharmaceutical compositions for oral administration are formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. With such a carrier, the pharmaceutical composition can be prepared into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like for ingestion by a patient.

Pharmaceutical preparations for oral administration are prepared by mixing the active ingredient with solid excipients and (optionally after grinding), treating the resulting mixture of granules in tablets or dragee cores.
gee core). If necessary, suitable additives can be added. Suitable excipients include sugar or protein excipients, such as sugars including lactose, sucrose, mannitol or sorbitol, starch from corn, wheat, rice, potatoes, etc., methylcellulose, hydroxypropylmethylcellulose or carboxyl. Cellulose, such as sodium methylcellulose; gums, such as gum arabic or tragacanth; and proteins, such as gelatin or collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof such as sodium alginate.

Dragee cores are used with suitable shells, such as concentrated sugar solutions, and include gum arabic, talc, polyvinylpyrrolidone, carbopol gel.
l), polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures and the like. Dyestuffs or pigments may be added to the tablets or dragees for tablet identification or to characterize the amount of active ingredient (ie, dosage).

Formulations that can be administered orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a tablet, such as glycerol or sorbitol. Push-fit capsules can contain the active ingredient in admixture with excipients or binders such as lactose or starch, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquids, or liquid polyethylene glycol, with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active ingredients may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or excipients include fatty oils such as sesame oil, and synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Also, non-lipid polycationic amino polymers are used for delivery.
If desired, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

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

The pharmaceutical composition of the present invention can be prepared by a known method, for example, a usual mixing treatment, dissolution treatment, granulation treatment, sugar coating formation treatment, wet grinding treatment (levigating), emulsification treatment, encapsulation treatment,
It is manufactured by entrapping or freeze-drying. The pharmaceutical composition may be provided as salts and may 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 tend to be more soluble in aqueous or protonic solvents than the corresponding free base forms. In another case, a suitable formulation would be 1 mM to 50 mM histidine, 0.1% to 2% sucrose, 2% to 7% mannitol, buffered before use, in the range of pH 4.5 to 5.5. A freeze-dried powder containing any or all of them may be used.

After the pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. In the case of DRASP administration, such labels indicate the dosage, frequency of administration, and method of administration.

Pharmaceutical compositions suitable for use in the present invention include those that contain the active ingredient in an effective amount to achieve the desired purpose. Determination of an effective amount can be made well within the capabilities of those skilled in the art.

For any compound, the therapeutically effective amount can be estimated initially either in animal models, such as, for example, mice, rabbits, dogs, pigs, or in cell culture assays of neoplastic cells. In addition, animal models can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine dosages and routes for administration in humans.

A therapeutically effective amount refers to, for example, the amount of the active ingredient of DRASP or a fragment thereof, a DRASP antibody, a DRASP agonist, a DRASP antagonist, or a DRASP inhibitor that ameliorates a symptom or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED ₅₀ (therapeutically effective dose in 50% of the population) or LD ₅₀ (lethal dose for 50% of the population Amount) can be determined by calculating the statistic. Dose ratio of therapeutic to toxic is the therapeutic index and it can be expressed as the ED ₅₀ / LD ₅₀ ratio. Preferably, the pharmaceutical composition exhibits a large therapeutic index. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for human use. The dosage of such components is less toxic or no toxicity is preferably in the range of concentrations circulating containing ED _50. Depending on the dosage form employed, the sensitivity of the patient and the route of administration, the dosage will vary within this range.

The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient, ie, to maintain the desired effect. Factors to consider include disease severity, overall subject health, age, weight, gender, diet, time and frequency of administration, concomitant medications, sensitivity, and response to treatment. It is. Long acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.

Typical dosage amounts range from 0.1 to 100,000 μg, depending on the route of administration, up to a total dose of about 1 g. Guidance on specific dosages or methods of delivery is provided in the literature and is generally available to physicians in the art. One skilled in the art can employ different formulations for nucleotides than for proteins and inhibitors. Similarly, delivery of a polynucleotide or polypeptide can be specific for a particular cell, condition, location, etc.

Diagnosis In another embodiment, an antibody that specifically binds to DRASP has been diagnosed with a condition or disease characterized by expression of DRASP, and has been treated with DRASP or an agonist, antagonist, or inhibitor of DRASP. It can be used in assays for patient monitoring. Antibodies useful for diagnostic purposes can be made in a manner similar to that for therapeutics described above. Assays for the diagnosis of DRASP include methods that use antibodies and labels to detect DRASP in extracts of human body fluids, cells or tissues. Antibodies can be used with or without modification,
In addition, labeling can be performed by binding to a reporter molecule through either a covalent bond or a non-covalent bond. A variety of reporter molecules known to those of skill in the art may be used, some of which are described above.

A variety of protocols for measuring DRASP, including ELISA, RIA and FACS, are well known in the art and provide the basis for diagnosing changes in DRASP expression or levels of abnormalities . A normal, or standard, value for DRASP expression can be established by combining a DRASP antibody with a body fluid or cell extract obtained from a normal mammal, preferably a human, under conditions suitable for complex formation. . Standard complex formation can be quantified using various methods, preferably using photometric means. The amount of DRASP expressed in control and diseased samples from the subject's biopsy tissue is compared to a standard value. The parameter for disease diagnosis is established by the deviation between the standard value and the value of the subject.

In another embodiment of the present invention, a polynucleotide encoding DRASP can be used for diagnostic purposes. Polynucleotides used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. This polynucleotide is used to detect and quantify gene expression in biopsy tissues where DRASP expression may be associated with disease. Diagnostic assays can be used to identify whether DRASP is present, absent, or overexpressed, and to monitor modulation of DRASP levels during therapeutic treatment.

In one embodiment, nucleic acid sequences encoding DRASP can be identified using hybridization with a PCR probe capable of detecting a polynucleotide sequence, including a genomic sequence, encoding DRASP or a closely related molecule. . The specificity of the probe, ie, whether the probe is derived from a very highly specific region (eg, the 5 'regulatory region) or a less specific region (eg, a conserved motif). Depending on the stringency of the hybridization or amplification (maximum, high, medium, or low)
A determination is made whether to identify only the naturally occurring sequence encoding ASP, or to identify alleles and related sequences.

Probes can also be used to detect related sequences, and should preferably have at least 50% sequence identity to any sequence encoding DRASP. The hybridization probe of the present invention is DNA or RNA, or derived from the sequence of SEQ ID NO: 6-10, or an intron of the DRASP gene,
It may be derived from a genome sequence including an enhancer and a promoter.

Means for generating specific hybridization probes for DNA encoding DRASP include methods in which a polynucleotide sequence encoding DRASP or a DRASP derivative is cloned into a vector to generate an mRNA probe. is there.
Such vectors are well known to those of skill in the art and are commercially available, and can be used to generate RNA in vitro by adding an appropriate RNA polymerase or appropriate labeled nucleotides.
It can be used to synthesize a probe. Hybridization probes can be labeled by various reporter groups, for example, the label can be from a radionuclide such as ³² P or ³⁵ S, or an enzyme such as alkaline phosphatase that binds to the probe via an avidin / biotin binding system. And the like.

[0158] A polynucleotide sequence encoding DRASP can be used for diagnosis of a disease associated with expression of DRASP. Examples of such diseases include, but are not limited to, tubular acidosis, anemia, achondroplasia dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, dysplasia, Smith-Magenis syndrome, Spinal dysplasia syndrome, hereditary mucosal epithelial dysplasia, hereditary keratosis, hypothyroidism, hydrocephalus, seizures (eg, chorea and cerebral palsy), spina bifida, anencephaly, cranial spondylolysis, congenital glaucoma , Cataracts and other disorders, as well as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (
MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and adenocarcinoma, leukemia (including Bloom syndrome), lymphoma, melanoma, myeloma, multiple Sexual endocrine tumors (Werner syndrome), sarcomas, teratocarcinomas, and especially adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary, Proliferative disorders such as cancer of the pancreas, parathyroid, penis, prostate, salivary gland, skin, spleen, testis, thymus, thyroid, and uterus, as well as acquired immunodeficiency syndrome (AIDS), Addison's disease, and adult respiratory distress syndrome Allergy, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic Dermatitis, skin muscle , Diabetes mellitus, emphysema, lymphocotoxin temporary lymphopenia, fetal erythroblastosis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, peracid Polycythemia, irritable bowel syndrome,
Multiple sclerosis, myasthenia gravis, inflammation of myocardium or pericardium, osteoarthritis, osteoporosis, pancreatitis, multiple myositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, systemic sclerosis Sickness, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, and complications of cancer, hemodialysis, and extracorporeal circulation, and viruses, bacteria, fungi, parasites, protozoa, and parasites Includes worm-like infections and autoimmune abnormalities / inflammatory diseases such as trauma. DRASP-encoding polynucleotide sequences can be obtained by Southern blot or northern analysis using patient biopsies or body fluids, dot blots or other membrane-based techniques,
Changes in DRASP expression can be detected using PCR techniques, dipstick, pins, ELISA assays, and microarrays. Such qualitative or quantitative test methods are well known to those skilled in the art.

In certain embodiments, nucleotide sequences encoding DRASP may be useful, particularly in assays that detect the presence of a related disorder as described above. The nucleotide sequence encoding DRASP can be labeled by standard techniques and added to body fluids or tissue samples from patients under conditions suitable for forming hybridization complexes. After an appropriate incubation period, the sample is washed and the signal is quantified and compared to a standard value. If the amount of signal in the patient's sample has changed significantly relative to the control sample, a change in the level of the nucleotide sequence encoding DRASP in the sample is indicative of the presence of the associated disease. Such assays can also be used to evaluate the effectiveness of a particular therapeutic treatment in animal studies, clinical trials, or monitoring the treatment of an individual patient.

To provide a basis for the diagnosis of diseases associated with the expression of DRASP, normal,
That is, a standard expression profile is established. This is accomplished by combining a bodily fluid or cell extract from a normal subject, either an animal or a human, with a DRASP-encoding sequence or fragment thereof under conditions suitable for hybridization or amplification. Standard hybridization can be quantified by comparing values obtained from a normal subject to values obtained from experiments using known amounts of substantially purified polynucleotides. The standard values thus obtained from a normal sample can be compared to values obtained from a sample of a patient with signs of disease. Deviation from standard values is used to prove the presence of the disease.

Once the presence of the disease has been confirmed and the treatment protocol initiated, the hybridization assay is repeated periodically to determine whether the level of expression in the patient has begun to approach that observed in normal patients. Can be evaluated. The results from successive assays can be used to show the efficacy of treatment over days to months.

For cancer, the presence of abnormal amounts of transcripts (insufficient or over-expressed) in biopsy tissue from an individual indicates a predisposition to the development of the disease, ie, the actual clinical symptoms are It can be a means to detect disease before it appears. This type of more definitive diagnosis allows health professionals to take preventive measures and provide more aggressive treatment earlier, thus preventing the development and further progression of cancer .

[0163] Uses for oligonucleotides designed from sequences encoding DRASP for further diagnosis include the use of PCR. These oligomers may be chemically synthesized, made with enzymes, or made in vitro . The oligomer is preferably a fragment of a polynucleotide encoding DRASP, or DRA.
It contains a fragment of a polynucleotide that is complementary to the polynucleotide encoding SP, and is used under optimized conditions to identify a particular gene or condition. Oligomers can also be used under mild stringency conditions for the detection and / or quantification of closely related DNA or RNA sequences.

Methods used to quantify DRASP expression also include the use of radiolabeled or biotinylated nucleotides, the use of co-amplification of control nucleic acids,
And use of experimental results interpolated with standard calibration curves (eg, Melby, PC
(1993) J. Immunol. Methods, 159: 235-244; Duplaa, C. et al. (1993) Anal. Bio
chem. 229-236). The rate of quantification of large numbers of samples is such that the oligomer of interest is present in various dilute solutions and is quickly quantified by spectrophotometric or colorimetric response
It can be accelerated by performing an ELISA format assay.

In another embodiment, oligonucleotides or longer fragments from any of the polynucleotide sequences disclosed herein can be used as targets in a microarray. Microarrays can be used to simultaneously monitor the expression levels of many genes and identify mutated sequences, mutations, and polymorphisms in genes. This information is useful in determining gene function, understanding the genetic basis of disease, diagnosing disease, and developing therapeutics and monitoring their activity.

Microarrays may be prepared and used to analyze by methods well known to those skilled in the art (eg, Brennan, TM et al. (1995) US Patent NO. 5,474,796; Schena, M., et al.
(1996) Proc. Natl. Acad. Sci. 93: 10614-10619; Baldeschweiler et al. (1995) P
CT application WO95 / 251116; Shalom D. et al. (1995) PCT application WO95 / 355
05: Heller. RA et al. (1997) Proc. Natl. Acad. Sci. 94: 2150-2155; and Hel.
ler. MJ et al. (1997) US Patent No. 5,605,662).

In another embodiment of the invention, a nucleic acid sequence encoding DRASP can be used to generate hybridization probes useful for mapping naturally occurring genomic sequences. This sequence can be mapped to a particular chromosome, a particular portion of the chromosome, or an artificial chromosome product, wherein the artificial chromosome product comprises:
For example, there are human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1 constructs or single-stranded chromosomal cDNA libraries (eg, H
arrington.JJ et al. (1997) Nat Genet. 15: 345-355; Price, CM (1993) Blood.
Rev. 7: I27-134; and Trask. BJ (1991) Trends Genet. 7: 149-154)
.

Fluorescent in situ hybridization (FISH) may be associated with other physical chromosome mapping techniques and genetic map data (eg, Heinz-Ulrich et al. (1995) in M.
eyers, supra , pp. 965-968). Examples of genetic map data can be found in various scientific journals or in Online Mendelian Inheritance in Man (OMIM). The location of the DRASP-encoding sequence on the physical chromosomal map and its association with a particular disease, or with a predisposition to a particular disease, helps to define the region of DNA involved in the disease.
The nucleotide sequence of the present invention can be used to detect differences in gene sequences between normal individuals, carriers, and affected individuals.

To expand the genetic map, physical mapping techniques such as in situ hybridization of chromosomal preparations and linkage analysis using established chromosomal markers can be used. In many cases, even if the number or arm of a particular human chromosome is unknown, the placement of the gene on the chromosome of another mammal, such as a mouse, will reveal relevant markers. By physical mapping, new sequences can be assigned to chromosomal arms, or parts thereof. This can provide valuable information to researchers searching for disease genes using positional cloning or other gene discovery techniques. Once a disease or syndrome has been identified in a particular genomic region (eg, 11q2
Incomplete positioning by genetic linkage to ataxia telangiectasia 2-23), any sequence mapped to that region represents a relevant or regulatory gene for further study. (Eg, Gatti, RA et al. (1988
) See Nature 336: 577-580). In addition, using the nucleotide sequence of the present invention, it is also possible to detect a difference between the position of a chromosome of a normal person and the position of a chromosome caused by translocation, inversion or the like of a carrier or an affected individual.

In another embodiment of the present invention, DRASP, its catalytic or immunogenic fragments, or oligopeptides thereof, are used for screening libraries of compounds in various drug screening techniques. Can be. The fragments used in such a screen can be free in solution, attached to a solid support, attached to a cell surface, or located intracellularly. The formation of a binding complex between DRASP and the agent to be tested can be measured.

[0171] Alternative drug screening techniques provide high-throughput screening for compounds with appropriate binding affinity for the protein of interest (eg,
(See Geysen et al. (1984) PCT application WO 84/03564). In this method, many different small test compounds are synthesized on a solid substrate. The test compound is reacted with DRASP or a fragment thereof and washed. Next, the combined DRASP is prepared by methods known in the art.
Is detected. Also, purified DRASP can be directly coated on a plate for use in the aforementioned drug screening techniques. Alternatively, the peptide can be captured and immobilized on a solid support using a non-neutralizing antibody.

In another example, a competitive drug screening assay can be used in which neutralizing antibodies capable of binding to DRASP specifically compete with a test compound for binding of DRASP. In this way, antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with DRASP.

In yet another embodiment, the new technology is based on characteristics of currently known nucleotide sequences, including, but not limited to, the triplet genetic code and specific base pair interactions. If so, the nucleotide sequence encoding DRASP can be used for molecular biology techniques that have not yet been developed.

Those skilled in the art will be able to utilize the present invention with the foregoing description without further explanation. Accordingly, the specific examples described below are merely illustrative and do not limit the present invention.

The patent applications, patents, publications, and US patent applications (attorney specification # PF-0563P; filed on August 28, 1998) and US patent applications described above and all patent applications described below (Attorney's Statement # PF-0580P; filed on August 7, 1998) is hereby incorporated by reference.

[0176]

【Example】

1 Preparation of cDNA library RNA was purchased from Clontech or isolated from the tissues shown in Table 4. Some tissues are homogenized and dissolved in a guanidinium isothiocyanate solution, while other tissues are homogenized and dissolved in phenol or TRIZOL (Life
Technologies), a single phase solution of guanidinium isothiocyanate and phenol. The resulting lysate was centrifuged on a CsCl cushion or extracted with chloroform. RNA was precipitated from the lysate with either isopropanol or sodium acetate and ethanol, or another conventional method.

[0177] Phenol extraction and precipitation of the RNA were repeated as necessary to increase the purity of the RNA.
In some cases, RNA was treated with DNase. For most libraries, olig
od (T) -bound paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatswo
rth CA) or OLIGOTEX ^™ mRNA purification kit (QIAGEN) to isolate poly (A +) RNA. Alternatively, for example, POLY (A) PURE mRNA purification kit (Ambion, Austin TX)
RNA was isolated directly from tissue lysates using another RNA isolation kit such as.

In some cases, RNA was provided to Stratagene to create a corresponding cDNA library. Otherwise, UNIZAP ^™ vector system (Stratagene)
Alternatively, cDNA was synthesized using the SUPERSCRIPT ^™ plasmid system (Life Technologies) by a recommended method known to those skilled in the art or by a similar method to prepare a cDNA library. (See, for example, Ausubel, 1997, supra , units 5.1-6.6). oligo d (
Reverse transcription was initiated using T) or random primers. After binding of the synthetic oligonucleotide adapter to the double-stranded cDNA, the cDNA was digested with the appropriate restriction enzyme (s). Most libraries use SEPHACRYL S1000, SEPHAROSE CL2B or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech),
Or size of cDNA by agarose gel electrophoresis for separation (300-1000bp)
Was selected. Suitable plasmids (eg, pBLUESCRIPT (Stratagene), pSPORT1
Plasmid (Life Technologies) or pINCY (Incyte Pharmaceuticals Inc., Pa.
lo Alto CA), etc.) to the compatible restriction enzyme site of the polylinker. The recombinant plasmid is, for example, XL1-Blue, XL1-BlueMRF, or SOLR (Stratagen
e) or transformed into competent E. coli cells such as DH5α, DH10B, or ElectroMAX DH10B (Life Technologies).

2 Isolation of cDNA Clones Plasmids were recovered from host cells by excision in vivo using the UNIZAP vector system (Stratagene) or cell lysis. Magic or WIZARD Mini
preps DNA Purification System (Promega), AGTC Miniprep Purification Kit (Edge Biosyste
ms, Gaithersburg MD) and QIAGEN's QIAWELL 8 Plasmid, QIAWELL 8 Plus P
Plasmids were purified using at least one of lasmid, QIAWELL 8 Ultra Plasmid purification system or REAL Prep 96 plasmid kit (QIAGEN). After precipitation, they were resuspended in 0.1 ml of distilled water and stored at 4 ° C. with or without lyophilization.

Alternatively, plasmid DNA was amplified from host cell lysates by direct binding PCR in a high-throughput format (Rao, VB (1994) Anal. Blochem. 216: 1-1).
Four). The lysis and thermal cycling process of the host cells was performed in a single reaction mixture. Samples are processed and stored in 384-well plates and the concentration of amplified plasmid DNA is measured using PICOGREEN dye (Molecular Probes, Inc., Eugene, OR) and a Fluoroskan II fluorescence scanner (Labsystems Oy, Helsinki, Finland). It was measured quantitatively.

3 Sequencing and Analyzes cDNA for ABI CATALYST 800 (Perkin-Elmer) or HYDRA microdispenser (Robbins Scientific, Sunnyvale CA) or MICROLAB 2
200 (Hamilton, Reno, NV) system and PTC-2000 thermal cyclers (MJ Research
, Watertown MA). cDNA was sequenced using the ABI PRISM 373 or 377 sequencing system (Perkin-Elmer) and standard ABI protocols, base pairing software, and kits. In one embodiment, the MEGABACE 1000 Capillary Electrophoresis Sequencing System (Molecular Dyna
mics, Sunnyvale CA). In another embodiment, the ABI PRISM BIGDYE Terminator cycle swquencing ready reaction kit (P
erkin-Elmer) for cDNA amplification and sequencing. In yet another embodiment, cDNA sequencing was performed using Amersham Pharmacia Biotech solutions and dyes. Reading frames for ESTs were determined using standard methods (Ausubel, 1997, supra , a review of unit 7.7). Some of the DNA sequences were selected and extended as described in 5 of this example.

The cDNA, extension, and polynucleotide sequences obtained from the shotgun sequencing were assembled and analyzed using a combination of software programs utilizing algorithms well known to those skilled in the art. Table 5 summarizes the software programs used, descriptions, references, and threshold parameters. The first column of Table 5 is the tools, programs, and algorithms used, the second column is a brief description of them, and the third column is a reference (all of which are incorporated herein by reference. Part of)
And the fourth column is the appropriate score, probability value, and other parameters used to assess the degree of correspondence between the two sequences (higher established values indicate higher homology). The array is MACD
Analysis was performed using NASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).

Confirmation of the polynucleotide sequence can be accomplished by removing vectors, linkers, and polyA sequences using programs and algorithms based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis to remove ambiguous base pairs. Performed by masking. The sequence was then converted to GenBan using programs based on BLAST, FASTA, and BLIMPS.
k primates, rodents, mammals, vertebrates, and eukaryote databases and BLOCK
Annotations were obtained by querying sequences selected from public databases such as the S database. Sequences were assembled into full-length polynucleotides using programs based on Phred, Phrap, and Consed, and screened against open reading frames using programs based on GeneMark, BLAST, and FASTA. The full-length polynucleotide sequence is translated to derive the corresponding full-length amino acid sequence, which is then translated into the GenBank database (described above), SwissProt, BLOCKS,
Analyzed by querying protein family databases such as PRINTS, Prosite, and PFAM.

The programs described above for the construction and analysis of full-length polynucleotide and amino acid sequences were also used to identify fragments of the polynucleotide sequence from SEQ ID NOs: 8-14. Fragments of about 20 to about 4000 nucleotides useful for hybridization and amplification have been described in the " Invention " section above.

4 Northern Analysis Northern analysis is an experimental technique used to detect the presence of a gene transcript, in which a membrane bound to RNA from a particular cell type or tissue is labeled and labeled. involving hybridization with nucleotide sequences (e.g., Sambrook et al., supra, ch.7; supra and Ausubel, FM et al., see ch.4 and 16.).

Using a similar computer technology using BLAST, GenBank or LIFESEQ ^™
Search for identical or related molecules in a nucleotide database such as a database (Insight). This analysis is faster than many membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is classified as an exact match or similar. The search criteria are based on the product sco
re). (% Sequence identity x% max 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 accurate to within 1-2% error, and for a product score of 70, it is an exact match. Similar molecules are usually identified by selecting those molecules that exhibit a product score between 15 and 40, but lower scores identify them as related molecules.

[0187] The results of the Northern analysis are reported as a distribution of the percentage of libraries in which transcripts encoding DRASP occurred. Analyzes include classification of cDNA libraries by organ / tissue as well as disease. Organ / tissue categories include cardiovascular, skin, developmental,
Includes endocrine, gastrointestinal, hematopoietic / immune, musculoskeletal, neural, reproductive, urine. Disease / disorder / symptom categories include cancer, inflammation / trauma, cell proliferation, neurology, and retention (poo
led) is included. For each category, the libraries expressing the sequence of interest were counted and divided by the total number of libraries across categories. Table 3 shows the tissue specificity as well as the percentage of expressed diseases.

5 Extension of the Polynucleotide Encoding DRASP The full length nucleic acid sequence (SEQ ID NOs: 6 and 7) was extended using the oligonucleotide primers based on these fragments to extend the clones described in the "Invention" section. Generated by One primer was synthesized to initiate extension of the antisense polynucleotide and the other primer was synthesized to initiate extension of the sense polynucleotide. The use of primers facilitated "extending" the known sequence and generated amplicons containing new unknown nucleotide sequences for the region of interest. Use the initial primers in OLIGO 4.06 software (Na
(National Biosciences, Plymouth, MN) or other suitable program.
~ 30 nucleotides in length, having a GC content of 50% or more, and about 6
The cDNA was designed to anneal to the target sequence at a temperature of 8-72 ° C. Hairpin structure and any extension of nucleotides resulting in primer-primer dimer was avoided.

The sequences were extended using a selected human cDNA library (Life Technologies). If more than one extension is necessary or desired, another set of primers is designed to further extend the known region.

Following the instructions for the XL-PCR kit (The Perkin Elmer Corp., Norwalk, Conn.), Thorough mixing of the enzyme and reaction mixture resulted in highly compatible amplification.
PCR was performed using a PTC-200 thermal cycler (MJ Research, Inc., Watertown, Mass.), With the following parameters starting at 40 pmol of each primer and all other components of the kit at recommended concentrations. Step 1 94 ° C for 1 minute (initial denaturation) Step 2 65 ° C for 1 minute Step 3 68 ° C for 6 minutes Step 4 94 ° C for 15 seconds Step 5 65 ° C for 1 minute Step 6 68 ° C for 7 minutes Step 7 Step 4 Repeat Steps 6 to 15 for further 15 cycles Step 8 94 ° C for 15 seconds Step 9 65 ° C for 1 minute Step 10 68 ° C for 7 minutes 15 seconds Step 11 Repeat Steps 8 to 10 for 12 cycles Step 12 72 ° C for 8 minutes Step 134 5 ° C. (hold) An aliquot of 5-10 μl of the reaction mixture was analyzed by electrophoresis on a low-concentration (about 0.6-0.8%) agarose minigel to determine which reaction was successful in extending the sequence. The band believed to contain the largest product was excised from the gel and QIA Quic
It was purified using a k purification kit (QIAGEN Inc.) and the overhang was excised using Klenow enzyme to facilitate religation and cloning.

After ethanol precipitation, the product was redissolved in 13 μl ligation buffer and 1 μl T
4-DNA ligase (15 units) and 1 μl of T4 polynucleotide kinase were added and the mixture was incubated for 2-3 hours at room temperature or overnight at 16 ° C. Competent E. coli cells (in 40 μl of the appropriate culture) were transformed with 3 μl of the ligation mixture and cultured in 80 μl of SOC culture (eg, Sambrook, supra, Appendix A, p. 2).
reference). After incubating at 37 ° C. for 1 hour, the E. coli mixture was purified from Luria Bertani (LB) -agar containing carbenicillin (2 × carb) (eg, Sambrook, supra, Append).
ix A, p. 1). The following day, several colonies were randomly selected from each plate and cultured in 150 μl of liquid LB / 2x carb medium in individual wells of an appropriate commercially available sterile 96-well microtiter plate. The following day, 5 μl of each overnight culture was transferred to a non-sterile 96-well plate, diluted 1:10 with water, and then 5 μl from each sample was transferred to a PCR array.

For PCR amplification, 18 μl containing vector primers, one or both gene-specific primers used for the extension reaction, and 4 units of rTth DNA polymerase
Of concentrated PCR reaction mixture (3.3x) was added to each well. Amplification was performed under the following conditions. Step 1 94 ° C for 60 seconds Step 2 94 ° C for 20 seconds Step 3 55 ° C for 30 seconds Step 4 72 ° C for 90 seconds Step 5 Repeat steps 2 to 4 for another 29 cycles Step 6 72 ° C for 180 seconds Step 7 4 ° C An aliquot of the PCR reaction (retained) was run on an agarose gel with a molecular weight marker. The size of the PCR product was compared to the original partial cDNA, the appropriate clone was selected, ligated to the plasmid and sequenced.

Similarly, using the nucleotide sequences of SEQ ID NO: 6 and SEQ ID NO: 7, the above procedure, oligonucleotides designed for 5 ′ extension, and 5 ′ regulatory sequences using appropriate genomic libraries Is obtained.

The full-length nucleic acid sequence of SEQ ID NOs: 8-10 was created by extending an appropriate fragment of the full-length molecule and designing oligonucleotide primers from this fragment. One primer was synthesized to initiate a 5 'extension of the known fragment and the other primer was synthesized to initiate a 3' extension of the known fragment. The starting primer is OLIG
O Using 4.06 software (National Biosciences) or other suitable program, a length of about 22-30 nucleotides, with a GC content of 50% or more, and a temperature of about 68-72 ° C. Was designed from the cDNA to anneal to the target sequence. Hairpin structure and any extension of nucleotides resulting in primer-primer dimer was avoided.

This sequence was extended using a selected human cDNA library. If more than one step extension is necessary or desirable, additional or nested sets of primers are designed.

High fidelity amplification was performed by PCR using methods well known to those skilled in the art. PCR is
Performed in a 96-well plate using a PTC-200 thermal cycler (MJ Research, Inc.). The reaction mixture contains a DNA template, 200 nmol of each primer, a reaction buffer containing Mg ²⁺ , (NH ₄ ) ₂ SO ₄ , and β-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE Includes enzymes (Life Technologies) and Pfu DNA polymerase (Stratagene). Primer set PCI A and
The following parameters were used for PCI B: 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 Store at 4 ° C. or use the following parameters for primer set T7 and SK +. 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 is determined by adding 100 μl of PICO GREEN quantification reagent (0.25% (v / v) PICOGREEN; Molecular Probes, E) in 1 × TE and 0.5 μl of undiluted PCR product.
ugene OR) was dispensed into each well of an opaque fluorometer plate (Coming Costar, Acton MA), and the DNA was measured to allow binding to the reagent. Add this plate to Flu
Scan with oroskan II (Labsystems Oy, Helsinki, Finland) to measure sample fluorescence and quantify DNA concentration. An aliquot of 5-10 μl 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, transferred to a 384-well plate, and
Cholera virus endonuclease (Molecular Biology Research, Madison WI
) And sonication or shearing was performed before religation into the pUC18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on a low concentration (0.6-0.8%) agarose gel, the fragments were excised, and the agar was digested with Agar ACE (Promega). Expanded clones into T4 ligase (New
PUC18 vector (Amersham Pharmacia) using England Biolabs, Beverly MA
Biotech), treated with Pfu DNA polymerase (Stratagene) to fill in restriction site overhangs, and transfected into competent E. coli cells. Transfected cells are selected on medium containing antibiotics, individual colonies are selected and LB / 2
The cells were cultured in a 384-well plate of X carb medium at 37 ° C. overnight.

Cells were lysed and Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu
DNA was amplified by PCR using DNA polymerase (Stratagene) 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 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 low DNA recovery were re-amplified under the above conditions. Dilute the sample with 20% dimethylsulphoxide (1: 2, v / v) and use DYENAMIC energy trans
fer sequencing primer and DYENAMIC DIRECT kit (Amersham Pharmacia
Biotech) or ABI PRISM BIGDYE Terminator cycle sequencing ready reac
The sequence was determined using the tion kit (Perkin-Elmer).

Similarly, using the nucleotide sequence of SEQ ID NOs: 8-10, 5 ′ using the above procedure, oligonucleotides designed for 5 ′ extension, and appropriate genomic libraries
A regulatory sequence is obtained.

6 Labeling and Use of Individual Hybridization Probes Using the hybridization probes obtained from SEQ ID NOs: 6-10, cDN
A, Screen genomic DNA or mRNA. Although the labeling of oligonucleotides of about 20 base pairs is specifically described, generally the same procedure is used for larger nucleotide fragments. Oligonucleotides can be transferred to OLIGO 4.06 software (Nationa
Designed using software in the art such as Biosciences, 50 pmol of each oligomer, 250 μCi of [γ- ³² P] adenosine triphosphate (Amersham Pharmacia Bi
otech) and T4 polynucleotide kinase (DuPont NEN, Boston MA). Labeled oligonucleotides are applied to SEPHADEX G-25 ultrafine molecular size exclusion dextran bead columns (Amersham Pharmacia Biotech)
Substantially purify using. Aliquots containing 10 ⁷ counts of labeled probe per minute were prepared with the following endonucleases, Ase I, Bgl II, Eco RI, Pst I, Xba1,
Alternatively, used for typical membrane-based hybridization analysis of human genomic DNA digested with one of Pvu II (DuPont NEN).

[0201] DNA from each digest was fractionated on a 0.7% agarose gel and run on a nylon membrane (NyD
RASPlus, Schleicher & Schuell, Durham NH). Hybridization is performed at 40 ° C. for 16 hours. The blot is washed continuously at room temperature under increasingly stringent conditions up to 0.1x sodium citrate saline and 0.5% sodium dodecyl sulfate to remove non-specific signals. XOMAT-AR film (Eastman Koda
k, Rochester NY) are exposed to the blots and the hybridization patterns are visualized and compared.

7 Array elements can be synthesized on the surface of a substrate by using a microarray chemical coupling procedure and an inkjet device. (For example, Baldeschweiler
, Supra). Also, using a predetermined array similar to dot blot or slot blot methods, utilizing thermal, UV, chemical or mechanical binding procedures,
Elements may be arranged and coupled to the surface of the substrate. Standard arrays can be manufactured by hand or using convenient methods and machines, and include any suitable number of elements. After hybridization, unhybridized probe is removed and the level and pattern of fluorescence are determined using a scanner. The degree of complementarity and relative abundance of each probe that hybridizes to an element on the microarray is evaluated by analyzing an image read by a scanner.

[0203] Full-length cDNAs, expressed sequence tags (ESTs), or fragments thereof, can include elements of a microarray. Suitable fragments for hybridization can be selected using software such as LASERGENE software (DNASTAR) well known to those skilled in the art. A full-length cDNA, EST, or a fragment thereof corresponding to one of the nucleotide sequences of the present invention, or a random selection from a cDNA library relevant to the present invention, may be obtained by a suitable method such as a glass slide. Place on the substrate. Its cDNA
Are fixed to glass slides, for example, by thermal and chemical treatment after UV crosslinking and further drying (eg, Schena, M. et al. (1995) Science 270: 46).
7-470; and Shalon, D. et al. (1996) Genome Res. 6: 639-645). A fluorescent probe is prepared and used for hybridization with an element on a substrate. The substrate is analyzed by the method described above.

8 Complementary Polynucleotides The sequence complementary to the sequence encoding DRASP, or any portion thereof,
Used to detect and reduce, ie, suppress, RASP expression. Although the use of oligonucleotides containing about 15 to about 30 base pairs is described, essentially the same method is used for smaller or larger sequence fragments. OLIGO 4.
Design appropriate oligonucleotides using 06 software and coding sequences of DRASP. To repress transcription, complement oligonucleotides with the most unique 5
'Designed from sequence and used to prevent binding of the promoter to the coding sequence. Design complementary oligonucleotides to suppress translation
Prevents ribosome binding to the transcript encoding

9 Expression of DRASP Expression and purification of DRASP is performed using a bacterial or viral based expression system. For expression of DRASP in bacteria, the cDNA is subcloned into an appropriate vector containing an antimicrobial resistance gene and an inducible promoter that increases the level of cDNA transcription. Examples of such promoters include, but are not limited to, the T5 or T7 bacteriophage promoter associated with the lac operator regulatory element and the trp-lac (tac) hybrid promoter. The recombinant vector is transformed into a suitable bacterial host, such as BL21 (DE3). Antibiotic-resistant bacteria express DRASP by induction with isopropyl β-D thiogalactopyranoside (IPTG). Expression of DRASP in eukaryotic cells can be expressed in insect or mammalian cell lines.
It is performed by infecting Autographica californica nucleopolyhedrovirus (AcMNPV), commonly known as baculovirus. The non-essential polyhedrin gene of the baculovirus is replaced with the cDNA encoding DRASP by either homologous recombination or bacterial-mediated gene transfer involving a transfer plasmid intermediate. Virus infectivity is maintained and high levels of cDNA are achieved by the strong polyhedrin promoter.
Is performed. In many cases, the recombinant baculovirus is used to infect Spodoptera frug iperda (Sf9) insect cells, but in some cases to infect human hepatocytes. The latter infection requires additional genetic alterations to the baculovirus. (For example, Engelhard. EK et al. (1994) Proc. Natl. Acad. S
ci. USA 91: 3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7: 1937-1945.).

In most expression systems, DRASP is, for example, glutathione S-transferase (
GST), or synthesized as a fusion protein with a peptide epitope tag such as FLAG or 6-His, which allows for rapid, one-step affinity-based purification of recombinant fusion proteins from crude cell lysates. 26 from Schistosoma japonicum
GST, a kilodalton enzyme, allows for the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (
Amersham Pharmacia Biotech). After purification, the GST moiety can be proteolytically cleaved from DRASP at the specific engineered site. It is a peptide of 8 amino acids
FLAG allows for immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues, allows for purification on metal chelating resins (QIAGEN). Ausubel has described the method of protein expression and purification (1995,
Supra , ch 10 and 16). Purified D obtained by these methods
RASP can be used directly in the following assays.

10 Demonstration of DRASP activity The activity of DRASP was determined by incorporation of [ ³² P] dATP into plasmids treated with DNA damage mediators such as cisplatin or ultraviolet radiation relative to control untreated plasmids. Measured (F. Coudore, et al. (1997) FEBS Lett. 414: 581-584).
Purify cell extracts from S. cerevisiae , E. coli , or mammalian cell lines that have endogenous repair activity that has been compromised due to mutations in the repair enzyme. Cell extracts are prepared by centrifugation at 300,000 xg after hypotonic lysis of the cells. The extract is treated with 63% ammonium sulfate to minimize non-specific nuclease activity. The repair synthesis assay is based on 200 μg protein, 300 n
g damaged plasmid, 300 ng control plasmid, 4 μM dATP, 20 μM dCTP each
Performed in a 50 μl reaction volume containing dTTP, dTTP, 0.2 μM [ ³² P] dATP, 20 mM HEPES-KOH (pH 7.8), 2.5 μg creatine phosphokinase, 7 mM MgCl ₂ , and 2 mM EGTA I do. The same reaction was performed with or without purified DRASP. 30
After incubation for 3 hours at 0 ° C., the reaction mixture is treated with 200 μg / ml protein kinase K and 0.5% SDS. Plasmid DNA is purified from the reaction mixture by phenol-chloroform extraction and ethanol precipitation. Data were obtained after gel electrophoresis of the linearized plasmid, densitometry of the gel negative to normalize for plasmid DNA recovery, autoradiography, and excised.
Quantification is performed by performing a scintillation count of the DNA band.

Alternatively, DRASP or a biochemically active fragment is ligated with ¹²⁵ I Bolson-Hunter reagent (Bol
(1973) Biochem. J. 133: 529). Candidate molecules previously placed in the wells of a multiwell plate are incubated with the labeled DRASP, washed, and all wells containing the labeled DRASP complex are assayed. Using the data obtained at the various DRASP concentrations, values for the association, affinity, and number of DRASP with the candidate molecule are calculated.

11 Functional Assays The function of DRASP is to demonstrate that DASP at physiologically elevated levels in mammalian cell culture systems.
Assay by expression of sequence encoding RASP. The cDNA is subcloned into a mammalian expression vector containing a strong promoter that expresses the cDNA at high levels. Selected vectors include pCMV SPORT (Life Technologie) and pCR3.1 (Inv
itrogen, Carlsbad, CA), each of which contains the cytomegalovirus promoter. 5-10 μg of the recombinant vector is transiently transfected into a human cell line, preferably endothelial or hematopoietic, by liposome preparation or electroporation. Also, 1-2 μg of additional 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 and to reliably predict expression of cDNA from a recombinant vector. Selected labeled proteins include green fluorescent protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.
Flow cytometry (FCM), a technology based on automated laser optics
Are used to identify transfected cells expressing GFP or CD64-GFP and to evaluate properties such as apoptotic status. FCM detects and quantifies the uptake of fluorescent molecules that diagnose 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, altered cell surface and intracellular protein expression as measured by reactivity with specific antibodies, and cells with fluorescein-bound annexin V protein Includes changes in plasma membrane composition as measured by binding to the surface. For flow cytometry methods, see Ormerod, MG (1994) Flow Cytometry, Oxford,
New York, NY.

[0210] The effect of DRASP on gene expression can be assessed using DRASP coding sequences and highly purified cell populations transfected with either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on the surface of transformed cells and bind to conserved regions of human immunoglobulin G (IgG). The transformed cells are
Magnetic beads coated with either human IgG or CD64 antibodies can be used to separate from untransformed cells (DYNAL, Lake Success, NY). m
RNA can be purified from cells by methods well known to those skilled in the art. Expression of mRNA encoding DRASP and other genes of interest can be analyzed by Northern analysis or microarray technology.

12 Production of DRASP-Specific Antibodies Polyacrylamide gel electrophoresis (PAGE) (eg, Harrington, MG (19
90) Methods Enzymol. 182: 488-495), or rabbits are immunized using substantially purified DRASP using other purification techniques according to standard protocols to produce antibodies.

Alternatively, the amino acid sequence of DRASP is analyzed using LASERGENE software (DNASTAR) to determine a region having high immunogenicity, and a corresponding oligopolypeptide is synthesized, and this is used to be known to those skilled in the art. The antibody is produced by the method. Details of how to select suitable epitopes, such as those near the C-terminus or within the hydrophilic region, are described in the literature of those skilled in the art (see, for example, Ausubel, 1995, supra , ch. 11).

Typically, an oligopeptide 15 residues in length is converted to ABI 431 using the chemistry of the fmoc method.
A Peptide Synthesizer (Perkin-Elmer) was used for synthesis and MBS (N-maleimidoben)
The reaction using zoyl-N-hydroxysuccinimide ester) resulted in KLH (Sigma, St. Louis,
MO) to enhance immunogenicity (see, eg, Ausubel, supra ). Rabbits are immunized with the oligopeptide-KLH complex in Freund's complete adjuvant. The resulting antiserum is, for example, a peptide bound to plastic, 1%
Tested for anti-peptide activity by blocking with BSA, reacting with rabbit antiserum, washing and reacting with radioiodine-labeled goat anti-rabbit IgG.

13 Purification of Naturally Occurring DRASP Using Specific Antibodies The naturally occurring or recombinant DRASP is substantially purified by immunoaffinity chromatography using an antibody specific for DRASP. The immunoaffinity column uses DRASP antibody with CNBr-activated Sepharose (Amersham Pharmacia Biotech)
It is prepared by covalently binding to an activated chromatography resin as described above. After bonding, block and wash the resin according to the manufacturer's instructions.

The culture solution containing DRASP is passed through an immunoaffinity column, and the column is washed under conditions that allow DRASP to be preferentially adsorbed (eg, a buffer having a high ionic strength in the presence of a surfactant). Conditions that disrupt antibody / DRASP binding (eg, p
The column is eluted with a buffer of H2-3 or a high concentration of chaotrope such as urea or thiocyanate ions and the DRASP is recovered.

14 Identification of Molecules That Interact with DRASP Label DRASP or a biologically active fragment thereof with ¹²⁵ I Bolton-Hunter reagent (eg, Bolton et al. (1973) Biochem. J. 133: 529). reference). Candidate molecules previously placed in the wells of a multi-well plate are incubated with labeled DRASP, washed, and any wells with labeled DRASP complexes are assayed.
Using the data obtained at the various DRASP concentrations, values for the association, affinity, and number of DRASP with the candidate molecule are calculated.

All of the above publications and patent applications are incorporated herein by reference. Those skilled in the art will be able to make various modifications of the methods and systems described herein without departing from the scope and spirit of the invention. Although the invention has been described with respect to particular preferred embodiments, it is to be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the claims.

[0218] BRIEF DESCRIPTION Table 1 Table, ID number (SEQ ID NO) of the polypeptide sequence and nucleotide sequence used to construct the full length sequence encoding DRASP, clone ID number, cDNA
A library and cDNA fragments are shown.

Table 2 shows the characteristics of each polypeptide sequence, including potential motifs and homologous sequences,
And methods and algorithms used to identify DRASP.

Table 3 shows the tissue-specific expression pattern of each nucleic acid sequence determined by Northern analysis, the disease, disorder, or condition associated with these tissues, and the vector into which each DNA was cloned.

Table 4 shows the tissues used for preparing a cDNA library from which a cDNA clone encoding DRASP was isolated.

Table 5 shows the programs used for DRASP analysis, their descriptions, references and parameter thresholds.

[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 5/06 A61P 7/00 4C084 5/14 7/06 4H045 7/00 9/10 101 7/06 11 / 00 9/10 101 11/06 11/00 13/00 11/06 13/12 13/00 15/00 13/12 17/00 15/00 19/00 17/00 19/08 19/00 21 / 00 19/08 21/04 21/00 25/00 21/04 27/06 25/00 27/12 27/06 29/00 27/12 101 29/00 31/04 101 31/10 31/04 31 / 12 31/10 31/18 31/12 33/00 31/18 35/00 33/00 35/02 35/00 37/00 35/02 37/08 37/00 43/00 111 37/08 C07K 14 / 47 43/00 111 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 A 5/10 G01N 33 / 53 M C12P 21/02 33/566 C12Q 1/68 G01N 33/15 Z G01N 33/53 33/50 Z 33/566 (C12P 21 / 02 C // G01N 33/15 C12R 1:91) 33/50 (C12P 21/02 C (C12P 21/02 C12R 1:19) C12R 1:91) C12N 15/00 ZNAA (C12P 21/02 5 / 00 A C12R 1:19) A61K 37/02 (81) Designated country 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, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), 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, ZW Neil Sea, CA 94040, Mountain View # 30, Dale Avenue 1240 (72) Inventor Bogun, Mariah Earl California, USA 94577 San Leandro Santiago Road 14244 (72) Inventor Lady, Loopa California, United States 94086 Sunnyvale West McKinley Dr. Eve 1233 (72) Inventor Gegler, Karl Jay, USA Menor Park Oakland Ave. 94025, California 1048 (72) Inventor Yue, Henry 94087, California, United States Sunnyvale Lewis Avenue 826 F-term (reference) 2G045 AA40 DA12 DA13 FB02 FB03 FB07 4B024 AA01 AA11 AA12 BA45 BA80 CA02 DA02 DA06 EA GA11 GA18 HA01 HA04 HA12 HA15 4B063 QA01 QA18 QA19 QQ42 QQ52 QR32. ZA55 ZA59 ZA66 ZA68 ZA75 ZA81 ZA89 ZA94 ZA96 ZA97 ZB02 ZB11 ZB13 ZB15 ZB21 ZB26 ZB27 ZB33 ZB35 ZB37 ZC06 ZC55 4H045 AA10 AA11 AA20 AA30 BA10 CA40 EA22 EA28 EA50 EA50 EA50 EA50 EA50 EA50 EA50

Claims (20)

[Claims] 1. The method of claim 1, wherein SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO.
: 5 and a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of fragments thereof. 2. A substantially purified variant having at least 90% amino acid sequence identity to the amino acid sequence of claim 1. 3. An isolated and purified polynucleotide encoding the polypeptide of claim 1. 4. A mutant sequence of an isolated and purified polynucleotide having at least 90% or more polynucleotide sequence identity to the polynucleotide of claim 3. 5. An isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide of claim 3. 6. An isolated and purified polynucleotide having a sequence complementary to the polynucleotide sequence of claim 3. 7. A method for detecting a polynucleotide, comprising: (a) hybridizing the polynucleotide of claim 6 with at least one nucleic acid of a sample to form a hybridization complex; (b) A method for detecting the hybridization complex, comprising a detection step in which the presence of the hybridization complex is correlated with the presence of a polynucleotide in the sample. 8. The method of claim 7, further comprising amplifying the polynucleotide before said hybridization. 9. An isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 6-10 and fragments thereof. 10. An isolated and purified polynucleotide variant having at least 90% or more polynucleotide sequence identity to the polynucleotide of claim 9. 11. An isolated and purified polynucleotide having a sequence complementary to the polynucleotide of claim 9. 12. An expression vector comprising at least a fragment of the polynucleotide of claim 3. 13. A host cell comprising the expression vector according to claim 12. 14. A method for producing a polypeptide, comprising: (a) culturing the host cell according to claim 13 under conditions suitable for expression of the polypeptide; and (b) medium for the host cell. And recovering the polypeptide from the protein. 15. A pharmaceutical composition comprising the polypeptide of claim 1 together with a suitable pharmaceutical carrier. 16. A purified antibody that specifically binds to the polypeptide of claim 1. 17. A purified agonist of the polypeptide of claim 1. 18. A purified antagonist of the polypeptide of claim 1. 19. A method for treating or preventing a disease associated with reduced activity or expression of DRASP, which comprises administering an effective amount of the pharmaceutical composition of claim 15 to a patient in need of such treatment. A method comprising the step of administering. 20. A method for treating or preventing a disease associated with increased activity or expression of DRASP, wherein an effective amount of the antagonist of claim 18 is administered to a patient in need of such treatment. A method that includes a process.