JP2002517243A - Cell cycle regulatory proteins - Google Patents

Abstract

  (57) [Summary] The present invention provides human cell cycle regulatory proteins (CECRPs) and polynucleotides that identify and encode CECRPs. The present invention also provides expression vectors, host cells, antibodies, agonists and antagonists. The present invention also provides a method for diagnosing, treating or preventing a disorder associated with the expression of CECRP.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Technical Field The present invention relates to nucleic acid and amino acid sequences of the cell cycle regulatory proteins,
It relates to the use of these sequences in the diagnosis, treatment and prevention of cell proliferative disorders and immune disorders.

[0002] cell division, is a fundamental process for all of the organisms to grow and breed.
In unicellular organisms such as yeasts and bacteria, each cell division doubles the number of organisms, while in multicellular species it produces new tissues and organs and replaces cells lost by aging and programmed cell death. Requires a number of cell divisions.
Although the details of the cell division cycle can vary, the basic process consists of three main steps: The first event is the intercellular phase, which involves preparation for cell division, DNA replication and production of essential proteins. The second process is mitosis, in which nuclear material is split and separated on both sides of the cell. The last process is cytokinesis, where the cytoplasm divides and divides. Division and division may involve the formation of a contractile, longitudinal, annular septum around the cells that allows the mother / daughter cells to separate. The flow and timing of these cell cycle events are under the control of a cell cycle control system that regulates the process at various checkpoints.

[0003] Regulating cell cycle progression relies on integrating growth control pathways with basic cell cycle machinery. Cell cycle regulators have been identified in humans and yeast by selecting cDNAs that, when overexpressed, block or activate cell cycle arrest signals within the yeast mating pheromone pathway.
Known regulators include the human CPR (cell division progression) genes, such as CPR8 and CPR2, and yeast CDs, such as CDC91, that block repressive signals.
There is a C (cell division control) gene. The CPR gene expresses various proteins including cyclins, tumor suppressor binding proteins, chaperones, transcription factors, translation factors, and RNA binding proteins (Edwards, MC et al. (1997) Genetics 147: 1063-
1076).

[0004] Several cell cycle transitions, including the entry and exit of cells from mitosis, depend on the activation and repression of cyclin-dependent kinases (Cdks). Cdka consists of the kinase subunit Cdk and the activating subunit, cyclin, and is regulated at multiple levels in its complex. Budding yeast (S. Cerevisiae
) And fission yeast ( S. pombe ) appear to be one Cds, but mammals have a variety of unique Cdks. Cyclin acts by binding to and activating cyclin-dependent protein kinase, which phosphorylates and activates selected proteins involved in the mitotic process. The Cdk-cyclin complex is activated by phosphorylation and by targeted degradation involving molecules such as CDC4 and CDC53.
It is adjusted to both positive and negative. Furthermore, Cdk is an inhibitor,
And is regulated by binding to other proteins, such as Suc1, which alters its specificity and alters accessibility to the regulator (Patra, D., and Danphy,
WG10 (1996) Genes Dev. 10: 1503-1515; and Mathias, N. et al. (1996) Mo
l. Cell Biol. 16: 6634-6643).

[0005] The regulation of mRNA processing plays an important role in the cell cycle when many transcripts are differentially regulated in individual cell types and tissues. mR
NA processing involves the differential, ie, alternative, splicing of exons, which allows the expression of functionally distinct proteins from similar mRNAs. Constitutive and alternative splicing involves small nuclear ribonucleoproteins (
(snRNP) and non-snRNP proteins in spliceosomes. The SR (serine and arginine-rich) family of non-snRNP splicing factors is required in the early stages of spliceosome assembly and has different but overlapping properties for various pre-mRNAs,
Splicing site selection can be changed. Several mammalian SR proteins have been characterized, such as HRS, ASF / SF2 and SC-35.
The sequence of the HRS contains human ASF / SF2 protein and Drosophila B52 /
Related to SRp55 protein. The latter has been shown to function in vitro to regulate 5-prime splice site selection (Diamond, RH et a).
l. (1993) J. Biol. Chem. 268: 15185-15192).

[0006] Progression of the cell cycle also requires coordinated expression of other proteins. B
The limp-1 regulator appears during the differentiation of B lymphocytes into immunoglobulin-secreting cells. Blimp-1 contains a Kruppel-type Zn finger motif and a proline-rich acidic region similar to regions of known transcription factors.
By stable or transient transfection of Blimp-1 into B lymphoma cell lines,
Many phenotypic changes associated with B cell differentiation into early plasma cell stages are expressed, such as induction of chain messages and immunoglobulin secretion, up-regulation of syndecan-1, and increased cell size and granularity. Inhibin, a number of transforming growth factor β-like families, has been shown to regulate cell differentiation and proliferation by antagonizing the DNA synthesis activity of activin (Turner,
CA et al. (1994) Cell (1994) 77: 297-306; and Matzuk, MM et al. (199
4) Proc.5 Nat. Acad. Sci. 91: 8817-8821).

[0007] The progress of the cell cycle and thus cell proliferation is governed by the complex interactions of protein complexes consisting of cyclins, cyclin-dependent protein kinases, cell growth factors, binding of inhibitory polypeptides, and related proteins. Cancer and immune disorders are characterized by uncoordinated cell growth. Certain cell proliferative disorders can be identified by changes in protein complexes that normally control cell cycle progression. The main therapeutic strategy involves re-establishing control over the course of the cell cycle by manipulating proteins involved in cell cycle regulation (Nigg, EA (1995) BioEssays 17: 471-480). ).

[0008] The discovery of novel cell cycle regulatory proteins and polynucleotides encoding them can provide novel compositions useful in the diagnosis, treatment and prevention of cell proliferative disorders and immune disorders, a need in the art. To be satisfied.

SUMMARY OF THE INVENTION The present invention is collectively referred to as "CECRP" and is referred to individually as "CECRP-1", "CECRP",
"CECRP-2", "CECRP-3", "CECRP-4" and "CECRP"
-5, characterized by a substantially purified polypeptide, a cell cycle regulatory protein. In one aspect, the present invention provides a method comprising SEQ ID NO: 1, SEQ ID NO:
NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO
: 5 (SEQ ID NO: 1 to SEQ ID NO: 5), and fragments thereof, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of:

[0010] The present invention further provides substantially purified variants having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 5, or a fragment of any of these sequences. I do. The present invention also relates to SEQ
Provided is an isolated and purified polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of ID NO: 1 to SEQ ID NO: 5, and fragments thereof. In addition, the present invention provides SEQ ID NO: 1 to S
At least 70% for a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of EQ ID NO: 5 and fragments thereof
Includes isolated and purified polynucleotide variant sequences having polynucleotide sequence identity.

Further, the present invention hybridizes under stringent conditions to a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5, and fragments thereof. An isolated and purified polynucleotide, as well as SEQ ID NO: 1 through SEQ ID NO:
5, and a sequence complementary to a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of:
Provide a purified polynucleotide.

[0012] Also, the present invention provides a method according to SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 7,
NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 (SEQ ID N
O: 6 to SEQ ID NO: 10), and fragments thereof, provided isolated and purified polynucleotides comprising a polynucleotide sequence selected from the group consisting of: Further, the present invention relates to SEQ ID NO: 6 to SEQ ID NO: 1.
An isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO.
: 6 to SEQ ID NO: 10, and fragments thereof, wherein the isolated polynucleotide has a sequence complementary to a polynucleotide comprising a polynucleotide sequence selected from the group consisting of:

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 NO: 1 to SEQ ID NO: 5, and fragments thereof. I do. In another aspect, the expression vector is contained in a host cell.

The present invention also provides a method for producing a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5, and fragments thereof, the method comprising: (A) culturing a host cell containing an expression vector having at least a fragment of a polynucleotide encoding the polypeptide under conditions suitable for expressing the polypeptide; and (b) culturing the polypeptide from the host cell culture. Recovering.

[0015] The present invention also relates to a method for preparing a pharmaceutical composition, comprising the steps of:
A pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of ID NO: 5 and fragments thereof.

Further, the present invention provides a purified antibody that binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5, and fragments thereof, and purification of the polypeptide. Agonists and purified antagonists are provided.

The invention also provides an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5, and fragments thereof. For treating or preventing a cell proliferative disorder, comprising administering to a subject in need thereof.

The invention also provides a method of treating an effective amount of an antagonist of a substantially purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 5, and fragments thereof. Provided is a method for treating or preventing a cell proliferation disorder, which comprises a step of administering to a subject in need thereof.

The present invention also provides an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 5, and fragments thereof. A method for treating or preventing an immune disorder, comprising administering to a subject in need thereof.

The invention also provides a method of treating an effective amount of an antagonist of a substantially purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 5, and fragments thereof. Provided is a method for treating or preventing an immune disorder, which comprises the step of administering to a subject in need thereof.

The present invention also relates to a biological sample containing nucleic acid, wherein SEQ ID NO: 1 to SEQ ID NO: 1
Provided is a method for detecting a polynucleotide encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and fragments thereof, comprising: (a) SEQ ID NO: 1 SEQ ID N
O: hybridizing a complementary sequence of a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of 5 and fragments thereof to at least one of the nucleic acids of the biological sample, and thereby a hybridization complex And (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of the polynucleotide encoding the polypeptide in the biological sample. I have. In one aspect, the nucleic acids of the biological sample are amplified by the polymerase chain reaction before hybridizing.

[0022] Embodiments of the invention the protein, before describing the nucleotide sequence and method, if the present invention is the particular method described, the protocol is not limited to cell lines, vectors and agents are modified Please understand that there is also. Also, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention, which is defined solely by the appended claims. It should be understood that it is limited.

As used herein, in the appended claims, the singular articles "
It is noted that "the" (or above) includes a plurality of indications unless the context clearly dictates otherwise. Thus, for example, reference to "a host cell" includes a plurality of such host cells, and "the antibody" refers to one or more antibodies and their equivalents known to those of skill in the art. And so on.

Unless defined differently, technical and scientific 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 verification of the present invention, the preferred methods, devices and materials are described herein. All publications described herein are hereby incorporated by reference to describe and disclose the cell lines, vectors, and methodologies reported in the publications that may be used in connection with the present invention. And part of it. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by prior invention.

Definitions "CECRP" refers to any species from any source, whether natural, synthetic, semi-synthetic or recombinant, in particular from mammals, including cows, sheep, pigs, rats, horses and, preferably, humans. 4 is the amino acid sequence of the resulting substantially purified CECRP.

“Agonist” refers to a molecule that, when bound to CECRP, increases the amount of CECRP or prolongs the period of activity. Agonists are proteins,
It may include nucleic acids, carbohydrates or any other molecule that binds to CECRP and modulates its action.

“Allelic variant” is an alternative form of the gene encoding CECRP. An allele results from at least one mutation in a nucleic acid sequence, resulting in an altered mRNA or polypeptide, with or without altering its structure or function. Any given gene may have one or many allelic forms,
Sometimes they don't have it at all. Common mutations that cause alleles generally result from natural deletions, additions or substitutions of nucleotides. Each of these types of changes, alone or in combination with the others, may be performed once or twice in a given sequence.
May occur more than once.

An “altered” nucleic acid sequence encoding CECRP includes various nucleotide deletions, insertions or substitutions resulting in a polynucleotide encoding the same or a functionally equivalent CECRP. This definition includes polymorphisms that may or may not be readily detectable using a particular oligonucleotide probe of a polynucleotide encoding CECRP and normal chromosomal locations relative to the polynucleotide sequence encoding CECRP. Inappropriate or unexpected hybridization to an allele having a different position than The encoded protein may also be "altered", including deletions, insertions or substitutions of amino acid residues that produce a silent change and result in a functionally equivalent CECRP. Deliberate amino acid substitutions can be made based on similarities in residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and also amphipathicity, as long as the biological activity of CECRP is retained.
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 are leucine; Includes isoleucine and valine, glycine and alanine, asparagine and glutamine, serine and threonine, phenylalanine and tyrosine.

“Amino acid” or “amino acid sequence” refers to oligopeptide, peptide, polypeptide or protein sequences and fragments thereof, as well as naturally occurring or synthetic molecules. Fragments of CECRP are from about 5 to about 15 amino acids in length, most preferably 14 amino acids, and preferably retain the biological or immunological activity of CECRP. "Amino acid sequence" is expressed as indicating the amino acid sequence of a naturally occurring protein molecule, although terms such as amino acid sequence mean that the amino acid sequence is limited to the complete natural amino acid sequence associated with the indicated protein molecule. do not do.

“Amplification” refers to the generation of additional copies of a nucleic acid sequence, which is generally performed using polymerase chain reaction (PCR) techniques well known in the art (eg,
Dieffenbach, CW and GS Dveksler (1995) PCR Primer, a Lab.oratory Ma
nual, Cold Spring Harbor Press, Plainview, NY, pp. 1-5.).

“Antagonist” refers to a molecule that reduces the biological or immunological activity of CECRP when bound to CECRP. Antagonists may include proteins, nucleic acids, carbohydrates or any other molecule that binds to CECRP and reduces the effects of CECRP.

An “antibody” is a Fa, F (ab ′) that can bind an epitope determinant. _Two And intact molecules such as Fv and fragments thereof. CECRP
Antibodies that bind polypeptides contain small peptides of interest as immunizing antigens
It can be made using intact polypeptides or fragments. Animals (e.g.
Polypeptides used to immunize mice, rats or rabbits)
Oligopeptides are derived from the translation of RNA or chemically synthesized
If desired, it can be linked to a carrier protein. Chemically linked to the peptide
Commonly used carriers include bovine serum albumin and thyroglobulin,
Contains lulimpet hemocyanin (KLH). And then use the coupled peptide
To immunize the animal.

An “antigenic determinant” is the portion of a molecule that makes contact with a particular antibody (ie, an epitope)
That is. When immunizing a host animal with a protein or fragment thereof, many regions of the protein may trigger the production of antibodies that specifically bind to a given region or three-dimensional structure on the protein. These regions or structures are called antigenic determinants. The antigenic determinant converts the intact antigen (
That is, it may compete with an immunogen used to induce an immune response).

“Antisense” is any composition that contains a nucleotide sequence that is complementary to a specific DNA or RNA sequence. The term "antisense strand" is used to indicate a nucleic acid strand that is complementary to the "sense" strand. Antisense molecules include peptide nucleic acids and can be produced by any method, including synthetic or transcription. Once introduced into the cell, the complementary nucleotide binds to the native sequence produced by the cell, forms a duplex, and blocks either transcription or translation. Symbol "negative (minus)"
Is sometimes used to indicate an antisense strand, and “positive (plus)” is sometimes used to indicate a sense strand.

“Biological activity” refers to a protein that has structural, regulatory, or biochemical functions of a naturally occurring molecule. Similarly, "immunological activity" refers to the ability of natural, recombinant or synthetic CECRP, or any of its oligopeptides, to elicit a specific immune response in a suitable animal or cell and to bind to a specific antibody. is there.

“Complementary” or “complementarity” refers to the natural association of polynucleotides under permissive salts and temperature conditions by base pairing. For example, in the case of the sequence "AGT",
It binds to the complementary sequence "TCA". The complementarity between two single-stranded molecules is "partial", where only some of the nucleic acids bind, or "complete" if perfect complementarity exists between the single-stranded molecules In some cases. The degree of complementarity between nucleic acid strands is
It significantly affects the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions that rely on the binding between nucleic acid strands and the design and use of peptide nucleic acid (PNA) molecules.

“Composition comprising a given polynucleotide sequence” or “composition comprising a given amino acid sequence” is widely used for any composition comprising a given polynucleotide or amino acid sequence. The composition may be in a dry or aqueous solution. C
A composition comprising a polynucleotide sequence encoding ECRP or a fragment thereof can also be used as a hybridization probe. The probe is stored lyophilized and may be associated with a stabilizing agent such as a carbohydrate. For hybridization, the probe may be dispersed in an aqueous solution containing salts (eg, NaCl), detergents (eg, SDS) and other compositions (eg, Denhardt's solution, milk powder, salmon sperm DNA, etc.).

A “consensus sequence” is a nucleic acid sequence that has been rearranged to decompose unnecessary bases or XL-PCR (Perkin Elmer, Norwal) in the 5 ′ or 3 ′ direction.
k, CT) or a computer program for constructing fragments (eg, GELVIEW Fragment Assembly System, GCG, Madis
on WI) is constructed from overlapping sequences of two or more Incyte clones. Certain sequences have been both extended and assembled to generate a consensus sequence.

“Correlated with polynucleotide expression” refers to CECRP by Northern analysis.
Detection of the presence of a ribonucleic acid similar to the polynucleotide encoding indicates the presence of the mRNA encoding CECRP in the sample, thereby indicating that the CECR
4 shows a correlation with expression of transcripts from polynucleotides encoding P.

A “deletion” changes in either the amino acid sequence or the nucleotide sequence,
Lack of one or more amino acid residues or nucleotides.

“Derivative” refers to a chemical modification of the nucleic acid sequence encoding CECRP or the complementary sequence of CECRP or the encoded CECRP. Illustrative of such modifications are the replacement of hydrogen by an alkyl, acyl or amino group. Nucleic acid derivatives encode polypeptides that retain the biological and immunological functions of the natural molecule. A derivative polypeptide is a polypeptide that is modified by glycosylation, polyethylene glycol formation (pegylation), or any similar process that retains the biological or immunological function of the derived polypeptide.

“Similarity” refers to the degree of complementarity. There is partial similarity or complete similarity. The term “identity” may be used in place of “similarity”. A partially complementary sequence is a sequence that at least partially inhibits the same sequence from hybridizing to a target nucleic acid, and "substantially similar" is used. Inhibition of hybridization of completely similar (identical) sequences to the target sequence can be tested using hybridization assays (Southern or Northern blot, solution hybridization, etc.) under conditions of low stringency. Substantially similar sequences or probes will compete with and inhibit the binding of completely similar sequences and probes to the target sequence under conditions of low stringency. It goes without saying that low stringency conditions are conditions under which non-specific binding is tolerated. Low stringency conditions require that the binding of the two sequences to each other be a unique (ie, selective) interaction. The lack of non-specific binding may be tested by the use of a second target sequence in which even a partial degree of complementarity is not present (eg, less than about 30% identity). In the absence of non-specific binding, the probe will not hybridize to a second non-complementary target sequence.

The expression “percent identity” or “% identity” is the percentage of sequence similarity in comparing 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 uses different methods, such as the cluster method (eg, Higgins, DG and PM).
Sharp (1988) Gene 73: 237-244) can be used to make an alignment of two or more sequences. In the algorithm of the cluster method, a sequence group is divided into clusters (collections) by checking the distance between both sequences for all pairs of sequences. This cluster group is aligned for each pair, and then the group is aligned. The percent similarity between two amino acid sequences, for example, sequence A and sequence B, is (length of sequence A-number of gap residues in sequence A-number of gap residues in sequence B) / (sequence A and sequence B Total number of residue matches between B) × 100. Cases where the similarity difference between the two amino acid sequences is low or not are not included in the percent similarity calculation. The percent identity between nucleic acid sequences can also be determined by other well-known methods, such as the Jotun Hein method (eg, Hein J. (1990) Metho
ds Enzymol. 183: 626-645).
Identity between sequences can also be determined by other well-known methods, for example, by changing hybridization conditions.

“Human artificial chromosomes” (HACs) are DNA sequences of 6 kb to 10 Mb in size, and are linear, small elements containing all the elements required for stable mitotic chromosome isolation and retention. Chromosomes (e.g., Harrington, JJ et al. (1997) Nat Genet.
15: 345-355).

“Humanized antibody” refers to an antibody molecule in which amino acids have been substituted in the non-antigen binding region to make it more like a human antibody, while retaining the original binding ability.

“Hybridization” refers to any process by which a strand of nucleic acid associates with a complementary strand through base pairing.

A “hybridization complex” is a complex formed between two nucleic acid sequences by forming hydrogen bonds between complementary G and C bases and between complementary A and T bases. It is about the body. These hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. The hybridization complex is composed of one nucleic acid sequence and a solid support (eg, paper, membrane, filter, chip, pin, or glass slide) present in or in solution (eg, C ₀ t or R ₀ t analysis). Or another nucleic acid sequence that is immobilized on a cell or any other suitable support on which the nucleic acid is immobilized.

“Insertion” or “addition” refers to a change in the amino acid or nucleotide sequence at which one or more amino acid residues or nucleotides are added, respectively, as compared to a naturally occurring molecule.

“Immune response” refers to a condition associated with inflammation, trauma, immune disease, or infectious or genetic disease. These conditions can be characterized by the production of various factors that activate cells and the system of defense, including cytokines, chemokines, and other signaling molecules.

“Microarray” refers to individual polynucleotides or oligos arranged on a support, such as paper, nylon or other types of membranes, filters, chips, glass slides or any other suitable solid support. An array consisting of nucleotides.

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

“Modulation” is an alteration of the biological activity of CECRP. For example, modulation results in an increase or decrease in the protein activity, binding properties, or biological, functional or immunological properties of CECRP.

The “nucleic acid” or “nucleic acid sequence” may be an oligonucleotide, a nucleotide or a polynucleotide and a fragment thereof, single-stranded or double-stranded, and represents a sense strand or an antisense strand. RNA,
Peptide nucleic acid (PNA) or any DNA-like or RNA-like substance. A “fragment”, when translated, produces a polypeptide that retains some functional characteristics, such as antigenic or structural domain properties, such as an ATP-binding site of a full-length polypeptide. Nucleic acid sequence.

As used herein, the terms “functionally related” or “operably linked” refer to functionally related nucleic acid sequences. A promoter is operably linked or operably linked to a coding sequence if the promoter regulates the transcription of the encoded polypeptide. Functionally related or operably linked nucleic acid sequences can be in close reading frame, but certain genomic sequences, such as repressor genes, are in close proximity to the encoded polypeptide. But also binds to an operator sequence that regulates the expression of the polypeptide.

An “oligonucleotide” is a nucleic acid sequence of at least about 6 to 60 nucleotides, preferably a nucleic acid sequence of about 15-30 nucleotides, more preferably a nucleic acid sequence of 20-25 nucleotides, for PCR amplification or hybridization assays. Method. As used herein, oligonucleotides refer to the terms "amplima,""a" generally as defined in the art.
Substantially equivalent to "primer", "oligomer" and "probe".

“Peptide nucleic acid” (PNA) is an antisense molecule or anti-gene agent comprising an oligonucleotide consisting of at least 5 nucleotides in length, which binds to the peptide backbone of amino acid residues terminating in lysine. ) That is. PNAs are polyethylene glycolated and extend the lifespan of cells, preferentially binding complementary single-stranded DNA and RNA therein, and stop transcription elongation (eg, Nielsen PE et al. (1993) Anticancer Drug Des 8:53). -63).

“Sample” is used in a broad sense. A biological sample suspected of containing the nucleic acid encoding CECRP or a fragment thereof or CECRP itself,
Body fluids, extracts from cells, chromosomes, membranes isolated from cell organs or cells, cells, which are present in solution or bound to a solid support, tissue, tissue print, etc. Includes genomic DNA, RNA or cDNA.

“Specific binding” or “specifically binds” refers to the interaction between a protein or peptide and an agonist, and an antibody and an antagonist. The interaction depends on the presence of a particular structure (ie, antigenic determinant or epitope) of the protein identified by the binding molecule. For example, if the antibody is specific for epitope "A", the presence of epitope A or free, unlabeled A and the protein containing the antibody in a reaction involving the labeled "A" will be reflected by the antibody. Will reduce the amount of labeled A bound to

“Strict conditions” are conditions that permit hybridization between a polynucleotide sequence and the claimed polynucleotide sequence. Appropriate levels of stringent conditions can be determined, for example, by the concentration of salts or organic solvents (eg, formamide) in the prehybridization and hybridization regions, or the hybridization temperature, and are well known in the art. In particular, stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or increasing the hybridization temperature.

“Substantially purified” refers to a nucleic acid or amino acid sequence that has been removed, isolated, or separated from the natural environment, which contains at least other components with which it is naturally associated. It does not contain 60%, preferably 75%, most preferably 90%.

“Substitution” is the replacement of one or more nucleotides or amino acids with different nucleotides or amino acids, respectively.

“Transformation” refers to the process by which foreign DNA enters and changes a recipient cell. It can be generated under natural or artificial conditions using various methods well known in the art. Transformation may be based on any known method for inserting foreign nucleic acid sequences into prokaryotic or eukaryotic host cells. The method is selected based on the host cell to be transformed and may include, but is not limited to, viral infection, electroporation, heat shock, lipofection, and particle irradiation. Such "transformed" cells include those that have been stably transformed, in which the inserted DNA is either an automatically replicating plasmid or part of its host chromosome. Can be duplicated. The cells also include cells that temporarily express DNA or RNA inserted for a limited period.

As used herein, a “variant” of CECRP refers to an amino acid sequence that is altered by one or more amino acids. A variant may be “conservatively” altered, in which the substituted amino acid has similar structural and chemical properties, eg, when replacing leucine with isoleucine. More rarely, variants may change "non-conservatively", such as when replacing glycine with tryptophan. Similar minor variants may also include amino acid deletions or insertions, or both. Indicators for determining whether an amino acid residue is substituted, inserted or deleted without abolishing biological and immunological activities can be found using a computer program well known to those skilled in the art, for example, LASERGENE software. Can be.

The term “variant” when used in the context of a polynucleotide sequence NHA
Polynucleotide sequences related to P may be included. This definition also includes, for example, "allelic variants" (as defined above), "splice variants,""speciesvariants," or "polymorphic variants." A splice variant may have significant identity to a reference molecule, but will have a greater or lesser number of polynucleotides due to a change in the splicing site of an exon during mRNA processing. The corresponding polypeptide may have additional functional domains or may lack domains. Species variants are polynucleotide sequences that differ between species. The polypeptides resulting therefrom usually have significant amino acid identity with each other. Polymorphic variants differ by one base in the polynucleotide sequence.
"Single nucleotide polymorphisms" (SNPs) are also included. The presence of SNPs may indicate, for example, a population, a disease state, or a propensity for a disease state.

[0065] This invention relates to novel cell cycle regulatory proteins (CECRP), polynucleotides encoding CECRP, and diagnosis of cell proliferative disorders and immune disorders, the discovery of the use of these compositions for the treatment and prevention based on.

In Table 1, the first and second columns are the sequence identification numbers (S
EQID NO :), the third column shows the clone ID of the Incyte clone from which the nucleic acid encoding each CECRP was first identified, and the fourth column shows the cDNA library of this clone. Column 5 lists the fragments, for example, the Incyte clone (and library) and shotgun sequences that are useful as fragments in hybridization techniques and are part of the consensus nucleotide sequence of each CECRP.

The columns of Table 2 show various properties of the polypeptide of the present invention. The first column is SEQ
Column 2 refers to the amino acid residue number, column 3 refers to a potential phosphorylation site, column 4 refers to a potential glycosylation site, and column 5 refers to ID NO: Column 6 indicates the sequence and / or structural motif, and column 6 shows the sequence homology and analysis method used to identify the protein through the protein motif.

The columns in Table 3 show the tissue expression of each nucleic acid sequence by Northern analysis, diseases or disorders associated with this tissue expression, and the vectors into which each cDNA was cloned.

The present invention also includes CECRP variants. A preferred CECRP variant is CECR
A CECRP variant that is at least about 80%, more preferably at least about 90%, and most preferably a PEC amino acid sequence is at least about 95% amino acid identical to CECRP and comprises at least one functional or structural feature of CECRP. is there.

The present invention also includes a polynucleotide encoding CECRP. In certain embodiments, the invention includes a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO: 6 to SEQ ID NO: 10.

The present invention also includes a mutant sequence of the polynucleotide sequence encoding CECRP. In particular, such a polynucleotide variant sequence will have at least about 80%, more preferably at least about 9%, of the polynucleotide sequence encoding CECRP.
It will have a polynucleotide sequence identity of 0%, most preferably at least about 95%. Certain aspects of the present invention relate to SEQ ID NO: 6 to SEQ ID NO.
SEQ ID NO: 6 to SEQ ID NO: having a polynucleotide sequence identity of at least about 80%, more preferably at least about 90%, and most preferably at least about 95% to a nucleic acid sequence selected from the group consisting of: A mutant sequence of a nucleic acid sequence selected from the group consisting of Any one of the above-described polynucleotide variants can encode an amino acid sequence that includes at least one of the functional or structural features of CECRP.

As a result of the degeneracy of the genetic code, a large number of nucleotide sequences encoding CECRP are generated, some of which exhibit minimal homology to the nucleotide sequence of any known naturally occurring gene. Will be understood by those skilled in the art. Thus, the present invention contemplates all possible variant sequences of the nucleotide sequence that will be formed by selecting combinations based on possible codon choices. These combinations are formed according to the standard triplet genetic code as applied to the nucleotide sequence of naturally occurring CECRP, and all such variations are to be considered as being explicitly disclosed.

The nucleotide sequence encoding CECRP and its mutant sequences are preferably capable of hybridizing under appropriately selected stringent conditions to the nucleotide sequence of naturally occurring CECRP, but may have substantially different codon usage. It may be advantageous to generate a nucleotide sequence that encodes or a derivative thereof. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host, depending on how frequently that particular codon is utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding CECRP and its derivatives without altering the encoded amino acid sequence include properties that are more desirable than transcripts generated from naturally occurring sequences, such as longer half-lives. RNA with
Generating a transcript.

The present invention also includes the generation of a DNA sequence encoding CECRP and its derivatives or fragments thereof completely synthetically. After generation, the synthetic sequences can be inserted into any of the many available expression vectors and cell lines using agents well known in the art at the time of filing this patent application. Further synthetically,
Mutations can be introduced into the sequence encoding CECRP or any fragment thereof.

The nucleotide sequences claimed under various stringency conditions as taught in the present invention, in particular SEQ ID NO: 6 to SEQ ID NO: 10, and fragments thereof, Polynucleotide sequences capable of hybridizing to the sequence are included (eg, Wahl, GM and SLBerger (1987; Me.
thods Enzymol. Vol. 152: 399-407) and Kimmel, AR (1987; Methods Enzymol. V
ol 152: 507-511)).

[0076] 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, SEQUE.
NASE (US Biochemical, Cleveland, OH), Taq polymerase (Perkin Elmer),
Use enzymes such as thermostable T7 polymerase (Amersham, Pharmacia Biotech, Piscataway NJ) or 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 (Hamilt
on, Reno, NV), Peltier Thermal Cycler 200 (PTC200; MJ Research, Watertown,
It is preferred to automate by equipment such as MA) and ABI CATALYST 800 (Perkin Elmer). Next, use the ABI 373 or 377 DNA sequencing system (Perk
in-Elmer), MEGABACE 1000 DNA Sequencing System (Molecular Dynamic
s, Sunnyvale CA) or other well-known systems. 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 , J.
ohn Wiley & Sons, New York NY, unit 7.7; Meyers, RA (1995) Molecular B
iology and Biotechnology , Wiley VCH, New York NY, pp. 856-853).

The nucleic acid sequence encoding CECRP can be extended utilizing partial nucleotide sequences and using various methods known in the art for detecting upstream sequences such as promoters and regulatory elements. . For example, one method that may be used, "restriction site" PCR, uses a universal primer to recover unknown sequences flanking known positions (eg, Sarkar. G (1993) PCR Methods Applic.
2: 318-322). In particular, genomic DNA is first amplified in the presence of a primer for the linker sequence and a primer specific for a known region. The amplified sequence is then subjected to a second round of PCR using the same linker primer and another unique primer internal to the first primer. The products of each round of PCR are transcribed using an appropriate RNA polymerase and sequenced using reverse transcriptase. Alternatively, the sequence may be amplified or extended using a divergent primer based on a known region, using inverse PCR. The template is derived from restriction fragments containing known genomic locations and surrounding sequences (Trigli
a T et al. (1988) Nucleic Acids Res 16: 8186). A third method is capture PCR (Lagerstrom M et al. (1991) PCR Methods Applic 1: 111-19) which involves PCR amplifying DNA fragments adjacent to known sequences of human and yeast artificial chromosomal DNA. In this way, multiple restriction enzyme digestions and ligation can be used to insert the engineered double-stranded sequence into the region of the unknown sequence before performing the PCR.
Other methods that may be used to recover unknown sequences are known in the art (see, eg, Parker JD et al. (1991; Nucleic Acids Res 19: 3055-3060). Can walk genomic DNA using nested primers and PromoterFinder library (Clontech, Palo Alto CA)
. This process eliminates the need to screen the library and is useful in finding intron / exon junctions. For all PCR-based methods, the primer is OLIGO 4.06 Primer Analysis Software (National Biosciences
Inc., Plymouth MN) or another suitable program, using commercial software such as 22-30 nucleotides in length, having a GC content of 50% or more,
It can also be designed to anneal to the template at a temperature of about 68-72 ° C.

When screening for full-length cDNAs, it is preferable to use libraries that have been selected in size to include longer cDNAs. Randomly primed libraries are also preferred in that they contain larger sequences, including the 5 'and upstream regions of the gene. The use of a randomly primed library is particularly preferred in situations where the oligo d (T) library does not produce full-length cDNA. Genomic libraries may be useful for extending sequence into a 5 'non-transcribed control region.

A commercially available capillary electrophoresis system can also be used to determine the sequence or size of the PCR product or confirm its nucleotide sequence. In particular, capillary sequencing involves the detection of emission wavelengths by a flowable polymer for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) activated by a laser, and a CCD camera. May be used. The output / light intensity is converted to electrical signals using appropriate software (eg, Genotyper and Sequence Navigator, Perkin Elmer), and the entire process from sample loading to computer analysis and electronic data display is computer controlled. Capillary electrophoresis is particularly preferred for sequencing small pieces of DNA that may be present within a limited amount of a particular sample.

In another embodiment of the present invention, a polynucleotide sequence encoding CECRP, or a fragment thereof, is incorporated into a recombinant DNA molecule to effect expression of CECRP, a fragment thereof, or a functional equivalent thereof, in a suitable host cell. May be used. Due to the inherent degeneracy of the genomic code, other DNA sequences encoding substantially identical or functionally equivalent amino acid sequences are generated, which can be used to generate CE sequences.
CRP can also be expressed.

The nucleotide sequences of the present invention may vary in CEC for a variety of reasons including, but not limited to, cloning, processing and also altering expression of the gene product.
To alter the sequence encoding RP, genetic engineering can be performed using methods well known in the art. Nucleotide sequences can be engineered using random fragmentation of gene fragments and synthetic oligonucleotides and DNA mixing by PCR reassembly. For example, site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, alter codon preferences, generate splicing variant sequences or introduce mutations.

In another embodiment, the sequence encoding CECRP can be synthesized in whole or in part using chemical methods well known in the art (eg, Caruthers
MH et al. (1980) Nuc Acids Res Symp Ser 215-23, Horn T et al. (1980) Nuc Acids Res
See Symp Ser 225-232). Alternatively, the protein itself may be CECR
It may be produced using chemical methods to synthesize the amino acid sequence or a portion of P. For example, peptide synthesis can be performed using various solid-phase techniques (eg, Roberge JY et al. (1995)
Science 269: 202-204), and automated synthesis can be achieved, for example, by using the ABI 431A Peptide Synthesizer (Perkin Elmer). Further, the amino acid sequence of CECRP, or any portion thereof, may be altered during direct synthesis, or may also be combined by chemical methods using sequences from other proteins or any portion thereof to produce the mutant polypeptide. It can also be generated.

The peptide can be substantially purified by preparative high performance liquid chromatography (eg, Chiez, RM and FZ Regnier (1990) Methods Enzymol.
182: 392-421). The composition of the synthetic peptide can be confirmed by amino acid analysis or sequencing (eg, Creighton, T. (1984) Proteins,
Structures. And. Molecular Prop..erties, WH Freeman and Co., New York,
See NY).

In order to express biologically active CECRP, the nucleotide sequence encoding CECRP or a derivative thereof is regulated in the transcription and translation of a coding sequence inserted in a suitable expression vector, ie, a suitable host. Into a vector containing the necessary sequence. These sequences include, for example, in vectors and CEC
Includes enhancers, constitutive and inducible promoters, and regulatory sequences such as 5 'and 3' untranslated regions in the polynucleotide sequence encoding RP. The strength and specificity of the action of such elements can vary. Specific initiation signals are also required for more efficient translation of the sequence encoding CECRP. Such signals include the ATG initiation codon and adjacent sequences, such as the Kozak sequence. If CECRP and its initiation codon and upstream control sequences are inserted into the appropriate expression vector, no other transcriptional or translational control signals are required. However, if only the coding sequence or a fragment thereof is inserted, an exogenous translational control signal, including the ATG start codon, must be provided. In addition, the start codon must be in the correct reading frame to ensure transcription of the entire insert. Exogenous transcriptional elements and initiation codons can be from a variety of sources, both natural and synthetic. Inclusion of appropriate enhancers in the particular cell line used can increase the efficiency of expression (see, eg, Scharf, D. et al. (1994) Results P.
robl. Cell Differ. 20: 125-162).

[0085] Expression vectors containing sequences encoding CECRP and appropriate transcription or translation control elements can be prepared using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques as well as in vivo genomic recombination (eg, Sambrook, et al . (1989) Molecular Cloning. A Laborator ) .
y Manual , Cold Spring Harbor Press, Plainview, NY, ch. 4, 8, and 16-17;
Ausubel, FM, etc. (1995, and periodic supplements) Current Protocols
in Molecular Biology , John Wiley & Sons, New York, NY, ch. 9, 13, and 16
Please refer to).

[0086] Various expression vector / host systems may be utilized to contain and express sequences encoding CECRP. These include, but are not limited to, bacteria transformed with a recombinant bacteriophage, plasmid or cosmid DNA expression vector, yeast transformed with a yeast expression vector, insect cell lines infected with a viral expression vector (eg, baculovirus). , A plant cell line transformed with a viral expression vector (eg, cauliflower mosaic virus CaMV, tobacco mosaic virus T
MV) or a plant cell line transformed with a bacterial expression vector (eg, Ti
Or pBR322 plasmid) or a microorganism such as an animal cell line. The invention is not limited by the host cell used.

[0087] In bacterial systems, a variety of cloning and expression vectors may be selected depending upon the use of the polynucleotide encoding CECRP. For example, routine cloning, subcloning, and propagation of polynucleotides encoding CECRP can be performed, eg, by BLUSCRIPT (Stratagene, La Jolla CA) or pSP.
This can be achieved using a multifunctional E. coli vector such as the ORT1 plasmid (Life Technologies). Incorporating sequences encoding CECRP into the various cloning sites of the vector to disrupt the lacZ gene allows colorimetric screening to identify transformed bacteria containing the recombinant molecule. In addition, these vectors are used for in vitro transcription, dideoxy sequencing, single strand rescue by helper phage.
), And may be useful for the generation of nested deletions in cloned sequences (eg, Van Heeke, G. and SM Schuster (1989) J. Biol. Chem. 264: 5503-55).
09). For example, if a large amount of CECRP is required for antibody production, CECRP
A vector that induces expression at a high level can be used. For example, vectors containing a strong inducible T5 or T7 bacteriophage promoter can be used.

For the production of CECRP, a yeast expression system can also be used. For example α factor, a variety of vectors, including alcohol oxidase and constitutive or inducible promoters such as PGH,, the yeast Saccharomyces cerevisiae (Saccharomy
ces cerevisiae ) and Pichia pastoris ( Pichia pastoris )
Can be used. In addition, such vectors induce either the extracellular secretion of the expressed protein or the retention inside the cell, allowing the integration of foreign sequences into the host genome for stable expression. (See, eg, Ausubel, supra; and Scorer, CA et al. (1994) Bio / Technology 12: 181-184).

When a plant expression vector is used, expression of the sequence encoding CECRP is driven by any of several promoters. For example, CaMV 35S
And a viral promoter such as the 19S promoter can be used alone or with the ω-leader sequence from TMV (Takamatsu et al. (1987) EMBO J 6
: 307-311). Alternatively, plant or heat shock promoters such as the small subunit of RUBISCO (Coruzzi et al. (1984) EMBO J 3: 1671-1680; Brog
lie et al. (1984) Science 224: 838-843 and Winter J and Sinibaldi RM (1991) Re.
sults Probl Cell Differ 17: 85-105). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. The technology is described in several generally available reviews (eg, Hobbs S or Murry LE in McGraw Hill Yearbook of Science and Technolog
y (1992) McGraw Hill New York NY, pp. 191-196).

In mammalian host cells, a number of viral expression systems may be utilized.
When an adenovirus is used as the expression vector, the sequence encoding CECRP contains an adenovirus transcription / adhesion consisting of the late promoter and tripartite leader sequence.
May be bound to the translation complex. Insertion into the non-essential E1 or E3 region of the viral genome can result in a live virus capable of expressing CECRP in infected host cells (eg, Logan and Shenk (1984) Proc Natl.
Acad Sci 81: 3655-3659). In addition, Rous sarcoma virus (RSV)
) A transcription enhancer, such as an enhancer, can be used to increase expression in a mammalian host cell. High levels of protein can also be expressed using SV40 or EBV vectors.

[0091] Human artificial chromosomes (HACs) can also be used to deliver larger fragments of DNA that can be contained and expressed in a plasmid. For treatment, a 6 kb to 10 Mb HAC is constructed and delivered using conventional delivery methods (liposomes, polycation amino polymers or vesicles).

For long-term, high-yield production of recombinant proteins in mammalian systems,
Stable expression is desirable. For example, a cell line that stably expresses CECRP can be transformed with an expression vector that includes the viral origin of the replication or endogenous expression element and a selectable marker gene on the same or another vector. Following the introduction of the vector, the cells are allowed to grow for 1-2 days in an enriched medium before switching to a selective medium. The purpose of a selectable marker is to confer resistance to selection, the presence of which allows cells that effectively express the introduced sequence to grow and be recovered. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate for the cell type.

[0093] Any number of selection systems can be used to recover transformed cell lines.
These include, but are not limited to, respectively tk ^- cells or aprt ^- herpes simplex virus thymidine kinase that can be used in cells (Wigler, M, etc. (1977) C
ell 11: 223-32) and adenine phosphoribosyl convertase gene (Lowy I et al.
1980) Cell 22: 817-23). Antimetabolites, antibiotics or herbicide tolerance can also be used as criteria for selection. For example, dhfr confers resistance to methotrexate (Wigler M et al. (1980) Proc Natl Acad Sci 77: 3567-70), n
pt confers resistance to aminoglycoside neomycin and G-418 (Colber
e-Garapin F et al. (1981) J Mol Biol 150: 1-14), als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Further selectable genes have been described, for example, trpB allows cells to utilize indole instead of tryptophan, and hisD allows cells to use histinol (histinol instead of histidine).
histinol) (Hartman SC and RC Mulligan (1988) Proc)
Natl Acad Sci 85: 8047-51). Visible markers such as anthocyanins, green fluorescent protein (GFP); Clonetech, Palo Alto, CA
), Β-glucuronidase and its substrate, β-D-glucuronoside, or luciferase and its substrate, luciferin. These markers are widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression due to specific vector systems (Rhodes CA et al. (19)
95) Methods Mol Biol 55: 121-131).

The presence or absence of marker gene expression indicates that the gene of interest is present, but its presence and expression need to be confirmed. For example, if the sequence encoding CECRP is inserted within a marker gene sequence, recombinant cells containing the sequence encoding CECRP can be identified by a lack of marker gene function. Alternatively, the marker gene is placed in tandem with a sequence encoding CECRP under the control of a single promoter. Expression of the marker gene in response to induction and selection usually indicates expression of the tandem gene as well.

[0095] In general, host cells that contain a nucleic acid sequence encoding CECRP and that express CECRP can be identified by a variety of procedures known to those of skill in the art. This procedure includes, but is not limited to, DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassay techniques, including membrane, solution or chip-based techniques for the detection and / or quantification of nucleic acids or proteins.

Using either specific polyclonal or monoclonal antibodies, C
Immunological methods for detecting and measuring ECRP expression are well known to those skilled in the art. For example, enzyme-linked immunoassay (ELISA), radioimmunoassay (RIA)
), Fluorescence-activated cell sorting (FACE), and the like. A two-site monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes in CECRP is preferred, but a binding protein competition assay may be used. These and other assays are known in the art. (Eg, Hampton, R. et al. (1990) Serological Methods, a Laboratory M
anual , APS Press, St Paul.MN, Section IV; Coligan, JE et al. (1997 and perio
dic supplements) Current Protocols in Immunology, Greene Pub. Associates
and Wiley-Interscience, New York, NY; and Maddox, DE, etc. (1983) J. Exp.
Med. 158: 1211-1216).

[0097] A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for generating labeled hybridization or PCR probes to detect sequences related to the polynucleotide encoding CECRP include oligo-labeling, nick translation, end-labeling or PCR amplification using labeled nucleotides. Including. Alternatively, CE
The sequence encoding CRP, or any portion thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art and are commercially available, and can be used to synthesize RNA probes in vitro by adding a suitable RNA polymerase such as T7, T3 or SP6 and labeled nucleotides. it can. These procedures include various commercially available kits (Pharmacia & upjohn (Kalamazoo, MI), Promega (Madison WI) and US
Biochemical Corp (Cleveland OH)). Appropriate reporter molecules or labels may be used, including radionuclides, enzymes, fluorescent agents, chemiluminescent or dye-producing agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

[0098] Host cells transformed with a nucleotide sequence encoding CECRP can be cultured under conditions suitable to allow the protein to be expressed in and recovered from cell media. Proteins produced by the transformed cells are secreted or retained intracellularly, depending on the sequence and / or vector used. As will be appreciated by those skilled in the art, expression vectors containing a polynucleotide encoding CECRP can be designed to include a signal sequence that directs CECRP secretion through the cell membrane of a prokaryotic or eukaryotic cell.

In addition, host cell strains can be 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, which separates the “prepro” part of the protein,
It can be used to identify protein targeting, folding, and / or action. A variety of host cells (eg, CHO, HeLa, MDCK, 293, and WI38) that have specific cellular machinery and characteristic mechanisms for post-translational action are available from American T.
It can be obtained from the Ype Culture Collection (ATCC; Bethesda, MD) and can be selected from these to ensure the correct modification and processing of the foreign protein to be introduced.

In another embodiment of the invention, a natural, modified or recombinant nucleic acid sequence encoding CECRP is ligated to a heterologous sequence to translate the fusion protein in any of the host systems described above. Can be caused. For example, a chimeric CECRP protein containing a heterologous molecule that can be recognized by a commercially available antibody may facilitate the selection of inhibitors of CECRP activity from a peptide library. Such molecules include, but are not limited to, glutathione S transferase (GS
T), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His allow purification of immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and their cognate fusion proteins on metal chelating resins, respectively. Become. FLAG, c-myc, and hemagglutinin (HA) allow immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize their epitope tags. By recombining the fusion protein to include a proteolytic cleavage site between the sequence encoding the CECRP and the heterologous protein sequence, the CECRP can be separated from the heterologous molecule after purification. Become. Methods for expression and purification of fusion proteins are described in Ausubel, FM et al. (1995 and periodic supplements).
Protocols in Molecular Biology, John Wiley & Sons, New York, NY, ch10. Various commercially available kits may be used to rapidly perform expression and purification of the fusion protein.

In yet another embodiment of the present invention, radiolabeled CECRP can be synthesized in vitro using the TNT ^™ rabbit reticulocyte lysate or wheat germ extract system (Promega, Madison, WI). . These systems are T7, T3, or SP6
Coupling transcription and translation of a protein coding sequence functionally related to a promoter. Translation takes place in the presence of a radiolabeled amino acid precursor, preferably ³⁵ S-methionine.

The production of fragments of CECRP can be performed not only by recombinant production but also by direct peptide synthesis using solid phase technology (eg, Cr
eighton, supra, pp. 55-60). Protein synthesis can be performed manually or automatically. Automation of synthesis can be achieved, for example, using an Applied Biosystems 431A peptide synthesizer (The Perkin-Elmer). CECRP
Can be synthesized separately and ligated to create a full-length molecule.

There are chemical and structural similarities between therapeutic CECRP and cell cycle regulatory proteins. In addition, CECRP expression is closely linked to cell proliferation. Therefore, CECRP is an activator or enhancer, and it is desirable to reduce the expression of CECRP in cancers or immune disorders that promote cell proliferation. CECRP
However, in cancers or immune disorders that are inhibitors or suppressors of the cellular processes that regulate cell growth, it is desirable to provide the protein or increase CECRP expression.

In one embodiment where CECRP is an inhibitor of cell proliferation, CECRP or a fragment or derivative thereof can be administered to a subject to treat or prevent a cell proliferation disorder. Such disorders include, but are not limited to, actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (M
CTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma; In detail, adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary gland, It may include skin, spleen, testis, thymus, thyroid, and uterine cancer.

In another embodiment, a pharmaceutical composition comprising purified CECRP can be used to treat or prevent a cell proliferation disorder, including, but not limited to, the above disorders.

In another embodiment, a specific agonist of CECRP can be administered to a subject to treat or prevent a cell proliferation disorder, including, but not limited to, the above disorders.

In yet another embodiment, CECRP or a fragment or derivative thereof can be administered to a subject to treat or prevent a cell proliferation disorder, including, but not limited to, the above disorders.

In another embodiment, where CECRP is an inhibitor of cell proliferation, CECRP or a fragment or derivative thereof can be administered to a subject to treat or prevent an immune disorder. Such disorders include, but are not limited to, acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis,
Amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes , Emphysema, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, lupus erythematosus, multiple sclerosis, Myasthenia gravis, inflammation of the myocardium or pericardium, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome,
Rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, ulcerative colitis, Werner syndrome, and cancer, hemodialysis, extracorporeal complications, viral infection, Bacterial, fungal, parasitic, protozoan, and helminth infections; and may include trauma.

In another embodiment, a pharmaceutical composition comprising purified CECRP can be used to treat or prevent an immune disorder, including, but not limited to, the above disorders.

In another embodiment, a specific agonist of CECRP can be administered to a subject to treat or prevent an immune disorder, including, but not limited to, the above disorders.

In yet another embodiment, CECRP or a fragment or derivative thereof can be administered to a subject to treat or prevent an immune disorder, including, but not limited to, the above disorders.

In yet another embodiment, wherein CECRP is a promoter of cell growth, CECR
An antagonist that decreases the expression or activity of P can be administered to a subject to treat or prevent a cell proliferation disorder, including, but not limited to, the above disorders. In one embodiment, an antibody that specifically binds CECRP can be used directly as an antagonist or indirectly as a targeting or delivery mechanism for delivering a pharmaceutical agent to cells or tissues that express CECRP.

In another embodiment, a vector expressing the complement of a polynucleotide encoding CECRP is administered to a subject to treat or prevent a cell proliferative disorder, including, but not limited to, the above disorders. it can.

[0114] In yet another embodiment, where CECRP is a promoter of cell proliferation, the CECR
An antagonist that reduces the expression or activity of P can be administered to a subject to treat or prevent an immune disorder, including, but not limited to, the above disorders.

In another embodiment, a vector that expresses the complement of a polynucleotide encoding CECRP can be administered to a subject to treat or prevent an immune disorder, including, but not limited to, the above disorders. .

In yet another embodiment, CECRP or a fragment or derivative thereof can be added to cells to stimulate cell proliferation. In particular, adding CECRP to cells in culture or to cells in vivo using a delivery mechanism such as liposomes, viral vectors or electroinjection to promote cell growth and tissue or organ regeneration. Can be. In particular, CECRP can be added to cells, cell lines, tissues or organ cultures in vitro or ex vivo to stimulate cell growth for use in xenografts or autotransplants. In some cases, cells may be preselected to resist infection or cancer or to correct genetic defects in diseases such as sickle cell anemia, beta thalassemia, cystic fibrosis, and Huntington's chorea. it can.

In another embodiment, a specific agonist of CECRP can be administered to cells to stimulate cell proliferation as described above.

In yet another embodiment, a vector capable of expressing CECRP, or a fragment or derivative thereof, can be administered to cells to stimulate cell proliferation as described above. In some embodiments, any of the proteins, antagonists, antibodies, agonists, complementary sequences or vectors of the invention may be administered in combination with other suitable therapeutic agents. Selection of suitable agents for use in combination therapy can be made by one of ordinary skill in the art based on conventional pharmaceutical principles. The combination of therapeutic agents acts interdependently, affecting the treatment and prevention of the various diseases mentioned above. Using this approach, therapeutic efficacy can be achieved with lower doses of the drug, thereby reducing the risk for adverse side effects.

[0119] Antagonists of CECRP can be generated using methods well known to those skilled in the art. Specifically, antibodies can be generated using purified CECRP, or a library of pharmaceutical agents can be screened to identify those that specifically bind to CECRP.

[0120] Antibodies to CECRP can be produced using methods well known to those skilled in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library. Neutralizing antibodies (antibodies that inhibit dimer formation) are particularly suitable for therapeutic use.

When producing antibodies, various hosts, including goats, rabbits, rats, mice, humans, etc., can be immunized by injecting CECRP or any fragment thereof or an oligopeptide having immunogenic properties. . Depending on the host species, 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, and surface actives such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol. Contains substances. Among adjuvants used in humans, BCG (
Calmette-Guerin bacilli) and Corynebacterium parvum are particularly preferred.

The oligopeptide, peptide, or fragment used to elicit antibodies to CECRP has an amino acid sequence of at least 5 amino acids, and more preferably has an amino acid sequence of at least 10 amino acids. They are preferably identical to part of the amino acid sequence of the native protein and include the complete amino acid sequence consisting of small naturally occurring molecules. Short stretches of CECRP amino acids can also be fused with stretches of another protein, such as KLM, and antibodies raised against the chimeric molecule.

[0123] Monoclonal antibodies to CECRP can be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, hybridoma technology, human B cell hybridoma technology and E.
Including BV-hybridoma technology (Koehler et al. (1975) Nature 256: 495-497, Kozb
or et al. (1985) J. Immunol Methods 81: 31-42, Cote et al. (1983) Proc Natl Acad Sci
80: 2026-2030, Cole, SP et al. (1984) Mol. Cell Biol. 62: 109-120).

In addition, techniques developed to generate “chimeric antibodies”, splicing of mouse antibody genes into human antibody genes to obtain molecules with appropriate antigen specificity and biological activity can be used (Morrison (1984) Proc Natl Acad Sci 8
1: 6851-6855: Neuberger et al. (1984) Nature 312: 604-608; Takeda et al. (1985) Natu
re 314: 452-454). Alternatively, the techniques described for generating single-chain antibodies may be adapted using methods known in the art to generate CECRP-specific single-chain antibodies. Antibodies with related specificity, but consisting of distinct idiotypic compositions, can also be generated by chain mixing from randomly combined immunoglobin libraries (Burton DR (1991) Proc Natl Acad Sci 88: 10134 -10137).

Antibodies can also be produced by inducing production in lymphocyte populations in vivo or by using recombinant immunoglobulin libraries or articles (Orlandi et al. (1989, Proc Natl A.
cad Sci 86: 3833-3837, Winter G and Milstein C (1991; Nature 349: 293-299)
Can be generated by screening a panel of highly specific binding agents as disclosed in US Pat.

[0126] Antibody fragments which contain specific binding sites for CECRP can also be generated. For example, such fragments include, but are not limited to, F (ab ') 2 fragments and F (ab') 2 fragments that can be generated by pepsin digestion of an antibody molecule.
(Ab ') Fab fragments that can be generated by reducing the disulfide bridges of the two fragments. Alternatively, a Fab expression library may be generated so that monoclonal Fab fragments with the desired specificity can be quickly and easily identified (Huse WD et al. (1989) Science 256: 1275-1281).
.

[0127] A variety of immunoassays can be used to screen for antibodies having the desired specificity. Various protocols for binding protein competition assays or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificity are well known in the art. Such immunoassays typically involve measuring the complex formed between CECRP and its specific antibody. A two-site monoclonal immunoassay utilizing monoclonal antibodies reactive to two non-interfering CECRP epitopes is preferred, but binding protein competition assays can also be used (Maddox, supra).

In another embodiment of the invention, a polynucleotide encoding CECRP or any fragment or complement thereof may be used for therapy. In one aspect, a complementary sequence to a polynucleotide encoding CECRP is used in situations where it is desirable to block transcription of mRNA. Specifically, the cells are C
It can be transformed with a sequence complementary to a polynucleotide encoding ECRP. As described above, the complementary molecule or fragment can be used to regulate CECRP activity or to regulate gene function. Such techniques are well known in the art, and sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or regulatory region of the sequence encoding CECRP.

Organs targeted by retroviruses, adenoviruses, herpes viruses or vaccinia viruses, and expression vectors derived from various bacterial plasmids,
It may be used to deliver a nucleotide sequence to a tissue or cell population. Vectors expressing a nucleic acid sequence complementary to the polynucleotide of the gene encoding CECRP can be prepared using methods well known to those skilled in the art (eg, Sa).
See mbrook et al. (supra) and Ausubel et al. (supra).

[0130] The gene encoding CECRP may be turned off by transforming a cell or tissue with an expression vector that expresses high levels of polynucleotides or fragments thereof encoding CECRP. Such constructs are used to introduce non-translatable sense or antisense sequences into cells. DN
Even if not integrated into A, such vectors can continue to transcribe RNA molecules until disabled by an endogenous nuclease. Transient expression may last for a month or more with non-replicating vectors, and will persist longer if the appropriate replication element is part of the vector system.

As described above, modification of gene expression can be achieved by controlling the gene encoding CECRP, 5
Or a complementary sequence to a regulatory region (signal sequence, promoter, enhancer and intron) or an antisense molecule (DNA, RNA or PNA) is designed. Oligonucleotides derived from the transcription initiation site, eg, between positions -10 and +10 from the start site, are preferred. Similarly, suppression can be achieved using "triple helix" base pairing techniques. Triple helix pairs are useful because inhibition of the ability of the duplex to become sufficiently open to bind polymerases, transcription factors or regulatory molecules. Recent advances in therapy using triple DNA have been described in a paper (Gee JE et al. (1994) In: Huber BE and BI Carr, Molecular and Imm.
unologic Approaches , Futura Publishing Co. Mt Kisco NY, pp. 163-177 ). Complementary sequences or antisense molecules can also be designed to block translation of mRNA by preventing transcripts from binding to ribosomes.

A ribozyme, an enzymatic RNA molecule, may be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, which involves nucleotide strand breakage cleavage. Examples used include engineered hammerhead motif ribozymes that can specifically and effectively catalyze the cleavage of nucleotide strand breaks in the sequence encoding CECRP.

The unique ribozyme cleavage site within any potential RNA target is first identified by scanning the target molecule for ribozyme cleavage sites GUA, GUU and GUC containing sequences. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site are evaluated for substructural features that render the oligonucleotide inoperable. Can be. The suitability of a candidate target can also be assessed by examining its ease of hybridization to complementary oligonucleotides using a ribonuclease protection assay.

The complementary ribonucleic acid molecules and ribozymes of the present invention can be prepared by any method known in the art for synthesizing nucleic acid molecules. These include methods for chemically synthesizing oligonucleotides, such as solid phase phosphoramidite chemical synthesis. Alternatively, the RNA molecule is an in vitro DNA sequence encoding CECRP.
And in vivo transcription. Such a DNA sequence is T7
Alternatively, they can be incorporated into various vectors using a suitable RNA polymerase promoter such as SP6. Alternatively, these cDNA constructs have complementary RN
A can be synthesized and introduced constitutively or inducibly into cell lines, cells or tissues.

[0135] RNA molecules can also be modified to improve intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5 'and also 3' ends of the molecule, or the use of phosphorothionate or 2'O-methyl instead of a phosphodiesterase linkage in the backbone of the molecule. . This concept is unique to the production of PNAs and is similar to inosine, cyeosin, and wibutosin, as well as adenine, cytidine, guanine, thymine, and uridine, which are not easily distinguished by acetyl-, methyl-, thio-, and endogenous endonucleases. These molecules can be extended throughout by including unconventional bases, such as modified formers.

[0136] Many methods for introducing a vector into cells or within the organization are available, in
Also suitable for use in vivo , in vitro and ex vivo . In the case of ex vivo treatment, the vector can be introduced into stem cells removed from a patient and propagated like a clone against an autograft returned to the same patient. Transfection, liposome injection or delivery by polycation amino polymer can be achieved using methods well known in the art (eg, Goldman, CK et al. (1997) Nature Biot.
echnology 15: 462-466).

[0137] Any of the above methods of treatment can be applied to a subject in need of treatment, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably humans.

Yet another embodiment of the present invention relates to the administration of a pharmaceutical composition used with a pharmaceutically acceptable carrier for any of the above therapeutic effects. Such pharmaceutical compositions can consist of CECRP, antibodies to CECRP, mimetics, agonists, antagonists or inhibitors of CECRP. The composition may be administered alone or in any sterile biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose and water, at least one such as a stabilizing compound, such as a stabilizing compound. It can be administered in combination with other agents. These compositions may be administered to the patient alone or in combination with other drugs, drugs or hormones.

Pharmaceutical compositions used in the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, subarachnoid, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, Administration can be by any route, including enteral, topical, sublingual, rectal means.

In addition to the active ingredient, these pharmaceutical compositions may contain a suitable pharmaceutically-acceptable excipient and adjuvants that facilitate the processing of the active compound into pharmaceutically usable preparations. In some cases. Further details on formulation and administration techniques can be found in Remington's Pharmaceutical Sciences (Maack Publis
hing Co. Easton PA).

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

Pharmaceutical preparations for oral administration are prepared by mixing the active compound with a solid excipient and, optionally, grinding the mixture to give a tablet or dragee core, if desired. After the addition of suitable auxiliaries, the mixture of granules is obtained by processing. Suitable excipients are sugars including lactose, sucrose, mannitol or sorbitol, corn, wheat, rice, potato or other plants, cellulose such as methylcellulose, hydroxypropylmethylcellulose or sodium carboxymethylcellulose, and gum arabic. And gums including tragacanth and carbohydrates or protein excipients including proteins such as gelatin and collagen. If necessary
Disintegrating or solubilizing agents may be added, including cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate.

Dragee cores are used with suitable coatings, such as concentrated sugar solutions, which are prepared from gum arabic, talc, polyvinylpyrrolidone, carboporgel (carbopol gel).
ol gel), polyethylene glycol and also titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the amount of active compound, ie, dosage.

Pharmaceutical preparations for oral administration include push-fit capsules made of gelatin, as well as soft, sealed soft seal capsules made of gelatin and a coating, such as glycol 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 compounds may be dissolved or suspended in suitable solutions, such as fatty oils, paraffin oils, or with or without stabilizers, such as liquid polyethylene glycols.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds. For injection, the pharmaceutical compositions of the invention are prepared in aqueous solutions, preferably in biocompatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. Aqueous injection suspensions may contain substances that thicken the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or solvents include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Optionally, 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 preparation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention may be prepared by means of conventional mixing, dissolving, granulating, sugar-coating, microparticulating, emulsifying, encapsulating, entrapping or lyophilizing processes known in the art. Manufactured by the method.

The pharmaceutical compositions are provided as salts and can be formed with a number of acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, and the like. Salts tend to be more soluble in aqueous or protic solvents than in the corresponding free base form. In other cases, a preferred formulation is any of 1 mM-50 mM histidine, 0.1% -2% sucrose, 0.1% -2% sucrose, 2% -7% mannitol, combined with a buffer prior to use. Lyophilized powder containing all or all.

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 administration of CECRP, such labeling will include the amount, frequency and manner of administration.

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to serve the purpose. Administering an effective amount can be accomplished within the ability of those skilled in the art.

For any compound, the pharmaceutically effective amount is initially estimated, for example, in any of the cell culture assays for neoplastic cells, or usually in mouse, rabbit, dog or pig animal specimens. The desired enrichment range and route of administration are also determined using animal specimens. The information can then be used to determine effective doses and routes for administration to humans.

A pharmaceutically effective amount is one that ameliorates the symptoms or condition, eg, CECRP or a fragment thereof, an antibody, agonist, antagonist, or CE of CECRP.
The amount of the main component of the inhibitor of CRP. The therapeutic efficacy and toxicity of such compounds is determined by standard pharmaceutical procedures in cell culture or laboratory animals, eg, E.
It can be determined by D ₅₀ (the amount pharmaceutically effective in 50% of the population) and LD ₅₀ (the amount that kills 50% of the population). Dose ratio between therapeutic and toxic effects is the therapeutic index and it can be expressed as the ratio LD ₅₀ / ED _50. Pharmaceutical compositions that exhibit large therapeutic indices are desirable. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human administration. The dosage of such compounds lies preferably toxicity within a range of circulating concentrations that include very little or absolutely no ED _50. Dosage will vary within this range depending upon the dosage form employed, sensitivity of the patient and the route of administration.

The exact dosage will be determined by a physician, depending on factors related to the patient requiring treatment. The amount and administration may be adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors that may be considered are the severity of the disease state, the health of the subject, the age, weight and sex of the patient, diet, time and frequency of administration, drug combinations, sensitivity to response, and Includes resistance / response. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week or once every two weeks depending on the half-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from 0.1 μg to 100,000 μg, with a total dosage up to about 1 g, depending on the route of administration. Guidance on detailed dosages and methods of delivery is provided in the literature and is generally available to practitioners in the art. Those skilled in the art
Different formulations will be used for nucleotides than for proteins or their inhibitors. Similarly, delivery of a polynucleotide or polypeptide will be specific to a particular cell, condition, location, etc.

Diagnosis In another embodiment, an antibody that specifically binds CECRP may be used to diagnose a condition or disease characterized by expression of CECRP, or to treat a patient being treated with a CECRP, agonist, antagonist or inhibitor. Can be used in assays to monitor Antibodies useful for diagnosis can be prepared in the same way as described above for therapy. Diagnostic assays for CECRP use antibodies and labels to detect CCERP in human body fluids or extracts of cells or tissues.
Includes a method for detecting ECRP. The antibodies can be used with or without modification, and can be labeled by binding, either covalently or non-covalently, to a reporter molecule. Various reporter molecules known in the art can be used, some of which are described above.

A variety of protocols are known in the art, including ELISA, RIA and FACS for measuring CECRP, and provide methods for diagnosing altered or abnormal levels of CECRP expression. A normal or standard value for CECRP expression is to bind a body fluid or cell extract taken from a normal mammalian subject, preferably a human, to an antibody against CECRP under conditions suitable for complex formation. Is established by The standard complex formation amount can be quantified by various methods, but a means by optical measurement is preferable. The amount of CECRP expressed in the subject, control and disease, samples from biopsy tissue are compared to standard values. Deviation between standard and subject values establishes parameters for diagnosing disease.

In another embodiment of the invention, a polynucleotide encoding CECRP can be used for diagnosis. Polynucleotides used include oligonucleotide sequences, antisense RNA and DNA molecules and PNA. The polynucleotides can be used to detect and quantify gene expression in biopsy tissues where CECRP expression may correlate with disease. Diagnostic assays can be used to discern the presence and overexpression of CECRP and to monitor modulation of CECRP levels during therapeutic treatment.

In one aspect, the nucleic acid sequence encoding CECRP can be identified by hybridization using a PCR probe capable of detecting a polynucleotide sequence comprising a genomic sequence encoding CECRP or a closely related molecule. . Specificity of a probe consisting of a highly specific region, for example, 10 unique nucleotides in the 5 'regulatory region, or a region of low specificity, for example, especially the 3' coding region, and its hybridization or amplification The stringency (maximum, high, middle or low) will determine whether the probe identifies only naturally occurring sequences that encode CECRP, allelic variants, or related sequences.

Probes can also be used to detect related sequences, and preferably comprise at least 50% of the nucleotides from any of the sequences encoding CECRP. The hybridization probe of the present invention is DNA or RNA,
SEQ ID NO: 6 to SEQ ID NO: 10 or the promoter
It is derived from a genomic sequence containing enhancer elements and introns of naturally occurring CECRP.

Means for generating a specific hybridization probe for DNA encoding CECRP include the steps of cloning a nucleic acid sequence encoding CECRP or a CECRP derivative into a vector for the production of an mRNA probe. Such vectors are well known in the art and are commercially available, and can be used to synthesize RNA probes in vitro by adding an appropriate RNA polymerase and appropriate labeled nucleotides. Hybridization probes can be labeled with various reporters, for example, radionuclides such as ³² P or ³⁵ S, or enzyme labels such as alkaline phosphatase, which are bound to the probe via an avidin / biotin binding system or the like. it can.

[0161] The polynucleotide sequence encoding CECRP can be used for diagnosis of a disorder associated with expression of CECRP. Such obstacles include, but are not limited to,
Actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, Primary thrombocythemia and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, in particular, adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, Ganglion, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary gland, skin, spleen, testis, thymus, thyroid and uterus cancer, and acquired immunodeficiency syndrome ( AIDS), Addison's disease, 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, dermatomyositis, diabetes, emphysema, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel Syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, inflammation of the myocardium or pericardium, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome , Systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, ulcerative colitis, Werner syndrome, and cancer, hemodialysis, complications of extracorporeal circulation, viral infection, bacterial infection, fungal infection, parasite infection, Protozoal and helminth infections; and trauma. CE
The polynucleotide sequence encoding CRP may be analyzed by Southern or Northern analysis,
For use in dot blots, or other membrane-based techniques, or in PCR techniques, or in dipsticks, pins, ELISA assays or microarrays that utilize body fluids or tissues from patient biopsies to detect altered CECRP expression be able to. Such qualitative or quantitative methods are well known in the art.

In certain embodiments, the nucleotide sequence encoding CECRP is useful in assays to detect activation or induction of various cancers, particularly those cancers as described above.
The nucleotide sequence encoding CECRP can be labeled in a standard manner and added to a body fluid or tissue sample from a patient under conditions suitable for forming hybridization complexes. After incubation for an appropriate time, the sample is washed, its signal is quantified and compared to a standard value. If the amount of signal in the biopsied or extracted sample is significantly altered from the amount of signal in the comparative control sample, then the nucleotide sequence has been hybridized to the nucleotide sequence in the sample and The presence of altered levels of the nucleotide sequence encoding CECRP indicates the presence of the associated disease. Such assays can also be used to evaluate the effectiveness of a particular treatment regimen in monitoring animal studies, clinical trials, or treatment of an individual patient.

Normal or standard values for expression are established to provide criteria for diagnosing a disease associated with CECRP expression. It binds a body fluid or cell extract from a normal subject, either an animal or a human, to a CECRP-encoding sequence or fragment thereof under conditions suitable for complex formation, well known in the art. Given by Standard hybridization can be quantified by comparing the value obtained from a normal subject to a value obtained from an experiment in which a known amount of a substantially purified polynucleotide is used. . Standard values obtained from normal samples are compared to values obtained from samples of subjects with symptoms of the disease. Deviation between standard and subject values is used to establish the presence of a disease state.

[0164] Once the disease is confirmed and the treatment protocol is initiated, hybridization assays are performed to assess whether the patient's expression levels begin to approach those observed in normal patients. It is repeated regularly. Results from continuous assays can be used to indicate the efficacy of treatment over a period of days to months.

In the case of cancer, the presence of abnormal amounts of transcripts in biopsy tissue from individual patients may be indicative of a predisposition to the development of the disease, or the disease may be detected before actual clinical symptoms occur. A means for detecting is provided. This more definitive diagnosis allows the physician to take precautionary measures or aggressive treatment in the early stages, thereby preventing or preventing the development of cancer.

Additional diagnostic uses for oligonucleotides designed from sequences encoding CECRP may include the use of PCR. Such oligomers may be chemically synthesized, produced enzymatically, or produced in vitro . Oligomers, including fragments of a polynucleotide encoding CECRP, or fragments of a polynucleotide complementary to the polynucleotide encoding CECRP, may be used under conditions optimized for the identification of specific genes or conditions. Is preferred. Closely related DNA or RNA, even if they are two identical oligomers, a nested set of oligomers, or a degenerate pool of oligomers
It can be used under low stringency conditions to detect and also quantify sequences.

Methods that can be used to quantify CECRP expression also include radiolabeling or biotin labeling of nucleotides, or cross-amplification of control nucleic acids, and standard curves to which experimental results are written (Melby, PC et al. (1993). ) J. Immunol. Methods,
159: 235-244; Duplaa, C. et al. (1993) Anal. Blochem. 229-236). The rate of quantification of large numbers of samples can be accelerated by performing an ELISA format assay, in which the oligomers of interest are expressed in various dilutions, and the spectral photometric or colorimetric reaction is rapidly quantified. Is brought about.

In yet another embodiment, oligonucleotides or long fragments thereof from any of the polynucleotide sequences described herein can be used as targets in a microarray. Microarrays can be used to simultaneously monitor the expression levels of a large number of genes (to generate a transcript image) and identify gene mutation sequences, mutations and polymorphisms. This information determines gene function,
It can be used to understand the genetic basis of the disease, to diagnose the disease, and to generate and monitor the activity of therapeutic agents.

Microarrays are prepared, used, and analyzed using methods well known 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) PCT application WO95 / 251116; Shalon, D. et al. (1995) PCT applicat
ion WO95 / 35505; Heller, RA et al. (1997) Proc. Natl. Acad. Sci. 94: 2150
-2155; and Heller, MJ et al. (1997) US Patent No. 5,605,662).

In another embodiment of the invention, nucleic acid sequences encoding CECRP may be used to generate hybridization probes useful for mapping naturally occurring genomic sequences. The sequence can be on a specific chromosome or on a specific region of the
Alternatively, it may be mapped to an artificial chromosome structure such as, for example, a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a bacterial P1 construct or a single chromosomal cDNA library (eg, Price, CM (1993) B
lood Rev. 7: 127-134; and Trask, BJ (1991) Trends Genet. 7: 149-154).

Fluorescence in situ hybridization correlates with other physical chromosome mapping techniques and genetic map data (eg, Heinz-Ulrich, et al. (1995) in Me
yers, RA (ed.) Molecular.Biology and Biotechnology, VCH Publishers New
York, NY, pp. 965-968). Examples of genetic map data can be found in various scientific journals or in Online Mendelian Inheritance in Man (OMIM). Correlation between the location of the gene encoding CECRP on the physical chromosome map and a particular disease or predisposition to a particular disease makes it possible to demarcate the region of DNA associated with that genetic disease. The nucleotide sequences of the present invention can be used to detect differences in gene sequences between normal and carrier, ie, infected individuals.

[0172] Physical mapping techniques, such as in situ hybridization of chromosomal preparations and linkage analysis using established chromosomal markers, can be used to extend the genetic map. In some cases, the arrangement of genes on the chromosome of another mammalian species, such as the mouse, may reveal relevant markers even if the number or arm of a particular human chromosome is unknown. New sequences can be assigned to chromosome arms or parts thereof by physical mapping. This provides valuable information to researchers searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome is naturally located by genetic linkage from a particular genomic region, for example, from AT to 11q22-23, any sequence mapping to that region may be relevant or regulated for further study. Genes can be represented (see, for example, Gatti et al. (1998) Nature 336: 577-580). Using the nucleotide sequence of the present invention, differences in chromosomal positions can be detected by translocation, inversion, and the like between normal individuals and carriers, that is, infected individuals.

In another embodiment of the present invention, CECRP, its catalytic or immunogenic fragments or oligopeptides, can be used to screen libraries of compounds in any of a variety of drug screening techniques. The fragments used in such a screen may be free in solution, attached to a solid support, supported on a cell surface, or located intracellularly. In some cases, the binding complex formed between CECRP and the factor under test is measured.

Another technique that may be used for drug screening provides for high-throughput screening of compounds with appropriate binding affinity for the protein of interest (see, eg, Geysen, et al. (1984) PCT application WO84 / 03564). I want to.) In this method, a number of different small test compounds, as applied to CECRP, are synthesized on a solid support such as a plastic pin or some other surface. The test compound reacts with CECRP or a fragment thereof and is washed. The bound CECRP is then detected by methods well known in the art. Also purified CECR
P can also be coated directly on a plate for use in the drug screening techniques described above. Alternatively, a non-neutralizing antibody can be used to capture the peptide and immobilize it on a solid support.

In another example, a neutralizing antibody capable of specifically binding CECRP is CE
A competitive drug screening assay is used that competes with the test compound for binding CRP. In this way, antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with CECRP.

In yet another embodiment, where the novel technology relies on properties of currently known nucleotide sequences, including but not limited to triplet genetic code and properties such as specific base pairing interactions. Can use the nucleotide sequence encoding CECRP in any molecular biology technology that will be developed in the future.

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 foregoing and all patent applications, patents, publications mentioned below, and in particular US application Ser. No. 60 / 088,695, are hereby incorporated by reference.

[0179]

【Example】

1 Preparation of cDNA Library RNA was purchased from Clontech (Palo AltO. CA) or isolated at Insight from the tissues listed in Table 4. The tissue is homogenized,
Dissolved in guanidinium isocyanate and the lysate was centrifuged on a CsCl cushion. Alternatively, the tissue is homogenized and phenol,
Alternatively, it was dissolved in a suitable mixture of a denaturing agent such as TRIZOL reagent (Life Technologies), a single-phase solution of phenol, and guanidine isocyanate, and the lysate was extracted with chloroform (1: 5 v / v). RNA was precipitated from the lysate using either isopropanol or sodium acetate and ethanol. Alternatively, preparative agarose gel electrophoresis can be used to convert R
NA was purified and recovered from Whatman P81 paper (Whatman, Lexington. MA). Phenol extraction and precipitation of RNA was repeated as necessary to increase RNA purity and keep RNA in RNase-free solution. In some cases,
RNA was treated with DNase. For most libraries, poly (A +) R
NA was isolated using oligo d (T) -bound paramagnetic particles (Promega. Madison. WI), Oiigotex resin, or Oligotex kit (QIAGEN Inc. Chatsworth. CA). Alternatively, RNA can be obtained from RNA Isolation kit (Stratagene) or Ambion PolyA Quick
Kits (Ambion. Austin. TX) were used for direct isolation from tissue products.

[0180] RNA is prepared using UNIZAP vector (Stratagene. La Jolla. CA) or SuperScript.
Following the procedure recommended by the plasmid system (Life TechnologieS. Inc)
Used for cDNA synthesis and cDNA library construction. Both procedures are based on methods well known in the art ((Ausubel, 1997,
units 5.1-6.6)). Alternatively, a cDNA library was constructed by Stratagene using RNA provided by Insight. Reverse transcription was initiated using oligo d (T) or random primer. A synthetic oligonucleotide adapter was ligated to the double-stranded DNA, and the cDNA was digested with appropriate restriction enzymes. For most libraries, the cDNA is Sephacryl S 1000 or Sepharose CL2B
Alternatively, the size was selected (300-1000 bp) using CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis.
). The cDNA is, for example, pBluescript (Stratagene), pSPORT1 (Life Technolog
ies), pINCY (Incyte Pharmaceuticals Inc, Paio Alto. CA) and ligated to a compatible restriction enzyme of a polylinker of a suitable plasmid. PINCY is JM10
Amplified in 9 cells and purified using QiaQuick column (QIAGEN Inc). The recombinant plasmid is, for example, XL1-Biue, XL1-Blue MRF or SOLR (Stratagene) or D
Competent E.H.H50t. DHIOB or ElectroMAX DH 10B (Life Technologies) . E. coli cells were transformed.

2 Isolation and Sequencing 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, REAL Prep 96 plasmid kit. After precipitation, they were resuspended in 0.1 ml of distilled water and stored at 4 ° C. with or without lyophilization.

Alternatively, plasmid DNA was amplified from host cell lysates by high-throughput format direct binding PCR (Rao, VB (1994) Anal. Blochem. 216: 1).
-14). 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 fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and Fluoroskan II fluorescence scanner (Labsystems Oy, Helsinki, Finland) did.

3 Homology search for cDNA clones and their analogous proteins ABI Catalyst 800 () or Hamilton Micro Lab 2200 (Hamilton, Reno, NV
) Was used in combination with Peltier Thermal Cyclers (PTC200 from MJ Research, Watertown, Mass.) To prepare cDNA for sequencing. Sequencing of this cDNA was performed using the standard ABI protocol, base-calling software and kit, according to the method of Sanger and AR Coulson (1975, J. Mol. Biol. 94:44).
1-448) in an ABI 373 or 377 DNA Sequencing System (Perkin Elmer). Alternatively, using a solution and dye from Amersham Pharmacia Biotech,
The DNA was sequenced. Reading frames were determined using standard methods (Ausubel. Supra).

Using the full-length nucleotide and / or amino acid sequences of this cDNA sequence and sequence listing as query sequences, GenBank primates, rods,
Database of mammals (mamp), vertebrates (vrtp), and eukaryotes (eukp),
Databases were searched, such as SwissProt, BLOCKS, and other databases in which previously identified motifs and sequences were annotated. Smith Waterman (Smith, T. et al. (1992) P.) to process primary sequence patterns and secondary structure gap penalties
algorithms such as rotein Engineering 5: 35-51) and BLAST (Basic Local)
Alignment Search Tool; Altschul, SF (1993) J. Mol. Evol 36: 290-300; and Altschui et al. (1990) J. Mol. Biol. 215: 403-410), and HMM (Hidden Markov Models) Eddy, SR (1996) Cur. Opin. Str. Biol. 6)
: 361-365 and Sonnhammer, ELL et al. (1997) Proteins 28: 405-420) using a program and algorithm to assemble and analyze nucleotide and amino acid sequences. These databases, programs, algorithms, methods and tools are already available and are available from Ausubel (supra, unit 7.7), Meyer
s, RA (1995; Molecular Biology and Biotechnology , Wiley VCH, Inc, New
York NY, p 856-853), software (Genetics Computer Group (GCG), Madis
on Wl) and in documents on Internet websites. Two comprehensive websites listing, describing and linking many databases and tools are: 1) the www resource in practical se
quence analysis (http://genome.wustl.edu/eddy/bio5495/online_resources.h
tml), and (2) the bibliography of computational gene recognition (ht
tp: //linkage.rockefeiler.edu/wli/gene/programs.html). For example,
The first website uses PFAM (http://genome.wustl.edu/Pf) as its database.
am /), and an HMM search tool (http://genome.wustl.edu/eddy/cgi)
-bin / hmm_page.cgi).

Table 5 outlines the databases and tools used here. The first column of Table 1 describes the tool, program or algorithm, the second column of the table describes the database name, the third column describes a brief description, and the fourth column describes (there (If described in), a score that determines the degree of coincidence between the two sequences (the higher the value, the higher the homology).

4 Northern Analysis Northern analysis is an experimental technique used to detect the presence of a transcript of a gene, in which a labeled nucleotide sequence is bound to a membrane to which RNA from a particular cell type or tissue is bound. Hybridization is performed (see, eg, Sambrook, supra, ch. 7 and Ausubel, FM, supra, ch. 4 and 16).

Similar computer techniques using BLAST (Altschul, SF 1993 and 1990, supra) were used to search for identical or related molecules in databases such as the GenBank or LIFESEQ ^™ database (Incyte Pharmaceuticals). This analysis
Very fast compared to many membrane-based hybridizations. In addition, the sensitivity of the computer search can be altered to determine whether a match is an exact match or homologous.

The reference value of the search is a product score, which is defined by the following equation. (% Sequence identity x maximum BLAST score (%)) / 100 This product score takes into account both the degree of similarity between the two sequences and the length match of the sequences. For example, if the product score is 40, the match is accurate within an error of 1-2%, and if the score is 70, the match is exact. Homologous molecules are usually identified by selecting those that show a product score of 15-40, but those with low scores can also be identified as related molecules.

The results of the Northern analysis are reported as a list of libraries in which transcripts encoding CECRP are generated. Abundance and abundance of sequence
A bundance list is also reported. Abundance directly reflects the number of detections of a particular transcript, and abundance is the abundance divided by the total number of sequences detected in the cDNA library.

5 Extension of Sequence Encoding CECRP Clone 037377, 162871, 236062, 1596
Using the nucleic acid sequences of 581 and 1853196, oligonucleotide primers were designed to extend the partial nucleotide sequence to full length. One primer was synthesized to initiate elongation in the antisense direction, and the other was synthesized to elongate the sequence in the sense direction. Primers were used to facilitate extension of a known sequence to generate an "outside" amplicon containing a new, unknown nucleotide sequence for the region of interest. Initial primers are designed from cDNA using OLIGO 4.06 (National Biosciences) or other suitable program, are approximately 22-30 nucleotides in length, have a GC content of 50% or more, and Anneal to the target sequence at a temperature of 72 ° C. Extension of nucleotides that would result in hairpin structure and primer-primer dimerization was avoided.

The sequence was extended using a selected human cDNA library (Gibco / BRL).
If more than one extension is needed or desired, an additional set of primers is designed to further extend the known region.

High fidelity amplification was obtained by thoroughly mixing the enzyme and reaction mixture according to the instructions for the XL-PCR kit (Perkin Elmer). 40 pm for each primer
If all other components of the kit are started at the recommended concentrations, PCR
Step 1 1 minute 94 ° C (initial denaturation) Step 2 1 minute 65 ° C Step 3 6 minutes 68 ° C Step 4 15 seconds 94 ° C Step 5 1 minute 65 ° C using ier Thermal Cycler (PTC200; MJ Research, Watertown MA) Step 6 7 minutes 68 ° C Step 7 Repeat Steps 4-6 for another 15 cycles Step 8 15 seconds 94 ° C Step 9 1 minute 65 ° C Step 10 7 minutes 15 seconds 68 ° C Step 11 12 cycles repeat Step 8-10 Step The procedure was performed at a parameter of 72 ° C. for 128 minutes and a step of 134 ° C. (hold).

A 5-10 μl aliquot of the reaction mixture was analyzed by electrophoresis on a low concentration (approximately 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 QI
Purified using A Quick ^™ (QIAGEN Inc. Chatsworth, CA) and excised from overhangs using Klenow enzyme to facilitate recombination and cloning.

After ethanol precipitation, the product was redissolved in 13 μl ligation buffer and 1 μl
1T4-DNA ligase (15 units) and 1 μl T4 polynucleotide kinase were added and the mixture was incubated for 2-3 hours at room temperature or at 16 ° C. overnight. Competent E. E. coli cells (in 40 μl of the appropriate medium) were transformed with 3 μl of the ligation mixture and cultured in 80 μl of SOC medium (see, eg, Sambrook, supra, Appendix A, page 2). After incubation for 1 hour at 37 ° C., E. E. coli mixture contains 2xCarb Luria Bertan
i (LB) -agar (see Sambrook, supra, Appendix A, page 1). The next day, several colonies were picked at random from each plate and placed in individual wells of a suitable commercially available sterile 96-well microtiter plate with 150 μl of liquid LB / 2.
Cultured in xCarb medium. The following day, 5 μl each of the overnight cultures were transferred to a sterile 96-well plate, diluted 1:10 with water, and then 5 μl of each sample was transferred to a PCR array.

For PCR amplification, 18 μl of the enriched PCR reaction mixture (3.3 ×) containing 4 units of rTth DNA polymerase, vector primers and one or both gene-specific primers used for the extension reaction were added to each well. Added. Amplification: 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 step 2-4 for an additional 29 cycles Step 6 72 ° C for 180 seconds Step 74 ° C (Retained).

An aliquot of the PCR reaction was run on an agarose gel with molecular weight markers. The size of the PCR product was compared to the original partial cDNA, the appropriate clone was selected, ligated into a plasmid and sequenced.

Similarly, using the nucleotide sequences of SEQ ID NO: 6 to SEQ ID NO: 10, the procedure described above, using oligonucleotides designed for 5 ′ extension and 5 ′ regulatory sequences using appropriate genomic libraries Get.

6 Labeling and Use of Individual Hybridization Probes cDNA, mRNA and genomic DNA are screened using hybridization probes based on SEQ ID NO: 8 to SEQ ID NO: 14. Although specifically described for labeling oligonucleotides of about 20 base pairs, the same procedure is used for larger cDNA fragments. The oligonucleotides were designed using the latest software, such as OLIGO 4.06 (National Bioscience), and the oligomer of 50pmol, [γ- ^{3 2} P] adenosine triphosphate (Amersham) and T4 polynucleotide kinase 250μCi (DuPo
nt NEN, Boston MA). The labeled oligonucleotide is applied to a Sephadex G-25 ultra-fine resin column (Pharmacia & Upjohn).
). Aliquots containing 10 ⁷ counts of labeled probe per minute were used for endonuclease (AseI, BglII, EcoRI, PstI, Xba1 or PvuII; D
uPont NEN, Boston, Mass.) in a typical membrane-based hybridization analysis of human genomic DNA that has been cut.

The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to a nylon membrane (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is performed at 40 ° C. for 16 hours. To remove non-specific signals, blots are washed sequentially at room temperature under increasingly stringent conditions up to 0.1x sodium citrate and 0.5% sodium dodecyl sulfate. XOMAT AR ^TM film (Kodak, Rochester, NY) is transferred to a Phosphoimager cassette (Molecular Dynamics
Hybridization patterns are visually compared after exposure to the blots for several hours in Sunnyvale, CA).

7 Array elements can be synthesized on the surface of a substrate using microarray chemical coupling procedures and inkjet devices (see, eg, Baldeschweiler, supra). Elements can be arranged using arrays similar to dot or slot blots, and the elements can be bound to the substrate surface by thermal, UV, mechanical or chemical bonding procedures, or vacuum systems. A typical array is made by hand or using available methods and machines, and may include any suitable number of elements. After hybridization, the microarray is washed to remove unhybridized probes and the level and pattern of fluorescence is determined using a scanner. The scanned images are examined to determine the degree of complementarity and the relative amount of each oligonucleotide sequence on the microarray.

Full-length cDNA or Expressed Sequence Tag (EST)
Contains the elements of the microarray. A full-length cDNA or EST corresponding to one of the nucleotide sequences of the present invention or randomly selected from a cDNA library related to the present invention is arranged on a suitable substrate, for example, a glass slide. The cDNA is immobilized on a glass slide by drying it thermally and chemically, for example using UV cross-linking (Schena, M. et al. (1995) Science).
270: 467-470, and Shalon, D. et al. (1996) Genome Res. 6: 639-645). A fluorescent probe is prepared and used to hybridize to an element on the substrate. The substrate is analyzed according to the procedure described above.

8 The sequence complementary to the complementary polynucleotide CECRP, or any sequence thereof,
Used to reduce or inhibit the expression of naturally occurring CECRP. About 15 to about 3
Although the use of oligonucleotides containing zero base pairs is specifically described, generally the same method can be used for smaller sequences or larger sequence fragments. The appropriate oligonucleotides can be designed using the Oligo 4.06 software and the coding sequence of CECRP. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5 'sequence and used to inhibit the promoter from binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed so that the ribosome is
Prevents binding to transcripts encoding RP.

9. Expression of CECRP Expression and purification of CECRP can be performed using a bacterial or viral based expression system. Due to the expression of CECRP in bacteria, antibiotic resistance and cD
Suitable vectors containing an inducible promoter to increase the level of transcription of NA
A is subcloned. Such promoters include the T5 or T7 bacteriophage promoter associated with the lac operator regulatory element and tr.
Includes, but is not limited to, the p-lac (tac) hybrid promoter. The recombinant vector is transformed into a suitable bacterial host, such as BL21 (DE3).
Bacteria with antibiotic resistance are isopropyl β-D thiogalactopyranoside (IPTG)
Induces HLOR when induced. Expression of CECRP in eukaryotic cells, Auto commonly known as baculovirus scan insect cell lines or mammalian cell lines
It is performed by infecting graphica californica nuclear pleiotropic virus (AcMNPV). The non-essential polyhedrin gene of the baculovirus is replaced with cDNA encoding CECRP by either homologous recombination or bacterial-mediated gene transfer with transfer plasmid-mediated. The virus's infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription. Recombinant baculovirus is often used to infect Spodoptera frugiperda (Sf9) insect cells, but may also be used to infect human hepatocytes. In the latter case, further genetic modification of the baculovirus is required. (For example, Engelhard. EK et al. (199
4) Proc. Natl. Acad. Sci. USA 91: 3224-3227; Sandig, V. et al. (1996) Hum. Ge
ne Ther. 7: 1937-1945.).

[0204] In most expression systems, CECRP is a fusion protein synthesized with, for example, glutathione S-transferase (GST) or a peptide epitope tag such as FLAG or 6-His. Affinity-based purification of the recombinant fusion protein can be performed quickly and in one go. GST, a 26 kilodalton enzyme from Schistosoma japonicum, allows purification of the fusion protein with immobilized glutathione while maintaining protein activity and antigenicity. (Pharmacia, Piscataway, NJ). After purification, the GST moiety is replaced with H
It can be proteolytically cleaved from LOR. FLA, a peptide of 8 amino acids
G allows for immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies. 6-His with six consecutively extending histidine residues allows for purification on metal chelating resins (QIAGEN Inc, Chatsworth, CA). Methods for protein expression and purification are described in Ausubel. FM et al. (1995, and periodically supplemented) Current Protocols in Molecular Biology (John Wiley & Sons, N.
ew York, NY, ch 10, 16). HLOR purified by these methods
Can be used directly to perform the following assays.

10 Demonstration of CECRP Activity CECRP activity is measured by measuring terminal differentiation or induction of cell cycle progression when CECRP is expressed at physiologically elevated levels in a mammalian cell culture system. Is shown. The cDNA is subcloned into a mammalian expression vector containing a strong promoter that increases the level of cDNA expression. The vectors selected are pCMV SPORTTM (Life Technologies, Gaithersburg, MD) and pCRTM 3.1
(Invitrogen, Carlsbad, CA), both of which include the cytomegalovirus promoter. 5-10 μg of the recombinant vector is transiently transfected into a human cell line, preferably endothelial or hematopoietic, using either liposome preparation or electroporation. It is co-transfected with 1-2 μg of an additional plasmid containing the sequence encoding the marker protein. Marker protein expression provides a means to distinguish transfected cells from non-transfected cells and is a reliable predictor of cDNA expression from recombinant vectors. The marker protein to be selected is, for example, green fluorescent protein (GFP; C).
lonetech, Palo Alto, CA) or CD64 or CD64-GFP fusion protein. Flow cytometry detects and quantifies the uptake of fluorescent molecules that diagnose events that precede or coincide with cell cycle progression or terminal differentiation. These events include changes in nuclear DNA content as measured by staining of the DNA with propidium iodide, changes in cell size and particle size as measured by forward light scatter and 90 ° side light scatter, Up-regulation or down-regulation of DNA synthesis as measured by a decrease in incorporation of origin, changes in cell surface and intracellular protein expression as measured by reaction with specific antibodies, fluorescein-conjugated annexin V protein to the cell surface Includes changes in plasma membrane composition as measured by binding. Flow cytometry is available from Ormerod, MG (
1994) described in Flow Cvtometry , Oxford, New York, NY.

11 Functional Assays The function of CECRP is assessed by the expression of sequences encoding CECRP at physiologically elevated levels in mammalian cell culture systems. cDNA is converted to cD
It is subcloned into a mammalian expression vector containing a strong promoter that expresses NA at high levels. Such vectors include pCMV SPORT ^TM (Life Technolog
ies. Gaithersburg. MD) and pCR ^TM 3.1 (Invitrogen, Carlsbad, CA).
Both contain the cytomegalovirus promoter. 5-10 μg of the recombinant vector is preferably transiently transfected into human cell lines, preferably of endothelial or hematopoietic origin, by liposome preparation or electroporation. In addition, 1-2 μg of plasmid containing the sequence encoding the labeled protein is co-transfected. Expression of the labeled protein allows one to distinguish between transfected and non-transfected cells. Further, the expression of the cDNA from the recombinant vector can be accurately predicted by the expression of the labeled protein. Such labeled proteins include green fluorescent protein (GFP
) (Clontech, Palo Alto, CA), and CD64 or CD64-GFP fusion proteins. Transfected cells expressing GFP or CD64-GFP are identified using automated flow cytometry (FCM) using a technique based on laser optics, and properties such as apoptotic status are evaluated. In addition, the FCM detects and measures the incorporation of a fluorescent molecule that diagnoses the phenomenon of preceding or simultaneous cell death. These phenomena include changes in nuclear DNA content as measured by staining DNA with propidium iodide, down-regulation of DNA synthesis as measured by reduced uptake of bromodeoxyuridine, and specific antibody and And changes in plasma membrane composition as measured by the binding of the fluorescent complex annexin V protein to the cell surface, as measured by the reactivity of E. coli. Flow cytometry is described in Ormerod, MG (1994) Flow Cytometry (Oxford, New York, NY.).

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

Production of 12 CECRP-Specific Antibodies For immunization of rabbits and production of antibodies using standard protocols, see PAGE electrophoresis (see, eg, Harrington, MG (1990) Methods Enzymol. 182: 488-495). Or CECRP substantially purified using other purification techniques.

In another method, the amino acid sequence of CECRP is analyzed using LASERGENETM software (DNASTAR) to determine a region having high immunogenicity, and the corresponding oligopeptide is synthesized by a method known to those skilled in the art, and Used to raise antibodies by methods well known to those skilled in the art. Methods for selecting appropriate epitopes, such as those in the hydrophilic region near or adjacent to the C-terminus, are well known in the art (eg, Ausubel et al., Supra, ch. 1).
See 1).

Typically, oligopeptides are 15 residues in length, using Applied Biosystems Peptide Synthesizer Mod using fmoc (fluorenylmethoxycarbonyl) chemistry.
It is synthesized using el 431A, and M-maleimidobenzoyl-N-hydroxysuccinimide ester (
MBS: bound to keyhole limpet hemocyanin (KLH, Sigma, St. Louis, MO) by reacting with Ausubel et al., Supra. Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. The resulting antiserum can be prepared, for example, by binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antiserum, washing, and reacting with radioiodine-labeled goat anti-rabbit IgG. Tested for peptide activity.

13 Purification of Spontaneous CECRP Using Specific Antibodies Spontaneous or recombinant CECRP is substantially purified by immunoaffinity chromatography using antibodies specific for CECRP. Immunoaffinity columns
CECRP antibodies are constructed by covalently linking them to an activated chromatographic resin such as CnBr-activated Sepharose (Pharmacia & Upjohn). After bonding, the resin is blocked and washed according to the manufacturer's instructions.

The medium containing CECRP was passed over an immunoaffinity column and the column was
The substrate is washed under conditions that allow selective absorption of P (for example, a detergent containing a high ionic strength buffer). The column was used under conditions that disrupted antibody / CECRP binding (pH 2
CECRP is eluted with ~ 3 buffers or a high concentration of chaotrope such as urea or thiocyanate ions).

13 Identification of Molecules Interacting with CECRP CECRP or a biologically active fragment thereof was analyzed using ¹²⁵ I Bolton-Hunter reagent.
(Bolton AE et al. (1973) Biochem J 133: 529). Candidate molecules previously sequenced in the wells of the multiwell plate are incubated with labeled CECRP, washed, and any wells with labeled CECRP complexes are assayed. Using data obtained from various CECRP concentrations, the number, affinity and CE
A value indicating the relationship between CRP and the candidate molecule is calculated.

All patent applications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described with reference to certain preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be considered within the scope of the following claims. .

[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 43/00 111 C07K 16/18 4H045 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 C12N 15/00 ZNAA C12P 21/02 A61K 37/02 C12Q 1/68 C12N 5/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA ( M, 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, GE, GH, GM, HR, HU, ID, IL, 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 (72) Inventor Lal, Pleity 95054 Santa Clara Las Drive, California 2382 (72) Inventor Tang, Wy Thom 95118, California, United States San Jose Runwick Co 4230 (72) Inventor Korey, Neil Sea, USA 40940, Mountain View # 30, Dale Avenue 1240 (72) Inventor Gegler, Carl Jay, 94025, California, United States Menlo Park Oakland Avenue 1048 (72) Inventor Bogung, Mariah Earl 94577 San Leandro Santiago Road, California, USA 14244 (72) Inventor Patterson, Chandra 94025 Menlo Park, California, USA Menlo Park # 1 Sherwood Way 490 F-term (reference) 4B024 AA01 AA11 BA80 CA04 DA01 DA02 DA05 DA11 EA04 GA11 4B063 QA01 QA13 QA18 QQ02 QQ42 QQ53 QR08 QR62 QS25 4B064 AG01 AG26 CA19 CC24 DA03 DA13 4B065 AA01X AA57X AA87X AA93Y AB01 AC14 BA02 CA24 CA25 CA44 CA46 4C084 CA18A23 CA53 CA23 CA15 CA15 CA15 CA15 CA15 CA15 CA15 CA15 CA15 CA15 CA15 CA15 CA15 CA18 CA12 CA15 CA15 CA15 CA15 CA15 CA15 CA15 CA12 CA15 CA15 CA15 CA15 CA15 CA15 CA15 CA15 CA18 CA12 CA23 CA15 ZA552 Z A592 ZA662 ZA682 ZA752 ZA812 ZA892 ZA942 ZA962 ZA972 ZB052 ZB072 ZB112 ZB132 ZB152 ZB262 ZB272 ZB332 ZB352 ZB372 ZB382 ZB392 ZC022 ZC062 ZC352 ZC552 4H045 AA10 AA11 AA20 FA10 FA10

Claims (23)

[Claims] 1. SEQ ID NO: 1, SEQ ID NO: 2, SEQ I
A substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of D NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, or a fragment thereof. 2. A substantially purified variant having at least 90% amino acid sequence identity to the sequence of claim 1. 3. An isolated and purified polynucleotide encoding the polypeptide of claim 1. 4. An isolated and purified polynucleotide variant having at least 90% 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. SEQ ID NO: 6, SEQ ID NO: 7, SEQ I
An isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of D NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, or a fragment thereof. 8. An isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide of claim 7. 9. An isolated and purified polynucleotide having a sequence complementary to the polynucleotide of claim 7. 10. An expression vector comprising at least a fragment of the polynucleotide of claim 3. 11. A host cell comprising the expression vector according to claim 10. 12. SEQ ID NO: 1, SEQ ID NO: 2, SEQ
A method for producing a polypeptide comprising an amino acid sequence selected from the group consisting of ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, or a fragment thereof, comprising: 12. A method comprising: culturing the host cell of claim 11 under suitable conditions; and (b) recovering said polypeptide from said host cell culture. 13. A pharmaceutical composition comprising the polypeptide of claim 1 together with a suitable pharmaceutical carrier. 14. A purified antibody that specifically binds to the polypeptide of claim 1. 15. A purified agonist of the polypeptide of claim 1. 16. A purified antagonist of the polypeptide of claim 1. 17. A method for treating or preventing a cell proliferation disorder, comprising administering an effective amount of the pharmaceutical composition of claim 13 to a subject in need of treatment. 18. A method for treating or preventing a cell proliferation disorder, comprising administering an effective amount of the antagonist of claim 16 to a subject in need of treatment. 19. A method for stimulating cell proliferation comprising administering to a cell an effective amount of the pharmaceutical composition of claim 13. 20. A method for treating or preventing an immune disorder, comprising administering an effective amount of the antagonist of claim 16 to a subject in need of treatment. 21. A method for treating or preventing an immune disorder, comprising administering an effective amount of the pharmaceutical composition of claim 13 to a subject in need of treatment. 22. In a biological sample containing nucleic acids, SEQ ID NO:
1, for detecting a polynucleotide encoding a polypeptide containing an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 or a fragment thereof (A) hybridizing the polynucleotide of claim 6 to at least one of the nucleic acids of the biological sample to form a hybridization complex; and (b) the hybridization complex. Detecting the presence of the hybridization complex correlates with the presence of a polynucleotide encoding the polypeptide in the biological sample. 23. The method of claim 22, wherein said nucleic acids of said biological sample are amplified by a polymerase chain reaction prior to said hybridization step.