JP2002534116A - Circadian rhythm-related proteins - Google Patents

Abstract

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

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Technical Field The present invention relates to nucleic acid sequence and amino acid sequence of a protein associated with the circadian rhythm, and reproductive disorders, gastrointestinal disorders, diagnosis of neurological disorders and developmental disorders, related to usage of these sequences in the treatment and prevention It is.

[0002] The most important external cue to the brain resulting in the establishment of background biological rhythm of the invention (cue) is a light. Mammals tend to be either diurnal or luminous, and their periods of rest and activity are divided according to boundaries corresponding to dawn and sunset. The dominant cycle of day and night is called circadian rhythm. The circadian rhythmic nature of biological processes
It is a fundamental property of all eukaryotes and some prokaryotes. These rhythms are regulated by an internal time keeping system. Changes in the external environment, in particular changes in the light-dark cycle, regulate this biological clock. Time cue
Under constant environmental conditions without rhythm, the rhythm regulated by the clock exhibits a period of approximately 24 hours. When mammals are placed in an environment that is deprived of sensation, they exhibit a rhythm with an approximate 24-hour cycle, which varies from species to species, with larger or smaller cycles. For example, humans exhibit a rhythm that averages one hour longer than a normal 24-hour cycle. After a period of segregation from normal signaling factors, most humans establish a cycle of rest and activity of 25 hours. However, in some species, it shifts to 48-50 hours, twice the normal rhythm. Isolation from normal signaling factors can stress physical and mental health. In the case of humans,
It is believed that the normal rhythm of arousal and sleep concentrated around 24 hours has a stabilizing effect on physical and mental health and psychological health. Due to the mess of this rhythm,
As a result, different body rhythms can occur and take new patterns out of normal synchrony (Reviewed in Restak, RM (1984) The Brain , Bantam Books, New Y
ork NY, pp. 101-111).

The suprachiasmatic nucleus (SCN) of the hypothalamus contains the dominant circadian clock that regulates most, if not all, rhythms in mammals. The name is SC just above the chiasm
It comes from the N position. Although the SCN receives information about light and darkness from the eyes, it has its own neural pathway, the hypothalamic tract (RHT), which is distinct from the major nerve bundles that transmit visual information to the brain. When SCN is destroyed in laboratory animals by surgical pinpoint damage to the brain, the sleep and wake rhythms as well as many other rhythms are lost. Interestingly, animals lacking the SCN run and feed in equal amounts in each 24 hour period, but their activity is irregularly distributed day and night. Melatonin, the major pineal hormone, can alter the circadian rhythm of mammals. The circadian effects of melatonin are thought to be mediated by melatonin receptors on SCN.

[0004] In general, there are many unknowns about the characteristics and degree of circadian rhythm-regulated gene expression. In the case of Drosophila melanogaster , it is known that only the period (per) gene, an essential component of the circadian pacemaker, exhibits rhythmic mRNA expression. By evaluation of the control of circadian gene expression in D. melanogaster, the gene depends on the period of a circadian expression as D. melanogaster rhythmic expression gene 2 (Deg-2) have been identified. Some mRNAs are known to show maximum expression in the morning, while other mRNAs are known to be most abundantly expressed in the evening. Each of the "morning-type" cDNAs is known to exhibit a unique dependence on the presence of a light-dark cycle, timed feeding, and the function of the per gene on periodic fluctuations.
Thus, diurnal control of gene expression is an important phenomenon in the fly head, which contains many uncharacterized genes. A subset of circadian expressed genes depends primarily on their rhythmic cycle of expression (Van Gelder, RN et al. (1995)
Curr. Biol. 5: 1424-1436). Another interesting molecule involved in biological timing is the product of the clk-1 gene, a 187 residue CLK-1 protein. The activity of clk-1 in the nematode Caenorhabditis elegans regulates the rate of development, the rate of behavior and the time of death of the insect. In the C. elegans clk-1 mutant, the timing of its physiological processes is deregulated, suggesting the existence of a timing mechanism that affects timed processes throughout the life of the insect. CLK-1 is known to be highly conserved among nematodes, rodents and humans, and it is structurally similar to the yeast metabolic regulator COQ7. The human, rat and mouse homologues of the clk-1 gene span 177 residues.
Shows 33% identity. The level of identity between the rat and human sequences strongly supports the idea of functional conservation of clk-1 from yeast to human. Furthermore, C
It is possible to split and align the sequence of the LK-1 homolog and reveal the presence of the 82 residue domain of the tandem repeat core (TRC) with some conserved hydrophobic residues (Ewbank , JJ et al. (1997) Science 275: 980-983).

[0005] Seasonal affective disorder (SAD) is a form of recurrent depressive disorder or bipolar disorder with manifestations of varying degrees of severity. Often, patients with seasonal affective disorder (SAD) have abnormalities in resetting their circadian clock. "Winter depression", the best known form of seasonal affective disorder, is characterized by recurrent depression, hypersomnia, carbohydrate cravings, and weight gain starting in autumn and continuing throughout winter . Current studies on SAD postulate that melatonin is a major mediator of the human circadian system in the CNS, suggesting retinal loss, internal circadian rhythm phase-disturbance, and neuroendocrinology. Expression disorder (neuroendoc
rinologically expressed disorders). Neurotransmitter abnormalities, such as serotonin, have also been linked to SAD. The prevalence of SAD is even higher among patients treated for depression (Gysin, F. et al. (1997) A
cta. Med. Port. 10: 887-893).

[0006] The disclosure of novel circadian rhythm-related proteins and polynucleotides encoding them provides new compositions useful for the diagnosis, prevention and treatment of reproductive, gastrointestinal, neurological and developmental disorders. It meets the needs of those skilled in the art.

SUMMARY OF THE INVENTION The present invention features a circadian rhythm-related protein that is a purified polypeptide, collectively referred to as "CIRYP", individually referred to as "CIRYP-1" and "CIRYP-2". . In one embodiment, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2. A naturally occurring amino acid sequence having at least 90% sequence identity, (c) SEQ ID NO.
: A biologically active fragment of an amino acid sequence selected from the group consisting of: 1-2, or (d
C) providing an isolated polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-2.

Furthermore, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2. A naturally occurring amino acid sequence having at least 90% sequence identity, (c) SEQ ID NO: 1-2
A biologically active fragment of an amino acid sequence selected from the group consisting of: or (d) SE
Provided is an isolated polynucleotide encoding a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of Q ID NOs: 1-2. Alternatively, the polynucleotide is selected from the group consisting of SEQ ID NO: 3-4.

Furthermore, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2. A naturally occurring amino acid sequence having at least 90% sequence identity, (c) SEQ ID NO: 1-2
A biologically active fragment of an amino acid sequence selected from the group consisting of: or (d) SE
Provided is a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of Q ID NO: 1-2. . Alternatively, the invention provides a cell transformed with the recombinant polynucleotide. In another alternative, the invention provides a genetic transformant comprising the recombinant polynucleotide.

The present invention also relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2. A naturally occurring amino acid sequence having at least 90% sequence identity, (c) SEQ ID NO: 1-2
A biologically active fragment of an amino acid sequence selected from the group consisting of: or (d) SE
Provided is a method for producing a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of Q ID NO: 1-2. The method includes (a) culturing cells under conditions suitable for expression of a polypeptide, the cells comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide. Culturing steps such as transformation with the recombinant polynucleotide, and (b) recovering the polypeptide so expressed.

Furthermore, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2. A naturally occurring amino acid sequence having at least 90% sequence identity, (c) SEQ ID NO: 1-2
A biologically active fragment of an amino acid sequence selected from the group consisting of: or (d) SE
Provided is an isolated antibody that specifically binds to a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of Q ID NO: 1-2.

Further, the present invention provides (a) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 3-4, and (b) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 3-4. A naturally occurring polynucleotide sequence having at least 70% sequence identity to (c) a polynucleotide sequence complementary to (a), or (d) (b)
Provided an isolated polynucleotide comprising a polynucleotide sequence complementary to Alternatively, the polynucleotide comprises at least 60 contiguous nucleotides.

Further, the present invention provides (a) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 3-4, and (b) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 3-4. A naturally occurring polynucleotide sequence having at least 70% sequence identity to (c) a polynucleotide sequence complementary to (a), or (d) (b)
Comprising a polynucleotide sequence comprising a polynucleotide sequence complementary to
Methods are provided for detecting a target polynucleotide in a sample. The method includes the steps of (a) hybridizing a sample to a probe comprising at least 16 contiguous nucleotides comprising a sequence complementary to a target polynucleotide in the sample, wherein the probe and the target A hybridization process in which the probe specifically hybridizes to the target polynucleotide under conditions where a hybridization complex is formed between the polynucleotides; and (b) whether the hybridization complex exists This includes a step of detecting whether or not the amount is present, and, if present, optionally detecting the amount thereof. Alternatively,
The probe contains at least 30 contiguous nucleotides. Alternatively, the probe comprises at least 60 contiguous nucleotides.

Further, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2. A naturally occurring amino acid sequence having at least 90% sequence identity, (c) SEQ ID NO: 1-2
A biologically active fragment of an amino acid sequence selected from the group consisting of: or (d) SE
Provided is a pharmaceutical composition comprising an effective amount of a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of Q ID NO: 1-2, and a pharmaceutically acceptable excipient. . Further, the present invention provides a method of treating a disease or condition associated with reduced expression of functional CIRYP, comprising administering the pharmaceutical composition to a patient in need of such treatment. I do.

Further, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2. A naturally occurring amino acid sequence having at least 90% sequence identity, (c) SEQ ID NO: 1-2
A biologically active fragment of an amino acid sequence selected from the group consisting of: or (d) SE
Provided is a method for screening a compound containing an immunogenic fragment of an amino acid sequence selected from the group consisting of Q ID NO: 1-2 for the effectiveness of a polypeptide as an agonist. The method includes (a) exposing a sample containing the polypeptide to the compound, and (b) detecting the activity of the agonist in the sample. Alternatively, the present invention provides a pharmaceutical composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. Alternatively, the present invention provides a method of treating a disease or condition associated with reduced expression of functional CIRYP, the method comprising administering the pharmaceutical composition to a patient in need of such treatment. I do.

Further, the present invention relates to (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, and (b) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2. A naturally occurring amino acid sequence having at least 90% sequence identity, (c) SEQ ID NO: 1-2
A biologically active fragment of an amino acid sequence selected from the group consisting of: or (d) SE
Provided are methods for screening compounds for antagonistic efficacy of a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of Q ID NO: 1-2. The method includes (a) exposing a sample containing the polypeptide to the compound, and (b) detecting the activity of the antagonist in the sample. Alternatively, the invention provides a pharmaceutical composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. Alternatively, the present invention provides a method of treating a disease or condition associated with overexpression of functional CIRYP, comprising administering the pharmaceutical composition to a patient in need of such treatment. .

Further, the present invention provides a method for screening a compound for efficacy in altering the expression of a target polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 3-4, comprising the steps of: The methods include (a) exposing a sample containing the target polypeptide to the compound, and (b) detecting a change in expression of the target polypeptide.

[0018] Proteins form the present invention for carrying out the invention, nucleic acid sequences, and before describing how the present invention,
It should be understood that the invention is not limited to the particular devices, materials, and methods disclosed herein, but that embodiments thereof can be varied. The terminology used herein is used for the purpose of describing specific embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Please also understand.

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

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

The definition term "CIRYP" is obtained from any species, particularly mammalian species, including cows, sheep, pigs, mice, horses, and humans, and is either natural, synthetic, semi-synthetic, or Refers to the substantially purified amino acid sequence of CIRYP obtained from any source, such as recombinant.

The term “agonist” refers to a molecule that enhances or mimics the biological activity of CIRYP. The agonists include proteins, nucleic acids, carbohydrates, small molecules or any other compounds or compositions that interact directly with CIRYP or act on a component of the biological pathway in which CIRYP is involved. Regulates the activity of CIRYP.

The term “allelic variant” refers to another form of the gene encoding CIRYP. An allelic variant has a mutated mRN due to at least one mutation in the nucleic acid sequence.
A or polypeptide occurs, but their structure or function may or may not change. Genes that do not have a naturally occurring allelic variant, 1
There is one that exists or many that exist. Common mutations that generally result in allelic variants are due to natural deletions, additions or substitutions of nucleotides.
Each of these forms of change, alone or in combination with other changes, can occur one or more times in a given sequence.

As used herein, an “altered” nucleic acid sequence encoding CIRYP includes a variety of nucleotides that result in a polynucleotide encoding a polypeptide comprising the same CIRYP or at least one functional feature of CIRYP. And those sequences with deletions, insertions or substitutions of This definition may include inappropriate or unexpected hybridization of the polynucleotide sequence encoding CIRYP to an allelic variant at a different position than the normal chromosomal location, or specific oligonucleotide probes of the polynucleotide encoding CIRYP. It includes polymorphisms that are easily detectable or difficult to detect. The encoded protein may also be "mutated" and may contain substitutions, deletions or insertions of amino acid residues that effect a silent change and result in a functionally equivalent CIRYP. Intentional amino acid substitutions may result in similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the residues, as long as the biological or immunological activity of CIRYP is retained. May be performed based on For example, negatively charged amino acids include aspartic acid and glutamic acid, and positively charged amino acids include lysine and arginine. Amino acids having uncharged polar side chains with similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; and phenylalanine and tyrosine.

The term “amino acid” or “amino acid sequence” is an oligopeptide, peptide, polypeptide or protein sequence, or any fragment thereof, and refers to a natural or synthetic molecule. When "amino acid sequence" is used to refer to the amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and similar terms refer to the complete, intact amino acid sequence associated with the described protein molecule. It is not limited to an array.

The term “amplification” refers to the production of additional copies of a nucleic acid sequence, usually performed using polymerase chain reaction (PCR) techniques well known to those skilled in the art.

The term “antagonist” is a molecule that inhibits or attenuates the biological activity of CIRYP. Antagonists include proteins, such as antibodies, nucleic acids, carbohydrates, small molecules or any other compounds or compositions, which either directly interact with CIRYP or are components of a biological pathway in which CIRYP is involved. Regulates the activity of CIRYP by acting on

The term “antibody” refers to whole molecules, Fa, F (ab ′) ₂ , and Fv that can bind antigenic determinants.
Refers to those fragments, such as fragments. Antibodies that bind CIRYP polypeptides can be prepared using untreated polypeptides or using fragments thereof containing small peptides that participate as antigens to immunize. The polypeptide or oligopeptide used to immunize an animal (eg, a mouse, rat, or rabbit) is obtained from translated or chemically synthesized RNA and can be coupled to a carrier protein if necessary. Commonly used carriers that bind chemically to the peptide include bovine serum albumin and thyroglobulin, keyhole limpet hemocyanin (KLH). The animal is then immunized with the conjugated peptide.

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

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

The term “biologically active” refers to a protein that has a structural, regulatory or biochemical function of a naturally occurring molecule. Similarly, "immunologically active" refers to the natural CIRY that triggers a specific immune response in
P, refers to the ability of recombinant CIRYP or synthetic CIRYP or any oligopeptide thereof.

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

The terms “composition comprising a given polynucleotide sequence” and “composition comprising a given amino acid sequence” generally refer to any composition comprising a given polynucleotide or amino acid sequence. The composition may include a dry formulation or an aqueous solution. A composition containing a polynucleotide encoding CIRYP or a fragment of CIRYP can be used as a hybridization probe. The probe can be stored in a lyophilized state and can be conjugated to a stabilizing agent such as a carbohydrate.
In hybridization, the probe is placed in an aqueous solution containing a salt (eg, NaCl), a surfactant (eg, sodium dodecyl sulfate (SDS)) and other substances (eg, Denhardt's solution, dried milk, salmon sperm DNA, etc.). Can be dispersed.

The term “consensus sequence” refers to a nucleic acid sequence in which unnecessary bases have been separated by resequencing or a 5 ′ direction and / or 3 ′ position using an XL-PCR kit (The Perkin Elmer Corp., Norwalk, Conn.). Or a computer program for assembling fragments (eg, GELVIEW Fragment Assembly system,
GCG, Madison WI) is a nucleic acid sequence assembled from two or more insight clones and, optionally, one or more overlapping sequences of one or more proprietary ESTs. Some sequences are extended and combined to create a consensus sequence.

The term “conservative amino acid substitution” refers to a substitution that substantially impairs the properties of the original protein, that is, the substitution preserves the structure and, in particular, the function of the protein without significantly altering it. . The following shows conservative amino acid substitutions where the original amino acid of the protein is replaced with another amino acid. Original residue Conservative substitution Ala Gly, Set Arg His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln , Glu Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile , Leu, Thr In general, in conservative amino acid substitution, (a) the main chain structure of the polypeptide in the substitution region, for example, β-sheet or α-helix conformation, (b) the charge or hydrophobicity of the molecule at the substitution site and And / or (c) most of the side chains are retained.

[0036] The term "deletion" refers to a change in the nucleotide or amino acid sequence that results in the deletion of one or more nucleotide or amino acid residues.

The term “derivative” refers to a chemically modified polypeptide or polynucleotide sequence. Chemical modification of a polynucleotide sequence can include, for example, replacement of hydrogen with an alkyl, acyl, hydroxyl, or amino group. A polynucleotide derivative encodes a polypeptide that retains the biological or immunological functions of the native molecule. Polypeptide derivatives have been modified by glycosylation, polyethylene glycolation (pegylation), or any other process that preserves the biological or immunological function of the original polypeptide.

The term “fragment” refers to a unique portion of CIRYP or a polynucleotide encoding CIRYP that is identical to, but shorter in length than, its parent sequence. "Fragments" can include those of a given sequence length minus one nucleotide / amino acid residue. For example, some fragments contain 5-100
Zero consecutive nucleotides or amino acid residues may be included. Probes, primers, antigens, therapeutic molecules or fragments used for other purposes are at least 5, 10
, 15, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from particular regions of the molecule. For example, a polypeptide fragment may contain the first 250 or 500 amino acids as shown in the given sequence (or the first
(25% or 50%). These lengths are exemplary, and examples of the invention may include any of the lengths described in the specification, including the sequence listings, tables and figures.

Certain fragments of SEQ ID NO: 3-4 include, for example, regions of a unique polynucleotide sequence that specifically identify SEQ ID NO: 3-4, which are different from other sequences in the same genome. Is included. Certain fragments of SEQ ID NO: 3-4 are useful, for example, in hybridization and amplification techniques, and similar methods that distinguish SEQ ID NO: 3-4 from related polynucleotide sequences. The exact length of a fragment of SEQ ID NO: 3-4 or a region of SEQ ID NO: 3-4 that corresponds to a fragment can be routinely determined by well known techniques based on the purpose of the fragment.

A fragment of SEQ ID NO: 1-2 is encoded by a fragment of SEQ ID NO: 3-4. Certain fragments of SEQ ID NO: 1-2 include a region of a unique amino acid sequence that specifically identifies SEQ ID NO: 1-2. For example, one fragment of SEQ ID NO: 1
It is useful as an immunogenic peptide for the production of an antibody that specifically recognizes -2.
The exact length of a fragment of SEQ ID NO: 1-2 or a region of SEQ ID NO: 1-2 that matches a fragment can be routinely determined by known techniques based on the purpose of the fragment.

The term “similarity” refers to a degree of complementarity. Similarity includes partial similarity and complete similarity. “Identity” may be used instead of the term “similarity”.
Partially complementary sequences that at least partially inhibit the same sequence from hybridizing to the target nucleic acid are referred to as "substantially similar." Inhibition of hybridization between a completely complementary sequence and the target sequence can be assayed under mild stringency conditions using a hybridization assay (such as Southern blot, Northern blot or solution hybridization). Substantially similar sequences or hybridization probes can compete for and inhibit the binding of perfectly similar (identical) sequences to the target sequence under mildly stringent conditions.
This does not mean that non-specific binding is tolerated under mildly stringent conditions, and under mildly stringent conditions, the mutual binding of the two sequences is specific (ie, (Selective) interaction. The absence of non-specific binding does not even have partial complementarity (
That is, less than about 30% similarity or identity) can be tested by using a second target sequence. In the absence of non-specific binding, a substantially similar sequence or probe will not hybridize to a second non-complementary target sequence.

The terms “percent identity” or “percent identity” for a polynucleotide sequence refers to the identity of residue matches between at least two or more polynucleotide sequences aligned using standard algorithms. Means percentage. Such algorithms use gaps in the compared sequences to make a more significant comparison between the two sequences in a standard and reproducible way to optimize the alignment between the two sequences be able to.

The percent identity between polynucleotide sequences can be determined using the default parameters of the CLUSTAL V algorithm incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package and includes a suite of molecular biology analysis programs (DNASTAR, Madison
WI). This CLUSTAL V is described in Higgins, DG and PM Sharp (198
9) CABIOS 5: 151-153 and Higgins, DG et al. (1992) CABIOS 8: 189-191. For alignment of pairs of polynucleotide sequences, the default parameters are set as Ktuple = 2, gap penalty = 5, window = 4 and “diagonals saved” = 4. The "weighted" residue weight table is selected as the default.
CLUSTAL V reports the percent identity as the "percent similarity" between a pair of aligned polynucleotide sequences.

Alternatively, a commonly used set of freely available sequence comparison algorithms is:
NCBI, Bethesda, MD, and the Internet (http: // www.ncbi.nlm.nih.gov/BLAS T /) National Center available from several sources, including such as for Biotechn
ology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Alt
schul, SF et al. (1990) J. Mol. Biol. 215: 403-410). BLA
The ST software suite includes a variety of sequence analysis programs, including "blastn", used to align known polynucleotide sequences with other polynucleotide sequences from various databases. A tool called "BLAST 2 Sequences" is available and is used to directly compare pairs of two nucleotide sequences. "BLAST 2 Sequences" is available interactively by accessing http://www.ncbi.nlm.nih.gov/gorf/b12.html . The “BLAST 2 Sequences” tool uses b
It can be used for both lastn and blastp (described below). The BLAST program is
Generally used with gaps and other parameters set to default settings. For example, when comparing two nucleotide sequences, the "BLAST 2 Sequences" tool Version 2.0.9 (May-07-199) set to default parameters
9) can be used with blastn. Such default parameters are, for example,
It can be as follows. Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch: −2 Open Gap: 5 and Extension Gap: 2 penalties Gap x drop-off: 50 Expect: 10 Word Size: 11 Filter: on Or a fragment obtained from a shorter sequence, such as a fragment obtained from a larger predetermined sequence (eg, at least 20, 30, 40, 50, 70). , 100 or 200 contiguous nucleotide fragments). Such lengths are merely exemplary, and the length at which percent identity can be determined using fragments of any length of the sequences set forth in the specification, including the sequence listing, tables and figures. Should be understood.

[0045] Despite nucleic acid sequences that do not show high identity, they may encode similar amino acid sequences due to the degeneracy of the genetic code. Nucleic acid sequences can be varied using degeneracy to create multiple nucleic acid sequences such that they all encode substantially the same protein.

The term “percent identity” or “percent identity” for a polypeptide sequence
By means the percentage of residue matches between at least two or more polypeptide sequences aligned using standard algorithms. Methods for polypeptide sequence alignment are well known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, as detailed above, usually preserve the acidity and hydrophobicity of the substitution site and preserve the structure (and thus function) of the polypeptide.

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

Alternatively, the NCBI BLAST software suite can be used. For example, when comparing pairs of two polypeptide sequences, "BLAS" set with default parameters is used.
The T2 Sequences tool version 2.0.9 (May-07-1999) can be used with blastp. Such default parameters can be, for example, as follows. Matrix: BLOSUM62 Open Gap: 11 and Extension Gap: 1 penalties Gap x drop-off: 50 Expect: 10 Word Size: 3 Filter: on Or fragments obtained from shorter sequences, eg, larger predetermined polypeptide sequences (eg, at least 15, 20, 30,
(A fragment of 40, 50, 70 or 150 contiguous residues).
Such lengths are merely exemplary, and the length at which percent identity can be determined using fragments of any length of the sequences set forth in the specification, including the sequence listing, tables and figures. Should be understood.

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

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

The term “hybridization” refers to an annealing process by which a single-stranded polynucleotide base pairs with a complementary strand under predetermined hybridization conditions. Specific hybridization means that two nucleic acid sequences have a high degree of identity. Specific hybridization complexes are formed under conditions that permit annealing, and remain hybridized after the "washing process." The washing step is particularly important in determining the stringency of the hybridization process; under more stringent conditions, non-specific binding (ie, pair binding between nucleic acid strands that are not perfectly matched) is reduced. The conditions under which annealing between nucleic acid sequences is permissible are routinely determined by those skilled in the art and are constant during a hybridization experiment, while washing conditions can be varied during the experiment for the desired stringency Thus, hybridization specificity is obtained. Conditions that allow annealing include, for example, at about 68 ° C., about 6 × SSC, about 1% (w / v) SDS, and about 100 μg / ml denatured salmon sperm DNA.
Holds in the presence of

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

High stringency conditions for hybridization between polynucleotides of the present invention include a 1 hour wash step at about 68 ° C. in the presence of about 0.2 × SSC and about 1% SDS. Alternatively, it is performed at a temperature of 65 ° C, 60 ° C, 55 ° C or 42 ° C. The concentration of SSC can vary from about 0.1 to 2 × SSC in the presence of about 0.1% SDS. Usually, blocking agents are used to block non-specific hybridization. Such blocking agents include, for example, denatured salmon sperm DNA at about 100-200 μg / ml. Organic solvents such as formamide at a concentration of about 35-50% v / v can be used in certain cases, such as, for example, hybridization of RNA and DNA. Useful alterations of these washing conditions will be readily apparent to those skilled in the art. Hybridization under particularly high stringency conditions may indicate evolutionary similarity between the nucleotides.
Such similarities strongly suggest a similar role for their nucleotides and encoded polypeptides.

The term “hybridization complex” refers to a complex formed between two nucleic acid sequences by the force of hydrogen bonding between complementary bases. A hybridization complex may either be formed in solution (e.g., C ₀ t or R ₀ t analysis), or one of the nucleic acids present in solution and the solid support (e.g., paper, membranes, filters,
A pin or glass slide, or other suitable nucleic acid immobilized on a cell or other suitable substrate on which the nucleic acid is immobilized.

The term “insertion” or “addition” refers to a change in a nucleotide or amino acid sequence that adds one or more nucleotides or amino acid residues, respectively.

The term “immune response” refers to a condition associated with inflammation, trauma, immune disorders or infectious or inherited diseases. These conditions are characterized by the expression of various factors that affect cells and the systemic defense system, such as cytokines, chemokines and other signaling molecules.

The term “microarray” refers to an arrangement of various polynucleotides on a substrate.

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

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

The term “nucleic acid” or “nucleic acid sequence” refers to a nucleotide, oligonucleotide, polynucleotide or any fragment thereof. These terms also refer to single-stranded or double-stranded, sense or antisense strands of genomic or synthetic origin.
Alternatively, it refers to RNA, peptide nucleic acid (PNA), or any DNA-like or RNA-like substance.

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

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

“Probe” refers to a nucleic acid sequence encoding CIRYP, its complementary sequence or a fragment thereof, which is used to detect the same nucleic acid sequence, allelic nucleic acid sequence or related nucleic acid sequence. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioisotopes, ligands, chemiluminescent reagents and enzymes. "Primers" are short nucleic acids (usually DNA oligonucleotides) which are
The target polynucleotide can be annealed by forming complementary base pairs. The primer can then be extended along the target DNA strand by a DNA polymerase enzyme. The primer set can be used, for example, for amplification (and identification) of a nucleic acid sequence by a PCR method.

The probes and primers used in the present invention usually have at least 15
Of consecutive nucleotides. To increase specificity, longer probes and primers can be used, including, for example, at least 20, 25, 30, 40, 50, 60 contiguous sequences of the disclosed nucleic acid sequences. , 70
, 80, 90, 100 or 150 nucleotides. Probes and primers
It should be understood that they may be considerably longer than these examples, and that any length of nucleotides provided herein, including tables, figures and sequence listings, may be used.

For preparation and use of probes and primers, see, for example, Sambrook,
J. et al., 1989, entitled Molecular Cloning: A Laboratory Manual , 2
Volume 1-3 of the edition (Cold Spring Harbor Press, Plainview NY) or Ausubel, FM
1987, entitled " Current Protocols in Molecular Biology " (Greene
Pubi. Assoc. & Wiley-Intersciences, New York NY) and Innis et al., 1990, entitled " PCR Protocols, A Guide to Methods and Applications " (Ac
ademic Press, San Diego CA). A primer set for PCR is, for example, Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Rese
arch, Cambridge MA) can be obtained from the known sequence using a computer program for that purpose.

Oligonucleotides used as primers are selected by computer programs for selecting primers well known in the art. For example, OLIGO 4.06 software allows the selection of primer pairs for PCR up to 100 nucleotides each,
Useful for analysis of oligonucleotides up to 5,000 nucleotides and larger polynucleotides from input polynucleotide sequences up to the kilobase. Similar primer selection programs incorporate additional features to enhance capacity. For example, PrimOU primer selection program (Genome Center at Unive
rsity of Texas South West Medical Center, available from Dallas TX), which allows you to select specific primers from megabase sequences and
(genome-wide scope). Primer3 primer selection program (Whitehead Institute / MIT Center for Genome Research, Ca
(available from mbridge MA1) allows the user to specify a sequence to be avoided as a primer binding site.
libaray) ". Primer3 is also particularly useful for selecting oligonucleotides for microarrays. (The source code for the latter two primer selection programs can be obtained from their respective sources to meet the specific needs of the user. Can be changed to). PrimerGen program (UK Human
Genome Mapping Project Resource Centre, available from Cambridge UK)
To design primers based on multiple sequence alignments, primers can be selected that hybridize to either the most conserved or the least conserved regions of the aligned nucleic acid sequences. Thus, this program is useful for identifying unique conserved oligonucleotide and polynucleotide fragments. Oligonucleotide and polynucleotide fragments identified by any of the selection methods described above, for example, identify polynucleotides that are completely or partially complementary in PCR or sequencing primers, microarray elements, or nucleic acid samples. It is useful in hybridization technology as a specific probe. The method for selecting the oligonucleotide is not limited to the method described above.

The term “recombinant nucleic acid” is not a naturally-occurring sequence, but a sequence that is an artificial combination of two or more different, distinct segments of sequence. This artificial combination
Although it is often carried out by chemical synthesis, more generally, for example, the above-mentioned Samb
Artificially manipulate spaced apart segments of nucleic acid by genetic engineering techniques as described in rook ( supra ). The “recombinant nucleic acid” also includes a nucleic acid that is changed simply by adding, substituting or deleting a part of the nucleic acid. A recombinant nucleic acid can include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid can be, for example, part of a vector used to transform a cell.

Alternatively, such a recombinant nucleic acid may be used for vaccination of a mammal expressing the recombinant nucleic acid, and a part of a vaccinia virus-based viral vector that elicits a protective immune response in the mammal. Can be

The term “sample” is used in its broadest sense. Samples suspected of containing CIRYP-encoding nucleic acids or fragments thereof, or CIRYP itself, may include bodily fluids,
Chromosomes isolated from cells, extracts from organelles or cell membranes, and cells, genomic DNA, RNA or cDNA in solution, or bound to a solid support, tissues, tissue prints, etc. .

The term “specific binding” or “specifically binds” refers to the interaction between a protein or peptide and an agonist, antibody, antagonist, small molecule or any natural or synthetic binding component. This interaction means that it depends on the presence of a particular structure of the protein (eg, an antigenic determinant or epitope recognized by the binding molecule). For example, if the antibody is specific for the epitope "A", then in the reaction involving the labeled free "A" and the antibody, the epitope "A" (ie, the free, unlabeled "A") The amount of labeled “A” that binds to the antibody is reduced by the presence of the polynucleotide containing

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

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

The term “substrate” refers to a film, filter, chip, slide, wafer, fiber,
Refers to any suitable solid or semi-solid support, including magnetic or non-magnetic beads, gels, tubes, plates, polymers, microparticles, capillaries. Substrates can take a variety of surface forms, such as walls, grooves, pins, channels, and pores to which polynucleotides and polypeptides bind.

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

The “variant sequence” of a specific nucleic acid sequence is referred to as the default parameter setting “BLAS
Using blastn with the "T2 Sequences" tool Version 2.0.9 (May-07-1999), it has been shown to have at least 40% sequence identity to a particular nucleic acid sequence at a given length. Nucleic acid sequence. Such pairs of nucleic acids may exhibit, for example, at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 98% or more identity at a given length. Certain mutant sequences are, for example, "alleles" (described above).
, "Splice", "species" or "polymorphic" variant sequences. A splice variant may have significant identity to a reference molecule, but may have more or fewer polynucleotides due to alternative splicing of exons during mRNA processing. The corresponding polypeptide may have additional functional domains or may lack domains present in the reference molecule. Species variant sequences are polynucleotide sequences that vary from species to species. The resulting polypeptides usually have significant amino acid identity with each other. Polymorphic variant sequences are those in which the polynucleotide sequence of a particular gene differs among individuals of a given species. Also, a polymorphic variant sequence can include a "single nucleotide polymorphism" (SNP) in which one nucleotide of a polynucleotide sequence is mutated. The presence of the SNP is, for example, a predetermined population,
It may indicate a feature of the condition or condition.

A “variant” of a particular polypeptide sequence is defined as the presence of a nucleic acid sequence using a blastp with the “BLAST 2 Sequences” tool Version 2.0.9 (May-07-1999) with default parameter settings. A nucleic acid sequence for which the identity of the particular polypeptide sequence to length has been determined to be at least 40%. A polypeptide sequence that has been determined to have at least 40% sequence identity to a particular polypeptide sequence at a given length. Such pairs of polypeptides may exhibit, for example, at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 98% or more identity at a certain length. .

[0077] This invention relates to novel human circadian rhythm-related protein (CIRYP), polynucleotides encoding CIRYP, and reproductive disorders, gastrointestinal disorders, diagnosis of neurological disorders and developmental disorders, treatment or of these for the prevention Based on discovery of use of the composition.

The nucleic acid encoding CIRYP-1 of the present invention can be obtained from a duodenal cDNA library (DUODNOT0) using computer search for nucleic acid and / or amino acid sequence alignment.
Identified in Incyte clone 1581573H1 from 1). The consensus sequence, SEQ ID NO: 3, is the duplicated and / or extended nucleic acid sequence listed below, Insights clones 1581573H1 (DUODNOTO1), 2604921H1 (LUNGTUT07), 816118R1 (OVAR
TUT01), 3253364H1 (OVARTUN01), 879455T1 (THYRNOT02) and 1312037F1 (COLNFET
02).

In one embodiment, as shown in FIGS. 1A-1D, the invention includes a polypeptide having the amino acid sequence of SEQ ID NO: 1. CIRYP-1 is 251 amino acids long and has three potential phosphorylation sites by casein kinase II at residues S43, T120 and S135 and two potential sites by protein kinase C at residues T70 and T120. And a phosphorylation site. As shown in FIGS. 3A and 3B, CIRYP-1 is a CIRP-1.
YP-2 (SEQ ID NO: 2), Dreg-2 (GI 1561732; SEQ ID NO: 5) and CLK-1 (GI 3415
017; SEQ ID NO: 6) with chemical and structural similarities. Especially CIRYP-1 and Dreg-
2 share 32% identity. The lengths of CIRYP-1 and Dreg-2 are roughly equivalent, 252 and 255 amino acids, respectively. CIRYP-1, CIRYP-2, Dreg-2 and
CLK-1 shares 11 conserved amino acid residues. These residues in CIRYP-1 are M1, R22, W111, F137, R140, L150, F156, K167, A176, Y198 and L210.
It is. Furthermore, CIRYP-1 shares conserved hydrophobic residues with CIRYP-2, Dreg-2 and CLK-1. These residues in CIRYP-1 are F83, G87, V94, L101, I145,
M182 and F209. SEQ ID NO: about 417 to 476 nucleotides:
Fragments 3 can be used, for example, to distinguish SEQ ID NO: 3 from related sequences,
Useful in hybridization and amplification techniques to identify O: 3. The encoded polypeptide is useful, for example, as an immunogenic peptide. Northern analysis shows expression of this sequence in various libraries, at least 58% of which is associated with cancer and at least 18% is associated with inflammatory and immune responses. Of particular note is the expression of CIRYP-1 in gastrointestinal tissue.

The nucleic acid encoding CIRYP-2 of the present invention can be obtained by using a computer search for nucleic acid and / or amino acid sequence alignment using a uterine cDNA library (UTRSNOT02).
From Incyte clone 2267905H1. The consensus sequence, SEQ ID NO: 4, is the following duplicated and / or extended nucleic acid sequence, Insights clones 2267905H1 (UTRSNOT02), 2267905R6 (UTRSNOT02), 1550606H1 (PROSN
OT06), 1683318F6 (PROSNOT15), 4594189H1 (PROSTUT18) and 2733567H1 (OVARTU
TO4) as well as the shotgun sequence SAEB01465R1.

In one embodiment, as shown in FIGS. 2A-2C, the present invention includes a polypeptide having the amino acid sequence of SEQ ID NO: 2. CIRYP-2 is 217 amino acids long, has one potential N-glycosylation site at residue N43 and a cA at residue S31.
One potential phosphorylation site by MP and cGMP-dependent protein kinase, plus residue T
It has one potential phosphorylation site by casein kinase II at 155 and two potential phosphorylation sites by protein kinase C at residues S31 and S214. As shown in FIG. 3A and FIG. 3B, CIRYP-2 is CLK-1 (GI 3415017; SEQ ID NO:
6) has chemical and structural similarities to CIRYP-1 (SEQ ID NO: 1) and Dreg-2 (GI 1561732; SEQ ID NO: 5). In particular, CIRYP-2 and CLK-1 share 85% identity. CIRYP-2 and CLK-1 are both 217 amino acids long. As mentioned earlier, the CIR
YP-1, CIRYP-2, Dreg-2 and CLK-1 share 11 conserved amino acid residues. These residues in CIRYP-2 are M1, R16, W87, F104, R107, L114, F120, K
131, A140, Y164 and L168. Further, as described above, CIRYP-2 is CLK-1, CIRYP
-1 and Dreg-2 share conserved hydrophobic residues. These residues in CIRYP-2 are Y67, M71, V79, M86, L111 and Y149. TRC in both CIRYP-2 and CLK-1 generally spanning residues A58 to L129 and repeating almost residues A140 to L217
Note the existence of the domain. S of nucleotides 77 to 148
Fragments of EQ ID NO: 4 are useful, for example, in hybridization and amplification techniques to distinguish SEQ ID NO: 4 from related sequences and to identify SEQ ID NO: 4. The encoded polypeptide is useful, for example, as an immunogenic peptide. Northern analysis shows expression of this sequence in various libraries, at least 67% of which are associated with cancer and at least 19% are associated with inflammatory and immune responses. Of particular note is the high expression (48%) of CIRYP-2 in reproductive tissues.

The present invention also includes CIRYP variants. Preferred CIRYP variants have at least about 80%, or at least about 90%, or even at least about 95% of the CIRYP amino acid sequence.
% Amino acid sequence identity and includes at least one functional or structural characteristic of CIRYP.

Further, the present invention includes a polynucleotide encoding CIRYP. In certain embodiments, the invention includes a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 3-4 encoding CIRYP.

The present invention further includes mutant sequences of the polynucleotide encoding CIRYP. In particular, such polynucleotide variant sequences have at least about 70%, or at least about 85%, or even at least about 95%, polynucleotide sequence identity to the polynucleotide sequence encoding CIRYP. In certain embodiments of the present invention, there are provided SEQ.
A variant of the polynucleotide sequence comprising a sequence selected from the group consisting of ID NO: 3-4, which comprises at least about 70% relative to a nucleic acid sequence selected from the group consisting of Alternatively, it has at least about 85%, and even at least about 95%, polynucleotide sequence identity. Mutant sequences of the above polynucleotides are all CI
It is possible to encode an amino acid sequence comprising at least one of the functional or structural features of RYP.

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

It is preferred that the nucleotide sequence encoding CIRYP and its mutant sequences are capable of hybridizing to the nucleotide sequence of naturally occurring CIRYP under appropriately selected stringency conditions, but not It is advantageous to create a nucleotide sequence encoding CIRYP or a derivative thereof that has substantially different codon usage, such as including the occurrence codon. Depending on the frequency with which particular codons are utilized by the host, codons can be selected to increase the rate at which expression of the peptide occurs in eukaryotic or prokaryotic hosts. Another reason for substantially altering the nucleotide sequence encoding CIRYP and its derivatives without altering the encoded amino acid sequence is that it has a longer half-life than transcripts generated from naturally occurring sequences. This is to create an RNA transcript having the desired properties.

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

Also included in the invention are hybrids, under various stringent conditions, to the claimed polynucleotide sequences, and particularly to SEQ ID NO: 3-4 and fragments thereof. Includes soyable polynucleotide sequences (eg, Wahl, GM
And SL Berger (1987) Methods Enzymol. 152: 399-407; Kimmel, AR (1987) M
ethods Enzymol. 152: 507-511). Hybridization conditions include the annealing and washing conditions described in the Definitions .

[0089] DNA sequencing methods are well known and can be used in any of the embodiments of the present invention. The method is based on the Klenow fragment of DNA polymerase I, SEQUEN
ASE (US Biochemical, Cleveland, OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Pharmacia Biotech, Piscataway NJ), or
Enzymes are used, such as a combination of a proofreading exonuclease and a polymerase as found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD). Prepare the array using MICROLAB 2200 liquid transfer sy
stem (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown)
It is preferred to automate by equipment such as MA) and ABI CATALYST 800 thermal cycler (Perkin-Elmer). Next, ABI 373 or 377 DNA sequencing system (Perkin-Elmer), MEGABACE 1000 DNA sequencing system (Molecul
Sequencing is performed using ar Dynamics, Sunnyvale CA) or other well-known equipment. The resulting sequence is analyzed using various algorithms well known to those of skill in the art (eg, Ausubei, FM (1997) Short Protocols in Molecular Biolog y , John Wiley & Sons, New York NY, unit 7.7; Meyers, RA (1995) Molecular Biology and Biotechnology , Wiley VCH, New York NY, pp. 856-853).

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

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

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

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

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

MOLECULARBREEDING (Maxygen Inc., Santa Clara CA;
described in US Patent Number 5,837,458; Chang, C.-C. et al. (1999) N
at. Biotechnol. 17: 793-797; Christians, FC et al. (1999) Nat. Biotechn.
ol. 17: 259-264; Crameri, A. et al. (1996) Nat. Biotechnol. 14: 315-319).
Such DNA shuffling techniques can be applied to alter or improve the biological properties of CIRYP, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds. DNA shuffling is the process of making a library of gene variants using PCR-mediated recombination of gene fragments. Next, the library is selected or screened to identify gene variants having the desired properties. These suitable mutants can then be pooled and further subjected to repeated DNA shuffling and selection / screening. Thus, genetic diversity is created by "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing irregularly mutated mutations can be recombined, screened, and re-shuffled until the desired properties are optimized. Alternatively, a given gene fragment is recombined with a homologous gene fragment of the same gene family of the same or a different species to maximize the diversity of multiple native genes in a controlled and regulatable manner. be able to.

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

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

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

Expression vector containing CIRYP coding sequence and appropriate transcriptional and translational regulatory regions
In order to produce, methods well known to those skilled in the art are used. These methods include:in v itro Recombinant DNA technology, synthetic technology, andin vivoIncludes genetic recombination techniques (eg,
See, Sambrook, J. et al. (1989)Molecular Cloning, A Laboratory Manual, Cold Spr
ing Harbor Press, Plainview, NY, ch. 4, 8, and 16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biology , John Wiley & Sons, New York, NY,
 ch. 9, 13 and 16).

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

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

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

[0103] Plant systems can also be used for expression of CIRYP. Transcription of the sequence encoding CIRYP can be facilitated by a viral promoter, such as the CaMV 35S and 19S promoters alone or in combination with the omega leader sequence from TMV (Takamatsu, N. et al. (1987)).
EMBO J. 6: 307-311). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. (For example, The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York; pp.191-1
96).

[0104] In mammalian cells, a number of viral-based expression systems are available. When an adenovirus is used as an expression vector, the sequence encoding CIRYP can be ligated into an adenovirus transcription / translation complex consisting of the late promoter and tripartite leader sequence. Insertions at non-essential E1 or E3 regions of the viral genome result in infectious viruses that express CIRYP in host cells (see, eg, Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. . 81:36
55-3659). In addition, transcription enhancers such as the Rous sarcoma virus (RSV) enhancer can be used to increase expression in mammalian host cells. Also, for high level expression of proteins, vectors based on SV40 or EBV can be used.

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

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

[0107] Any number of selection systems can be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, respectively, tk ^- and apr ^-. As used in the cell (e.g., Wigler, M et al (1977) Cell 11:
223-32; see Lowy, I. et al. (1980) Cell 22: 817-23). Also, resistance to antimetabolites, antibiotics or herbicides can be used as a basis for selection, for example, dhfr confers resistance to methotrexate, neo is aminoglycoside neomycin and G-418.
Als or pat are chlorsulfuron, phosphinotricin acetyltransferase
se) (eg, Wigler, M. et al. (1980) Proc. Natl. Acad. Sc
i. 77: 3567-70; see Colberre-Garapin, F. et al. (1981) J. Mol. Biol. 150: 1-14). Additional selectable genes are described in the literature, for example, trpB and hisD, which alter the cellular need for metabolites (eg, Hartman, SC and R).
.C. Mulligan (1988) Proc. Natl. Acad. Sci. 85: 8047-8051). For example, visible markers such as anthocyanin, green fluorescent protein (GFP; Clontech), β-glucuronidase and its substrate β-glucuronide, or luciferase and its substrate luciferin can be used. These markers are widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression by a particular vector system (eg, Rhodes, CA et al. (1995) Methods Mol. Bio
l. 55: 121-131).

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

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

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

A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic and amino acid assays. Oligolabeling methods (oligolab) are used to generate labeled hybridization or PCR probes to detect sequences related to the polynucleotide encoding CIRYP.
eling), nick translation, end-labeling, or PCR amplification using labeled nucleotides. Or an array encoding CIRYP,
Alternatively, any fragment thereof can be cloned into a vector for the production of an mRNA probe. Such vectors are well known to those of skill in the art and are commercially available, and are used to synthesize RNA probes in vitro by adding a suitable RNA polymerase, such as, for example, T7, T3 or SP6, and labeled nucleotides. can do. These methods are based on various commercially available kits (Amersham Pharmacia Biotech, Promega (M
adison WI) and US Biochemical). Suitable reporter molecules or labels used to facilitate detection include radionuclides, enzymes, fluorescent agents, chemiluminescent or dye-producing agents, substrates, cofactors, inhibitors, magnetic particles, and the like.

[0112] Host cells transformed with a nucleotide sequence encoding CIRYP can be cultured under conditions suitable for the recovery and expression of the protein from cell culture. Proteins produced by the transformed cells may be secreted or retained intracellularly depending on the sequence and / or the vector used. As will be appreciated by those skilled in the art, expression vectors containing polynucleotides encoding CIRYP can be designed to include signal sequences that direct CIRYP secretion through prokaryotic or eukaryotic cell membranes.

In addition, host cell strains are chosen for their ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation,
Glycosylation, phosphorylation, lipidation, and acylation are included. Post-translational processing that cleaves the "prepro" form of the protein can be used to identify the target, folding and / or activity of the protein. A variety of host cells (eg, CHO, HeLa, M
DCK, MEK293, and WI38) are American Type Culture Collection (ATCC, Mana
ssas, VA) and can be selected to ensure correct modification and processing of the foreign protein.

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

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

[0116] By direct peptide synthesis using solid phase technology, as well as recombinant production,
CIRYP fragments can be created (see, for example, Creighton, supra, pp. 55-60). Protein synthesis can be performed manually or automatically. Automated synthesis can be performed, for example, using an ABI 431A peptide synthesizer (Perkin Elmer). The various fragments of CIRYP can also be synthesized separately and then combined to create the full length molecule.

Between the region of CIRYP and a protein that plays a role in biological timing, such as Dreg-2 (GI 1561732) and mouse CLK-1 (GI 3415017) of therapeutic D. melanogaster , for example, the sequence And chemical and structural similarities in the context of motifs. In addition, expression of CIRYP has implications for gastrointestinal, reproductive and nervous tissues. Therefore, CIRYP is thought to play a role in reproductive disorders, gastrointestinal disorders, neurological disorders and developmental disorders. In the treatment of diseases associated with increased expression or activity of CIRYP, it is desirable to reduce the expression or activity of CIRYP. In the treatment of a disease associated with decreased expression or activity of CIRYP, it is desirable to increase the expression or activity of CIRYP.

Thus, in one embodiment, CIRYP or a fragment or derivative thereof can be administered to a patient for the treatment or prevention of a disease associated with reduced expression or activity of CIRYP. Examples of such diseases include, but are not limited to, abnormal prolactin production, infertility including tubal abnormalities, ovulation abnormalities, and endometriosis, disrupted estrous cycle, disrupted menstrual cycle, polycysts Ovarian syndrome, ovarian hyperstimulation syndrome, endometrial or ovarian tumors, uterine fibroids, autoimmune abnormalities, ectopic pregnancy, and teratogenesis, breast cancer, mammary fibrocystic disease and hypertrophy, and disrupted spermatogenesis Reproductive disorders such as abnormal sperm physiology, testicular cancer, prostate cancer, benign prostatic hyperplasia, prostatitis, Peyronie's disease, male breast and gynecomastia; and dysphagia Esophagitis,
Esophageal spasm, esophageal stricture, esophageal cancer, indigestion, digestive disorders, gastritis, gastric cancer, anorexia, nausea, vomiting, gastroparesis, sinus or pyloric edema, abdominal angina, heartburn, gastroenteritis,
Ileus, intestinal infection, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic cancer, biliary tract disease, hepatitis, hyperbilirubinemia, cirrhosis, passive congestion of the liver, hepatome, infectious colon Inflammation, ulcerative colitis, ulcerative proctitis, Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, colon cancer, colonic obstruction, irritable bowel syndrome, short small bowel syndrome, diarrhea, constipation, gastrointestinal bleeding and acquired immunodeficiency syndrome (AIDS) gastrointestinal disorders; restlessness, Alzheimer's disease, amnesia, amyotrophic lateral sclerosis, bipolar disorder, catatonia, brain tumor, dementia, depression, diabetic neuropathy, Down's syndrome, extrapyramidal terminal defects Syndrome, dystonia, epilepsy, Huntington's disease, peripheral nervous disease, multiple sclerosis, pyramidism, Parkinson's disease, paranoid psychosis, postherpetic neuralgia, schizophrenia, seasonal emotional disorders (Seasonal affective disorder; SAD) and neuropathy such as Tourette's disease; and tubular acidosis, anemia, Cushing's syndrome, dwarfism of chondrodysplasia, Duchenne-Becker muscular dystrophy, epilepsy, gonad dysplasia, WAGR syndrome (Wilms tumor, aniridia, genitourinary disorders, mental retardation), Smith-Magnenis syndrome, myelodysplastic syndrome, hereditary mucosal epithelial dysplasia, hereditary keratoderma, Charcot-Marie-Tooth Disease and hereditary neuropathy such as neurofibromatosis, hypothyroidism, hydrocephalus, chorea (Syndenham's c
horea) and seizure disorders such as cerebral palsy, spinal cord fission, anencephaly, cranial spondylolysis,
Includes developmental disorders such as congenital glaucoma, cataracts, and sensory nerve hearing loss.

In another embodiment, a vector capable of expressing CIRYP or a fragment or derivative thereof for the treatment or prevention of a disease associated with reduced expression or activity of CIRYP, including but not limited to those described above. It may be administered to a patient.

In another embodiment, purified CIRYP together with a suitable pharmaceutical carrier is used to treat or prevent a disease associated with decreased expression or activity of CIRYP, including but not limited to those described above. May be administered to a patient.

In another embodiment, an agonist that modulates the activity of CIRYP is administered to a patient for the treatment or prevention of a disease associated with decreased expression or activity of CIRYP, including but not limited to those described above. May be.

In yet another embodiment, an antagonist of CIRYP may be administered to a patient for the treatment or prevention of a disease associated with increased expression or activity of CIRYP. Examples of such diseases include, but are not limited to, the reproductive disorders, gastrointestinal disorders, neurological disorders and developmental disorders described above. In one embodiment, an antibody that specifically binds CIRYP can be used directly as an antagonist, or indirectly as a delivery mechanism or targeting to bring the agent to cells or tissues that express CIRYP.

In another embodiment, the complement of a polynucleotide encoding CIRYP can be expressed for the treatment or prevention of a disease associated with increased expression or activity of CIRYP, including but not limited to those described above. The appropriate vector may be administered to the patient.

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

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

The oligopeptides, peptides or fragments thereof used to elicit antibodies for CIRYP have an amino acid sequence consisting of at least 5 amino acids, and generally of at least 10 amino acids. It is also preferred that these oligopeptides, peptides or fragments are identical to part of the amino acid sequence of the natural protein and contain the entire amino acid sequence of the small natural molecule. The short extension part of CIRYP amino acid is K
Fusion to an extension of another protein, such as LH, can produce chimeric molecule antibodies.

[0127] Monoclonal antibodies to CIRYP can be prepared using any technique that allows the production of antibody molecules by continuous cell lines in culture. Such techniques include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and EBV-hybridoma technology (eg, Kohler, G. et al. (1975) Nature 256: 495-49).
7; Kozbor, D. et al. (1985) Immunol. Methods 81: 31-42; Cote, RJ et al. (1983) P
roc. Natl. Acad. Sci. 80: 2026-2030; Cole, SP et al. (1984) Mol. Cell Biol.
62: 109-120).

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

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

[0130] Antibody fragments which contain specific binding sites for CIRYP can also be generated. For example, such fragments include, but are not limited to, F (ab ') ₂ fragments that can be generated by pepsin digestion of antibody molecules, and F (ab') ₂ fragments that are generated by reducing disulfide bridges. Fab fragments that can be used. Alternatively, a Fab expression library can be constructed so that monoclonal Fab fragments with the desired specificity can be identified quickly and easily (see, for example, Huse, WD et al. (1989) Science 246: 1275-1281).

[0131] A variety of immunoassays can be used for screening to identify antibodies with the desired specificity. Numerous protocols for competitive binding assays or immunoradiometric assays using either monoclonal or polyclonal antibodies with established specificity are well known in the art. Such immunoassays generally involve the measurement of the formation of a complex between CIRYP and its specific antibody. A two-site monoclonal antibody-based immunoassay using monoclonal antibodies reactive to two non-interfering CIRYP epitopes.
Ased immunoassay) is usually used, but competitive binding assays can also be used (Pound, supra ).

Using various methods such as Scatchard analysis with radioimmunoassay techniques,
Evaluate the affinity of the YP antibody. Affinity is expressed as the binding constant, Ka, defined as the molar concentration of the OP antibody complex at equilibrium divided by the molar concentrations of free antibody and free antigen. The Ka measured for a polyclonal antibody reagent with heterogeneous affinities for a number of CIRYP epitopes represents the average affinity or avidity of the CIRYP antibody. The Ka measured for a monoclonal antibody reagent monospecific for a particular CIRYP epitope represents a true measure of affinity. High affinity antibody reagents with Ka values of 10 ⁹ to 10 ¹² L / mol are preferably used in immunoassays where the CIRYP antibody complex must withstand rigorous manipulations. Low-affinity antibody reagents with a Ka value of 10 ⁶ to 10 ⁷ L / mol can be used for immunopurification (
It is preferably used for immunopurification and similar treatments. (Catty, D. (1988
) Antibodies, Volume I: A Practical Approach .IRL Press, Washington, DC;
Liddell, JE and Cryer, A. (1991) A Practical Guide to Monocional Anti bodies , John Wiley & Sons, New York NY).

The titer and avidity of the polyclonal antibody reagents are further evaluated to determine the quality and suitability of such reagents for certain downstream applications. For example, a polyclonal antibody reagent containing at least 1-2 mg / ml of specific antibody, preferably 5-10 mg / ml of specific antibody, is commonly used in methods requiring precipitation of the CIRYP antibody complex. . Guidelines for antibody specificity, titer, and avidity in various applications, as well as guidelines for antibody quality and use, are generally available
(See e.g., Catty, supra, and Coligan et al., Supra).

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

Expression vectors derived from retroviruses, adenoviruses, herpes or vaccinia viruses, or from various bacterial plasmids, are used for delivery of nucleotide sequences to target organs, tissues, or groups of cells. obtain. Vectors for expressing nucleic acid sequences complementary to a polynucleotide encoding CIRYP can be produced using methods well known to those of skill in the art (see, eg, Sambrook, supra , and Ausubel, 1995, supra) . ).

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

As described above, by designing a sequence complementary to the control, 5 ′, or regulatory region of the gene encoding CIRYP, ie, an antisense molecule (DNA, RNA or PNA), it is possible to alter gene expression. it can. Transcription start point (eg, +1 from start site)
Oligonucleotides derived from positions 0 to -10) are preferred. Similarly, inhibition can be achieved using triple helix base-pairing methodology. Polymerase,
Triple helix pairing is useful because it inhibits the ability of the double helix to unwind sufficiently for binding of transcription factors or regulatory molecules (eg, Gee, JE et al. (1994) In:
Huber, BE and BI Carr, Molecular and Immunologic Approaches , Futura
Publishing Co., Mt. Kisco, NY, pp. 163-177). Complementary sequences, ie, antisense molecules, can be designed to inhibit mRNA transcription by blocking transcript binding to ribosomes.

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

A specific ribozyme cleavage site within any potential RNA target is first identified by scanning the target molecule for a ribozyme cleavage site that includes the following sequences GUA, GUU and GUC. Once identified, a short RNA sequence between 15-20 ribonucleotides corresponding to the region of the target gene containing the cleavage site is a secondary structural feature that renders the oligonucleotide inoperable. Can be evaluated. The suitability of the candidate target can also be assessed using a ribonuclease protection assay by testing for accessibility to hybridization with the complementary oligonucleotide.

[0140] Complementary ribonucleic acid molecules and ribozymes of the present invention can be produced by methods well known in the art for synthesizing nucleic acid molecules. These include techniques for the chemical synthesis of oligonucleotides, such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules can be created by in vitro and in vivo transcription of a DNA sequence encoding CIRYP. Such a DNA sequence can be incorporated into a variety of vectors with an appropriate RNA polymerase promoter, such as T7 or SP6. Alternatively, these cDNA constructs that synthesize constitutively or inducibly complementary RNA can be introduced into cell lines, cells or tissues.

[0141] RNA molecules can be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5 'and / or 3' end of the molecule, phosphorothioate or 2'O instead of phosphodiesterase linkages in the backbone of the molecule. -Including using methyl. This concept is unique in PNA production, as well as acetyl-, methyl-, thio-, and similar modified forms of adenine, cytidine, guanine, thymine, and uridine that are not readily recognized by endogenous endonucleases. It can be extended to all these molecules by including less commonly used bases such as, inosine, queosin, and wibutosin.

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

[0143] Any of the above treatment methods can be applied to any subject in need of treatment, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.

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

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

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

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

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

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

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

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

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

The pharmaceutical composition of the present invention can be produced by a known method, for example, a usual mixing treatment, dissolution treatment, granulation treatment, sugar coating formation treatment, wet grinding treatment (levigating), emulsification treatment, encapsulation treatment,
It is manufactured by entrapping or freeze-drying. The pharmaceutical composition may be provided as salts and may be formed with a number of acids including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. Salts tend to be more soluble in aqueous or protonic solvents than the corresponding free base forms. In another case, a suitable formulation would be 1 mM to 50 mM histidine, 0.1% to 2% sucrose, 2% to 7% mannitol, buffered before use, in the range of pH 4.5 to 5.5. A freeze-dried powder containing any or all of them may be used.

After the pharmaceutical composition has been prepared, it can be placed in a suitable container and labeled for treatment of an indicated condition. In the case of CIRYP administration, such a label would indicate the dose, frequency, and method of administration.

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

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

A therapeutically effective amount is, for example, CIRYP or a fragment thereof that ameliorates a symptom or condition.
, CIRYP antibody, CIRYP agonist, CIRYP antagonist, or CIRYP antibody
Refers to the amount of the active ingredient of the inhibitor. Therapeutic efficacy and toxicity can be determined by cell culture or
Standard pharmaceutical methods in animals, such as ED₅₀(Therapeutic in 50% of the population
Effective dose) or LD₅₀(50% lethal dose in the population)
Can be determined. The dose ratio of toxic to therapeutic effects is the therapeutic index and the LD ₅₀ / ED₅₀It can be expressed as a ratio. Pharmaceutical compositions exhibit a large therapeutic index.
Is preferred. Data obtained from cell culture assays and animal studies were
It can be used to summarize the range of dosage for use. Such ingredients
The dose of ED is low or no toxicity₅₀Including circulating concentrations of
It is preferably within the range. Depending on the dosage form used, patient sensitivity and route of administration
Thus, dosages will vary within this range.

The exact dosage will be determined by the practitioner, in light of factors related to the patient that requires treatment.
Dosage and administration are adjusted to provide sufficient levels of the active ingredient, ie, to maintain the desired effect. Factors to consider include disease severity, general patient health, age, weight, gender, diet, time and frequency of administration, concomitant medications,
Includes response sensitivity, and response to treatment. Long-acting pharmaceutical compositions may be given every 3 to 4 days, every week, or 2 or 3 days depending on half-life and clearance rate of the particular formulation.
It may be administered once a week.

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

Diagnosis In another embodiment, an antibody that specifically binds to CIRYP may be used to diagnose a disease characterized by expression of CIRYP or to treat a patient receiving treatment with CIRYP or an agonist, antagonist or inhibitor of CIRYP. It can be used in assays for monitoring. Antibodies useful for diagnostic purposes can be made in a manner similar to that for therapeutics described above. Assays for the diagnosis of CIRYP include methods using antibodies and labels to detect CIRYP in extracts of human body fluids, cells or tissues. Antibodies can be used with or without modification, and can be labeled by binding, either covalently or non-covalently, to a reporter molecule. A variety of reporter molecules known to those of skill in the art may be used, some of which are described above.

Various protocols for measuring CIRYP are known in the art, including ELISA, RIA and FACS, and provide the basis for diagnosing changes in CIRYP expression or levels of abnormalities . The normal, or standard, value of CIRYP expression is the binding of CIRYP antibodies to body fluids or cell extracts obtained from normal mammalian (eg, human) patients under conditions suitable for complex formation. Can be established. The standard amount of complex formation can be quantified using various methods such as photometric means. The amount of CIRYP expressed in control and diseased samples from the patient's biopsy tissue was
Compare with standard value. Establish parameters for disease diagnosis by deviation between standard values and patient values.

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

In one embodiment, the nucleic acid sequence encoding CIRYP is identified using hybridization with a PCR probe capable of detecting a polynucleotide sequence, including genomic sequences, encoding CIRYP or a closely related molecule. it can. The specificity of the probe, ie, whether the probe is derived from a very highly specific region (eg, the 5 'regulatory region) or a less specific region (eg, a conserved motif). And the stringency of hybridization or amplification (maximum, high, medium, or low)
It is determined whether only naturally occurring sequences encoding CIRYP are identified, or alleles and related sequences are also identified.

[0164] Probes can also be used to detect related sequences and should preferably have at least 50% sequence identity to any sequence encoding CIRYP. The hybridization probe of the present invention may be DNA or RNA, or derived from the sequence of SEQ ID NO: 3-4, or derived from the sequence of the genome including introns, enhancers, and promoters of the CIRYP gene. .

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

[0166] The polynucleotide sequence encoding CIRYP can be used for diagnosis of a disease associated with expression of CIRYP. Examples of such diseases include, but are not limited to, abnormal prolactin production, infertility including tubal abnormalities, abnormal ovulation, and endometriosis, disrupted estrous cycle, disrupted menstrual cycle, Cystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial and ovarian tumors, uterine fibroids, autoimmune abnormalities, ectopic pregnancy, and teratogenesis, breast cancer, breast fibrocystic disease and hypertrophy, and spermatogenesis Confusion, abnormal sperm physiology, testicular cancer, prostate cancer, benign prostatic hyperplasia, prostatitis, Peyronie's disease (P
eyronie's disease), reproductive disorders such as male breast and gynecomastia; and dysphagia, peptic esophagitis, esophageal spasm, esophageal stricture, esophageal cancer, indigestion, digestive disorders, gastritis, stomach cancer, anorexia, nausea Vomiting, gastroparesis, edema of sinus or pylorus, abdominal angina, heartburn, gastroenteritis, ileus, intestinal infection, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic cancer, biliary disease, hepatitis Hyperbilirubinemia, cirrhosis, passive congestion of the liver, hepatome, infectious colitis, ulcerative colitis, ulcerative proctitis, Crohn's disease, Whipple disease, Mallory-Weiss syndrome, colon cancer, colonic obstruction, Irritable bowel syndrome, short small bowel syndrome, diarrhea, constipation, gastrointestinal bleeding and acquired immunodeficiency syndrome (
AIDS) gastrointestinal disorders; immobility, Alzheimer's disease, amnesia, amyotrophic lateral sclerosis, bipolar disorder, catatonia, brain tumor, dementia, depression, diabetic neuropathy, Down syndrome, extrapyramidal terminal defect syndrome Dystonia, epilepsy, Huntington's disease, peripheral nervous disease, multiple sclerosis, neuropyramidosis, Parkinson's disease, paranoid psychosis, postherpetic neuralgia, schizophrenia, seasonal affective disorder;
SAD) and neurological disorders such as Tourette's disease; and tubular acidosis, anemia,
Cushing's syndrome, chondrodysplasia dwarfism, Duchenne-Becker muscular dystrophy, epilepsy, gonad dysplasia, WAGR syndrome (Wilms' tumor, aniridia, genitourinary dysfunction, mental retardation), Smith-Magenis syndrome syndrome),
Syndrome dysplasia syndrome, hereditary mucosal epithelial dysplasia, hereditary keratosis, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, chorea (Syndenham's chorea) And seizure disorders such as cerebral palsy, spinal cord fission,
Includes developmental disorders such as anencephaly, cranial spondylolysis, congenital glaucoma, cataract, and sensory nerve hearing loss. The polynucleotide sequence encoding CIRYP can be analyzed by Southern blot or northern analysis using patient biopsy tissue or body fluids, dot blot or other membrane-based techniques, PCR techniques, dipstick assays, etc. )
Changes in CIRYP expression can be detected using, pin and multiformat ELISA-like assays and microarrays. Such qualitative or quantitative test methods are well known to those skilled in the art.

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

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

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

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

[0171] Additional diagnostic uses for oligonucleotides designed from the sequence encoding CIRYP include the use of PCR. These oligomers may be chemically synthesized, made with enzymes, or made in vitro . The oligomer is preferably a fragment of a polynucleotide encoding CIRYP, or CIRYP.
And a fragment of a polynucleotide complementary to the polynucleotide encoding is used under optimized conditions to identify a particular gene or condition. Oligomers can also be used under mild stringency conditions for the detection and / or quantification of closely related DNA or RNA sequences.

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

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

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

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

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

To enlarge the genetic map, physical mapping techniques such as in situ hybridization of chromosomal constructs and linkage analysis using established chromosomal markers can be used. In many cases, even if the number or arm of a particular human chromosome is unknown, the placement of the gene on the chromosome of another mammal, such as a mouse, will reveal relevant markers. By physical mapping, new sequences can be assigned to chromosomal arms, or parts thereof. This can provide valuable information to researchers searching for disease genes using positional cloning or other gene discovery techniques. Once a disease or syndrome has been identified in a particular genomic region (eg, 11q22-
Once incompletely positioned by the genetic linkage to telangiectasia to 23), any sequence mapped to that region may represent a relevant or regulatory gene for further study ( For example, Gatti, RA et al. (1988) Nat
ure 336: 577-580). In addition, using the nucleotide sequence of the present invention, it is also possible to detect a difference between the position of a chromosome of a normal person and the position of a chromosome caused by translocation, inversion or the like of a carrier or an affected individual.

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

[0179] Alternative drug screening techniques provide high-throughput screening for compounds with appropriate binding affinity for the protein of interest (eg,
(See Geysen et al. (1984) PCT application WO 84/03564). In this method, many different small test compounds are synthesized on a solid substrate. The test compound is reacted with CIRYP or a fragment thereof and washed. Next, combining CIRYP in a manner well known in the art.
Is detected. Also, purified CIRYP can be directly coated on a plate for use in the aforementioned drug screening techniques. Alternatively, the peptide can be captured and immobilized on a solid support using a non-neutralizing antibody.

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

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

[0182] Those skilled in the art will be able to utilize the present invention with the foregoing description without further explanation. Therefore, the specific preferred embodiments described below are merely illustrative and do not limit the invention.

All patent applications, patents, publications, and US patent applications [Attorney Specification No. PF-0659P (filed Jan. 15, 1999)] mentioned herein are referred to herein. Is a part of the present specification.

[0184]

【Example】

1. Preparation of cDNA Library The DUODNOT01 cDNA library was prepared using RNA isolated from duodenal tissue collected during radical pancreatoduodenectomy of a 41-year-old Caucasian woman. Family history included benign hypertension and skin tumors.

The UTRSNOT02 cDNA library was generated using RNA isolated from uterine tissue collected during vaginal hysterectomy of a 34 year old Caucasian woman. The patient's medical history included mitral valve disorders. Family history included gastric cancer, congenital heart abnormalities, irritable bowel syndrome, ulcerative colitis, colorectal cancer, cerebrovascular disease, type II diabetes and depression.

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

[0187] RNA phenol extraction and precipitation were repeated as necessary to increase RNA purity.
In some cases, RNA was treated with DNase. Most libraries use oligo
d (T) -bound paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatswort
hCA) or OLIGOTEX mRNA purification kit (QIAGEN) to isolate poly (A +) RNA. Alternatively, RNA was isolated directly from tissue lysates using another RNA isolation kit, such as the POLY (A) PURE mRNA purification kit (Ambion, Austin TX).

In some cases, RNA was provided to Stratagene to create a corresponding cDNA library. Otherwise, UNIZAP vector system (Stratagene) or
CDNA was synthesized using the SUPERSCRIPT plasmid system (Life Technologies) according to the recommended method known to those skilled in the art or a similar method to prepare a cDNA library. (See, for example, Ausubel, 1997, supra , units 5.1-6.6). Reverse transcription was initiated using oligo d (T) or random primers. Synthetic oligonucleotide adapter
After binding to the double-stranded cDNA, the cDNA was digested with the appropriate restriction enzyme (s). Most libraries use SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE
The size of the cDNA (300-1000 bp) was selected by CL4B column chromatography (Amersham Pharmacia Biotech) or agarose gel electrophoresis for separation. The cDNA was ligated to the compatible restriction enzyme site of a polylinker of an appropriate plasmid (eg, PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), or plNCY (Incyte Pharmaceuticals)). Recombinant plasmid, for example,
Competent E. coli cells such as XL1-Blue, XL1-BlueMRF or SOLR (Stratagene), or DH5α, DH10B or ElectroMAX DH10B (Life Technologies) were transformed.

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

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

3 Sequencing and Analysis The cDNA sequencing reaction was performed using standard methods or ABI CATALYST 800 (Perki
n-Elmer) High-throughput equipment such as thermal cycler or PTC-200 thermal cycler (MJ Research) and HYDRA microdispenser (Robbins Scientific)
Alternatively, the treatment was performed using MICROLAB 2200 (Hamilton) liquid transfer system. The cDNA sequencing reaction was performed using reagents provided by Amersham Pharmacia Biotech or ABI PRISM BIGDYE Terminator cycle sequencing ready.
It was prepared using the reagents supplied in an ABI sequencing kit such as a reaction kit (Perkin-Elmer). Electrophoretic fractionation of cDNA sequencing reactions and detection of labeled polynucleotides were performed using the MEGABACE 1000 DNA Sequencing System (Molecular Dynamics); ABI PRISM 373 or 377 Sequencing System (Perkin-Elmer) and standard ABI protocols. Combination with base pairing software; or other sequence analysis systems well known to those skilled in the art. cDNA
Reading frames in the sequence were identified using standard methods (Ausubel, 1997, supra , uni
t 7.7 review). Some of the cDNA sequences were selected and extended as described in 5 of this example.

Using a combination of software programs that utilize well-known algorithms,
Polynucleotide sequences obtained from NA sequencing were constructed and analyzed. Table 1 provides an overview of the tools, programs, and algorithms utilized, as well as appropriate descriptions, references, and threshold parameters. The first column of Table 1 is the tools, programs, and algorithms used, the second column is a brief description of them, and the third column is a reference (all of which are incorporated herein by reference. Column 4), and the fourth column contains the appropriate score, probability value,
And other parameters (the higher the score, the higher the homology between the two sequences).
The sequence was obtained from MACDNASIS PRO software (Hitachi Software Engineering, South S
an Francisco CA) and LASERGENE software (DNASTAR).
Polynucleotide and polypeptide sequence alignments are performed using the MEGALIGN multi-sequence alignment program (DNASTAR), which calculates the percent identity between aligned sequences.
) Was created using the default parameters specified by the clustal algorithm incorporated in.

Validation of polynucleotide sequences can be accomplished by removing vectors, linkers and polyA sequences and masking ambiguous base pairs using programs and algorithms based on BLAST, dynamic programming and dinucleotide nearest neighbor analysis. It was performed by doing. The sequences were then sequenced using GenBank primate, rodent, mammalian, vertebrate and eukaryotic databases using programs based on BLAST, FASTA and BLIMPS, and BLOCKS, PRINN.
Selected sequences from public databases such as TS, DOMO, PRODOM and PFAM were queried to obtain annotations. Sequences were assembled into full-length polynucleotides using programs based on Phred, Phrap and Consed and screened in reading frame using programs based on GeneMark, BLAST and FASTA. The full-length polynucleotide sequence is translated to derive the corresponding full-length amino acid sequence, which is then translated into the GenBank database (described above), SwissProt, BLOCKS, PRIN
Analyzed by querying protein family databases such as TS, DOMO, PRODOM, Prosite and PFAM based on Hidden Markov Model (HMM). HMM
Is a probabilistic approach to analyzing the common primary structure of gene families (see, eg, Eddy, SR (1996) Cur. Opin. Str. Biol. 6: 361-365).

The programs described above for the construction and analysis of full-length polynucleotide and amino acid sequences were also used to identify fragments of the polynucleotide sequence from SEQ ID NO: 3-4. Fragments of about 20 to about 4000 nucleotides useful for hybridization and amplification have been described above in the " Invention " section.

4 Northern Analysis Northern analysis is an experimental technique used to detect the presence of gene transcripts, where a membrane bound to RNA from a particular cell type or tissue is labeled with a labeled membrane. Involved in hybridization with nucleotide sequences (eg,
Sambrook et al., Supra, ch.7; supra and Ausubel, FM et al., See ch.4 and 16).

Using a similar computer technique using BLAST, GenBank or LIFESEQ (I
Search for identical or related molecules in nucleotide databases such as ncyte Pharmaceuticals). This analysis is faster than many membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is classified as an exact match or similar. The search criteria are based on the product score defined below.
). (% Sequence match x% max BLAST score) / 100 The product score takes into account both the degree of similarity between the two sequences and the length of the sequence match. For example, for a product score of 40, the match is accurate to within 1-2% error,
A product score of 70 is an exact match. Similar molecules are usually identified by selecting those molecules that exhibit a product score between 15 and 40, but lower scores identify them as related molecules.

The results of the Northern analysis are reported as a distribution of the percentage of libraries in which transcripts encoding CIRYP occurred. The analysis includes the classification of cDNA libraries by organ / tissue and disease. Organ / tissue classifications include cardiovascular, dermal, developmental, endocrine, gastrointestinal, hematopoietic / immune, musculoskeletal, nervous, reproductive and urinary. Disease / symptom classifications include cancer, inflammation / trauma, cell proliferation, nerves and pooled. For each category, the libraries expressing the sequence of interest were counted and divided by the total number of libraries across categories. The expression specific to each tissue and the ratio of expression specific to a disease, disease or condition are shown in the description of the "invention".

5 Extension of the polynucleotide encoding CIRYP The full-length nucleic acid sequence of SEQ ID NO: 3-4 was generated by extending an appropriate fragment of the full-length molecule and using oligonucleotide primers designed from this fragment. did. One primer was synthesized to initiate 5 'extension of the known fragment and the other primer was synthesized to initiate 3' extension of the known fragment. The starting primer is OLIGO 4.
Using software (National Biosciences) or other suitable program, is about 22-30 nucleotides in length and has a GC content of 50% or more;
The cDNA was designed to anneal to the target sequence at a temperature of about 68-72 ° C.
Hairpin structure and any extension of nucleotides resulting in primer-primer dimer was avoided.

This sequence was extended using a selected human cDNA library. If more than one step is necessary or desired, an additional or nested set of primers is designed.

High fidelity amplification was performed by PCR using methods well known to those skilled in the art. PCR is
Performed in a 96-well plate using a PTC-200 thermal cycler (MJ Research, Inc.). The reaction mixture contains a DNA template, 200 nmol of each primer, a reaction buffer containing Mg ²⁺ , (NH ₄ ) ₂ SO ₄ and β-mercaptoethanol, and Taq DNA polymerase (
Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies) and Pfu
DNA polymerase (Stratagene). Primer set PCI A and PCI B
The following parameters were used for Step 1 94 ° C for 3 minutes Step 2 94 ° C for 15 seconds Step 3 60 ° C for 1 minute Step 4 68 ° C for 2 minutes Step 5 Repeat steps 2, 3 and 4 20 times Step 6 68 ° C for 5 minutes Step 7 Store at 4 ° C. or use the following parameters for primer set T7 and SK +. Step 1 94 ° C for 3 minutes Step 2 94 ° C for 15 seconds Step 3 57 ° C for 1 minute Step 4 68 ° C for 2 minutes Step 5 Repeat steps 2, 3, and 4 20 times Step 6 68 ° C for 5 minutes Step 7 Store at 4 ° C. The DNA concentration in each well is determined using 0.5 μl of undiluted PCR product and 100 μl of PICOGREEN quantification reagent (0.25% (v / v) PICOGREEN; Molecular Probes, Eu) dissolved in 1 × TE.
gene OR) was distributed to each well of an opaque fluorometer plate (Coming Costar, Acton MA) and measured so that DNA could bind to the reagent. Put this plate in Fluor
Scan with oskan II (Labsystems Oy, Helsinki, Finland) to measure sample fluorescence and quantify DNA concentration. Aliquot 5-10 μl aliquots of the reaction mixture
Analyze by electrophoresis on a% agarose minigel to determine which reaction has successfully extended the sequence.

The extended nucleotides were desalted and concentrated, transferred to 384-well plates, and CviJI choleravirus endonuclease (Molecular Biology Research, Madison WI).
And sonication or shearing was performed before religation to the pUC18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on a low concentration (0.6-0.8%) agarose gel, the fragments were excised, and the agar was digested with Agar ACE (Promega). The expanded clone is used for T4 ligase (New Eng
land Biolabs, Beverly MA) using the pUC 18 vector (Amersham Pharmacia Bio
tech) and treated with Pfu DNA polymerase (Stratagene) to fill in restriction site overhangs and transfected into competent E. coli cells. Transfected cells are selected in media containing antibiotics, individual colonies are selected and LB
The cells were cultured overnight at 37 ° C. in a 384-well plate of a / 2 × carb culture solution.

The cells were lysed and Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu
DNA was amplified by PCR using DNA polymerase (Stratagene) with the following parameters. Step 1 94 ° C for 3 minutes Step 2 94 ° C for 15 seconds Step 3 60 ° C for 1 minute Step 4 72 ° C for 2 minutes Step 5 Repeat steps 2, 3, and 4 29 times Step 6 72 ° C for 5 minutes Step 7 Store at 4 ° C. DNA was quantified with PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recovery were re-amplified under the above conditions. Dilute the sample with 20% dimethylsulphoxide (1: 2, v / v) and use DYENAMIC energy trans
fer sequencing primer and DYENAMIC DIRECT kit (Amersham Pharmacia
Biotech) or ABI PRISM BIGDYE Terminator cycle sequencing ready reac
The sequence was determined using the tion kit (Perkin-Elmer).

Similarly, using the nucleotide sequence of SEQ ID NO: 3-4, the above procedure, oligonucleotides designed for 5 ′ extension, and the appropriate genomic library to obtain 5 ′ regulatory sequences.

6 Labeling and Use of Individual Hybridization Probes Using the hybridization probes obtained from SEQ ID NO: 3-4, cDNA
Genomic DNA or mRNA. Although the labeling of oligonucleotides of about 20 base pairs is specifically described, generally the same procedure is used for larger nucleotide fragments. Oligonucleotides can be transferred to OLIGO 4.06 software (National
Biosciences) and 50 pmol of each oligomer, 250 μCi of [γ- ³² P] adenosine triphosphate (Amersham Pharmacia Biot.
ech) and T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled oligonucleotide is substantially purified using a SEPHADEX G-25 ultrafine molecular size exclusion dextran bead column (Amersham Pharmacia Biotech). Aliquots containing 10 ⁷ counts of labeled probe per minute were digested with one of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba1, or Pvu II (DuPont NEN). Used for typical membrane-based hybridization analysis of human genomic DNA.

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. The blot is washed sequentially at room temperature under increasingly stringent conditions up to 0.1 × sodium citrate saline and 0.5% sodium dodecyl sulfate to remove non-specific signals. Hybridization patterns are visualized and compared using autoradiography or alternative imaging means.

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

The full-length cDNA, expressed sequence tags (ESTs), or fragments thereof can include elements of a microarray. Suitable fragments for hybridization can be selected using software such as LASERGENE software (DNASTAR) well known to those skilled in the art. A full-length cDNA, EST, or a fragment thereof corresponding to one of the nucleotide sequences of the present invention, or a random selection from a cDNA library relevant to the present invention, is applied to a suitable substrate such as a glass slide. To place. By treating the cDNA thermally and chemically after, for example, UV crosslinking and further drying,
Fix to glass slides (eg, Schena, M. et al. (1995) Science 270: 467-47).
0; and Shalon, D. et al. (1996) Genome Res. 6: 639-645). A fluorescent probe is prepared and used for hybridization with an element on a substrate. The substrate is analyzed by the method described above.

8. Complementary polynucleotide The sequence complementary to the sequence encoding CIRYP, or any portion thereof, may be replaced with a naturally occurring C
Used to detect and reduce, ie suppress, the expression of IRYP. Although the use of oligonucleotides containing about 15-30 base pairs is described, essentially the same method is used for smaller or larger sequence fragments. OLIGO 4.06
Design the appropriate oligonucleotides using the software and the coding sequence of CIRYP. To repress transcription, complement oligonucleotides with the most unique 5 '
Designed from sequence and used to prevent binding of the promoter to the coding sequence. To suppress translation, complementary oligonucleotides are designed to prevent ribosome binding to the transcript encoding CIRYP.

9. Expression of CIRYP Expression and purification of CIRYP is performed using a bacterial or virus based expression system. For expression of CIRYP in bacteria, the cDNA is subcloned into an appropriate vector containing an antimicrobial resistance gene and an inducible promoter that increases the level of cDNA transcription. Examples of such promoters include, but are not limited to, the T5 or T7 bacteriophage promoter associated with the lac operator regulatory element and the trp-lac (tac) hybrid promoter. The recombinant vector, BL
Transform into a suitable bacterial host such as 21 (DE3). Antibiotic-resistant bacteria express CIRYP by induction with isopropyl β-D thiogalactopyranoside (IPTG).
Expression of CIRYP in eukaryotic cells is achieved by infecting insect or mammalian cell lines with Autographica californica nucleopolyhedrovirus (AcMNPV), commonly known as baculovirus. The non-essential polyhedrin gene of the baculovirus is replaced with a cDNA encoding CIRYP by either homologous recombination or bacterial-mediated gene transfer involving a transfer plasmid intermediate. Virus infectivity is maintained and strong polyhedrin promoters drive high levels of cDNA transcription. In many cases, the recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells, but in some cases to infect human hepatocytes. The latter infection requires additional genetic alterations to the baculovirus. (For example, Engelhard. EK et al. (1994) Proc. Natl. Acad. Sci. US
A 91: 3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7: 1937-1945).

[0210] In most expression systems, CIRYP is, for example, glutathione S-transferase (
GST), or a peptide epitope tag such as FLAG or 6-His, which is synthesized as a fusion protein, allowing for rapid, one-step affinity-based purification of the recombinant fusion protein from crude cell lysates. GST, a 26 kilodalton enzyme from Schistosoma japonicum, allows the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amer
sham Pharmacia Biotech). After purification, GST from CIRYP at specific operating sites
The part can be proteolytically cut. FLAG, an eight amino acid peptide
Allows immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues, allows for purification on metal chelating resins (QIAGEN). Ausubel (1995, earlier ) describes methods for protein expression and purification.
Id , ch 10 and 16). Purified CIRY obtained by these methods
P can be used directly in the following assays.

10 Demonstration of CIRYP Activity The electromobility shift assay (EMSA) measures CIRP transcription by measuring activation of CIRYP transcription in response to changes in light and dark states.
Demonstrate YP activity. EMSA can be used to detect DNA-protein complexes, and radiolabeled or chemically labeled DNA sequences can be incubated with a particular protein. The resulting conjugate was separated on a polyacrylamide gel and visualized by autoradiography. If you keep the animals constantly in the dark,
DNA binding activity to the promoter region of a given circadian rhythm-related gene decreases. Thus, the binding of the transcriptional activator to the promoter region of CIRYP would be expected to vary depending on the amount of light exposure, which is directly proportional to CIRYP activity.

11 Functional Assays The function of CIRYP was measured at physiologically elevated levels of C in mammalian cell culture systems.
Assay by expression of the sequence encoding IRYP. The cDNA is subcloned into a mammalian expression vector containing a strong promoter that expresses the cDNA at high levels. Selected vectors include pCMV SPORT (Life Technologie) and pCR3.1 (Inv
itrogen, Carlsbad, CA), each of which contains the cytomegalovirus promoter. 5-10 μg of the recombinant vector is transfected instantaneously, for example, into an endothelial or hematopoietic cell line by liposome preparation or electroporation. Also,
1-2 μg of additional plasmid containing the sequence encoding the labeled protein is co-transfected. Expression of the labeled protein allows one to distinguish between transfected and non-transfected cells and to reliably predict expression of cDNA from a recombinant vector. Selected fluorescent proteins include green fluorescent protein (GFP;
Clontech), CD64, or CD64-GFP fusion protein. Automated laser optics based technology, flow cytometry (FCM), is used to identify transfected cells expressing GFP or CD64-GFP and to characterize the cells (eg,
Apoptotic status). FCM detects and quantifies the uptake of fluorescent molecules that diagnose the phenomenon of preceding or simultaneous cell death. These phenomena include:
Changes in nuclear DNA content as measured by staining DNA with propidium iodide, down-regulation of DNA synthesis as measured by reduced uptake of bromodeoxyuridine, cell surface as measured by reactivity with specific antibodies And changes in the expression of protein in cells, as well as changes in plasma membrane composition as measured by binding of fluorescein-bound annexin V protein to the cell surface. For flow cytometry methods, see Ormerod, MG (1994) Flow Cytometry, Oxford, New
York, NY.

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

Production of 12 CIRYP-specific antibodies Polyacrylamide gel electrophoresis (PAGE; see, eg, Harrington, MG (1990
Rabbits are immunized according to standard protocols to produce antibodies with CIRYP, which is substantially purified using Methods Enzymol. 182: 488-495) or other purification techniques.

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

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

13 Purification of Native CIRYP Using Specific Antibodies Natural or recombinant CIRYP is substantially purified by immunoaffinity chromatography using antibodies specific for CIRYP. Immunoaffinity columns are made by covalently linking the CIRYP antibody to an activated chromatography resin such as CNBr-activated Sepharose (Amersham Pharmacia Biotech). After bonding, block and wash the resin according to the manufacturer's instructions.

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

14 Identification of molecules that interact with CIRYP Label CIRYP or a biologically active fragment thereof with ¹²⁵ I Bolton-Hunter reagent (eg, Bolton AE and WM Hunter (1973) Biochem. 133: 52
9-539). Candidate molecules previously placed in the wells of a multiwell plate are incubated with labeled CIRYP, washed, and any wells with labeled CIRYP complexes are assayed. Using the data obtained at the various CIRYP concentrations, values for binding, affinity and number of CIRYP to the candidate molecule are calculated.

[0220] Those skilled in the art will be able to make various modifications of the methods and systems described herein without departing from the scope and spirit of the invention. Although the invention has been described with respect to particular preferred embodiments, it is to be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the claims.

Brief Description of the Tables Table 1 shows the tools, programs and algorithms used for the analysis of CIRYP, along with appropriate descriptions, references and parameter thresholds.

[Sequence list]

[Brief description of the drawings]

FIG. 1A shows the amino acid sequence (SEQ ID NO: 1) and the nucleic acid sequence (SEQ ID NO: 3) of CIRYP-1. MACDNASIS PRO software (Hitachi Software Enginee
ring, South San Francisco CA).

FIG. 1B shows the amino acid sequence (SEQ ID NO: 1) and the nucleic acid sequence (SEQ ID NO: 3) of CIRYP-1. MACDNASIS PRO software was used to create the alignment.

FIG. 1C shows the amino acid sequence (SEQ ID NO: 1) and the nucleic acid sequence (SEQ ID NO: 3) of CIRYP-1. MACDNASIS PRO software was used to create the alignment.

FIG. 1D shows the amino acid sequence (SEQ ID NO: 1) and the nucleic acid sequence (SEQ ID NO: 3) of CIRYP-1. MACDNASIS PRO software was used to create the alignment.

FIG. 2A shows the amino acid sequence (SEQ ID NO: 2) and the nucleic acid sequence (SEQ ID NO: 4) of CIRYP-2. MACDNASIS PRO software (Hitachi Software Enginee
ring).

FIG. 2B shows the amino acid sequence (SEQ ID NO: 2) and the nucleic acid sequence (SEQ ID NO: 4) of CIRYP-2. MACDNASIS PRO software was used to create the alignment.

FIG. 2C shows the amino acid sequence (SEQ ID NO: 2) and the nucleic acid sequence (SEQ ID NO: 4) of CIRYP-2. MACDNASIS PRO software was used to create the alignment.

FIG. 3A: CIRYP-2 (Incyte Clone ID 2267905; SEQ ID NO: 2), CLK-1 (GI 3415017; SE
QID NO: 6), CIRYP-1 (Incyte Clone ID 1581473; SEQ ID NO: 1) and Dreg-2 (
GI 1561732; SEQ ID NO: 5).
The alignment was prepared using a multi-sequence alignment program (DNASTAR, Madison WI) of LASERGENE software.

FIG. 3B: CIRYP-2 (Incyte Clone ID 2267905; SEQ ID NO: 2), CLK-1 (GI 3415017; SE
QID NO: 6), CIRYP-1 (Incyte Clone ID 1581473; SEQ ID NO: 1) and Dreg-2 (
GI 1561732; SEQ ID NO: 5).
The alignment was prepared using a multi-sequence alignment program (DNASTAR, Madison WI) of LASERGENE software.

[Table 1]

[Table 2]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 1/16 A61P 5/14 4C084 1/18 13/02 4C085 5/14 13/08 4H045 13/02 15 / 00 13/08 15/08 15/00 19/00 15/08 21/00 19/00 25/00 101 21/00 25/08 25/00 101 25/14 25/08 25/16 25/14 25 / 18 25/16 25/20 25/18 25/28 25/20 27/06 25/28 27/12 27/06 27/16 27/12 31/18 27/16 35/00 31/18 37/02 35/00 C07K 14/47 37/02 16/18 C07K 14/47 C12N 1/15 16/18 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 C12Q 1/68 A 5/10 G01N 33/15 Z C12P 21/02 33/50 Z C12Q 1/68 33/53 M G01N 33/15 33/566 33/50 C12N 15/00 ZNAA 33/53 5/00 A 33/566 A61K 37/02 (81) Designated country EP (AT, BE, CH, CY, D E, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ) , MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI , GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, G, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Tang, Wy Tom San Jose Runwick Court 95118, California, United States of America 4230 (72) Inventor Ryu, Dung Aina M. 95136, California, United States San Jose Park Belmont Place 55 (72) Inventor Azimzi, Yalda 94545, California, USA Hayward Rock Springs Drive 2045 F-term (reference) 2G045 AA25 AA35 BB20 CA26 CB03 DA12 DA13 DA14 DA36 FB02 FB03 FB06 FB07 4B024 AA01 AA11 BA80 CA04 DA02 EA04 FA02 GA11 HA12 4B063 QA19 QA20 QQ43 QR08 QR33 QR42 QR56 QS25 QS34 QX02 4B064 AG01 CA10 ACA4A02 ACO4 AA07 AA17 BA01 BA08 BA22 BA23 BA44 CA17 CA53 CA56 CA59 CA62 DC50 NA14 ZA022 ZA052 ZA062 ZA162 ZA182 ZA202 ZA 332 ZA342 ZA662 ZA682 ZA712 ZA752 ZA812 ZA942 ZA962 ZB072 ZB262 ZC062 ZC352 ZC552 4C085 AA03 CC01 CC21 DD23 EE01 GG01 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA75 EA20 EA50 FA72 FA74

Claims (23)

[Claims] 1. An isolated polypeptide, comprising: (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2; (b) an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2. A naturally occurring amino acid sequence having at least 90% sequence identity to the amino acid sequence to be determined; (c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2; Or (d) a polypeptide comprising an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2. 2. The isolated polypeptide of claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2. 3. An isolated polynucleotide encoding the polypeptide of claim 1. 4. The isolated polynucleotide of claim 3, comprising a sequence selected from the group consisting of SEQ ID NOs: 3 and 4. 5. A recombinant polynucleotide comprising a promoter sequence operably linked to the polynucleotide of claim 3. 6. A cell transformed with the recombinant polynucleotide of claim 5. 7. A genetic transformant comprising the recombinant polynucleotide according to claim 5. 8. The method for producing the polypeptide according to claim 1, wherein (a) culturing the cell under conditions suitable for expression of the polypeptide, wherein the cell comprises: Culturing, transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to the polynucleotide encoding the polypeptide of (b), and (b) recovering the polypeptide so expressed A method for producing a polypeptide, characterized in that: 9. An isolated antibody that specifically binds to the polypeptide of claim 1. 10. An isolated polynucleotide, comprising: (a) a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 3 and 4; (b) a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 3 and 4. A naturally occurring polynucleotide sequence having at least 70% sequence identity to the selected polynucleotide sequence, (c) a polynucleotide sequence complementary to (a), or (d) a polynucleotide sequence to (b) Isolated polynucleotide comprising a complementary polynucleotide sequence. 11. An isolated polynucleotide comprising at least 60 contiguous nucleotides of the polynucleotide of claim 10. 12. A method for detecting a target polynucleotide in a sample comprising the polynucleotide sequence of claim 10, wherein (a) the sample is complementary to the target polynucleotide in the sample. In the process of hybridizing with a probe comprising at least 16 contiguous nucleotides containing a unique sequence, under the conditions that a hybridization complex is formed between the probe and the target polynucleotide, Specifically hybridizes with the target polynucleotide, and (b) detecting whether or not the hybridization complex is present, and if present, optionally determining the amount thereof. And a detecting step. 13. The method of claim 12, wherein said probe comprises at least 30 contiguous nucleotides. 14. The method of claim 12, wherein said probe comprises at least 60 contiguous nucleotides. 15. A pharmaceutical composition comprising an effective amount of the polypeptide of claim 1 and a pharmaceutically acceptable excipient. 16. A method for treating a disease or condition associated with reduced expression of functional CIRYP, comprising administering the pharmaceutical composition of claim 15 to a patient in need of such treatment. . 17. A method for screening a compound effective as an agonist of the polypeptide of claim 1, comprising: (a) exposing a sample containing the polypeptide of claim 1 to the compound; and (b) Detecting the activity of the agonist in the sample. 18. A pharmaceutical composition comprising an agonist compound identified by the method of claim 17 and a pharmaceutically acceptable excipient. 19. A method for treating a disease or condition associated with reduced expression of functional CIRYP, comprising administering a pharmaceutical composition of claim 18 to a patient in need of such treatment. . 20. A method for screening a compound effective as an antagonist of the polypeptide of claim 1, comprising: (a) exposing a sample containing the polypeptide of claim 1 to the compound; and (b) Detecting the activity of the antagonist in the sample. 21. A pharmaceutical composition comprising an antagonist compound identified by the method of claim 20 and a pharmaceutically acceptable excipient. 22. A method of treating a disease or condition associated with overexpression of functional CIRYP, comprising administering to a patient in need of such treatment a pharmaceutical composition of claim 21. 23. A method for screening a compound effective for altering expression of a target polypeptide comprising the sequence of claim 4, comprising: (a) exposing a sample comprising said target polypeptide to said compound. A screening method, comprising: (b) detecting a change in expression of the target polypeptide.